The UK's First Case of BSE Was Identified in 1986, and the Outbreak

SCIENTIFIC SESSIONS 47th ANNUAL MEETING AMERICAN ASSOCIATION OF VETERINARY LABORATORY DIAGNOSTICIANS

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AAVLD First Plenary Session Saturday, October 23, 2004 8:00 a.m. – 11:15 a.m. Guilford B

TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES

Moderator: Gary Osweiler Page 8:00 WELCOME AND ANNOUNCEMENTS

8:05 BSE Surveillance in the UK – Angus Wear 25

8:45 Molecular Characterization of Prion Isolates from Livestock and Cervids – Juergen Richt 26

9:25 Break – 15 Minutes

9:40 Diagnosis of Transmissible Spongiform Encephalopathies – Tim Baszler 27

10:20 Ecology, Epidemiology and Control of CWD – Elizabeth Williams 28

11:00 Panel, Questions

11:15 AAVLD HOUSE OF DELEGATES

AAVLD First Plenary Session

TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES

Sponsored By

BIO-RAD LABORATORIES

1 Transmissible Spongiform Encephalopathy/Virology Scientific Session Saturday October 23, 2004 1:00 p.m. – 3:00 p.m. Auditorium III

Moderators: Tim Baszler and James Evermann Page 1:00 p.m. Development of an Automated High-Throughput Screening Enzyme-Linked 31 Immunosorbent Assay for Bovine Spongiform Encephalopathy in Cattle and Chronic Wasting Disease in Elk and Deer in Alberta Canada - J.T.Y. Wu, E.Y.W. Chow, L.S.Y. Wong, M. Longson, C. Onderka C. Wittmeier and E.E. Bowlby

1:15 p.m. Western-blot Techniques with Improved Sensitivities for Confirming the Diagnosis of 32 BSE, Scrapie and CWD – J.M. Bilheude, J.P. Bourgeois, M. Feyssaguet, G. Nespoulous, E. Comoy, J.P. Deslys, S. Simon, N. Morel, Y. Frobert and J. Grassi

1:30 p.m. A Second-Generation Method for Accurately Detecting Bovine Spongiform 33 Encephalopathy – V. Leathers, R. Toomik, K. Velek, L. Plourde, S. Koller, M. Ryle, C. Wong, L. Estey

1:45 p.m. Break

2:00 p.m. Polioencephalitis in Ruminants in the UK - S.F.E. Scholes, H. Ainsworth, C. Bidewell, A. 34 Colloff, A. Dawson, P. Duff, R.J. Higgins, P.J. Watson, M. Wessels, K. Whitaker, P.F. Nettleton

2:15 p.m. Evaluation of a Blocking ELISA for Detection of West Nile Virus Antibodies in Horses, 35 Chickens and Wild Ring-Necked Pheasants - E.M. Zhou, D.K. Kirby, K. Lin, D.L. Reynolds, J. Fabios, K.B. Platt, T. Bogenschutz and D.L. Garner

2:30 p.m. Detection and Characterization of Naturally Occurring West Nile Virus Infection in a Wild 36 Female Turkey - Z. Zhang, F. Wilson, R. Read, L. Pace, S. Zhang

2:45 p.m. Hemorrhagic Bowel Syndrome in Dairy Cattle: Preliminary Results from a Case Control 37 Study - D.C. Sockett, A.I. Brower, K.L. Woods, R.E. Porter, P.N. Bochsler, S.M. Godden, N.E. Forsberg, S.B. Puntenney and Y. Wang

2 Epidemiology Scientific Session Saturday October 23, 2004 1:00 p.m. – 3:00 p.m. Guilford C

Moderators: Francois Elvinger and Claudia Munoz-Zanzi Page 1:00 p.m. Serologic Response of Horses Vaccinated Against WNV and Those Recovering from 41 Naturally Occurring Disease - A. Davidson, J. Traub-Dargatz, R. Dewell, R. Rodeheaver, J. Stricklin, E. Ostlund, D. Pederson, S. Albers, R. Forde, T. Brigner, S. Roach, R.Long, R. Callan, R. Moorhead, and M. Salman

1:15 p.m. Characteristics and Efficiency of Diagnostic Tests Used in the 2002 LowPath Avian 42 Influenza Outbreak in Virginia - F. Elvinger, D. L. Ward, F.W. Pierson, B.L. Akey, B.A. Porter-Spalding, D. Senne

1:30 p.m. Use of Veterinary Diagnostic Laboratory Data to Rapidly Detect Infectious Disease 43 Outbreaks - H. Kassenborg, B. Miller, K. Green, M. Thurn, J. Griffith , and K. Smith

1:45 p.m. Effectiveness of Pooling Strategies for Detection of Johne’s Disease Infected Cattle 44 Herds - C. Munoz-Zanzi, S. Wells

2:00 p.m. The BioPortal Information System for Foot-and-Mouth Disease Surveillance - A.M. 45 Perez, M.C. Thurmond, W.O. Johnson, T.E. Carpenter, P.W. Grant, R.B. Garabed, B.A. Melbourne, Y-K. Choi, A.J. Branscum, L.D. Benning, T.W. Bates, C. Lynch, M. Eidson, M. Ascher, I. Gotham, and H. Chen

2:15 p.m. Epidemiological Models for Global Surveillance of Foot-and-Mouth Disease - A.M. 46 Perez, M.C. Thurmond, T.E. Carpenter, T.W. Bates, W.O. Johnson, B.A. Melbourne, S.A. Aslam, R.B.Garabed, Y.ku Choi, A.J. Branscum, M.L. Gallego, and P.W. Grant

2:30 p.m. Foot-and-Mouth Disease Surveillance Involving the Testing of Bulk Milk Tank 47 Samples - M.C. Thurmond and A.M. Perez

2:45 p.m. The 13th Edition of the UN Model Regulations on Transport of Dangerous Goods: 48 Effects on Veterinary Diagnostic Laboratories - L.J. Thompson

3 Pathology Scientific Session Saturday October 23, 2004 1:00 p.m. – 3:00 p.m. Auditorium II

Moderators: H. L. Shivaprasad and Donal O’Toole Page 1:00 p.m. Hair-loss Syndrome in Pacific Northwest Black-tailed Deer - R.J. Bildfell, J.W. Mertins, 51 J.A. Mortenson and D.F. Cottam

1:15 p.m. Avian Tuberculosis and Erysipelas Infection in Chukars - A.S. Dhillon - D. Schaberg; 52 S.K. Weber; D.V. Bandli and F. Wier

1:30 p.m. Hepatic Cirrhosis and Hemochromatosis in an Onager (Equus hemionus) - J.R. Hayes, R.A. 53 Gandolf, S. D. Grimes, J. Thilsted, and M. Atkinson

1:45 p.m. Pathology of West Nile Virus Disease in North Carolina Alligators - P.G. Moisan, L.F. 54 Humphries, S.J. Page, and J.M. Law

2:00 p.m. Successful Experimental Induction of Acute Malignant Catarrhal Fever in Bison Using 55 Aerosols of Ovine Nasal Mucus Containing Ovine Herpesvirus-2 (OvHV-2) - D. O' Toole, N.S. Taus, W.C. Davis, T.B. Crawford, and H. Li

2:15 p.m. Ulcerative Enteritis Associated with Clostridium perfringens Type A in Bobwhite Quail 56 (Colinus virginianus) - H.L. Shivaprasad, R. Kokka, R. Crespo, G. Songer, P. Cortes and F. Uzal

2:30 p.m. Tongue Is An Excellent Sample for Parvoviral Diagnosis in Dogs and Cats - C.A. 57 McKnight*, M. Kiupel, A. Wise, and R. Maes

2:45 p.m. Rangelia vitalli Infection in Dogs: Epidemiological, Clinicopathological and 58 Ultrastructural Findings - A. P. Loretti*, S.S. Barros

*Graduate Student Competition

4 Toxicology Scientific Session Saturday October 23, 2004 1:00 p.m. – 3:00 p.m. Auditorium IV

Moderators: Robert Poppenga and Catherine Barr Page 1:00 p.m. Brain Concentrations of Macrolide Endectocides Associated with Cases of Intoxication 61 or Suspected Intoxication - R.H. Poppenga - I. Rudik-Miksa, and M.R. Cummings

1:15 p.m. The Use of LC/MS Methodology in the Diagnosis of Paraquat Poisoning - B. Puschner, 62 M.S. Filigenzi, E. Pratt, E.R. Tor

1:30 p.m. Determination of Liver Vitamin E: Stability and Matrix Distribution - I. Rudik-Miksa, 63 R.H. Poppenga, and M.R. Cummings

1:45 p.m. Screening of Erythropoietin, Recombinant Human Erythropoietin and 64 Darbepoietin-alpha in Horse Plasma - A.K Singh, S. Gupta, and A. Sage

2:00 p.m. Diagnosis of Taxus (Yew) Poisoning in a Horse - A.K. Tiwary*, B. Puschner, H. Kinde, 65 J. Reagor and E.R. Tor

2:15 p.m. Paresis and Death in Elk (Cervus elaphus) Due to Lichen Intoxication in South Central 66 Wyoming - W.E. Cook, M.F. Raisbeck, T.E. Cornish, E.S. Williams, B. Brown, and G. Hiatt

2:30 p.m. Toxicologic Surveillance of Search & Rescue Dogs Deployed to the World Trade 67 Center, The Pentagon, and The Staten Island Fresh Kills Landfill Sites - W.K. Rumbeiha, S.D. Fitzgerald, W.E. Braselton, C.M. Otto, A.B. Downend

2:45 p.m. Proteomics Approaches to Biomedical Problems - S. B. Hooser, C.R. Wilson 68

* Graduate Student Competition

5 Virology Scientific Session Saturday October 23, 2004 1:00 p.m. – 3:00 p.m. Guilford B

Moderators: Robert Fulton and Jane Christopher-Hennings Page 1:00 p.m. Disinfection of Coronavirus and Orthomyxoviruses in Liquid and on Solid Surfaces 71 by Various Disinfectants - J.M. Bieker-Hawkinson*, J. Anderson, R.D. Oberst, and S. Kapil

1:15 p.m. Serologic Response to Inactivated Avian Influenza A Vaccine in Turkeys - K.L. 72 Pabilonia*, Van Campen, H., Wooming, B., Podell, B.K. and Salman, M.D.

1:30 p.m. Initial Results Toward a Collaborative Evaluation of a Real-time Assay Reagent 73 Specific for the Detection of West Nile Virus - S. F. Sells* - D.J. Johnson, J.D. Callahan, M.L. Vickers, A.K.Maloy, W.M. Nelson, E.N. Ostlund, I. Stewart, J. Roberts, M. Sebastian, K. Grandfield, C. Jackson, K.B. Poonacha, C.B. Hong, N. Williams, R.C. Giles, D. Bolin, and L. Harrison

1:45 p.m. First Report of Spring Viremia of Carp Virus in Wild Common Carp (Cyprinus 74 carpio) in North America - A.L. Dikkeboom , C. Radi, S. Marquenski , M. Engel, A.E. Goodwin , K Way , D.M. Stone , C Longshaw , and K. Toohey- Kurth

2:00 p.m. Persistence of Bluetongue Virus in the Insect Vector and Its Implications for Disease 75 Control - J.O. Mecham, D.M. White, B.S. Drolet and W.C. Wilson

2:15 p.m. Foot and Mouth Disease in Bison: Laboratory Analyses of Serum, Blood, 76 Oesophageal-Pharyngeal Fluid, and Tissue Samples - H. Wang, G.B. Ward, M.Y. Deng, J.C. Rhyan, M. Yeh, M.L. Berninger, B. Donahue, J. Dio, S.A. Shawky, S.H. Wainwright, T.S. McKenna

2:30 p.m. Severe Egg Production Reduction in Turkeys Due to Avian Influenza Virus H3N2 - 77 Y. Zhang, B. Byrum, M. Saif, S. Grimes, S. Rajeev, C. Sarver, and L. Dorman

2:45 p.m. RT-PCR in the Newcastle Disease and Avian Flu Diagnosis - A. Panshin, E. 78 Shihmanter, I. Gissin, Y. Tendler

* Graduate Student Competition

Bacteriology Scientific Session Sunday October 24, 2004 8:00 a.m. – 12:00 p.m. Guilford A

Moderators: Brenda Love and Melissa Libal Page 8:00 a.m. Use of a Real-Time Polymerase Chain Reaction Assay for Detection of 81 Mycobacterium avium subsp. paratuberculosis from Bovine Fecal Samples and Confirmatory Cultures - L.A. Estey, P.R. Andersen, A. Asp, T. Bezold, D. Eugene and P. I. Tyrrell

8:15 a.m. Evaluation of Six Decontamination Protocols for the Isolation of M. avium subsp. 82 paratuberculosis from Bovine Feces - K. A. Johansen and J.B. Payeur

8:30 a.m. Development of Real Time Quantitative PCR Technology for the Identification of 83 Mycoplasma bovis in Bovine Milk - B.C. Love, F. Zambito, S.E. Myers, D.P. Shaw

8:45 a.m. Direct Evaluation of Bovine Fecal Samples for Mycobacterium avium subsp. 84 paratuberculosis by a Real-Time PCR Commercial Assay - B.L. Mangold, S.R. Weeks, T.I. Calvin, W.M. Nelson, and J. Christopher-Hennings

9:00 a.m. Optimization of an Algorithm for Laboratory Diagnosis of Mycobacterium avium 85 subsp. paratuberculosis in Bovine Feces Utilizing a Liquid Culture System - S. Rajeev, Y. Zhang, and B. Byrum

9:15 a.m. Culturing Mycobacterium avium spp paratuberculosis from Sheep Feces in the 86 United States - S. Robbe-Austerman, J.R. Stabel, B.N. Harris, K. Johansen, M.V. Palmer, J.B. Payeur

9:30 a.m. Evaluating the Accuracy of the IFN-γ ELISA and the Skin Test to Detect 87 Mycobacterium avium ssp. paratuberculosis (MAP) Infection in Sheep – S. Robbe-Austerman, J.R. Stabel, M.V. Palmer

9:45 a.m. BREAK AND POSTER SESSION

10:15 a.m. Development and Evaluation of a New PCR Test for the Detection - 88 Mycobacterium avium subsp. paratuberculosis - J.R. Stabel, J.P. Bannantine, and M. Paustian

10:30 a.m. A Technique for Enhanced Detection of Acid Fast Bacilli (AFB) (Mycobacterium 89 subsp avium paratuberculosis-MAP) in Liquid Culture - R.H. Whitlock, A. Monson, B. Aksim, S, McAdams, T. Fyock and R. Sweeney

10:45 a.m. Research Challenges for Brucellosis Eradication - P.H. Elzer 90

11:00 a.m. Strain Typing Brucella abortus Isolated from California and Florida Cattle Herds 91 by Using “HOOF-Prints” – a New DNA Fingerprinting Technique for Brucella – B.J. Bricker and D.R. Ewalt

11:15 a.m. Investigation of Reported Resistance of Streptococcus equi ssp. zooepidemicus to Trimethoprim-sulfamethoxazole in Horses - D.J. Feary, J. Traub-Dargatz, S. Roach, D.R. Hyatt, R. Jones, C. C. Wu, and P. S. Morley 92

11:30 a.m. Characterization of a Highly Antimicrobial Resistant Escherichia coli from 93 Newborn Calves with Colibacillosis in North Dakota - P.S. Gibbs, J. Foster, S.R. Petermann, M. Theis, T. Solseng, L.P. Schaan, D.F. Krogh, and G.J.C. Carlson

11:45 a.m. Genetic Variability of Moraxella (subgenus Moraxella) bovis and Moraxella 94 (subgenus Branhamella) ovis Field Strains Recovered from Cattle with Infectious Bovine Keratoconjunctivitis - P.S. Nabity*, H.E. Cerny, P.D. Fey, S.Hinkley

* Graduate Student Competition

8 Microbiology Scientific Session Sunday October 24, 2004 8:00 a.m. – 12:00 p.m. Guilford C

Moderators: Lindsay Oaks, Jr. and Carole Bolin Page 8:00 a.m. Experimental Infection of Reindeer (Rangifer tarandus) with Mycobacterium bovis: 97 Pathological and Immunological Findings - M.V. Palmer, W.R. Waters, T.C. Thacker, W.C. Stoffregen, R.E. Slaughter, S.L. Jones, J.E. Pitzer, and F.C. Minion

8:15 a.m. Survey of Antibiotic Resistance in Mycobacterium tuberculosis Strains Isolated from 98 Elephants - N.B. Harris, R.A. Osorio, and J.B. Payeur

8:30 a.m. Use of a New Competitive ELISA Procedure for the Serodiagnosis of Glanders 99 (Burkholderia mallei) Infections in Horses - J. B. Katz, T. O. Bunn, D. R. Kinker

8:45 a.m. Competitive ELISA for the Detection of Antibodies Against Foot-and-mouth Disease 100 Virus Using a Biotinylated 3ABC Recombinant Protein - A. Clavijo, E-M. Zhou, P. Kitching

9:00 a.m. Immune Response Differences in Serums from Bovine Herpesvirus-1 Vaccinated 101 Cattle: Dependence of Viral Strain - R. W. Fulton, L. J. Burge, J.M. d’ Offay, R. Funk, G. D. Weaver, H. Van Campen, and B.J. Johnson

9:15 a.m. Sequencing-Based Identification of a Novel Coronavirus in Ferrets with Epizootic 102 Catarrhal Enteritis (ECE) and Development of Molecular Diagnostic Tests for ECE A.G. Wise, M. Kiupel, C. Isenhour and R.K. Maes

9:30 a.m. Use of Antemortem Tests to Identify Cattle with Disseminated Mycobacterium avium 103 ssp. paratuberculosis Infection Detected by Postmortem Culture of 15 Tissues - H.L. Hirst, J.E. Lombard, M.C. Antognoli, M.M. Dennis, M.D.. Salman, , and F.B. Garry

9:45 a.m. BREAK AND POSTER SESSION

10:15 a.m. Apparent Seroprevalence of Anaplasmosis in Wild Ungulates from the Northwestern 104 and Northcentral United States - J. B. Katz

10:30 a.m. Detection of Neospora caninum DNA from Central Nervous System of Deer by 105 Nested PCR - L.G. Corbellini *, L. Xie, J.A. Galeota, B.W. Brodersen, D.J. Steffen

10:45 a.m. Outbreak of West Nile Virus in Farmed Alligators (Alligator mississippiensis) in 106 Southern North Carolina - L.F. Humphries *, S.J. Page, P.G. Moisan, J.M. Law

11:00 a.m. The Use of Environmental Sampling As a Herd Diagnostic Tool for Detecting 107 Mycobacterium avium subsp paratuberculosis on Minnesota Dairy Farms - E.A Raizman*, S.J. Wells, S.M. Godden, R.F. Bey, M.J. Oakes, D.C. Bentley, K.E. Olsen

11:15 a.m. Evaluation of Fluorescent Focus Neutralization Assay in Comparison with SVN, 108 ELISA and IFA for Serological Diagnosis of Porcine Reproductive and Respiratory Syndrome Virus Infection - D. K. Kirby and E.-M. Zhou

11:30 a.m. Reconstruction and Analysis of Eradication Efforts During the 2002-03 Outbreak of Exotic Newcastle Disease - R. Speers, M. Webb, B. Howell, M. Grund, C. Hughes, E. Myrus and J. Silverman 109

11:45 a.m. Patterns of Relationship in Emergency Response: An Exotic Newcastle Disease Case 110 Study - R. Werge, L. Cooper, and C. Cardona

* Graduate Student Competition

Molecular Diagnostics Scientific Session Sunday October 24, 2004 8:00 a.m. – 12:00 p.m. Auditorium III

Moderators: Sharon Hietala and Steven Kleiboeker Page 8:00 a.m. Simultaneous Detection of North American and European Porcine Reproductive and 113 Respiratory Syndrome Virus Using Real-time Quantitative RT-PCR - S.B. Kleiboeker, S.K. Schommer, S-M Lee, S. Watkins, W. Chittick, and D. Polson

8:15 a.m. Quantification of Porcine Reproductive and Respiratory Syndrome Virus in Boar Serum 114 and Semen - J. Christopher-Hennings, J. D. Callahan, Y. Fang, A. Wasilk, M. Dammen , T.A. Gay, M.E. Reos, E.A. Nelson, and W.M. Nelson

8:30 a.m. Application of Molecular Epidemiological Data to Support Forensic Disease Outbreak 115 Investigations: Evaluation Using the 2002-2003 Exotic Newcastle Disease Outbreak - B.M. Crossley, P.J. Hullinger, S.L. Messenger, E. Skowronski, K. Smith, and S.K. Hietala

8:45 a.m. High Throughput Viral RNA Isolation for Molecular Diagnosis - X. Fang, R. C. Willis, W. 116 Xu, Q. Hoang and M. Bounpheng

9:00 a.m. Fast and Simple Viral RNA Isolation for BVDV Molecular Diagnostics - Q. Hoang, R.C. 117 Willis, W. Xu, M.A. Bounpheng, and X. Fang

9:15 a.m. High Throughput Detection of Bluetongue Virus by a Taqman Real-time RT-PCR - M. A. 118 Jimenez-Clavero, E. San Miguel, M. J. Ruano, M. C. López, E. R. Gómez-Tejedor, C. Gómez-Tejedor

9:30 a. Detection of Foot-and-Mouth Disease in Samples from Afghanistan by a Portable Real- 119 m. Time RT-PCR Assay - K.R. Schumann, T.R. Beckham, L. Jackson, D. Couch, A.J. Eberling, A.C. Giuffre, S.J. Pauszek, J.M. Robida, M.L. Berninger, T.M. Sigafoose, M.A. Kenney, L. Rodriquez, T.S. McKenna, B.M. Martin

9:45 a.m. BREAK AND POSTER SESSION

10:15 a.m. Phylogeny-Based Multiplex Real-Time RT-PCR for Rapid Detection of Vesicular 120 Stomatitis Viruses NJ and IN-1 from Diverse Geographical Regions - Luis L. Rodriguez, Steve J. Pauszek, George Smoliga and William C. Wilson

10:30 a.m. Validation of a Real-Time RT-PCR for Vesicular Stomatitis Virus - G.J. Letchworth, C. 121 Jiménez, M. Herrero, T.E. Cornish, W.C. Wilson, G. Smoliga, S. Pauszek, C. Dornak, M. George, L.L. Rodriguez

10:45 a.m. Diagnostic Evaluation of a Portable Real-time Reverse Transcriptase PCR Assay for the 122 Detection of Classical Swine Fever - G. Risatti, L.G. Holinka, Z. Lu, G. Kutish, J.D. Callahan, W.M. Nelson, E. Brea Tió, and M.V. Borca

11 11:00 a.m. Comparison of Six RNA Extraction Methods for the Detection of Classical Swine Fever Virus by Real-time and Conventional Reverse Transcription-Polymerase Chain Reaction - M.Y. Deng, H. Wang, G.B. Ward, T.R. Beckham, and T.S. McKenna 123

11:15 a.m. Development of an Optimized Multiplex PCR to Detect Virulence Genes in 124 Enterotoxigenic Escherichia coli (ETEC) - L.L. Ruesch and D.H. Francis

11:30 a.m. Quantitative Real-Time PCR for the Detection of Mycobacterium avium subsp. 125 paratuberculosis in Bovine Fecal Samples - R.W. Sweeney, B.L. Mangold, S. McAdams, T. Calvin, R.H. Whitlock

11:45 a.m. Identification of Escherichia coli Flagellar Types by Restriction Fragment Analysis of 126 Amplified fliC Gene - C. Deb Roy, E. Roberts and M. A. Davis

* Graduate Student Competition

Molecular Diagnostic Session

Sponsored by

CEPHEID

Pathology Scientific Session Sunday October 24, 2004 8:00 a.m. – 12:00 p.m. Auditorium IV

Moderators: Michael Yaeger and David Steffen Page 8:00 a.m. Current Intentional Microbial Threats to Animal Agriculture and Their Relevance to 129 the AAVLD and the Veterinary Pathologist - T.M. Wilson

8:15 a.m. Pathologic and Toxicologic Surveillance of Search & Rescue Dogs Deployed to the 130 World Trade Center & Pentagon: Preliminary Findings - S.D. Fitzgerald, W.K. Rumbeiha, C.M. Otto, and A.B. Downend

8:30 a.m. Canine Coronavirus – Associated Mortality Without Evidence of Concurrent Canine 131 Parvoviral Infection - J.F. Evermann, J.R. Abbott, and S. Han

8:45 a.m. Fatal Mycobacteriosis with Hepatosplenomegaly in a Young Dog Due to 132 Mycobacterium avium - D. O’ Toole, S. Tharp, B.V. Thomsen, E. Tan, and J.B. Payeur

9:00 a.m. New Pathological Observations on Naturally Infected Cats with Physaloptera 133 praeputialis (Nematoda: Spirurida, Physalopteridae) - S. Naem, A.A. Farshid, and V.T. Marand

9:15 a.m. Microscopic Lesions in Cats with Colonic Tritrichomonas Infection - M.J. Yaeger, 134 J.L. Gookin

9:30 a.m. The Role of Immunostimulation on the Pathology of Post-weaning Multisystemic 135 Wasting Syndrome (PMWS) in Pigs Under Field Conditions - J. Haruna*, P. Hanna, D. Hurnik, B. Ikede, L. Miller and C. Yason

9:45 a.m. BREAK AND POSTER SESSION

10:15 a.m. Hemolytic Uremic and Edema Disease-like Syndrome in a Mare and Foal Associated 136 with Escherichia coli 0103 - D.H. Gould, A.H. Davidson, C.E Dickinson, M.E. Legare, D.R. Hyatt and C. DebRoy

10:30 a.m. Gizzard Hemorrhage and Erosion in Broiler Chickens at Hatch - F. J. Hoerr, J. J. 137 Giambrone, L. Li, T. Dormitorio, and D. Poole

10:45 a.m. Utilization of Real-Time PCR for the Detection of Haemophilus parasuis from Swine 138 Tissues - D. Jordan, K. Harmon, and L. Hoffman

11:00 a.m. The Association of Clostridium perfringens alpha and beta2 Toxins with Microscopic 139 Lesions and Diarrhea in Neonatal Swine - M.J. Yaeger and R Ellis

11:15 a.m. Enterotoxemia by Enterotoxin and Beta 2 Toxin Positive Clostridium perfringens 140 Type D in a Goat Kid - F.A. Uzal, D.J. Fisher, S. Sayeed, B.A. McClane, G. Songer, H.T. Trinh and M.E. Fernandez Miyakawa

11:30 a.m. Sarcoptic Mange in Raccoons in Michigan - S.D. Fitzgerald, T.M. Cooley, A. Murphy, M.K. Cosgrove, and B.A. King 141

11:45 a.m. Hypocalcemia of Hypovitaminosis D in Mature and Immature Pigs - Y.L. Jones, M. 142 Kiupel

* Graduate Student Competition

Virology Scientific Session Sunday October 24, 2004 8:00 a.m. – 12:00 p.m. Auditorium II

Moderators: K-J Yoon and Edward Dubovi Page 8:00 a.m. Assessment of the Stability of PRRS Virus RFLP Pattern During In Vivo Replication - 145 S.-H. Cha* and K.-J. Yoon

8:15 a.m. Relative Prevalence of Reassortant H1N1 Swine Influenza Viruses with Avian 146 Polymerase Genes and Classic H1N1 Viruses with Swine Polymerase Genes - B.H. Janke, K.M. Harmon, K-J.Yoon, G.A. Erickson, and R.J. Webby

8:30 a.m. Humoral Immune Responses of Pigs to SIV Infection and Vaccination - W.-I. Kim*, 147 W.-H. Wu, B.H. Janke, K.-J. Yoon

8:45 a.m. Characterization of a Reassortant Human/Swine H1N2 Influenza Virus Isolated from 148 Pigs in a U.S. Swine Herd - M.L. Gramer*, C. Mahlum-Wees, K.D. Rossow, R.J. Webby and S.M. Goyal

9:00 a.m. Nested PCR Detection and Duration of Porcine Circovirus Type 2 in Semen Samples 149 from Naturally Infected Boars - K. A. McIntosh*, J.C.S. Harding, J.A. Ellis and G.D. Appleyard

9:15 a.m. Ulcerative and Hemorrhagic Typhlocolitis in an Angus Heifer Associated with Natural 150 Bovine Enterovirus Type-1 Infection - U. Blas-Machado, M.J Boileau, J.T. Saliki, S.L. Caseltine, S.D. Goens, J.C. Duffy, and R.D. Welsh

9:30 a.m. Bovine Viral Diarrhea Virus Persistent Infection in Two White-Tail Deer in 151 Southeastern South Dakota – C.C.L. Chase, L.J. Braun, P. Leslie-Steen, T. Graham, D. Miskimins and J.F Ridpath

9:45 a.m. BREAK AND POSTER SESSION

10:15 a.m. Bovine Viral Diarrhea Virus (BVDV) Subtypes in Diagnostic Laboratory Accessions 152 from Clinical and Necropsy Cases: Distribution of BVDV1a, 1b, and 2a Subtypes – R.W. Fulton, J. F. Ridpath, S. Ore, J.T. Saliki, L. J. Burge, and A.W. Confer

10:30 a.m. Genetically Identical Bovine Virus Diarrhea Viruses Isolated from an Aborted Ovine 153 Fetus and a Yearling Hereford Heifer on the Same Premises – B. Thompson, E. Dubovi, B. Njaa, J.F. Ridpath

10:45 a.m. Viral Antigen Distribution in the Respiratory Tract of Cattle Persistently Infected with 154 Bovine Viral Diarrhea Virus Subtype 2a - A.W. Confer, R.W. Fulton, D.L. Step, B.J. Johnson, and J.F. Ridpath

15 11:00 a.m. Comparison and Optimization of High Throughput Viral RNA Isolation Methods For 155 FMDV - A.G. Giuffre, J.M. Robida, X. Fang, M.A. Kenney, T.S. McKenna, B.M. Martin, and T.R. Beckham

11:15 a.m. Malignant Catarrhal Fever-like Disease in Sheep Following Intranasal Inoculation with Ovine Herpesvirus 2 - H. Li, D. O'Toole, O. Kim, J. L. Oaks, and T.B. Crawford 156

11:30 a.m. Avian Paramyxovirus Infection -1 in Pigeons – A Retrospective Study - H.L. 157 Shivaprasad, G. Cooper, F. Uzal, P. Woolcock, S. Hietala, B. Crossley, B. Charlton, F. Sommer, R. Crespo, R. Chin, M. McFarland, L. Woods, M. Anderson, H. Kinde, B. Daft, J. Odani, D. Read, A. Bickford, P. Hullinger and B. Barr

11:45 a.m. Isolation of Equine Influenza Virus from Racing Greyhounds with Fatal Hemorrhagic 158 Pneumonia - E.J. Dubovi, P.C. Crawford, R.O. Donis, W.L. Castleman, Iain Stephenson, and E.P.J. Gibbs

* Graduate Student Competition

16 USAHA/AAVLD Joint Plenary Session Monday, October 25, 2004 8:00 a.m. – 11:30 p.m. Guilford B

Animal Disease Surveillance in the 21st Century – Important Tools for Response, Protection of Public Health, and Trade

Moderators: Gary Osweiler and Rick Willer

8:00 a.m. Opening Remarks – Moderators

8:05 a.m. KEYNOTE ADDRESS Global Perspective - the World Organisation for Animal Health (OIE) – 161 Alex Thiermann

8:40 a.m. Importance of Surveillance to North America – Brian Evans 162

9:15 a.m. Emerging and Re-emerging Zoonotic Diseases – Importance of Veterinary 163 Surveillance for Protection of Public Health – Lonnie King

9:45 a.m. BREAK

10:00 a.m. Surveillance of Disease: Epidemiologic Perspectives- Wayne Martin 164

10:30 a.m. Characterization of the Recent U.S. BSE Case and Methods for Surveillance – 165 Juergen Richt

11:00 a.m. The Value and Risks of Surveillance for Avian Influenza – David Suarez 166

11:30 a.m. AAVLD House of Delegates

Joint Plenary Session

ANIMAL DISEASE SURVEILLANCE IN THE 21ST CENTURY

Sponsored By

BIO-RAD LABORATORIES RMS, RESEARCH MANAGEMENT SYSTEMS

17 Poster Session 47th Annual Meeting American Association of Veterinary Laboratory Diagnosticians Saturday October 23, 12:00 p.m. through Monday, October 25, 1:00 p.m. Guilford Prefunction Area 3rd Floor

No. Poster Title Page 1 Effects of Incubation Temperature, Decontamination Method and Regular Shaking on Diagnostic 171 Performance of a New Liquid Culture Method, the TREK ESP Culture System II and para-JEM Broth, for Detection of Mycobacterium avium subsp. paratuberculosis in Fecal Samples - C. van Maanen, M. G. J. Koene, V. Oosterhuis, T. von Banniseht, S.E. Allen, and N.M. Sullivan

2 In Vitro Evaluation of a Phenolic Disinfectant’s (Environ LpH®) Effect on Chronic Wasting 173 Disease-Associated Prion – K. L. Cramer, J. E. Jewell, C. T. Larsen, L. A. Baeten, M.W. Miller

3 Clinical, Virology, Histopathology, Serology, and PCR Findings of a Fatal Eastern Equine 174 Encephalomyelitis Infection in a Puppy - M.D. Farrar, D.L. Miller, S.L. Stiver, C.A. Baldwin, and C.L. Hall

4 Necropsy Findings and Arbovirus Surveillance in Mourning Doves (Zenaida macroura) from the 175 Southeastern United States - R. W. Gerhold, C. M. Tate, S. E. Gibbs, D. G. Mead, A. B. Allison, and J. R. Fischer

5 Use of a Microtiter Serum Neutralization Test to Detect West Nile Virus Antibodies - A.L. Glaser 176

6 Comparison of the Polymerase Chain Reaction (PCR), Virus Isolation and Electron Microscopy 177 for the Diagnosis of Orf Virus Infections - J. Guo; L.L. Logan and A. de la Concha-Bermejillo

7 Development of a Real-time PCR Panel for Detection of Ruminant Endemic Diseases that Mimic 178 Foot and Mouth Disease - K. Homb, D. Krueger, P. Bochsler, A. Brower, R. Porter, K. Woods and K. Toohey- Kurth

8 Evaluation of the BACTEC® 960 MGIT™ System for Growth of Mycobacterium bovis - N. 179 Hines* and J. Payeur

9 Wasting and Mortality in Beef Cattle Caused by Eurytrema coelomaticum in Southern Brazil 180 M.R.S. Ilha, A.P. Loretti

10 Effect of Hexadecylpyridium Chloride (HPC), Vancomycin and Natamycin on the Growth of 181 Mycobacterium avium subsp. paratuberculosis - K. A. Johansen, E. E. Hugen and J.B. Payeur

11 Diagnosis of Clostridium spiroforme in a Rabbit - D. Jordan, T. Klinefelter, J. Kinyon 182

12 Development and Validation of a PCR Test to Detect Lawsonia intracellularis in Tissues and 183 Feces from Swine - G.K.A. Josephson, H.Y. Cai, P. Bell-Rogers, M. Archambault, B. McEwen, M.G. Maxie 13 Antimicrobial Susceptibility Profiles of Recent Isolates of Mycoplasma bovis - Joann M. Kinyon, 184 Ricardo F. Rosenbusch, Lorraine J. Hoffman, and Michael Apley

14 BVDV Infection in Cell Culture-A Laboratory Disaster - J. G. Landgraf, J. V. Warg, L. G. Koster, 185 K. A. Eernisse, M. P. Emery, S. L. Swenson

15 Implementation of Strategy to Control Bovine Viral Diarrhea Virus in the United States - 186 R.L.Larson, D. M. Grotelueschen

16 Implementing the Standard: Personnel – Reading Between the Lines - B. Lawler and M. Pedersen 187

17 Diagnostic Lab Connectivity and Electronic Health Certificates for Equids - K. Maher and J.A. 188 Facchiano

18 PFGE on Campylobacter jejuni from Animals-Patterns and Clusters - W. Manley, A. Boerger- 189 Fields, K.W. Mills

19 Papillomavirus-associated Basosquamous Carcinoma in an Egyptian Fruit Bat (Rousettus 190

aegyptiacus) - C. McKnight,* M. Kiupel, A. Wise, C. Howe, and R. Maes

20 Correlation of Leptospira interrogans Fluorescent Antibody Test with Histology and 191 Immunohistochemistry in Aborted and Abattoir-Collected Bovine Fetuses - E. K. Meseck*, B. Njaa, B. Thompson, L. Warnick and P. McDonough

21 Postmortem Evaluation of Thirty-one Brucella-Reactor Cattle from Wyoming and Evidence of 192 Apparent Transmission from Elk - D. Montgomery, K. Mills, D. O’Toole, H. Edwards, W. Cook, S. Olsen, B. Bricker, D. Ewalt, A. Jensen, P. Yaeger, J. Logan, B. Combs

22 Aldosterone and Progesterone Producing Cortical Adrenal Tumor in a Cat - I.D.R. Pardo*, 193 G.Johnson, A. Declue, and M. Kiupel

23 Expression of Leptospiral Immunoglobulin-like Protein by Leptospira interrogans and Evaluation 194 of Its Diagnostic Potential in a Kinetic Enzyme Linked Immunosorbent Assay - R.U.M. Palaniappan, Y-F. Chang, F. Hassan, S. P. McDonough, M. Pough, S. C. Barr, K. W. Simpson, H. O. Mohammed, S. Shin, P. McDonough, and R. L. Zuerner

24 Evaluation of lig-based Conventional and Real Time PCR for the Detection of Pathogenic 195 Leptospires – R.U.M. Palaniappan, Y-F. Chang, P. Harpending, S. P. McDonough, E. Dubovi

25 Bacterological Characterization of Respiratory Disease in Calves and Antimicrobial Susceptibility 196 - Y. Pineda, S. Mora, J. Santander

26 The Distribution of Mycobacterium avium subsp paratuberculosis in Minnesota Dairy Farms 197 Using Bacterial Culture of Fecal Pools - Raizman E.A*, S.J. Wells, C. Muñoz Zanzi, S.M. Godden

27 Detection of c-Kit (CD 117) on Cutaneous Mast Cell Tumors of Ferrets by 198 Immunohistochemistry - J.A. Ramos-Vara, M.A. Miller, G.C. Johnson, C.M. Loiacono, and M. Kundu

28 Immunohistochemical Detection of Tryptase in Cutaneous Mast Cell Tumors of Ferrets - J.A. 199 Ramos-Vara, M.A. Miller, G.C. Johnson, C.M. Loiacono, and M. Kundu

29 Isolation of Persistently Infected P388D, a Mouse Macrophage Cell Line, with Ehrlichia risticii, 200 the Causative Agent of Potomac Horse Fever - S. Sahu, D.D. Pedersen, J. Stasko, and E. N. Ostlund

30 West Nile Virus Outbreak in Horses in North Dakota, 2002: A Characterization of the Equine 201 C ases - L.A. Schuler, M.L. Khaitsa, N. Dyer and C.L. Stoltenow 31 Characterization of a Porcine Teschovirus Isolated During an Emerging Disease Investigation in 202 Imported Pigs - J.V.Warg, J. G. Landgraf L. G. Koster, L. A. Anderson, S. L. Swenson, M. J. Yaeger

32 Differentiation of Mycobacterium bovis Infection of Cattle from M. avium subsp. avium and M. 203 avium subsp. paratuberculosis Infection Using a Recombinant ESAT-6:CFP-10 Fusion Protein - W.R. Waters, M.V. Palmer, B.J. Nonnecke, and F.C. Minion

33 Disseminated Melanoma and Osteogenic Tumor in a Rabbit - F. Williams III*, C.M. Loiacono, 204 L. Bloomfield

34 Effect of Delayed or Prolonged Fixation on Immunohistochemical Detection of Bovine Viral 205 Diarrhea Virus in Ear-Notch Biopsies - M.A. Miller, .A. Ramos-Vara, S.B. Kleiboeker, R.L. Larson

35 Evaluation of SEM, SE75.3 and SE18.7 in ELISAS for Serum Antibody to Streptococcus equi - 206 J.F. Timoney, A. Graves, S. Muthupalani, and J. Morrow

36 Investigation of Acute Nephrosis in a Herd of Beef Cattle in Nebraska - L.G. Corbellini *, M. 207 Carlson, B. W. Brodersen, A. Doster, D.G. Rogers, D.J. Steffen

37 Neosporosis in Cows Vaccinated with a Neospora caninum Vaccine - L.G.Corbellini, D.G. 208 Rogers, D.J. Steffen, B.W. Brodersen, and D.R. Smith

38 Evaluation of a Multiplex PCR Assay for Identification of Mycobacterium avium subsp. 209 paratuberculosis from Liquid Cultures - K.S. Anklam, E.J.B. Manning, S. Sreevatsan, and M.T. Collins

39 Detection of Mycobacterium avium subsp paratuberculosis in Bovine Feces and Milk using 210 Adiapure® Extraction Kit and Real Time Adiavet® PCR Kit – B. Blanchard, Y. Versmisse and B. Chevallier

40 Development of Multiplex Real-Time RT PCR for the Detection of Akabane and Aino Viruses 211 and Molecular Characterization of Israeli Isolates of Akabane Virus - Y. Stram, A. Levine, L. Kuznetzova, J. Brenner, Y. Braverman, M. Guinni.

41 Detection of Brachyspira hyodysenteriae and pilosicoli in Porcine Feces Using Real-Time PCR - 212 P. L. Bell-Rogers, M. Archambault, G. Josephson, B. McEwan, R. Friendship, J. Prescott, G. Maxie and H.Y. Cai

Transfer and Optimization of a Single-tube CSFV Real-time RT-PCR Assay to a High-throughput 213 42 96 Well Format - A.J. Eberling, B.M. Martin, T.S. McKenna , T.R. Beckham

43 Prevalence Study of Coxiella burnetii (Q-fever) in the United States Dairy Herds Based on Bulk 214 Tank Milk Testing by Trans-PCR - S.G. Kim, E.H. Kim, A.E. Cassano, E.J. Dubovi

20 44 Wildlife Disease Research at the APHIS National Wildlife Research Center - R.G. McLean, L. 215 Clark, M. R. Dunbar, and K. C. Vercauteren

*Graduate Student Competition

POSTER SESSION

Sponsored by

CEPHEID

SCIENTIFIC SESSIONS 47th ANNUAL MEETING AMERICAN ASSOCIATION OF VETERINARY LABORATORY DIAGNOSTICIANS

______

First Plenary Session Saturday, October 23, 2004 8:00 a.m. – 11:15 a.m. Guilford B

TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES

Moderator: Gary Osweiler Page

8:00 Opening Remarks

8:05 BSE Surveillance in the UK – Angus Wear 25

8:45 Molecular Characterization of Prion Isolates from Livestock and Cervids – Juergen 26 Richt

9:25 Break – 15 Minutes

9:40 Pathology and Testing Strategies for BSE – Tim Baszler 27

10:20 Ecology, Epidemiology and Control of CWD – Elizabeth Williams 28

11:00 Panel, Questions

11:15 AAVLD HOUSE OF DELEGATES

AAVLD First Plenary Session

TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES

Sponsored By

BIO-RAD LABORATORIES

24 BSE Surveillance in the UK

Angus Wear1

The UK’s first case of BSE was identified in 1986, and the disease was made notifiable in 1988. The outbreak reached its peak in 1992/93, with more than 36,000 confirmed cases in a twelve month period. Until 1999, monitoring of the progress of the outbreak was based solely on passive surveillance (i.e. histological confirmation of clinical cases). Following the association of bovine BSE with human vCJD, the sale of use of meat for human consumption from bovine animals over thirty months of age (OTM) was prohibited. By the late 1990s, rising public health concerns, a continued ban on UK beef exports, and the need to confirm that the decline in clinical cases genuinely reflected falling levels of infection, created a political impetus for more active surveillance.

The first active survey, conducted between January and May of 1999, examined the brainstems of 4,000 routinely slaughtered cattle >5years old, using conventional histopathology and the ‘Prionics’ PrP Western blot, and 0.5% of samples gave positive results. A larger survey carried out in 2000 on 10,000 >5year-old slaughter cattle gave similar results.

Such voluntary surveys have been overtaken by European Commission decisions requiring the UK to sample all bovine ‘Fallen stock’ >24months of age, all over thirty month (OTM) casualty cattle and a proportion of routinely slaughtered (OTM) animals. In addition, all >24month abattoir casualties must be tested before the carcase can be released for human consumption. The current surveillance programme currently involves annual testing of some 180,000 fallen stock and OTM casualties, and 200,000 OTM abattoir slaughtered cattle. All current rapid testing is by the BioRad TeSeE method.

Possible future relaxation of the current ban on OTM meat sales for human consumption in favour of release subject to testing will impose even larger volume testing with shorter turnaround times.

The original evaluation of rapid BSE tests (BioRad Platelia, Prionics Western Blot and ENFER) by the European Union in 1999 did not include any autolysed material. With all test methods being involved in the assay of fallen stock material, the possibility of false negative results became an increasing concern throughout the community. VLA conducted a study of autolysed material which indicated that all current methods were capable of testing such material. Subsequent EU evaluations have included both autolysed positive and negative material.

This talk will describe the methods used to put in place the large scale sampling and testing required by active BSE surveillance in the UK. The logistical problems of increasing the scale of testing and transfer to alternate test methods are discussed.

1Senior Scientific Officer, Veterinary Laboratories Agency, VLD Newcastle, Newcastle on Tyne, NE12 9SE, England.

25 Molecular Characterization of Prion Isolates from Livestock and Cervids

Juergen Richt Virus and Prion Disease Research Unit National Animal Disease Center, USDA, ARS, Ames, IA

26 Diagnosis of Transmissible Spongiform Encephalopathies

Tim Baszler

Accurate and sensitive methods for detection of transmissible spongiform encephalopathies (TSEs) are critical not only for disease diagnosis, but also for effective surveillance and eradication programs implemented to maintain world markets for animal agriculture. Diagnosis of transmissible spongiform encephalopathy has evolved from traditional approaches, based upon clinical disease and morphological changes in the brain, to molecular based assays that detect abnormal prion protein in tissues and fluids obtained from diseased animals. The formation of abnormal, disease-associated prion protein (PrPd) occurs only in TSEs and to date its presence serves as the only reliable and specific marker for animals with prion diseases. Several new molecular diagnostic techniques are aimed at increasing the sensitivity and specificity of PrPd detection, as well as identifying surrogate markers other than prion protein itself. These new tests are needed in order to detect TSE agents in body fluids, particularly blood and extraneural tissues so that practical, preclinical screening tests may be applied to live animals. New test development for prion diseases is a rapidly evolving field. Rapid screening tests for active surveillance of bovine spongiform encephalopathy (BSE), using commercial diagnostic kits based on postmortem detection of PrPd, have already greatly improved identification of infected cattle prior to entering the human food chain. The presentation will review traditional, current molecular, and potential future diagnostic methods for TSE diagnosis in animals.

Washington State University, Pullman, WA

27 Epidemiology and Control of Chronic Wasting Disease

Elizabeth Williams1 and Michael Miller2

Chronic wasting disease (CWD) is unique among the transmissible spongiform encephalopathies. In contrast to scrapie, bovine spongiform encephalopathy, and transmissible mink encephalopathy, CWD occurs in non-domestic species and in populations of cervids managed under agricultural conditions and in free-ranging populations under minimal human control. The state, provincial, and federal programs in place or soon to be implemented, guide management of CWD in the farmed cervid industries. These herd certification programs require individual animal identification, annual animal census, and surveillance; they have been successful and only a few CWD-affected cervid herd remain under quarantine in the United States. Management of CWD in free-ranging populations of mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus), and elk (Cervus elaphus) is extremely difficult and, with currently available techniques (population reduction, buffer zones, movement restrictions), is unlikely to be eradicated once it is established over large geographic areas. Movement in free-ranging deer herds in Colorado and Wyoming appears to be associated with traditional migration routes and interaction of animals on winter and summer ranges.

Chronic wasting disease is infectious and contagious. Animals probably are naturally exposed by the oral route, the CWD agent is taken up along the alimentary tract and accumulates in associated lymphoid tissues and the enteric nervous system, and probably enters the central nervous system via the nerves. Widespread distribution of the CWD prion protein occurs in lymphoid tissues during the course of infection and this may contribute to the apparent efficiency of transmission. No Pnrp genotype in elk and deer has been found to provide complete resistance to CWD infection, however, genotype may influence incubation times in elk (shortened incubation times in animals homozygous for methionine at codon 132 of the prion protein) and mule deer (shortened incubation times in animals homozygous for serine at codon 225 of the prion protein). Although the mechanisms are not fully understood, CWD agent probably exits an affected animal via excretions or secretions. Recently, horizontal transmission, environmental contamination adequate for transmission, and transmission associated with decomposed carcasses have been shown for CWD in mule deer. These findings have implications for management of possibly contaminated premises and movement of carcasses of cervids harvested in CWD endemic areas.

1Wyoming State Veterinary Laboratory and Department of Veterinary Sciences, University of Wyoming, Laramie, Wyoming 82070 2Colorado Division of Wildlife, 317 West Prospect, Fort Collins, Colorado 80526

28 Transmissible Spongiform Encephalopathy/Virology Scientific Session Saturday October 23, 2004 1:00 p.m. – 3:00 p.m. Auditorium III

1:30 p.m. A Second-Generation Method for Accurately Detecting Bovine Spongiform 33 Encephalopathy – V. Leathers, R. Toomik, K. Velek, L. Plourde, S. Koller, M. Ryle, C. Wong, L. Estey

1:45 p.m. Break

2:00 p.m. Polioencephalitis in Ruminants in the UK - S.F.E. Scholes, H. Ainsworth, C. Bidewell, A. 34 Colloff, A. Dawson, P. Duff, R.J. Higgins, P.J. Watson, M. Wessels, K. Whitaker, P.F. Nettleton

2:30 p.m. Detection and Characterization of Naturally Occurring West Nile Virus Infection in a Wild 36 Female Turkey - Z. Zhang, F. Wilson, R. Read, L. Pace, S. Zhang

30 Development of an Automated High-Throughput Screening Enzyme-Linked Immunosorbent Assay for Bovine Spongiform Encephalopathy in Cattle and Chronic Wasting Disease in Elk and Deer in Alberta Canada

J. T. Y. Wu1, E. Y. W. Chow1, L. S. Y. Wong1, M. Longson1, C. Onderka1 C. Wittmeier1 and E. E. Bowlby1

A commercial enzyme-linked immunosorbent assay (ELISA) (Bio-Rad Inc.) has been used to screen for the presence of Bovine Spongiform Encephalopathy (BSE) prions in cattle and Chronic Wasting Disease (CWD) prions in Elk and Deer in Europe. This is the only test that has been approved for screening both types of diseases in USA and Canada. Automating ELISA tests yield a cost effective procedures with a quick turn around times (TAT), however the Bio-Rad ELISA has not been automated. We have increased the efficiency of this manual ELISA by automating it using a robotic liquid-handling workstation. In developing this automated-ELISA we have chosen the Beckman Biomek 2000 (B2K) automation workstation. The automation software program Bioworks 2.1C allowed us to configure the B2K, the ELISA reader and the Stacker Carousel within one program. The pull-down menu permitted the programmer to arrange different transfer and dilution steps according to the ELISA protocol. Special attention was given to accommodate the multi-step and multi-equipment requirements of the immunoassay procedures. Incubation times were scheduled between steps. The degree of complexity of the working assay was kept to a minimum so that only one unique operation program had to be written for this test. The robotic machine was simple to operate, robust and easily maintained, thus eliminating forced downtime due to human and/or machine failure. The a-ELISA assay performed high-throughput screening for Transmissible Spongiform Encephalopathies (TSE) prions with at least a 10 to 20-fold increase in test capacity. The operation schedule allowed ample walk away time (WAT) in between the steps of the ELISA, enabling the technologist to perform other duties such as sample receiving, sample preparation, test validation and result interpretation. This lowered the cost per test by lowering the labour costs and decreased the TAT. Besides the automation of the essential steps specified by the protocol, we further automated our data reporting method. This was done by incorporation a unique 10-digit bar code number for each specimen, once it was received. The unique code allowed each specimen to be tracked throughout the test procedures. All results were captured by the workstation computer and, through the use of a spreadsheet program, were downloaded into our Animal Health Surveillance System (ANHSURS) database to further processing. The ability to continuously trace the different steps the specimen had gone through allowed us to satisfy ISO 17025 requirements. Parallel experiments comparing the manual and the automated ELISAs showed a 100% reproducibility of the diagnostic results.

1 Immunology and Virology Unit, Biology Section, Agri-Food Laboratories Branch, Food Safety Division, Alberta Agriculture, Food and Rural Development, Edmonton, Alberta, Canada.

31 Western-blot Techniques with Improved Sensitivities for Confirming the Diagnosis of BSE, Scrapie and CWD

J.M. Bilheude1, J.P. Bourgeois1, M. Feyssaguet1, G. Nespoulous1, E. Comoy2, JP Deslys2,S. Simon3, N. Morel3, Y. Frobert3 and J. Grassi3

Most tests in use today for the diagnosis of Transmissible Spongiform Encephalopathies (TSEs) are based on detection of the abnormal form of the prion protein (PrPSC), which accumulates in peripheral lymphoid tissues during the incubation period or in the brain of affected animals. Since the normal cellular form of PrP (PrPc) is always present in tissues containing PrPSC and in absence of antibodies specifically recognizing PrPSC, its specific detection is currently achieved after a proteinase K treatment which completely degrades the cellular form of PrP.

During the last years we developed ELISA based rapid tests for the post mortem diagnosis of BSE in cattle, Scrapie in sheep and goat and CWD in cervids based on a two step assay procedure. The first step consists in selective purification and concentration of PrPSC by means of a special process which comprises: homogenisation of brain tissue, treatment of the homogenate with proteinase K, concentration of PrPSC by centrifugation and resuspension of the resulting pellet using a denaturing treatment. During the second step, the "solubilised" PrPSC is finally measured by means of a two-site immunometric assay ("sandwich" immunoassay). These tests are now marketed by Bio-Rad Laboratories as TeSeE® and TeSeE® sheep/goat and are currently used for large scale active surveillance of BSE in cattle, scrapie in sheep and goat in Europe as well as for CWD in USA and Canada.

Given the critical consequences induced by the identification of TSE cases in flocks, a positive result provided by a rapid test has to be confirmed by an independent confirmatory technique including: Histopathology, Immunohistochemistry or western-blot.

We have developed two different western-blot aimed to detect PrPSc in central nervous system tissues and very well suited for confirming TSEs in ruminants. They both involve a purification procedure very similar to the one used in the Bio-Rad ELISA screening test, followed by an optimized electrophoretic separation and detection of blotted PrP by high affinity monoclonal antibodies. A first Western-blot is dedicated to BSE in cattle (TeSeE® Bovine Western-blot) while the second one (TeSeE® ovine/caprine Western-blot) is optimized for confirming scrapie in sheep and goat or CWD in cervids. Both Western- blots allow very sensitive and specific detection of PrPSC in tissues from TSE infected animals. They are sensitive enough to confirm all positive cases detected by the corresponding ELISA screening test and can be performed within a day (less than 6.5 hours). Interestingly, the sheep Western-blot can confirm a high proportion of unusual scrapie cases corresponding to new strains of scrapie recently identified in Europe. This includes the Nor-98 strain and the so-called "unclassified isolates" which are characterized in that they are associated with the production of PrPSC presenting a very low resistance to proteinase K treatments.

1Bio-Rad Laboratories, 3 boulevard Raymond Poincaré, 92430 Marnes-la-Coquette, France 2CEA, GIDTIP/DRM/DSV, CEA/FAR, 92265 Fontenay aux Roses, France 3CEA, Service de Pharmacologie et d'Immunologie, CEA/Saclay, 91191 Gif sur Yvette, France

32 A Second-Generation Method for Accurately Detecting Bovine Spongiform Encephalopathy

V. Leathers1, R. Toomik2, K. Velek1, L. Plourde1, S. Koller1, M. Ryle1, C. Wong1, L. Estey1

The bovine spongiform encephalopathy (BSE) epidemic was mainly confined to the United Kingdom and western Europe, with intermittent cases identified in other parts of the world. In December 2003, the first case of BSE was reported in Washington state in the U.S. As a result, the U.S. Department of Agriculture has increased surveillance to ensure that BSE-tainted products do not enter the market. Methods for rapid and accurate screening of postmortem samples are essential. The IDEXX HerdChek Bovine Spongiform Encephalopathy Antigen Test Kit is designed to rapidly identify samples containing disease-associated prion protein (PrPSc), providing dependable results in less than 4.5 hours.

The IDEXX HerdChek Bovine Spongiform Encephalopathy Antigen Test Kit (BSE EIA) is an antigen- capture enzyme-linked immunoassay (EIA) for detection of the abnormal conformer of PrPsc in postmortem brain tissues from bovines affected by BSE. The test utilizes a nonbiological PrPsc-selective capture technology developed by Microsens Biotechnologies (London, UK; EU patent application: WO 03/073106 A2). The basis of this technology is the use of Seprion affinity ligands for selective PrPsc capture through interactions between the polyionic Seprion ligand and the aggregated abnormal form of PrP. Nonaggregated normal PrPc does not bind to the Seprion ligand in the presence of competing polyanionic surfactant. The use of polyanions to capture PrPsc is well-documented in the scientific literature. Because the Seprion ligand-capture technology does not use a monoclonal antibody for specific capture, proteinase K is not required for the digestion of a monoclonal antibody’s epitope. A simple brain tissue-water homogenate is added directly to the Seprion ligand-coated plate. This technology eliminates the need for the complicated, time-consuming, and potentially error-prone proteinase K sample preparation required by first-generation tests.

The IDEXX BSE EIA is USDA-approved and has completed phase one of the current round of European Commission validation studies. The performance of the IDEXX BSE EIA has been compared to immunohistochemistry and EU-approved ELISAs. In a study of >8000 negative bovine brain samples collected in the United States and Europe, the IDEXX BSE test performed with 100% specificity. The mean S-N (sample OD – negative control OD) was >5.6 standard deviations from the cutoff, demonstrating that the samples are sufficiently removed from the test cutoff to provide an extremely low false-positive rate. The IDEXX BSE EIA correlated 100% with a current EU-approved ELISA in the detection of BSE from non-obex neural tissue (n=132). Additionally, evaluation of BSE homogenate dilution series have shown that the IDEXX BSE test sensitivity is equivalent to the EU-approved ELISA used for comparison. Tissue autolysis studies have demonstrated that the IDEXX BSE test will perform well on samples collected from fallen stock and samples left unrefrigerated.

The IDEXX HerdChek Bovine Spongiform Encephalopathy Antigen Test Kit is faster, easier to use and requires less hands-on time than other BSE test kits. Its 100% specificity provides dependable results, with no false-positives. The IDEXX BSE test has been validated for use in automated or manual formats, runs entirely at room temperature, and does not require incubators or centrifuges. The IDEXX BSE test combines a simple homogenization step with a straightforward EIA, revolutionizing transmissible spongiform encephalopathy (TSE) diagnostic ease-of-use and efficiency for TSE laboratories. This truly second-generation test revolutionizes BSE testing by offering an unmatched combination of performance and ease-of-use.

1 Research and Development, IDEXX Laboratories, Westbrook, Maine 2 IDEXX Scandinavia AB, Osterbybruk, Uppsala, Sweden

33 Polioencephalitis in Ruminants in the UK

S.F.E. Scholes1, H. Ainsworth2, C. Bidewell3, A. Colloff4, A. Dawson5, P. Duff6, R.J. Higgins1, P.J. Watson6, M. Wessels7, K. Whitaker7, P.F. Nettleton8

From 1994 – 2004, 21 sheep and cattle with polioencephalitis submitted to VLA Regional Laboratories tested negative for louping-ill by serology and/or immunohistochemistry. Polioencephalitis (PE) denotes predominantly mononuclear inflammation of grey matter with neuronal necrosis and neuronophagia. Major differential diagnoses in humans are neurotropic viral infections and paraneoplastic encephalitis (Love & Wiley, 2002). Louping-ill virus (a tick-borne flavivirus) and bovine herpesvirus 1 (BHV1) infections of ruminants are the only recognised causes of PE in the UK. The aim of this retrospective study was to map the neuroanatomical distribution of PE in these 19 sheep and 2 cattle to assess if one or more putative viral infections are likely to be involved. Anatomically defined sites within all the major neuroanatomical regions of brain and where available spinal cord were evaluated. Eighteen ovine cases could be assigned to one of 3 groups, based on lesion distribution and animal age. Group 1 consisted of 8 lambs (3 – 4 weeks of age from 4 flocks) in which neuronal necrosis involved red nuclei, vestibular complex, cerebellum (cortex and roof nuclei) and in 3/3 lambs, dorsal root ganglia. In the second group, 6 lambs (5 – 12 weeks of age from 3 flocks) had neuronal necrosis, mineralization and loss accompanied by gliosis and perivascular cuffing focused in the basal nuclei, hippocampus and cerebral cortex with variable involvement of brainstem nuclei and cerebellum. Group 3 comprised 4 adult sheep from 4 flocks with similar lesion distribution to group 1 sheep in addition to marked neuronal necrosis in the mesencephalic nucleus of the trigeminal nerve. One 3 weeks old lamb from another flock had lesions similar to group 3 sheep. The 2 cattle with PE were yearlings originating from different herds; one had lesions similar to group 1 sheep whereas neuronal necrosis was more widespread in the other. Joest Degen bodies characteristic of Borna disease and Negri bodies were not observed in any of these cases. In 7 recent cases IHC testing was negative for WNV and BHV1, and in one sheep from group 3 IHC for rabies antigen was negative. Histological findings similar to group 2 lambs and possibly group 1 lambs were reported by Duffell (1984) in 4-6 weeks old lambs in which no serological evidence of louping-ill, Border disease or maedi-visna was found. The lesions in the 2 cattle resemble those in Swiss cattle in which IHC for rabies virus, Borna disease virus and central European tickborne encephalitis virus was negative (Theil and others 1998). A full set of laboratory data is not available for many cases because these cases were identified retrospectively. However, preliminary laboratory data, immunohistochemical results and lesion distribution strongly suggest that the lesions observed in these sheep and cattle are unusual or new presentations of encephalitis caused by a known virus or one or more novel viruses. Further studies are required to investigate possible neurotropic viral infections in these cases.

Duffell, S.J. (1984) Idiopathic meningoencephalitis of sheep in England. Veterinary Record 115, 547 Love, S. Wiley, C.A. (2002) Viral diseases. In Greenfield’s Neuropathology, 7th Edition, Eds Graham, D.I. & Lantos, P.L., Arnold, Volume 2, 1-105 Theil, D., Fatzer, R., Schiller, I., Caplazi, P., Zurbriggen, A., Vandevelde, M. (1998) Neuropathological and aetiological studies of sporadic non-suppurative meningoencephalomyelitis of cattle. Veterinary Record 143, 244-249

1VLA-Lasswade, Pentlands Science Park, Bush Loan, Penicuik, Midlothian, Scotland, UK EH26 0PZ; 2VLA Bury St Edmunds; 3VLA Winchester; 4VLA Truro; 5RVC VLA Surveillance Centre; 6VLA Penrith; 7VLA Preston; 8Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian, Scotland, UK EH26 0PZ

34 Evaluation of a Blocking ELISA for Detection of West Nile Virus Antibodies in Horses, Chickens and Wild Ring-Necked Pheasants

E.-M. Zhou1, D.K. Kirby1, K. Lin1, D.L. Reynolds2, J. Fabios2, K.B. Platt2, T. Bogenschutz3 and D.L. Garner3

West Nile virus (WNV) has spread farther and faster than expected in the United States and caused significant morbidity and mortality in avian and mammalian species. Data from Center for Disease Control (CDC) indicates that WNV has been detected in dead birds of at least 138 species. Current serological diagnostic tests available for animal species are IgM capture ELISA and plaque reduction neutralization test (PRNT). The IgM capture ELISA is the only official serologic test for detection of equine IgM antibodies against WNV. The PRNT is to detect cross-species neutralizing antibodies. However, due to its complexity, involving live virus and cost effect, PRNT has limited application as a routine serologic test for WNV. A relatively simple and economic serologic test, therefore, is needed to detect anti-WNV antibodies from other species.

We have developed a blocking ELISA prototype with the available reagents from the IgM capture ELISA and antisera from 172 horses and 24 chickens. Horse sera were field samples in which 120 were WNV antibody negative and 50 were WNV positive as tested by the IgM capture ELISA. Chicken sera (total of 196 samples) were collected from 12 specific pathogen free chickens that received three vaccinations against equine WNV vaccine and 12 negative control chickens. By using the negative 120 equine and 98 SPF chicken serum samples, the cutoff inhibition value of 24.5% (average % inhibition +3SD) was determined for the blocking ELISA. With this cutoff value, the relative diagnostic specificity of the blocking ELISA was 100% for equine and chicken serum samples. We determined the relative diagnostic sensitivity of 96% and 83% for 50 equine and 12 chicken IgM capture ELISA positive samples, respectively.

Limited research results on WNV in pheasants were reported in comparison with other wild birds. According to CDC, ring-necked pheasants are susceptible to WNV infection with the average viremia of 2.7 days post infection. To evaluate the blocking ELISA, 80 pheasant serum samples collected in Iowa were tested using the blocking ELISA. With the cutoff value of 24.5%, 15 of 80 pheasant samples were tested positive by the blocking ELISA with the average inhibition value of 52.7%. To confirm these results, 15 positive and 15 negative pheasant samples determined by the IgM capture ELISA were tested by PRNT which showed that all 15 negative pheasant samples were negative by PRNT and 10 of 15 (66.7%) positive samples were PRNT positive.

In summary, the blocking ELISA showed usefulness as a routine test for serodiagnosis of WNV infection in pheasants. It requires further evaluation and validation using experimentally or naturally infected animal serum samples.

1Department of Vet Diagnostic and Production Animal Medicine, Iowa State University, Ames, IA. 2Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA. 3Wildlife Bureau, Iowa Department of Natural Resources, Des Moines, IA.

35 Detection and Characterization of Naturally Occurring West Nile Virus Infection in a Wild Female Turkey

Z. Zhang, F. Wilson, R. Read, L. Pace, S. Zhang

A recent study has suggested the possible direct transmission of West Nile Virus (WNV) from infected commercial turkeys to turkey farm workers based on epidemiological evidence. On the other hand, experimental infection of domestic turkeys with WNV resulted in low levels of viremia, but not encephalitis. Thus a paradox appears to exist between the transmission potential and the level of viremia and disease expression. In this article, we report a case of naturally occurring WNV infection in a wild turkey. Histopathologic evaluation, immunohistochemistry (IHC), viral isolation (VI), and reverse transcription polymerase chain reaction (RT-PCR) were used to detect and characterize the infection associated with WNV.

An adult wild female turkey was submitted to Mississippi Veterinary Research and Diagnostic Laboratory (VRDL) due to its atypical behavior. Gross pathological findings were unremarkable. Histopathologic evaluation revealed encephalitis, characterized by extensive multifocal perivascular lymphocytic infiltration or cuffing in the brain, marked heterophilic hyperplasia in bone marrow, and multifocal interstitial lymphocytic infiltration in the heart, pancreas, ventriculus, and skeletal muscles. IHC detected WNV antigen in numerous neurons and glial cells in the brain. Strong positive staining of the primitive myeloid cell populations in the bone marrow, the respiratory epithelial cells along bronchiole in lung, and epithelial cells in kidney was seen. Diffuse staining of the myocardium and the inflammatory cells infiltrating Purkinje fibers in the heart was also observed. WNV was isolated from the above tissues/organs using cultured Vero cells and cytopathogenic effect (CPE) induced by WNV was observed. Higher copies of viral RNA (equivalent to 1x104 PFU/100mg) were detected in lung, brain, or kidney than in heart, liver or spleen (equivalent to 1x103 PFU/100 mg) using quantitative RRT-PCR.

The results from this case demonstrated that WNV infection caused systemic pathological changes, including encephalitis and myocarditis, in the affected turkey. The virus targeted CNS, myocardium, multiple epithelial cell types (especially in lung/kidney), and bone marrow elements. The histopathological changes and viral tropism in this case were similar to those in naturally infected zoological birds, but not experimentally inoculated turkeys. Furthermore, the pathological changes and the presence of WNV antigen in the bone marrow have not been described prior to this case.

Mississippi Veterinary Research and Diagnostic Laboratory, Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, 2531 Northwest St, Jackson, MS 39216.

36 Hemorrhagic Bowel Syndrome in Dairy Cattle: Preliminary Results from a Case Control Study

D.C. Sockett1, A.I. Brower1, K.L. Woods1, R.E. Porter1, P.N. Bochsler1, S.M. Godden2, N.E. Forsberg3, S.B. Puntenney3 and Y. Wang3

Hemorrhagic bowel syndrome (HBS) is being recognized with increasing frequency by veterinary practitioners and diagnostic laboratories in the US. The disease is usually sporadic (1-3% of adult dairy cattle affected) but herd outbreaks involving up to 10% or more of the cows on a given dairy have been reported. The syndrome is reported more frequently on large dairies (≥ 100 cows) especially those feeding a total mixed ration. All ages of cows can be affected but the incidence of HBS tends to be higher in 2nd lactation and older animals particularly in the first 100 days of lactation.

The cause(s) of HBS is unknown but the acute nature of the disease and the pathological lesions observed strongly suggest that toxins (bacterial and/or fungal) are involved in the disease process. Initial reports speculated that Clostridium perfringens type A was associated with HBS and more recently Aspergillus fumigatus has been implicated as well. However, none of the published reports have included control samples obtained from cows that died of conditions other than HBS.

Beginning in the summer of 2003, the Wisconsin Veterinary Diagnostic Laboratory (WVDL) began sending tissue samples from HBS and non-HBS cases to Dr. Forsberg’s laboratory for real time PCR testing for A. fumigatus. All samples were sent blind to Dr. Forsberg’s laboratory. The PCR assay uses forward and reverse primers specific for A. fumigatus that targets the internally-transcribed spacer (ITS-1) domain between two ribosomal genes (18S, 5.8S). The WVDL did a full diagnostic work-up that included histopathology, aerobic and anaerobic bacteriologic culture, and genotyping of C. perfringens isolates plus virus isolation for BVDV. The data from the preliminary study is summarized below.

Cause of Death Number Number Number Number Number of C. perfringens Salmonella BVDV A. fumigatus Samples Type A Positive Positive Positive Positive

HBS 16 14 1 0 13

Other Gastrointestinal 9 6 4 0 0 Tract Diseases

Results indicate there is an association between A. fumigatus and HBS (χ2 = 12.15, p ≤ 0.001) but not for C. perfringens (χ2 =0.532, p ≤ 0.466). However if the trends observed in the preliminary data do not change, C. perfringens type A will be significantly associated with HBS in a larger data set. It is important to point out that association of an infectious agent with a particular disease does not prove disease causation.

1Wisconsin Veterinary Diagnostic Laboratory, University of Wisconsin, Madison, WI 2Department of Clinical and Population Sciences, University of Minnesota, St. Paul, MN 3Department of Animal Sciences, Oregon State University, Corvallis, OR

Epidemiology Scientific Session Saturday October 23, 2004 1:00 p.m. – 3:00 p.m. Guilford C

Moderators: Francois Elvinger and Claudia Munoz-Zanzi

Page 1:00 p.m. Serologic Response of Horses Vaccinated Against WNV and Those Recovering from 41 Naturally Occurring Disease - A. Davidson, J. Traub-Dargatz, R. Dewell, R. Rodeheaver, J. Stricklin, E. Ostlund, D. Pederson, S. Albers, R. Forde, T. Brigner, S. Roach, R.Long, R. Callan, R. Moorhead, and M. Salman

1:15 p.m. Characteristics and Efficiency of Diagnostic Tests Used in the 2002 LowPath Avian Influenza 42 Outbreak in Virginia - F. Elvinger, D. L. Ward, F.W. Pierson, B.L. Akey, B.A. Porter- Spalding, D. Senne

1:30 p.m. Use of Veterinary Diagnostic Laboratory Data to Rapidly Detect Infectious Disease Outbreaks 43 - H. Kassenborg, B. Miller, K. Green, M. Thurn, J. Griffith , and K. Smith

1:45 p.m. Effectiveness of Pooling Strategies for Detection of Johne’s Disease Infected Cattle Herds – 44 C. Munoz-Zanzi, S. Wells

2:00 p.m. The BioPortal Information System for Foot-and-mouth Disease Surveillance - A.M. Perez, 45 M.C. Thurmond, W.O. Johnson, T.E. Carpenter, P.W. Grant, R.B. Garabed, B.A. Melbourne, Y-K. Choi, A.J. Branscum, L.D. Benning, T.W. Bates, C. Lynch, M. Eidson, M. Ascher, I. Gotham, and H. Chen

2:15 p.m. Epidemiological Models for Global Surveillance of Foot-and-mouth Disease - A.M. Perez, 46 M.C. Thurmond, T.E. Carpenter, T.W. Bates, W.O. Johnson, B.A. Melbourne, S.A. Aslam, R.B.Garabed, Y.ku Choi, A.J. Branscum, M.L. Gallego, and P.W. Grant

2:30 p.m. Foot-and-mouth Disease Surveillance Involving the Testing of Bulk Milk Tank Samples - 47 M.C. Thurmond and A.M. Perez

2:45 p.m. The 13th Edition of the UN Model Regulations on Transport of Dangerous Goods: Effects on 48 Veterinary Diagnostic Laboratories - L.J. Thompson

40 Serologic Response of Horses Vaccinated Against WNV and Those Recovering from Naturally Occurring Disease

Ann Davidson1, Josie Traub-Dargatz1, Renee Dewell1, Racquel Rodeheaver1, Joe Stricklin1, Eileen Ostlund2, Doug Pederson2, Sara Albers2, Richard Forde3, Tiffany Brigner3, Susan Roach1, Rachel Long1, Rob Callan1, Ron Moorhead4, and Mo Salman1

This study included a total of 140 horses that were immunized with a killed WNV vaccine according to manufacturer recommendations and 37 laboratory confirmed equine clinical cases of WNV infection in Colorado and Nebraska. Serum was collected from immunized horses at the time of initial vaccination, 4 to 6 weeks post the initial vaccination series, and 5 to 7 months later. Serum was collected from clinical cases 4 to 6 weeks after laboratory diagnosis of WNV and 5 to 7 months later. Serology indicated that all equids surviving WNV infection developed Plaque Reduction Neutralization Titers (PRNT) at >1:100 in the 4 to 6 week period post disease and 90% maintained this level for at least 5 to 7 months. Of the vaccinated horses, 67% had a PRNT of >1:100, while 13.6% had no detectable neutralizing antibody titer (PRNT<1:10) after the initial series in the fall of 2002. Follow up samples in the vaccinated group (n=84) 5 to 7 months later in spring of 2003 revealed 33% with a PRNT of >1:100, while 28.6% had no detectable neutralizing antibody (PRNT<1:10). In summary while all recovered cases of WNV mounted a serologic response of >1:100 (PRNT), not all vaccinated horses mounted this high level of neutralizing antibody. Although the level of neutralizing antibody response required for resistance to WNV infection in equids is unknown this study would suggest that some vaccinated horses are poor responders.

Acknowledgement: funding for this study provided through a special grant from USDA, the Cooperative State Research Education and Extension Services of the CSU Program for Economically Important Infectious Animal Diseases (PEIIAD), a program within the Animal Population Health Institute (APHI).

1Colorado State University, College of Veterinary Medicine and Biomedical Sciences, Department of Clinical Sciences, Fort Collins, Colorado, 80523 2 National Veterinary Services Laboratories, USDA:APHIS:VS, Ames, IA, 50010 3 Colorado Department of Agriculture, Animal Industry Division-Veterinary Section, Rocky Mountain Regional Animal Health Laboratory, Denver, CO, 80211 4 Animal Clinic and Pharmacy, Ogallala, NE, 69153

41 Characteristics and Efficiency of Diagnostic Tests Used in the 2002 LowPath Avian Influenza Outbreak in Virginia

F. Elvinger, 1 D. L. Ward, 2 F.W. Pierson, 1 B.L.Akey, 3 B.A. Porter-Spalding, 4 D. Senne. 5

Low pathogenic avian influenza was first diagnosed in a Virginia flock on March 12, 2002, with the last positive flock being detected July 2, 2002. One hundred ninety-seven premises (125 commercial turkey (meat birds), 28 turkey breeder, 13 broiler, 29 broiler breeder and 2 layer farms) with 4.74 million birds were declared infected and were depopulated.

The Virginia Department of Agriculture and Consumer Services (VDACS), which had been conducting serologic testing (agar gel immunodiffusion test) at slaughter, implemented pre-slaughter testing of all breeder birds, commercial turkeys and broilers, as well as mandatory testing of any flocks with respiratory symptoms. Various tests were used to detect infected birds, including the Directigen® Flu A Test at the VDACS laboratory; real time reverse transcriptase - polymerase chain reaction (RRT-PCR) at the National Veterinary Services Laboratories (NVSL) and VDACS laboratory; and virus isolation at the NVSL.

The objective of this study was to provide post-hoc validation (estimation of diagnostic sensitivity and specificity) of the Directigen® Flu A Test and the RRT-PCR, which had not been validated for use in birds at the time of the outbreak, and determine their suitability for the intended purpose. However, concerns arose about the validity and integrity of collected data. Samples for all tests had been obtained via several sampling modes including 72 hour pre-slaughter testing of live birds and slaughter testing, weekly surveillance testing of daily mortality (barrel surveillance), convenience sampling of backyard flocks, and testing of flocks with clinical signs of respiratory disease. The incident command system did not provide for a unique and uniformly structured data collection and storage system, which resulted in changing and inconsistent data entry, undocumented editing of existing data, and proliferation of ‘master’- and other datafiles. This led to difficulties in particular in establishing the necessary and unequivocal link between a test result and the associated test, specimen, bird, specimen collection dates and other dates, and house and premise identification.

Consequently, only agreement beyond chance (K-coefficient) between Directigen, PCR and virus isolation was assessed. In 29,636 tracheal swabs from turkeys and chickens combined, when comparing PCR to Virus Isolation, K = 0.78 (95% confidence interval [0.72;0.85]; 3,519 swabs); Directigen to Virus Isolation, K = 0.78 ([0.71;0.85]; 3,536 swabs); and PCR to Directigen, K = 0.72 ([0.66;0.78]; 28,245 swabs). ‘Crude’ sensitivities and specificities, when assessed in reference to virus isolation only, were 0.81 and 0.993 for PCR and 0.68 and 0.999 for Directigen.

In conclusion, performances of Directigen and PCR were ‘similar,’ with substantial agreement between all test results. However, in future outbreaks, authorities have to establish guidelines and conventions for data entry, storage, evaluation and exchange, and provide documentation of any manipulation of collected data, in order to assure the integrity and validity of the data for immediate use during control operations of an outbreak, as well as for the later evaluation of control and eradication procedures.

1Department of Large Animal Clinical Sciences, VMRCVM, Virginia Tech, Blacksburg, VA. 2Office of Research and Graduate Studies, VMRCVM, Virginia Tech, Blacksburg, VA. 3NY State Dept. Agriculture & Markets, Albany, NY. 4USDA:APHIS:VS:ERO, Raleigh, NC. 5USDA:NVSL, Ames, IA.

42 Use of Veterinary Diagnostic Laboratory Data to Rapidly Detect Infectious Disease Outbreaks

H. Kassenborg1, B. Miller2, K. Green2, M. Thurn3, J. Griffith2 , and K. Smith2;

Weaknesses in bioterrorism-related or zoonotic disease surveillance in animal populations can have profound human and animal health and economic impacts. Rapid identification of an infectious disease outbreak is crucial to minimize agricultural economic consequences and reduce morbidity and mortality in humans. While all states require the reporting of OIE List A diseases, and the monitoring of others, relatively few states have the structure or capacity to analyze the reports and provide feedback to the reporting veterinarian, let alone communicate results to relevant public health authorities. These systems rely on the identification of specific diseases (e.g., anthrax, brucellosis) before an investigation and intervention is initiated. This may take weeks to months from the time of exposure on the farm to outbreak recognition. Unfortunately, the window of opportunity for an effective public health and veterinary response to bioterrorism -related and foreign animal diseases is short. Consequently, rapid, real-time, surveillance of agents of animal disease is needed.

Public health surveillance systems designed for the early detection of outbreaks in human populations have received significant attention for their potential to identify illness associated with a bioterrorism event. In Minnesota, we are integrating animal disease surveillance activities with those for humans. The Minnesota Veterinary Diagnostic Laboratory (MVDL) represents a potential source of vast amounts of animal health data that can potentially be used for the detection of public health related events. We patterned our laboratory-based electronic surveillance system on the one in use by the Minnesota Department of Health (MDH) as this system analyzes real-time data to determine if there is an early warning of a human health event.

Secure transfer of electronic accession data is sent by MVDL to MDH daily. As a proxy for an initial infectious disease diagnosis, we used the number of accessions by animal species that resulted in viral or bacterial culture. We applied the analysis algorithms that MDH has developed or human syndromic surveillance to these data to determine if this system can be part of our surveillance structure.

Initial analyses of this system indicate that these methods may be useful in detecting infectious disease outbreaks in animal populations. A retrospective time series analysis comparing viral and bacterial culture data to a known Erysipelothrix outbreak demonstrated acceptable system sensitivity (31%), specificity (99%) and positive predictive value (86%).

Conclusions: This system demonstrates that rapid, near real-time syndromic surveillance may be accomplished using existing veterinary data. Further refinement of the detection algorithm is needed to improve system sensitivity and outbreak detection.

1Minnesota Department of Agriculture, St. Paul, MN. 2Minnesota Department of Health (MDH), Minneapolis, MN. 3Minnesota Veterinary Diagnostic Laboratory, St. Paul, MN.

43 Effectiveness of Pooling Strategies for Detection of Johne’s Disease Infected Cattle Herds

C. Munoz-Zanzi1, S. Wells1

Johne’s disease is a very common and costly animal health problem on US dairy farms, occurring on at least 25% of Midwestern dairy farms, leading to losses to the U.S. dairy industry exceeding $200 million annually. In addition, controversy regarding potential public health associations between M. paratuberculosis and Crohn’s disease persists. ELISA-based testing strategies fail to detect most dairy herds at low prevalence of infection. On the other hand, preliminary evidence from bacterial culture of fecal pools and environmental sampling indicates these methods may be effective testing strategies for detection of infected herds. The initial testing for herd status classification is a critical first step in the Johne’s disease control program since it provides the direction of future efforts (on- farm control or Herd Status Program).

Theoretical modeling was used to elucidate the impact of various animal, test, sampling, and herd factors on the effectiveness of testing procedures involving pooling of fecal samples to detect herds with animals shedding M. paratuberculosis. Specific factors that were considered in the evaluation included within-herd prevalence of infection, expected distribution of shedding in infected cattle, number of randomly selected animals, pool size, and test detection limit. Herd-level sensitivity based on pooling (PHSE) was calculated as the probability that at least one pool tested positive given that a herd is truly infected. Preliminary results showed that within-herd level prevalence and number of animals sampled were positively associated with PHSE. An increase in pool size from 5 samples/pool to 10 samples/pool had a slight impact of HPSE if a low detection limit can be assumed for the test (1 CFU/0.1 gr). In addition, pooling resulted in a higher herd-level sensitivity compared with individual-sample testing, especially for low prevalences. Given the assumptions in this model, a sample size of 30 animals tested in pools of 5 samples yielded a herd-level sensitivity as high as individual-sample testing (approximately 0.94 for a prevalence of 0.1) for a much lower cost (6 tests vs. 30 tests).

Results from this study demonstrate the impact of factors influencing the effectiveness of pooling procedures for detection of infected herds and can be used to make recommendations for its application in the Johne’s disease control program.

1 Department of Veterinary Population Medicine, University of Minnesota, St. Paul, MN.

44 The BioPortal Information System for Foot-and-mouth Disease Surveillance

A.M. Perez,1 M.C. Thurmond,1 W.O. Johnson,2 T.E. Carpenter,1 P.W. Grant,1 R.B. Garabed,1 B.A. Melbourne,1 Y-K. Choi,2 A.J. Branscum,2 L.D. Benning,1 T.W. Bates,3 C. Lynch,4 M. Eidson,4 M. Ascher,3 I. Gotham,5 and H. Chen6

Key performance attributes of real-time infectious disease surveillance systems include the secure and rapid capture, management, and analysis of large amounts of data, and dissemination of accurate results. National and global surveillance systems necessarily will need to draw upon a wide array of state, national, or international disease and information databases across many jurisdictions, including federal and state diagnostic laboratories, for relevant diagnostic and epidemiologic information. These databases, however, typically are very diverse in their structure, language, format, and other design characteristics, which do not permit their interoperability in a single system. The inability of these databases to communicate in some standard way precludes the use or presentation of much of the information inherent in surveillance data.

We will present some of our initial work toward an integrated information sharing and data analysis environment being developed for global foot-and-mouth disease surveillance. This effort is part of a larger research project, referred to as the BioPortal system, which is being developed by the University of Arizona, in collaboration with the California and New York Departments of Health, for West Nile virus and botulism in those two state jurisdictions. The long-term objective of the project is to develop technologies and related standards and protocols needed for full implementation of a national infectious disease information infrastructure.

The FMD BioPortal is a web-based system for the real-time, confidential capture and dissemination of data, diagnostic results, and FMD-related risk information to and from countries, agencies, and laboratories. The aim of the portal is to offer a secure means for rapid transfer of data, analyses, and maps needed to assess changes in FMD risk for specified geographic locations and times. FMD-related data are obtained automatically or by ‘hand’ upload from websites of the OIE, FAO, CIA, and other organizations, as well as from individual country or agency databases. The database outputs are reformatted into a standardized XML structure via a data adapter specific to a particular messaging system. The data are subjected to a variety of epidemiological analyses and mapping, including methods for spatio-temporal clustering, anomaly detection, and spread or movement prediction.

We will illustrate some examples of output for FMD risk mapping, risk assessment, geographic clustering of cases, seasonal and cyclical occurrences of cases, anomaly detection, and movement risk prediction. Examples will be presented using data from middle eastern and South American countries and from California.

The FMD BioPortal is an information system that can be used nationally and globally for FMD and FMD risk surveillance, as well as for surveillance of other animal diseases.

1FMD Modeling and Surveillance Laboratory, Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 2Department of Statistics, University of California, Davis, CA 3Lawrence Livermore National Laboratory, Livermore, CA 4California Department of Health Services, Sacramento, CA 5New York State Department of Health, Albany, NY 6Artificial Intelligence Lab and Hoffman E-Commerce Lab, University of Arizona, Tucson

45 Epidemiological Models for Global Surveillance of Foot-and-mouth Disease

A.M. Perez1, M.C. Thurmond1, T.E. Carpenter,1 T.W. Bates,2 W.O. Johnson,3 B.A. Melbourne,1 S.A. Aslam,1 R.B.Garabed,1 Y.ku Choi,3 A.J. Branscum, 3 M.L. Gallego,1 and P.W. Grant1

Foot-and-mouth disease (FMD) is one of the most contagious and costly diseases of domestic livestock, the introduction of which into the U.S. would result in major destruction to animal agriculture, livestock markets, and critical infrastructures. The disease is distributed globally and has recently spread to countries with national biosecurity programs similar to that currently practiced by the U.S. In order to improve biosecurity against FMD, it will become increasingly important to have knowledge of the current status and risk of FMD in the world, including location, movements, and dynamics that may foster spread of FMD to the U.S. In this session, we will present briefly epidemiological models that that our team has developed to describe the global temporal-spatial distribution of FMD and of FMD risk, including factors that predict changes in FMD status or movement, as applicable to real-time global FMD surveillance.

Two general approaches were taken, one describing FMD in a global context and the other in a country or regional context. FMD data were obtained from the OIE FMD HandiSTATUS data bank, and surrogate and covariate information was obtained from Internet-accessible data sources for the countries examined. Regional models focused on the time, space (geographic location), and time-space evolution of FMD in specific countries. A probabilistic cokriging model of FMD in Pakistan used information from partial reporting, and a hybrid model with kernel density, spatial scan statistic, and time series methods predicted FMD and FMD cycles in Iran. Bayesian autoregressive, spatio-temporal models characterized FMD in Turkey, Iran, and Afghanistan. Anomaly detection models were developed to identify unexpected locations or numbers of cases. Markov chain methods and risk analysis, including gravity models, projected the likely introduction of FMD into FMD-free regions, such as California and areas in Colombia, and directional models were used to estimate the most likely direction of FMD transmission in Bolivia. Global models predicted current and future FMD status in regions and countries of the world. Bayesian polytomous response logistic regression models predicted country-specific FMD status based on measures of a country’s political, economic, and agricultural viability and stability, and a global ecological model identified ecological zones of the world that would favor FMD. Country-specific models were validated using methods of cross-validation and expert opinion, as solicited from veterinarians in each country.

Generally, the models identified several predictors of FMD, including cattle density, presence of major roads or highways, favorable temperature, moisture, and elevation, political and economic instability, seasonality changes, religious events, and livestock market price differential across borders. These models can be applied via web portals for real-time global risk surveillance of FMD to characterize changes in time, place, and transmission of FMD, and to identify new anomalous and unexpected FMD cases or precursor events. Real-time global risk surveillance for FMD, utilizing prediction, forecasting, and anomaly detection models, would improve our awareness and assessment of FMD globally and would provide fundamental information to develop and enhance national biosecurity.

1The FMD Modeling and Surveillance Laboratory, Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 2Lawrence Livermore National Laboratory, Livermore, CA 3Department of Statistics, University of California, Davis, CA

46 Foot-and-mouth Disease Surveillance Involving the Testing of Bulk Milk Tank Samples

M.C. Thurmond1 and A.M. Perez1

Effective control and eradication of foot-and-mouth disease (FMD) in the U.S. will depend on very early detection and destruction of infected animals, before the virus can be transmitted widely to uninfected herds, states, and countries. The current system for detecting FMD relies mainly on the observation of livestock and on recognition of FMD clinical signs. Moreover, subtle clinical manifestations can be overlooked and misdiagnosis is possible for the many common indigenous diseases with signs that mimic those of FMD. Historically, the sole reliance on clinical recognition has delayed FMD diagnosis by weeks (U.K. 2001) or even months (Taiwan 1997), resulting in catastrophic dissemination of the disease. Prerequisite to achieving effective, early control will be implementation of a surveillance system that permits early detection of the virus before clinical signs become apparent or are recognizable.

We present a modeled assessment of routine testing of bulk milk samples for the presence of FMD virus, considering 2x milking and twice-daily tank sampling for intensively managed, dry lot dairies of California. A simulation model for detection of the first (index) case of FMD was developed to include transmission of the virus to cows within a corral holding 100 head and among corrals, the concentration of virus in milk of infected cows, milk production decline as a consequence of FMD, and dilution of virus in the bulk tank. Two herd sizes of 100 and 1000 lactating cows were considered, each with an average milk production of 32 liters/cow/day. Other assumptions were that exposure at time zero was from one infectious cow in a corral holding 100 cows and 95% of the cows became infected within 3 days. Concentration of FMDV plaque forming units in milk, milk production decline, and time-to-appearance of clinical signs were estimated from data in the literature. The assumed detection threshold for the PCR milk assay was 100 virus particles/ml and the number of virus particles/pfu was 1000 (Dr. T. Beckham, personal communication). Locally conducted real-time PCR testing was assumed for milk and clinical samples.

The model indicated that virolactia would begin in a small proportion of cows by 3 days after exposure and would be present in a few cows for up to 12 days. Estimated log10 virus concentrations ranged from 3.82 to 9.1 per ml of milk secreted. Milk production declined by 40%, beginning 2 days before lesions first appeared. Clinical signs appeared 6 days after exposure, all cows had clinical signs by 9 days post exposure, and FMD was diagnosed by PCR testing of clinical samples 7 days after exposure. After 10,000 simulations of the model for a 100-cow herd, FMDV detection in the bulk milk was predicted as early as 3 days, and, on average, 5.3 days after exposure. For a 1000-cow herd, the model did not indicate FMDV would be detected in the bulk milk at all during the first 12 days after exposure. For large herds, an alternative approach that would lessen the dilution effect of a large volume of bulk milk, and possibly further improve detection time, would be to test bulk milk from cows in ‘hospital’ pens (50-75 cows) or from individual milking strings (100-125 cows each).

These results suggest that, depending on assay detection limits, routine testing of bulk tank milk for FMD virus in small herds or groups of cows could result in earlier detection of FMD than by relying on recognition of clinical cases, and that bulk tank milk testing should be explored as a possible FMD surveillance strategy. Improvements in assay detection limits and overall diagnostic sensitivity would likely further reduce the delay in FMDV detection in bulk milk.

1The FMD Modeling and Surveillance Laboratory, Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA

47 The 13th Edition of the UN Model Regulations on Transport of Dangerous Goods: Effects on Veterinary Diagnostic Laboratories

L.J. Thompson1

These Recommendations have been developed by the United Nations Economic and Social Council's Committee of Experts on the Transport of Dangerous Goods in the light of technical progress, the advent of new substances and materials, the exigencies of modern transport systems and, above all, the requirement to ensure the safety of people, property and the environment. They are addressed to governments and international organizations concerned with the regulation of the transport of dangerous goods.

These regulations form the basis of all international air transport of dangerous goods, including the ICAO and IATA regulations. Many individual countries use these UN Model Regulations as the basis of their dangerous goods transport regulations. The US Department of Transportation has stated their intention of harmonization with these regulations. The regulations are due to take effect after January 2005.

There are several general and specific points in the proposed regulations that will have direct effects on AAVLD member laboratory activities. The UN is abandoning the use of Risk Groups 1, 2, 3 and 4 to classify infectious substances for transport. The new classification scheme will divide infectious substances into Category A and Category B groups. Category A includes infectious substances which are transported in a form that, when exposure to it occurs, is capable of causing permanent disability, life threatening or fatal disease to humans or animals. Category B group will include all other infectious substances which do not meet the criteria for inclusion in Category A. This list was developed using a risk assessment approach on the individual infectious substance.

Category A infectious substances must be shipped using UN P620 packaging with accompanying shippers declaration of dangerous goods. Category B infectious substances must be shipped using UN P650 packaging. There is no requirement for a shipper’s declaration of dangerous goods for Category B items. Cultures of Category B infectious substances that are transported for the purpose of identification can be shipped as Diagnostic Specimens, using UN 3373. These will require UN P650 packaging. Substances which do not contain infectious substances or which contain substances unlikely to cause disease in humans or animals are not subject to these regulations.

There will be training requirements for all persons who package Category A or Category B infectious substances for shipment to assure proper selection and use of packaging material and proper completion of declarations and package markings.

1Veterinary Diagnostic Laboratory, University of Georgia College of Veterinary Medicine, Tifton, Ga.

Pathology Scientific Session

48 Saturday October 23, 2004 1:00 p.m. – 3:00 p.m. Auditorium II

Moderators: H. L. Shivaprasad and Donal O’Toole Page 1:00 p.m. Hair-loss Syndrome in Pacific Northwest Black-tailed Deer - R.J. Bildfell, J.W. Mertins, J.A. 51 Mortenson and D.F. Cottam

1:15 p.m. Avian Tuberculosis and Erysipelas Infection in Chukars - A.S. Dhillon - D. Schaberg; S.K. 52 Weber; D.V. Bandli and F. Wier

1:30 p.m. Hepatic Cirrhosis and Hemochromatosis in an Onager (Equus hemionus) - J.R. Hayes, R.A. 53 Gandolf, S. D. Grimes, J. Thilsted, and M. Atkinson

1:45 p.m. Pathology of West Nile Virus Disease in North Carolina Alligators - P.G. Moisan, L.F. 54 Humphries, S.J. Page, and J.M. Law

2:00 p.m. Successful Experimental Induction of Acute Malignant Catarrhal Fever in Bison Using Aerosols 55 of Ovine Nasal Mucus Containing Ovine Herpesvirus-2 (OvHV-2) - D. O' Toole, N.S. Taus, W.C. Davis, T.B. Crawford, and H. Li

2:15 p.m. Ulcerative Enteritis Associated with Clostridium perfringens Type A in Bobwhite Quail (Colinus 56 virginianus) - H.L. Shivaprasad, R. Kokka, R. Crespo, G. Songer, P. Cortes and F. Uzal

2:30 p.m. Tongue Is An Excellent Sample for Parvoviral Diagnosis in Dogs and Cats - C.A. McKnight*, M. 57 Kiupel, A. Wise, and R. Maes

2:45 p.m. Rangelia vitalli Infection in Dogs: Epidemiological, Clinicopathological and Ultrastructural 58 Findings - A. P. Loretti*, S.S. Barros

50 Hair-loss Syndrome in Pacific Northwest Black-tailed Deer

R.J. Bildfell1, J.W. Mertins2, J.A. Mortenson3 and D.F. Cottam4

A syndrome of hair loss (HLS) has affected Columbian black-tailed deer (Odocoileus hemionus columbianus) in western Oregon and Washington since 1996. Affected animals develop thin hair coats to completely alopecic areas over the thorax, flanks and hindquarters. Necropsy examinations of 21 HLS- affected black-tailed deer (BTD), including 11 natural mortalities and 10 euthanatized animals, revealed all animals to be in poor body condition. Juveniles were most frequently affected (18/21) and males were over-represented (76%). At least 17 of 21 deer carried large burdens of internal parasites and all animals had at least some degree of verminous pneumonia. A variety of other lesions such as subcutaneous abscesses or bacteria pneumonia were identified in a few animals. Virus isolation attempts on 5 deer were unrewarding.

Large numbers (tens of thousands) of chewing lice were present on all animals, yet with the exception of two juveniles with demodecosis, only small numbers of other ectoparasite species were found. Histopathology of alopecic areas was characterized mainly by orthokeratotic hyperkeratosis with most follicles in telogen/catagen phase. However, samples from non-alopecic to interface regions often revealed a superficial perivascular eosinophilic dermatitis, sometimes with eosinophilic erosions and parakeratotic crusts. The louse was identified at the National Veterinary Services Laboratories (Ames, Iowa) as an indeterminate species of Damalinia (Cervicola). Members of this subgenus typically parasitize Old World ungulates and have not been previously documented on North American cervids. We postulate that the surprising severity and breadth of this pediculosis outbreak in the Pacific Northwest BTD is largely due to ectoparasitism by a species of chewing louse that represents a new pathogen for the host. This loss of pelage and increased time spent grooming may also be important contributors to the poor body condition of these animals. The emergence of HLS in at least one Oregon wildlife management district has coincided with a drop in fawn survival rates but studies to document a causal relationship have not been performed.

1 Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR. 2 U.S. Department of Agriculture-Animal and Plant Health Inspection Service, Veterinary Services, National Veterinary Services Laboratories, Ames, IA. 3 U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services, Salem, OR. 4 Oregon Department of Fish and Wildlife, Newport, OR

51 Avian Tuberculosis and Erysipelas Infection in Chuckars

A. S. Dhillon, D. Schaberg; S. K. Weber; D. V. Bandli and F. Wier

Four chuckars were submitted for postmortem examination, with a history of a loss of 21 birds out of a breeder flock of 200 chuckars. Other adjoining breeder flocks were not affected. The livers of three chukars were severely enlarged, hard in texture, and some areas were pale. The liver of one chukar was not enlarged, however, had numerous pale white areas. The spleens of all chukars were enlarged and contained numerous pale white foci. The cecal wall contained several raised focal areas on the serosal surface. The mesenteric attachments near the ceca and serosal surface of the rectum of one chukar contained several raised focal areas. The results of aerobic cultures performed on one liver and two individual spleens were negative. Results of salmonella culture were negative on composite sample of all livers and intestines. Composite tissues of liver and spleen were positive for M. avium by isolation. The isolated organism was also identified by PCR to be M. avium.

Microscopically the hepatocytes of all four liver sections were replaced by multifocal, small to large, occasionally coalescing aggregates of hypereosinophilic, granular material, surrounded by a rim of macrophages and multinucleated giant cells, bordered by dense fibrous tissue or collagen, or small foci of epitheloid macrophages, lymphocytes, and plasma cells. The hypereosinophilic material often contained large numbers of acid-fast bacilli (presumptive Mycobacterium spp.). In the adjacent hepatocellular cord, 75 to 95% of the hepatocytes were widely separated or replaced by large amounts of pale eosinophilic, homogenous to finely granular material (presumptive amyloid) that often obscured sinusoids. Remaining hepatocytes were mildly to moderately swollen and contained numerous, indistinct, intracytoplasmic vacuoles. The sinuses and lymphoid aggregates in the spleen were replaced by multifocal, large aggregates of cellular debris surrounded by macrophages, multinucleated giant cells, and fibrous tissue, similar to those described in the liver. Small to large, multifocal granulomas were present in the intestine, similar to those described in the liver and spleen replaced the lamina propria, as well as the tunica muscularis. Similar, smaller granulomas were adhered to the serosal surface, and widened the coelomic air sacs. Histologic diagnosis was of hepatitis, granulomatous, multifocal, and severe with presence of intralesional Mycobacterium spp. Other lesions were of amyloidal splenitis, granulomatous, severe, with intralesional Mycobacterium spp. The intestine lesions were of enteritis, coelomitis, multifocal, severe, with intralesional Mycobacterium spp.

Three twelve-week-old, chukars were submitted for examination from a different farm with a history of mortality. Two out of three birds had mild hydropericardium. The hearts, livers and spleens were congested, mottled and enlarged. Focal, pale, hemorrhages were present in the pancreas. The kidneys were swollen, congested, and mottled. Urate deposits were present in the ureters in all birds. From three livers Erysipelothrix rhusiopathiae was isolated in large numbers and in pure culture. The results of Salmonella isolation were negative. Microscopically multiple sections of spleen had severe splenic necrosis and nearly no normal splenic parenchyma was present, and areas of necrosis occupied up to an estimated 30% of the sectional area. Additionally, many extravascular colonies of bacterial organisms were prominent within some section of spleen. Section of heart, liver, kidney, pancreas and intestine were generally well preserved, and altered by numerous bacterial organisms within small vascular spaces. Often these accumulations of bacteria were not associated with an inflammatory cellular response or any degeneration or necrotizing change in the adjacent tissues. Staining with Brown and Hopps stain confirmed the organisms as Gram-positive and often elongate rods. The final diagnosis was of hepatitis and splenitis of Erysipelothrix rhusiopathiae etiology

Avian Health and Food safety Laboratory, WADDL, Washington State University, 7613 Pioneer Way E. Puyallup, WA

52 Hepatic Cirrhosis and Hemochromatosis in an Onager (Equus hemionus)

J.R. Hayes 1, R. A. Gandolf 2, S. D. Grimes 1, J. Thilsted 1, M. Atkinson 2

A dead 17-year-old female Persian onager weighing 422 pounds was presented in February, 2004 with a history of mild depression for 2 days, which progressed over the next 2 days to severe depression and recumbency. The animal was chemically immobilized for examination, treatment and relocation to a barn. Physical examination revealed ulcers on the distal and lateral aspects of the tongue, and a distended mass was detected by rectal palpation. The animal died as anesthetic reversal drugs were being administered. Serum chemistry from a sample collected during immobilization revealed elevated levels of AST (1190 U/L), GGTP (899 U/L), alkaline phosphatase (723 U/L) and CPK (657 U/L). Also elevated were BUN and creatinine levels (50 and 2.8 mg/dl, respectively). Bilirubin levels were within normal range (1.7 mg/dl) and chloride levels were mildly decreased (90 mEq/L). There was an increased white blood count (35,000/uL) with neutrophilia (30,450) and monocytosis (1050), and red blood cells and hematocrit were decreased (4.24x106/uL and 28.5%, respectively). Serum ferritin was markedly elevated (19,122 ng/ml; reference range 43-761) although serum iron and total iron binding capacity levels were within equine reference ranges (113 and 397 ug/dl, respectively).

Necropsy revealed an enlarged firm liver that weighed 27 pounds (6.4% body weight), multiple fibrous adhesions bridging the convex surface of the liver to the diaphragm, and approximately 150 ml of serosanguineous fluid in the abdominal cavity. The liver contained areas that were orange, greenish brown or red, with multiple 2-15 mm grayish white firm lobulated areas of fibrous connective tissue within the parenchyma, which had a gritty consistency and was tough to cut.

Microscopic lesions in the liver included marked atrophy and loss of hepatocytes, with micronodular hepatocellular regeneration, bridging portal fibrosis, biliary hyperplasia, with widespread effacement of hepatic architecture and thickening of the hepatic capsule by bands of fibrous connective tissue (accompanied by proliferation of fibroblasts). Golden brown granular cytoplasmic pigment was abundant in hepatocytes, Kuppfer cells and within macrophages in connective tissue; this material stained positive for iron with Prussian blue. Multiple large bizarre cells were randomly distributed throughout sections, and 0-4 mitotic figures were observed at 400X (mean 0.67 in 10 fields). Other hepatic lesions included foci of coagulative necrosis, abscessation, acute hemorrhage and lymphocytes. Prussian blue positive iron pigment was also detected in macrophages in the heart, lung, kidney (within glomeruli), spleen and ovary.

Liver iron analysis revealed 5140 ppm (wet weight basis); reference range for horses is 50-600 ppm. Levels of other elements were within normal limits for equine liver.

Review of records at the ADDL revealed that tissues from three other onagers from this facility also contained cytoplasmic pigment compatible with iron, confirmed by Prussian blue staining. Serum samples from two of these onagers also contained markedly increased ferritin levels (4132 and 18, 270 ng/ml) but normal serum iron and TIBC levels.

This is the first report of hemochromatosis in an onager to our knowledge. Hemochromatosis is the most common heritable metabolic disease in humans, and has been reported in animals such as Myna birds, toucans, lories, Salers cattle and in horses. The pathogenesis of this condition in onagers is not known. Review of the diet did not suggest that oral iron overload was likely. Review of breeding records of onagers in the United States may be useful to determine if common recent ancestors may be a factor.

1 Animal Disease Diagnostic Laboratory, Ohio Department of Agriculture, Reynoldsburg, OH 2 The Wilds, Cumberland, OH Pathology of West Nile Virus Disease in North Carolina Alligators

P.G. Moisan1. L.F. Humphries2, S.J. Page2, J.M. Law2

West Nile Virus is a Flavivirus of the Family Flaviviridae that has caused disease in birds, mammals, and reptiles of multiple species since its introduction into North America in 1999. We describe an outbreak and pathological findings in an outbreak that occurred among housed alligators at a commercial alligator farm in January-February 2004.

The alligators were housed in group pens, separated by age and size, and had not been mixed for several months. The diet consisted of culled birds from local chicken farms. Four alligators from the first group of animals (1.5 years old) were presented in January, and 3 alligators from a separate group (4.5 years old) were presented in February. Death loss was 38 of 250 alligators from the first pen. No premonitory signs were exhibited in the first pen. A single moribund alligator was presented with the first group. In the second pen, 30 of 150 animals died after the entire pen was anorexic for 7-10 days.

Necropsy results for all 7 animals were similar, consisting of necrotizing stomatitis, laryngitis, esophagititis, enterocolitis, hepatitis, and splenitis.

Bacteriology was performed on various tissues, including larynx, kidney, liver, intestine, and lung. Isolates from various tissues of all 7 alligators consisted of Aeromonas hydrophila, Edwardsiella tarda, Morganella morganii, Plesiomonas shigelloides, and Proteus rettgeri. Salmonella hadar was isolated from the first group; S. senftenberg and S. worthington were isolated from animals in the second group.

Histopathology was performed on major tissues from each group of alligators. In addition to the lesions described at the time of gross examination, encephalitis was also identified histologically.

Immunohistochemistry performed on numerous formalin-fixed, paraffin-embedded tissues was positive for liver, spleen, kidney, gastrointestinal tissues, and lung. There was no reaction of the affected brain from a single animal tested by immunohistochemistry.

Viscera from 11 clinically healthy alligators were collected at slaughter during different slaughters from the month of March 2004. Samples from these were tested by polymerase chain reaction technique and were found to be negative for the West Nile virus. Bacterial isolates from these healthy alligators were similar or identical to those found in the animals from the 2 pens of sick animals.

The source of the virus was not determined.

We conclude that major death losses on alligator farms from West Nile Virus infection are possible during winter months, even while North Carolina mosquito populations are in the overwintering period.

References: 1. Miller DL, Mauel MJ, Baldwin C, Burtle G, Ingram D, Hines II MJ, Frazier KS. West Nile virus in farmed alligators. Emerg Infect Dis [serial online] 2003 Jul [date cited]. Available from: URL:http://www.cdc.gov/ncidod/EID/vol9no7/03-0085.htm 2. Brinton MA. The molecular biology of West Nile Virus: a new invader of the Western Hemisphere. Annu Rev Microbiol. 2002;56:371-401

1 Rollins Animal Disease Diagnostic Laboratory, North Carolina Department of Agriculture, Raleigh, NC 2 Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC

54 Successful Experimental Induction of Acute Malignant Catarrhal Fever in Bison Using Aerosols of Ovine Nasal Mucus Containing Ovine Herpesvirus-2 (OvHV-2)

D. O'Toole1, N. S. Taus2, W. C. Davis3, T. B. Crawford3, and H. Li2

Sheep-associated malignant catarrhal fever (MCF) is a diagnostic, epidemiological and experimental challenge, in large part because the causative agent, OvHV-2, has not been cultured. Information about the behavior of the virus in susceptible host species has, until recently, been derived from field studies of spontaneous outbreaks. Experimental transmission relied on the use of large volumes of fresh blood from natural cases of animals terminally affected with MCF, making replicate studies impossible. Following the recognition that aerosolized nasal mucus from sheep undergoing intense viral shedding could transmit infection to uninfected sheep (Kim et al, 2003, Virus Res 98(2):117-122), we conducted two studies to establish whether aerosol exposure induced MCF in bison. Bison were chosen due to their known high susceptibility to the disease.

In study 1, four yearling male bison seronegative for MCF viruses were aerosolized with nasal secretions from sheep experiencing intensive shedding events. Each of two animals received 2 ml of an inoculum containing ~4.7 x 107 OvHV-2 DNA copies from one sheep (#2698). Each of the other two bison received 2 ml of inoculum containing ~2.7 x 107 OvHV-2 DNA copies from another sheep (#2357). By 16 - 20 days post-aerosolization (DPA), the four bison were serologically positive. By 16 - 24 DPA, OvHV-2 DNA was detectable by nested PCR in peripheral blood leukocytes of all four animals. OvHV-2 DNA levels in PBLs of individual bison reached peak levels (59,000- 109,000 copies/2 g DNA) at 29, 29, 33, and 46 DPA, respectively. The four died 29, 29, 41 and 49 DPA. All four had gross and histological lesions typical of MCF, comprising colitis, cystitis, abomastitis and vasculitis. In study 2, eight yearling bison were used. Four served as inoculated principals, two were in-contacts, and two served as unexposed controls. The principals were inoculated intranasally with a mixture of nasal secretions (~2.35 x 107 copies from the same inoculum of sheep #2698 in study 1, plus ~3.23 x 106 copies from sheep #2609). The in-contact and unexposed bison were inoculated with nasal mucus collected from an OvHV-2 negative sheep. In the principles group, two animals (#5 and #8) became PCR-positive at 38 and 41 DPA, and remained PCR-positive. These animals seroconverted at 55 and 75 DPA, respectively. The remaining two bison (#2 and #6) in the principal group were PCR-positive at one-time point only (59 and 62 DPA), and remained negative by cELISA. One animal (#5) of four in the principal group developed clinical signs of MCF and died at 66 DPA. The remaining three principals survived to the end of the study (>100 DPA). All in-contacts and unexposed group animals remained PCR-negative, and none developed clinical signs or lesions of MCF. We conclude that: • Aerosolized nasal mucus derived from sheep during peak shedding events of OvHV-2 is a viable method for inducing clinical MCF in bison. • Developing pooled, standardizing inocula from sheep shedding OvHV-2 is an important goal in order to develop a challenge model of MCF, which is superior to the use of whole blood from field cases of MCF since it allows replicate studies. • The different outcomes in study 1 (100% incidence MCF) vs. study 2 (25% incidence of MCF) may be attributable to insufficient nebulization procedures due to a frozen nebulizer tube under severe weather conditions (-20 C) during study 2 • The dose of inoculum may be a factor determining whether and when bison develop MCF, or become latently infected. • This is the first report of successful transmission of MCF via a natural route in any species.

1Wyoming State Veterinary Laboratory, 1174 Snowy Range Road, Laramie, WY 82070. 2Animal Diseases Research Unit, USDA-ARS, Washington State University, Pullman, WA 99164. 3Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164.

55 Ulcerative Enteritis Associated with Clostridium perfringens Type A in Bobwhite Quail (Colinus virginianus)

H. L. Shivaprasad1, R. Kokka1, R. Crespo1, G. Songer3, P. Cortes1 and F. Uzal2

During the latter part of 2003 a producer lost more than 50 % of 1000 bobwhite quail between 8 and 16 weeks of age. Clinical signs ranged from sudden death to listlessness, depression, watery white droppings, ruffled feathers, loss of weight and increase in mortality. Necropsy of approximately 30 birds revealed multiple deep ulcers of the mucosa throughout the small intestine and ceca. The ulcers could be seen from the serosa of the intestine, and some caused perforation and subsequent peritonitis. The livers in some birds contained white foci of necrosis, and many birds had enlarged and congested spleens. Most of the birds were emaciated. Microscopic lesions consisted of multifocal severe fibrinosuppurative ulcerative enteritis associated with large numbers of rod-shaped bacteria, and necrotizing hepatitis with or without rod-shaped bacteria.

The clinical signs and lesions in these birds resembled ulcerative enteritis. Ulcerative enteritis also called Quail disease is one of the most common and important diseases of quail caused by the bacterium Clostridium colinum. However, extensive anaerobic culturing of the intestine and liver yielded almost pure cultures of only C. perfringens. C. colinum was not isolated in spite of using special media containing 8 % horse plasma. Further, Gram staining of the intestinal and liver impression smears from the submitted quail revealed bacteria consistent with the morphology of C. perfringens. Most of the C. perfringens isolated from liver and intestine were genotype A and were PCR positive for cpb2, the structural gene for β2 toxin.

Retrospective examination of records of quail submissions to the California Animal Health and Food Safety Laboratory System revealed many cases where C. perfringens was isolated from the liver and intestine and was associated with hepatitis and ulcerative enteritis in quail. Thus retrospective and current findings suggest that C. perfringens type A may cause ulcerative enteritis in quail, with clinical signs and lesions which resemble those of ulcerative enteritis caused by C. colinum. Final conclusions await experimental reproduction of the disease.

1Fresno Branch, 2San Bernardino Branch, California Animal Health and Food Safety Laboratory System, College of Veterinary Medicine, University of California, Davis, CA. 3Department of Veterinary Science and Microbiology, University of Arizona, Tucson, AZ.

56 Tongue Is An Excellent Sample for Parvoviral Diagnosis in Dogs and Cats

C.A. McKnight*1, M. Kiupel1, A. Wise2, and R. Maes2

Parvoviruses are non-enveloped single stranded DNA viruses, which require mitotically active cells for viral replication. The most commonly used tissues for the diagnosis of canine and feline parvovirus are small intestine, spleen, lymph nodes and bone marrow. Due to the rapid turnover of the epithelium of the tongue, this may also be a site of viral replication. Pseudocytoplasmic and intranuclear inclusions have been previously identified in the epithelium of the tonguea,b supporting our hypothesis. However, no comprehensive studies have been done to determine the sensitivity and specificity of the tongue as a sample for parvoviral detection.

To test our hypothesis, a protocol was designed for the collection of samples from dogs and cats that had died with clinical signs of a parvoviral infection. Gross necropsy examination was performed on 8 dogs and 8 cats submitted to the Michigan State University Diagnostic Center for Population and Animal Health with a clinical history suggestive of parvovirus. The tongues of all animals were carefully examined grossly for epithelial lesions. Selected tissues, including tongue and small intestine, were fixed in 10% neutral buffered formalin for microscopic examination. Sections of tissue were also stained for parvoviral antigen using immunohistochemistry (IHC). Fresh samples of small intestine and tongue were submitted for direct fluorescent antibody (FA) testing. RT-PCR was also done on DNA extracted from 5- 10 micron sections of formalin fixed, paraffin imbedded tissue.

Eleven out of 12 animals, which had suspect or positive FA staining in the small intestine, and correlating histologic evidence of crypt necrosis, also had positive FA and immunohistochemical staining in the tongue. One dog with microscopic evidence of crypt necrosis, which had been frozen for one month prior to necropsy, was suspect by FA in the small intestine, but the tongue tested positive by FA, IHC and RT- PCR. Our results to date indicate that tongue is an excellent sample for parvoviral testing in dogs and cats, especially in cases where postmortem autolysis may preclude optimal microscopic and immunohistochemical evaluation.

References aHullinger, G.A., M.E. Hines II, E.L. Styer, K.S. Frazier, and C.A. Baldwin. 1998. Pseudocytoplasmic inclusions in tongue epithelium of dogs with canine parvovirus-2 infections. Journal of Veterinary Diagnostic Investigation. 10: 108-111. b Matsui, J., J. Matsumoto, T. Kanno, T. Awakura, H. Taniyama, H. Ruuoka and H. Ishikawa. 1993. Intranuclear inclusions in the stratified squamous epithelium of the tongue in dogs and cats with parvoviral infection. Veterinary Pathology. 30: 303-305.

1Diagnostic Center for Population and Animal Health, Michigan State University, E. Lansing, MI. 2Department of Veterinary Microbiology, Michigan State University, E. Lansing, MI.

*To be considered for graduate student award.

57 Rangelia vitalli Infection in Dogs: Epidemiological, Clinicopathological and Ultrastructural Findings

A. P. Loretti*1, S. S. Barros2 This report describes the epidemiological, clinical, pathological and ultrastructural findings of a tickborne disease of dogs caused by the obligate intracellular organism Rangelia vitalli. The disease, first described in Brazil in 1908, has been referred to popularly as “nambiuvú” (bloody ears) or “peste de sangue” (bleeding plague). The life-cycle of R. vitalli consists of an intraerythrocytic developmental phase, and an exoerythrocytic phase occurring in the cytoplasm of endothelial cells. R. vitalli-parasitized erythrocytes are regarded as uncommon to rare findings in blood smears, especially in the chronic form of the infection. In this case series (2000 and 2002-3), the disease was diagnosed in 8 dogs from southern Brazil. It was characterized by anemia, jaundice, fever, splenomegaly, lymphadenomegaly, widespread petechiation on the oral and vaginal mucosa, persistent bleeding from tips and external surface of the pinnae and mouth, epistaxis, and bloody feces. The disease was observed in dogs from rural and suburban areas of 3 different couties in the state of Rio Grande do Sul (RS). Categories of dogs affected by the pathogen included hunting dogs, guard dogs and companion dogs. The disease occurred at any time of the year, although peak occurrence was observed during the summer and was associated with the presence of large populations of ticks. The ixodid ticks Rhipicephalus sanguineus and Amblyomma aureolatum were consistently found infesting affected dogs from suburban areas and rural areas, respectively. Laboratory findings included regenerative anemia, spherocytosis, icteric plasma and bilirubinuria. Four animals died spontaneously. Of those, 3 animals were jaundiced and died acutely after approximately 1 week. One animal that died after a long, protracted clinical course of approximately 2-3 months had anemia. One animal recovered after therapy with imidocarb dipropionate and blood transfusion, and another one recovered after treatment with doxycycline and corticotherapy. Two animals died after therapy with diminazene aceturate. Necropsy findings included diffuse pallor or yellowish discoloration of the carcass and internal organs, enlarged spleen, generalized lymph node enlargement and hemorrhage in the gastrointestinal tract. Microscopically, R. vitalli was seen in parasitophorous vacuoles in the cytoplasm of endothelial cells of blood capillaries. R. vitalli was not found in erythrocytes. Marked erythrophagocytosis was observed in the lymph nodes. Ultrastructurally, those organisms were also observed free in the lumen of the capillaries. The fine structure of both the parasite and its parasitophorous vacuole were similar to those protozoa of the phylum Apicomplexa. The disease was successfully reproduced in one experimental dog inoculated IV with blood sampled from an infected dog. Immunohistochemistry for L. chagasi, N. caninum and T. gondii was consistently negative. No other blood parasites or rickettsial agents were found in erythrocytes or leucocytes in both spontaneous and experimental cases. Gross and histological findings typical of diamidine poisoning i.e. symmetric bilateral hemorrhagic encephalomalacia affecting the brainstem were observed in those 2 animals treated with diminazene aceturate. In both cases, the gross and histological lesions were consistent with R. vitalli infection but no parasites were observed. Tentative clinical diagnosis of R. vitalli infection was based on the history, clinical picture, hemogram and favorable response to therapy. The diagnosis was confirmed through microscopic examination of smears from the bone marrow sampled during necropsy or histological sections from multiple tissues especially the lymph nodes where the organisms were most frequently found. Based on our ultrastructural findings, we suggest that R. vitalli be considered as a protozoan parasite of the phylum Apicomplexa. Spherocytosis and erythrophagocytosis suggest that R. vitalli causes an immune mediated hemolytic anemia as previously observed by other authors. Maybe wild animals are reservoirs of this parasite since in rural areas the tick A. aureolatum has been found infesting domestic dogs as well as wild carnivores.

1Section Vet Pathol, Dept Vet Clin Pathol, Fac Vet Med, Fed Univ Rio Gde Sul (UFRGS), Porto Alegre, RS, Brazil. Current Position: graduate student, PhD Pathobiol, Dept Pathobiol, Ontario Vet Coll (OVC), Univ Guelph, Guelph, Ontario, Canada. 2Dept Anim Pathol, Fac Vet Med, Fed Univ Pelotas (UFPel), Pelotas, RS, Brazil *To be considered for graduate student award

58 Toxicology Scientific Session Saturday October 23, 2004 1:00 p.m. – 3:00 p.m. Auditorium IV

Moderators: Robert Poppenga and Catherine Barr Page

1:00 p.m. Brain Concentrations of Macrolide Endectocides Associated with Cases of Intoxication or 61 Suspected Intoxication - R.H. Poppenga - I. Rudik-Miksa, and M.R. Cummings

1:15 p.m. The Use of LC/MS Methodology in the Diagnosis of Paraquat Poisoning - B. Puschner, M.S. 62 Filigenzi, E. Pratt, E.R. Tor

1:30 p.m. Determination of Liver Vitamin E: Stability and Matrix Distribution - I. Rudik-Miksa, R.H. 63 Poppenga, and M.R. Cummings

1:45 p.m. Screening of Erythropoietin, Recombinant Human Erythropoietin and Darbepoietin-alpha in 64 Horse Plasma - A.K Singh, S. Gupta, and A. Sage

2:00 p.m. Diagnosis of Taxus (Yew) Poisoning in a Horse - A.K. Tiwary*, B. Puschner, H. Kinde, J. 65 Reagor and E.R. Tor

2:15 p.m. Paresis and Death in Elk (Cervus elaphus) Due to Lichen Intoxication in South Central Wyoming 66 - W.E. Cook, M.F. Raisbeck, T.E. Cornish, E.S. Williams, B. Brown, and G. Hiatt

2:30 p.m. Toxicologic Surveillance of Search & Rescue Dogs Deployed to the World Trade Center, The 67 Pentagon, and The Staten Island Fresh Kills Landfill Sites - W.K. Rumbeiha, S.D. Fitzgerald, W.E. Braselton, C.M. Otto, A.B. Downend

2:45 p.m. Proteomics Approaches to Biomedical Problems - S. B. Hooser, C.R. Wilson 68

* Graduate Student Competition

60 Brain Concentrations of Macrolide Endectocides Associated with Cases of Intoxication or Suspected Intoxication

R.H. Poppenga1, I. Rudik-Miksa1, and M.R. Cummings1

Macrolide endectocides are compounds with activity against both internal and external parasites, specifically nematodes and arthropods. They are derivatives of fermentation products of soil-dwelling bacteria (Streptomyces spp.) and are biologically active at low concentrations. There are two major groups: the avermectins, including abamectin, ivermectin and doramectin and the milbemycins including milbemycin oxime and moxidectin. The macrolide endectocides have a wide margin of safety, although intoxication can occur following over-dosage, especially in sensitive dog breeds such as Collies. Our laboratory developed a simple, rapid and sensitive LC/MS method for detection of several macrolide endectocides (ivermectin, doramectin, selamectin, moxidectin and eprinomectin) in biological fluids and tissues in order to assist in the investigation and diagnosis of suspected intoxications.

To date, forty cases requesting macrolide endectocide detection and quantification have been received. Thirty-three cases involved testing of samples collected from exposed animals, four cases involved testing of drug products and three cases involved testing of food products (1 meat and 2 milk samples). Samples from a variety of animal species have been submitted including dogs (n = 18), horses (n = 4), sheep (n = 3), cats (n = 2), goats (n = 2), an iguana, a deer, a cow and a ferret. Most cases involved exposure to ivermectin (n = 19) followed by moxidectin (n = 7), selemectin (n = 5) and eprinomectin (n = 1). Ivermectin was the most common macrolide endectocide of concern in dogs (n = 10) followed by selemectin (n = 4) and moxidectin (n = 3).

Sufficient information was available for ten cases to categorize them as either a toxicosis or a suspected toxicosis. Seven of these cases involved ivermectin and three involved moxidectin. While a variety of tissues were tested, brain was believed to be the most relevant since clinical signs of intoxication are related to the central GABA-ergic effects of the drugs. Brain samples were available for nine of the ten cases. Based upon quantification of ivermectin or moxidectin in the submitted brain samples, concentrations associated with intoxication or suspected intoxication ranged from 91 ppb to 645 ppb for ivermectin and 100 ppb to 160 ppb for moxidectin.

1Department of Pathobiology and the Pennsylvania Animal Diagnostic Laboratory System, New Bolton Center, University of Pennsylvania, Kennett Square, PA

61 The Use of LC/MS Methodology in the Diagnosis of Paraquat Poisoning

B. Puschner1, M.S. Filigenzi1, E. Pratt2, E.R. Tor1

Paraquat is one of the most specific pulmonary toxins known. It is a contact herbicide, classified as a restricted use pesticide by the Environmental Protection Agency and therefore can only be used by licensed users. Successful management of paraquat poisoning requires treatment within hours of the exposure, because treatment has been ineffective in the chronic stages. Thus, rapid diagnosis is crucial for the successful outcome of a paraquat poisoned animal. Over a period of several weeks in the summer of 2003, a large number of dogs were presented to veterinary emergency clinics in the Portland area. All dogs had a common history of acute onset of vomiting and diarrhea after visiting a park in the city. Initially, clinical pathological results were unremarkable. One dog had a small piece of twisted wire in his stomach, but all other dogs had normal radiographs. All dogs were hospitalized for supportive care and an emergency celiotomy was performed to remove the foreign body in one dog. The dogs continued to exhibit gastrointestinal signs for several days. Within 1-3 days after the onset of clinical signs, several dogs developed renal failure. Subsequent to the resolution of gastrointestinal signs and/or renal failure, most dogs became tachypnic and developed respiratory failure. Based on the common history and the progression of clinical signs, malicious poisoning with paraquat was suspected. Urine samples from several dogs were submitted for testing, but many of these samples were obtained days after the suspected exposure to paraquat. Only one urine sample was suspect positive for paraquat when analyzed by a qualitative colorimetric method (method detection limit 1 ppm), while the remaining urine samples were negative. Because of the severity and extent of this case, a more sensitive and reliable method was needed for testing. A rapid and sensitive analytical method using liquid chromatography-tandem mass spectrometry for the analysis of low concentrations of paraquat in urine and bait samples was developed. Urine samples were extracted using Diazem SPE columns. An aliquot of the extract was filtered through a 0.45 µm filter into a plastic autosampler vial and analyzed by LC/MS operated in a positive electrospray ionization mode. The method detection limit of paraquat in urine when analyzed by LC/MS was 0.05 ppm. The urine samples that were negative by the colorimetric SPE column methodology were found to be positive using the more sensitive LC/MS method. Diagnosis of paraquat poisoning has improved significantly since the development of the analytical method described here. The newly developed method represents a vast improvement over the existing SPE colorimetric, spectrophotometric and HPLC post column derivatization methods previously used for paraquat analysis. LC/MS is the method of choice for the forensic-toxicological investigation of poisonings by paraquat. Confirmation of paraquat in biological samples provides invaluable information to the clinician in directing the clinical course and initiating adequate treatment in confirmed, non-fatal poisoning cases.

1 California Animal Health and Food Safety Laboratory System – Toxicology Laboratory, University of California, Davis, CA 2 Dove Lewis Animal Emergency Hospital, Portland, OR. Current address: Pfizer Company, Portland, OR.

62 Determination of Liver Vitamin E: Stability and Matrix Distribution

I. Rudik-Miksa1, R.H. Poppenga1, and M.R. Cummings1

Vitamin E (VitE) encompasses a group of fat-soluble antioxidants, with α-tocopherol being the most abundant and possessing the highest biological activity. Interest in the determination of VitE status of animals is due to the occurrence of deficiency diseases such as degenerative myeloencephalopathy. The importance of α-tocopherol in the normal growth and performance of animals has resulted in VitE screening becoming a routine test offered by many veterinary diagnostic laboratories (VDLs).

A round robin was conducted between five VDLs to compare and evaluate inter-laboratory α–tocopherol variations in liver VitE concentrations. All laboratories utilized liquid chromatography (HPLC) with either UV or fluorescence (Fl) detection. Liver samples from three different animal species were analyzed and results indicated a large interlaboratory variation. For example, the range reported for liver tissue from an elk was 8.7 to 66.5 µg/g wet weight. This outcome led us to re-evaluate our liver VitE procedure. Once optimized, we conducted an α–tocopherol stability and matrix distribution study in liver. The study was designed to answer the following questions:

1) Does the sample need to be handled in a dark environment (i.e., place the sample in foil) to avoid VitE loss? 2) Do VitE concentrations vary in the liver depending on the site of sampling? 3) Does the sample have to be kept frozen to preserve VitE concentrations? 4) Do repeated freeze-thaw cycles alter VitE concentrations?

Storage of either whole pieces of liver or their corresponding homogenized fractions at room temperature and at -20ºC for various lengths of time as well as the effect of the number of freeze-thaw cycles were evaluated for their effect on VitE concentrations. Distribution of Vit E between liver lobes (3 different lobes; n = 30 animals) and within lobes (3 samples per lobe) was analyzed to determine if variation in sampling site and homogenization can influence the amount of detected VitE. Results showed that repeated freeze-thaw cycles and storage temperature did not alter VitE concentrations. However, a large loss of VitE was observed after samples were homogenized. VitE distribution within and between lobes did not show any significant variation with an average %RSD of 6.49 and 14.1, respectively.

1Department of Pathobiology and the Pennsylvania Animal Diagnostic Laboratory System (PADLS), New Bolton Center, University of Pennsylvania, Kennett Square, PA.

63 Screening of Erythropoietin, Recombinant Human Erythropoietin and Darbepoietin-alpha in Horse Plasma

Ashok K Singh, Shveta Gupta, and Abby Sage

Erythropoietin (Epo), a 36 kDa glycoprotein, regulates mammalian erythrocyte and hemoglobin production. Human recombinent Epo (rhEpo) is used to treat anemia and blood loss. Darbepoietin-alpha (DAR)is a synthetic hyperglycosylated hormone that is more stable and effective than rhEpo. These glycopeptides are being misused in racing horses for achieving greater aerobic power and improved performance. However, the commonly used methods of drug detection in horses do not detect these peptides. Therefore, the aim of this investigation was to validate for analyzing horse samples different immunological tests that have been designed for human used. We have studied the following: (1) reactivity of rhEpo and DAR to antibodies raised against conserved and non-conserved regions of the glycoproteins, (2) relative distribution of carbohydrates by using the enzymes N-glycanase, O-glycanase and Sialidase A, and (3) trypsin fragmentation or glycosylated and deglycosylated moleculas and their analysis by MALDI-TOF mass spectrometry. This study indicated that DAR and rhEPO had approximately 55% and 40% glycosylation. Three commercial antibodies (from DPC, R & D and DiaSorin) crossreacted rhEpo and Dar, but the R&D antibody did not cross-react the horse Epo. Thus, an analysis of horse samples with R&D ELISA kit will detect the concentrations of rhEpo and/or Dar, and analysis of the samples with DSL-ELISA or RIA will detect total (endogenous horse Epo + rhEpo or Dar) concentrations. The MALDI-TOF-MS was used for confirmation of the ELISA results.

This work is supported by a grant from the Racing Medication and Testing Consortium.

Veterinary Diagnostic Laboratory & Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, St Paul, MN 55108.

64 Diagnosis of Taxus (Yew) Poisoning in a Horse

Asheesh K. Tiwary*1, Birgit Puschner1, Hailu Kinde2, John Reagor3 and Elizabeth R. Tor1

A 2-year-old bay thoroughbred colt was found dead overnight in its stall without a known history of any illness, or toxin exposure. No information on the clinical signs prior to this animal's death was reported. A full necropsy was performed the following morning and revealed a mild to moderate degree of endocardial hemorrhages in both ventricles. Microscopic examination of the heart showed an acute mild mutifocal necrosis of papillary muscles and ventricles.The stomach content contained approximately 2.25 % Taxus alkaloids as determined by GC/MS. In the past, diagnosis of Taxus poisoning has been mainly based on history of exposure and the presence of plant parts in the gastrointestinal tract. Feed microscopy of the stomach contents was performed and the stomatal pattern of the plant parts most closely resembled the Pacific/Western yew, although, the cellular pattern was slightly different and was suggestive of a hybrid, or another species, in the stomach content. Histopathological lesions associated with Taxus poisoning have not been published for horses. Therefore, this is the first report of cardiac lesions in a horse following lethal exposure to Taxus. Based on these findings, it is suggested that Taxus exposure needs to be considered in the differential diagnosis of horses that die suddenly or have cardiac lesions suggestive of Taxus exposure, even if intact plant parts are not found in the gastrointestinal tract by visual inspection. In addition, species other than the well-established Japanese and English varieties of Taxus should also be considered as a potential threat to animals.

1 California Animal Health and Food Safety Laboratory System, University of California, Davis, CA. 2 California Animal Health and Food Safety Laboratory System, University of California, San Bernadino, CA. 3 Texas Veterinary Medical Diagnostic Laboratory, Texas A&M University, College Station, TX

*To be considered for resident award

65 Paresis and Death in Elk (Cervus elaphus) Due to Lichen Intoxication in South Central Wyoming

W. E. Cook1, M. F. Raisbeck2, T. E. Cornish2, E. S. Williams2, B. Brown3, and G. Hiatt3

In February and March 2004, we documented 326 free-ranging elk (Cervus elaphus) that developed paresis, became recumbent, and died or were euthanized in the Red Rim habitat area southwest of Rawlins, Wyoming. The estimated total loss was 400 – 500 elk. Elk were found in sternal recumbency, alert and responsive, but unable to rise. They were afebrile with normal pulse and respiration. Orange- brown to red urine was observed adjacent to many affected elk. Over time elk progressed to lateral recumbency followed by dehydration, obtundation, and death. When provided feed and water or given a variety of treatments several elk remained alive and responsive but never became ambulatory. Most of the elk found alive were euthanized because survival and return to normal function seemed unlikely. We necropsied 12 elk from the field; these animals were in fair to good body condition. Elk recumbent for a day or more demonstrated gross evidence of degenerative myopathy, with pallor and streaking in skeletal muscles, particularly the semimembranosus, semitendinosus, and gastrocnemius muscles. Many had distended colons filled with feces of normal consistency. Microscopic examination of tissues from the majority of elk was unremarkable, with significant lesions most consistently observed in skeletal muscles. Affected muscles had degenerative lesions of varying duration, severity, and distribution, some with early mineralization and attempts at regeneration, and some associated with degenerating protozoal cysts (Sarcocystis sp.). Sporadic lesions were observed in other tissues from a small number of elk, including mild tubular degeneration/necrosis in kidneys, mild fibrinoid degeneration/change in small blood vessels of adrenal glands and a few other organs, and mild hepatocellular degeneration or, less frequently, apoptosis/necrosis.

Results from histopathology, virus isolation, bacterial culture, parasite analyses, and toxicology analyses ruled out common infectious, inflammatory, toxic, and traumatic causes of weakness, paresis, and recumbency. During field investigations, we noted large quantities of tumbleweed shield lichen (Xanthoparmelia chlorochroa) in the area of affected elk. This lichen also was identified in the rumen contents of several elk, in some cases contributing to 50% or more of rumen contents. We collected approximately 50 kg of lichen and fed it to 3 captive elk. Initially, elk were offered a diet of 100% lichen for 7 days. After 7 days, elk were offered free choice alfalfa hay and lichen. Two elk exhibited signs of ataxia, which rapidly progressed to weakness and recumbency after 7 and 10 days on this diet respectively. Both elk were euthanized and necropsied. Gross and microscopic lesions were consistent with lesions from the affected elk in the field and orange-brown to red urine was noted in the pens where the elk had been housed. The third elk ate little of the lichen diet, showed no clinical signs, and was returned to a normal diet after 10 days.

We identified tumbleweed shield lichen as the most likely cause of the paresis and recumbency syndrome that was eventually followed by death. In early to mid March, elk that were ambulatory migrated out of the area, and no subsequent losses have been documented in these elk. Interestingly, cattle, horses, mule deer, and pronghorn were observed on Red Rim and had access to the lichen during the elk mortality event but were unaffected. The toxic compound of the lichen has not yet been identified. Although previous researchers hypothesized that the toxic compound was usnic acid, this has not been confirmed. We plan to analyze the lichen for toxic compounds, to feed whole lichen to experimental captive ruminants to better characterize the syndrome, and to analyze the diets of the other herbivores in the area to determine if they ate the lichen.

1Wyoming Game & Fish Department, 1174 Snowy Range Road, Laramie, WY 2Wyoming State Veterinary Laboratory, University of Wyoming, 1174 Snowy Range Road, Laramie, WY 3Wyoming Game and Fish Department, 260 Buena Vista, Lander, WY

66 Toxicologic Surveillance of Search & Rescue Dogs Deployed to the World Trade Center, The Pentagon, and The Staten Island Fresh Kills Landfill Sites

W.K. Rumbeiha1, S.D. Fitzgerald1, W. Emmett Braselton2, C.M. Otto3, A.B. Downend3

Abstract: A prospective surveillance program was used to evaluate possible toxicant exposure in dogs utilized in search and rescue (SAR) efforts at the World Trade Center (WTC), Pentagon, and Staten Island Fresh Kills Landfill sites immediately following the September 11, 2001 terrorist attacks. A total of 70 dogs deployed to the sites were used in the study. An additional 51 non-deployed SAR dogs were enrolled as controls in the study. Of the 70 deployed dogs, 13 were deployed at the Pentagon, 11 at Staten Island, and 46 at the World Trade Center sites. This was an ante-mortem study on whole blood or blood plasma samples. Samples were drawn between October 2001 and June 2002. Toxicological analyses, included whole blood lead, whole blood mercury, plasma assays for polychlorinated biphenyls (PCBs), and gas chromatography/mass spectrometric (GC/MS) analysis of blood plasma for toxic organic compounds. Results indicated that all dogs tested negative for blood lead at a 0.05 parts per million (ppm) detection limit except for two dogs deployed at the WTC which tested positive at 0.06 and 0.07 ppm. All plasma samples tested negative for PCBs at a 0.05 ppm detection limit. All plasma samples tested negative for toxic organic compounds by GC/MS. No statistically significant differences were found in blood mercury concentration between control dogs and deployed dogs for any of the three study sites. These results indicate that for toxicants monitored in this study, there was no significant exposure of dogs deployed for SAR following September 11, 2001 terrorist attacks compared to non-deployed controls.

1 Department of Pathobiology and Diagnostic Investigation and the Diagnostic Center for Population and Animal Health, Michigan State University, East Lansing, MI

2 Department of Pharmacology and Toxicology and the Diagnostic Center for Population and Animal Health, Michigan State University, East Lansing, MI

3 Department of Clinical Studies, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA

67 Proteomics Approaches to Biomedical Problems

S. B. Hooser1, C.R. Wilson1

Proteomics refers to the study of the proteome (i.e., the protein complement of the entire genome; i.e., all of the proteins in a sample). Currently, the field of proteomics is experiencing an effort to develop high throughput methods to quantify changes in proteins when individuals are exposed to a variety of diseases. Identification and quantification of large numbers of proteins which undergo specific changes in response to specific disease states will likely lead to earlier and more specific detection and treatment of those diseases. The same assays may also allow evaluation of patient status and response to treatment. Neoplastic, inflammatory, degenerative, and nutritional diseases, along with toxicities, are some clinical conditions which can possibly be identified through proteomics analysis. Until relatively recently, high throughput separation, display, quantification, and rapid identification of large numbers of proteins in a sample was technically very difficult. However, recent advances in the development of these technologies have begun to overcome many of the previous problems. For instance, the application of proteomics technologies to the serum of ovarian cancer patients by scientists at the National Cancer Institute, has shown promise for earlier diagnosis of this disease. At Purdue University, in collaborations between the Department of Chemistry and the School of Veterinary Medicine, we have begun preliminary studies to develop quantitative, high-throughput liquid chromatography, mass spectrometry methods for clinical and diagnostic use.

In this talk, we will describe the utility of global protein identification and quantification in disease diagnosis. Briefly, to reduce complexity for separation and identification, a subset of proteins is selected from all of the serum proteins. For example, lectins can be used to select and separate glycoproteins from non-glycosylated serum proteins such as albumin. The selected proteins are then digested to short peptides with trypsin. To compare sera from diseased individuals to normal sera, the peptides in the diseased sample are labeled with a stable isotope which adds a known mass to the weight of each peptide. The peptides in the normal sample are labeled with an isotope of a different mass (commonly 6 mass units). The two samples are then mixed together. The combined peptides are separated by HPLC. Peptides from the same proteins resolve identically via HPLC even though the masses are different. Isolated peptides are then subjected to MALDI-TOF or ESI mass spectrometry. Peptides from the same protein, but from different samples, appear as doublets separated by six mass units. If the peptides increase or decrease more than two-fold, this is considered to be disease (or treatment) related and these changes are recorded. The peptide sequence can then be determined and the protein identified. Due to the relatively recent development of the instrumentation and reagents needed to perform quantitative diagnostic/clinical proteomics, these techniques are still in the methods development phase. However, because of the ability to quantify the global changes in serum proteins and then potentially identify the proteins which have changed in a diseased individual, proteomics holds great promise to provide a powerful tool for the diagnosis of disease.

1Animal Disease Diagnostic Laboratory, Purdue University, West Lafayette, IN

68 Virology Scientific Session Saturday October 23, 2004 1:00 p.m. – 3:00 p.m. Guilford B

Moderators: Robert Fulton and Jane Christopher-Hennings

Page 1:00 p.m. Disinfection of Coronavirus and Orthomyxoviruses in Liquid and on Solid Surfaces by Various 71 Disinfectants - J.M. Bieker-Hawkinson*, J. Anderson, R.D. Oberst, and S. Kapil

1:15 p.m. Serologic Response to Inactivated Avian Influenza A Vaccine in Turkeys - K.L. Pabilonia*, Van 72 Campen, H., Wooming, B., Podell, B.K. and Salman, M.D.

1:30 p.m. Initial Results Toward a Collaborative Evaluation of a Real-time Assay Reagent Specific for the 73 Detection of West Nile Virus - S. F. Sells* - D.J. Johnson, J.D. Callahan, M.L. Vickers, A.K.Maloy, W.M. Nelson, E.N. Ostlund, I. Stewart, J. Roberts, M. Sebastian, K. Grandfield, C. Jackson, K.B. Poonacha, C.B. Hong, N. Williams, R.C. Giles, D. Bolin, and L. Harrison

1:45 p.m. First Report of Spring Viremia of Carp Virus in Wild Common Carp (Cyprinus carpio) in North 74 America - A.L. Dikkeboom , C. Radi, S. Marquenski , M Engel, A.E. Goodwin , K Way , D.M. Stone , C Longshaw , and K. Toohey- Kurth

2:00 p.m. Persistence of Bluetongue Virus in the Insect Vector and its Implications for Disease Control - 75 J.O. Mecham, D.M. White, B.S. Drolet and W.C. Wilson

2:15 p.m. Foot and Mouth Disease in Bison: Laboratory Analyses of Serum, Blood, Oesophageal- 76 Pharyngeal Fluid, and Tissue Samples - H. Wang, G.B. Ward, M.Y. Deng, J.C. Rhyan, M. Yeh, M.L. Berninger, B. Donahue, J. Dio, S.A. Shawky, S.H. Wainwright, T.S. McKenna

2:30 p.m. Severe Egg Production Reduction in Turkeys Due to Avian Influenza Virus H3N2 - 77 Y. Zhang, B. Byrum, M. Saif, S. Grimes, S. Rajeev, C. Sarver, and L. Dorman

2:45 p.m. RT-PCR in the Newcastle Disease and Avian Flu Diagnosis - A. Panshin, E. Shihmanter, I. 78 Gissin, Y. Tendler

* Graduate Student Competition

70 Disinfection of Coronavirus and Orthomyxoviruses in Liquid and on Solid Surfaces by Various Disinfectants

J.M. Bieker-Hawkinson*1,2 , J. Anderson1, R.D. Oberst1, and S. Kapil1

Chemical disinfection and inactivation of viruses is largely understudied but is very important for rapid and successful containment in the case of viruses with low ID50 and thus, highly contagious. The objective of this research is to evaluate the efficacy of these formulations at various concentrations against highly infectious coronaviruses and orthomyxoviruses. Bovine coronavirus (BCV) is a member of antigenic group II of the Coronaviridae that causes both enteric and respiratory disease in cattle and related species. BCV was used as a surrogate of SARS for studying the viral inactivation. Swine influenza virus H1N1 (H1N1) was also selected for studying the efficacy of the various disinfectants.

The efficacy of the test disinfectants was evaluated using an in vitro cell culture system (Human Rectal Tumor 18, HRT-18, BCV or Madin-Darby Canine Kidney, MDCK, H1N1). The test disinfectants included Sandia National Laboratory developed DF-200d, bleach, ethanol, and Virkon® S. Efficacy testing was conducted with liquid cell culture propagated inoculum and in the presence of various organic challenges (ie. feces, soil, litter, etc) for various exposure times using the test disinfectants at various concentrations. Inactivation was determined by treating the inoculum for the specified contact time, immediately diluting and/or ultracentrifugation (remove toxicity), and infecting into appropriate cell line. In all assays, a positive control was conducted by using 0.1M PBS in place of the disinfectant. Inactivation was verified by the following: negative CPE, negative FA, negative HA. This work was repeated by inoculating the liquid virus onto various 1x1 cm2 coupons (anodized steel, polypropylene, butyl rubber) and allowing to dry followed by exposure to the various test disinfectants. The effect of disinfection on the virus structural proteins (ie. nucleocapsid protein, hemagglutinin) was determined by treating the viruses with the test disinfectants for various times at various concentrations followed by western blotting using monoclonal antibodies to the specified protein. Samples were also evaluated using RT-PCR to further characterize the effect of the disinfectant treatments on viral RNA. Negative results using these assays correlate with negative infectivity in the in vitro cell culture system.

Our in vitro experiments resulted in complete inactivation of BCV and H1N1 by all test disinfectants following 1 minute exposure at the following concentrations: 12.5% DF-200d, 10% bleach, 70% ethanol, and 1% Virkon® S. In determining the mechanism of action, only the DF-200d and bleach appeared to completely degrade viral RNA. In all cases the ethanol and in some trials the Virkon® S treated samples had some integral RNA remaining following a 1 minute exposure. The DF-200d, bleach, ethanol, and Virkon® S all appeared to degrade, or at least remove, the proteins evaluated based on the western blotting assays conducted. In developing methodology to better understand the mechanism of viral inactivation by evaluating specific effects on various components of the virus by the disinfectants we conclude that the inactivation is likely occurring due to one or a combination of the following: degradation of the lipid envelope, degradation of the receptor binding viral proteins, and degradation of the viral RNA with our test disinfectants.

1Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 2Sandia National Laboratories, Albuquerque, NM

*To be considered for graduate student award

71 Serologic Response to Inactivated Avian Influenza A Vaccine in Turkeys

Pabilonia, K.L.*, Van Campen, H., Wooming, B., Podell, B.K. and Salman, M.D.

In April 2002, a Colorado turkey farm experienced an outbreak of avian influenza virus (H8N4), characterized by a clinical signs of respiratory disease and a 1% increase in mortality. An autogenous inactivated whole virus vaccine was used to control losses. The purpose of this study was to asses the ability of three different serologic testing methods, agar gel immunodiffusion (AGID), enzyme-linked immunosorbent assay (ELISA) and hemagglutination-inhibition (HI), to detect the antibody response to an inactivated vaccine and challenge with live virus.

An initial study was conducted to determine the antibody response to avian influenza vaccination in turkeys on the farm. The turkeys were vaccinated twice, at four and eight weeks of age, and serum samples were collected at various time points pre- and post-vaccination. An antibody response to vaccination was detectable within three weeks after administration of the first vaccination. Peak HI titers were achieved at 14 days after administration of the second vaccination. Results suggest that the AGID test is best suited for avian influenza surveillance and the HI assay is best suited for quantitatively measuring the antibody response to vaccination.

The efficacy of this vaccine was then tested in an experimental setting. Three groups of fifty of turkeys each were evaluated: 1) Turkeys vaccinated at four and eight weeks of age, 2) Turkeys vaccinated at four weeks of age and 3) Sham-vaccinated controls. At ten weeks of age, all of the turkeys were challenged intranasally with avian influenza A virus (H8N4). Blood was collected weekly from each turkey to assess the serologic response to vaccination and challenge. An antibody response to vaccination was detectable three weeks after administration of the first vaccination in vaccinated groups. The twice-vaccinated group achieved higher titers than the once-vaccinated group. The control group seroconverted two weeks after challenge. The results of this experiment were similar to that of the previous field study.

*To be considered for graduate student award

72 Initial Results Toward a Collaborative Evaluation of a Real-time Assay Reagent Specific for the Detection of West Nile Virus

S. F. Sells1,2,* ; D. J. Johnson3; J. D. Callahan4, M.L. Vickers1; A.K.Maloy1; Wm. M. Nelson4; E. N. Ostlund3; I. Stewart5; J. Roberts1; M. Sebastian1; K. Grandfield1; C. Jackson1; K. B. Poonacha1; C. B. Hong1; N. Williams1; R. C. Giles1; D. Bolin1; and L. Harrison1

Two veterinary diagnostic facilities have participated in a parallel evaluation of a commercial real-time RT-PCR reagent for detecting West Nile virus. In 2001 Tetracore developed a real-time RT-PCR reagent for the detection of West Nile virus. Results of in-house testing for sensitivity, specificity, and from pilot collaborative studies with several veterinary centers appeared promising and were reported in 2003.

In this study the USDA National Veterinary Services Laboratories and the University of Kentucky Livestock Disease Diagnostic Center have tested a larger number of archived diagnostic specimens, mainly of equine neurologic tissue. The diagnostic assay has also been evaluated by spiking equine brain homogenates with known amounts of an equine origin reference virus. Multiple sampling has allowed better characterization of some of the assay critical control variables, such as viral load in the specimen and sample preparation. Using samples from 114 diagnostic specimens in comparison to a nested RT- PCR assay, considered here as the gold-standard, the real-time assay reagent exhibited 98% correlation, 100% specificity, and 96.7% sensitivity.

Two positive diagnostic specimens were repeatedly extracted to evaluate effects of different sample preparation techniques on the real-time assay. The specimens were chosen based on their low (high Number of Detectable Units) or high (low NDU) cycle threshold (Ct) values. Several commonly used silica adsorption and phenolic sample preparation methods were compared. Qualitatively, each kit was 100% effective at detecting the virus in the high NDU specimen. Initial results using the low NDU specimen found that some methods could cause the assay to fail in over 50% of prepared samples. When detected, the Ct value from these samples was usually in the 38-40 cycle range.

Variations in Ct were less apparent when different sample preparation techniques were applied to specimens of equine brain homogenates that had been spiked with a reference virus. As judged by a plaque reduction neutralization test, the undiluted titer was 105 pfu/ml. As judged by the nested RT-PCR assay or the real-time assay, the NDU was approximately 1010 per ml.

1University of Kentucky Livestock Disease Diagnostic Center, The University of Kentucky, Lexington Kentucky, 40501 2The New College, in affiliation with The University of Madras, Chennai, India 3USDA Animal and Plant Health Inspection Service, Veterinary Services, National Veterinary Services Laboratories, Ames, IA 50010 4Tetracore Inc. Gaithersburg, Maryland 20878, United States. 5Department of Biology, The University of Kentucky, Lexington, Kentucky 40506

*To be considered for graduate student award

73 First Report of Spring Viremia of Carp Virus in Wild Common Carp (Cyprinus carpio) in North America

A.L. Dikkeboom 1, C. Radi 1, S. Marquenski 2, M Engel 3, AE Goodwin 4, K Way 5, DM Stone 5, C Longshaw 5, and Kathy Toohey- Kurth 1

Spring viremia of carp virus (SVCV) causes a rhabdoviral disease of cultured common carp, Cyprinus carpio and has been isolated from other cyprinids under natural conditions. SVC disease outbreaks have been reported most often in carp farms in Europe but have also been reported, on one occasion, in the Middle East. Clinical signs of SVC include edema, inflammation of the swim bladder, ascites and petechial hemorrhages in the gill and skin. Moribund fish display lethargy, loss of equilibrium, uncoordinated swimming, exopthalmia and low respiration. Outbreaks of SVC generally occur when water temperatures are between 10 and 17°C; however fry can be affected at temperatures as high as 22- 23°C.

The first report of SVC disease in North America occurred in a private koi farm in North Carolina in April and May of 2002. In spring 2002, an estimated 1,500 common carp (Cyprinus carpio) died over a six week period in Cedar Lake which is located in Polk and St. Croix Counties, in northwestern Wisconsin. The lake is a natural water body with an inlet and an outlet stream seasonally controlled by a low head dam (0.61 m). The outlet stream discharges into the Apple River which flows into the St. Croix River which in turn joins the Mississippi River at Prescott, WI. Carp in this lake are self sustaining. In this area of the state, water temperatures were below 19°C from April to June 8, 2002. Carp ceased dying when water temperature was consistently above 19°C. Approximately 1,500 fish (over 10,000 kg of biomass) or 20 % of the population died. Dead fish ranged in size from 46 to 90 cm and larger fish weighed an average of 6 kg. The kill was specific to carp.

Three moribund carp were necropsied and had lesions consistent with spring viremia of carp (SVC) disease, including petechia and ecchymotic hemorrhages on the skin, ascites, and edema of the kidney and spleen. A virus was isolated on fathead minnow cells and was shown to be a rhabdovirus by electron microscopy. ELISA, immunocytochemistry, and serum neutralization assays were performed. In vitro transmission studies were conducted. RT-PCR and partial sequence analysis were also performed on this isolate.

ELISA and serum neutralization tests indicated that the rhabdovirus isolate was more closely related to SVCV than to pike fry rhabdovirus. Immunocytochemistry indicated that the Cedar Lake isolate did not share complete antigenic identity with the European reference SVCV strain. In vitro transmission studies showed that the isolate produced signs of SVC disease; however the isolate was of low virulence for juvenile carp. Sequence analysis showed that the Cedar Lake SVCV isolate was more closely related to a recently isolated North Carolina strain of SVCV (98.6% nucleotide identity) and to strains of Asian origin rather than the European reference strain of SVCV. This is the first report of a SVC epizootic in a wild common carp population in North America.

1 Wisconsin Veterinary Diagnostic Laboratory, University of Wisconsin-Madison, Madison, WI 2. Department of Natural Resources, Madison, WI 3 Wisconsin Department of Natural Resource, Baldwin, WI 4 Aquaculture/Fisheries Center, University of Arkansas at Pine Bluff, Pine Bluff, Arkansas 5 Centre for Environment, Fisheries and Aquaculture Science, Weymouth Laboratory UK

74 Persistence of Bluetongue Virus in the Insect Vector and Its Implications for Disease Control

J.O. Mecham1, D.M. White2, B.S. Drolet1 and W.C. Wilson1

Bluetongue virus (BTV) infects sheep, cattle and other ruminants and is transmitted by Culicoides spp. biting midges. The virus infection cycle is maintained by the insect vector taking a blood meal from an infected ruminant host and then transmitting the virus to an uninfected host during subsequent feeding. Virus transmission is interrupted in temperate climates during winter months when the insect vector is no longer active. Seasonal bluetongue disease outbreaks coincide with the re-emergence of the insect vector. Possible mechanisms for over wintering of BTV and the seasonal re-emergence cycles include 1) the spread from areas of year-round endemic activity to areas of epizootic seasonal activity; 2) prolonged persistence of virus in cattle or wild ruminants; and 3) persistence of virus in the insect vector. We present an overview of evidence for the latter mechanism. BTV can persist in the insect vector and this persistence may play an important role in over-wintering of the virus as well as explain the seasonal re- emergence of disease in susceptible ruminant hosts. Understanding the maintenance of BTV in nature, in the absence of apparent disease outbreaks, will help us develop more effective risk management and control strategies for bluetongue disease.

1 USDA, Agricultural Research Service, Arthropod-borne Animal Diseases Research Laboratory, Laramie, WY 2 CDC, Special Pathogens Branch, Atlanta, GA

75 Foot and Mouth Disease in Bison: Laboratory Analyses of Serum, Blood, Oesophageal-Pharyngeal Fluid, and Tissue Samples

H. Wang,1 G.B. Ward,1 M.Y. Deng,1 J.C. Rhyan,2 M. Yeh,1 M.L. Berninger,1 B. Donahue,1 J. Dio, 1 S.A. Shawky,1 S.H. Wainwright,2 T.S. McKenna,1

Recently, we demonstrated the susceptibility of American bison (Bison bison) to foot and mouth disease (FMD) via intra-species and inter-species transmissions (1). We report here the results of a comparison between reverse transcription-polymerase chain reaction (RT-PCR) and virus isolation (VI) for the detection of FMD virus (FMDV) in blood, oesophageal-pharyngeal (OP) fluid, and tissue samples; and results of a comparison between an enzyme-linked immunosorbent assay (ELISA) and an agarose gel immunodiffusion (AGID) assay for the detection of anti-FMDV antibodies in sera.

Total RNA in blood, OP fluid, and tissue homogenates was extracted by a TRIzol® LS Reagent (Invitrogen, Carlsbad, CA)-based procedure and subjected to RT-PCR. PCR products were analyzed by agarose gel electrophoresis. VI for blood, OP fluid and tissue samples was conducted using primary lamb kidney (LK) cells and Instituto Biologico Rim Suino (IBRS-2) pig kidney cells. VI results were confirmed by a serotype-specific antigen ELISA. Anti-FMDV antibodies in sera were detected by a non- structural FMDV protein 3ABC-based ELISA and the 3-D based AGID.

In an initial analysis, RNA extracts of blood and tissue samples taken from bison at the peak of clinical FMD manifestations were surprisingly negative for FMDV in RT-PCR. It was found that these RNAs were falsely negative due to RT-PCR inhibition since they became FMDV-positive after being diluted and re-tested by the RT-PCR. To establish the quality of extracted RNA, an RT-PCR for an internal gene, ACTB encoding for β-actin, was also performed. A negative result by RT-PCR for FMDV was accepted when the same sample was positive by RT-PCR for ACTB.

Using the optimized RT-PCR procedure, we analyzed 65 blood, OP fluid, and tissue samples collected from 5 bison that were either inoculated with FMDV or infected with the virus by contact with a virus- inoculated bison or cattle. Twenty-two of these samples were positive for FMDV by RT-PCR. Among these 22 samples, only 4 were positive by VI. FMDV RNA was widely detected in samples collected from bison on 6 days post-inoculation (DPI) or 6 days post-exposure (DPE). Positive RT-PCR tissues included blood, OP fluid, various lymph nodes, rumen, liver, lungs, kidney, male reproductive tissues and palentine tonsil. The virus was not detected by RT-PCR from most tissue samples collected from bison between 12 and 49 DPE . The virus, however, was detected by RT-PCR in 2 samples of coronary bands collected from a bison which was euthanized on 37 DPE. These 2 samples were negative by VI.

Nineteen sera from five bison were tested for the presence of antibodies to FMDV by ELISA and AGID. None of the samples collected prior to 13 DPI or DPE were positive. All five of the samples collected after 13 DPI or DPE were positive by ELISA. Four of the five samples collected after 13 DPI or DPE were positive by AGID.

1USDA, APHIS, Veterinary Services, NVSL, Foreign Animal Disease Diagnostic Laboratory, Greenport NY 11944; 2USDA, APHIS, Veterinary Services, National Wildlife Research Center, 4101 LaPorte Avenue, Fort Collins, CO 80521.

76 Severe Egg Production Reduction in Turkeys Due to Avian Influenza Virus H3N2

Y. Zhang1, B. Byrum1, M. Saif2, S. Grimes1, S. Rajeev1, C. Sarver1, and L. Dorman1

An outbreak resulting in severe reduction in egg production occurred in a turkey layer farm in Ohio. Within two weeks of the initial clinical disease in Barn 1, the disease spread to all five barns on the farm and caused greater than 95% drop in egg production. No other clinical signs were seen. Two out of five cloacal swab samples collected the first week after signs of clinical disease were positive for influenza A virus matrix gene by realtime RT-PCR. Twenty-four of sixty random sera (40%) collected at the same time were positive for influenza virus by commercial ELISA kit. All of the ELISA positive samples were positive for H3N2 by hemagglutination inhibition test. All five cloacal samples from barn 5 (representing 25 birds) randomly collected when the barn was experiencing the clinical disease of egg reduction were positive for influenza A virus by RT-PCR. Serum samples collected the fourth week after the outbreak were all positive for H3N2. Four weeks after the outbreak started, cloacal swabs were all negative for type A influenza by RT-PCR. Two birds were necropsied. Microscopically, a granulomatous salpingitis was prominent in one turkey. In another oviduct, follicular-like lymphoid foci were present in the mucosa. Small intestine, trachea, lung, and pooled tissue samples from the two birds were negative for AI by RT- PCR. However, electronmicroscopy revealed orthomyxovirus particles from pooled oviduct samples of two birds. The oviduct samples were also positive for influenza A virus by RT-PCR. Based on the clinical signs, pathologic lesions, EM, and RT-PCR results, we concluded that influenza virus H3N2 caused the localized infection in the oviduct that resulted in severe drop in egg production for the turkey layer farm.

1ADDL, Ohio Department of agriculture, Reynoldsburg, Ohio 43068 2OARDC, College of Veterinary Medicine, Wooster, Ohio

77 RT-PCR in the Newcastle Disease and Avian Flu Diagnosis

A. Panshin1, E. Shihmanter1, I. Gissin1, Ye. Tendler2.

Virulent strains of avian paramyxovirus type 1 (or Newcastle disease virus - NDV) and avian influenza viruses (AIV) are responsible for Newcastle disease and avian flu, highly contagious and economically important diseases primarily of poultry. The molecular basis for NDV pathogenicity is dependent on the F protein cleavage site amino acid sequence. Fewer basic amino acids are present in the F protein cleavage site of lentogenic NDV isolates than either mesogenic or velogenic strains, which have similar cleavage site sequences. Kant et al. (Avian Pathology, 1997, 26, 837-849) have developed reverse transcription coupled to polymerase chain reaction (RT-PCR) which detects and differentiates virulent and non-virulent NDV strains directly in tissue homogenate. Infections produced by most of avian influenza virus are asymptomatic, although a few highly pathogenic strains (from H5 or H7 subtype) can cause disease with high mortality in poultry. Although the pathogenicity of AIV is a polygenic trait, the hemagglutinin (HA) glycoprotein plays a central role. The RT-PCR procedure developed by Lee et al. (2001) is rapid and sensitive, and could be used for the identification and HA-subtyping of AIV. Moreover, RT-PCR followed by sequence analysis of HA cleavage site is used for rapid determination of the virulence potential of H5 and H7 viruses.

To evaluate diagnostic value of the above mentioned RT-PCR techniques the results obtained by means of the RT-PCR were compared to the data obtained by means of the classical methods of NDV and AIV characterization. Sixty NDV and fifty four AIV reference strains and field isolates were studied. In addition, 3 sets of universal primers for identification and characteristics of AIV were tested. In 11 cases the ability of AIV strains to induce cytokine production in human monocyte cultures were investigated.

It was shown that in NDV infection, the diagnosis by RT-PCR compared well with classical virological methods and our data confirmed the results of Kant et al. The results of RT-PCR were in good agreement (92%) with experimentally determined Intracerebral Pathogenicity Index (ICPI) values and nucleotide sequences of cleavage site regions. However in 4 out of 60, the virulence of NDV viruses could not be confirmed by RT-PCR. Where as all the 54 AI viruses, the results of RT-PCR with our primer sets and with sets of primers designed by Horimoto and Kawaoka (1995) gave an excellent correlation with the data obtained by means of the conventional methods. None of the 11 AI viruses induced an abnormal high cytokine synthesis in the human monocytes culture.

1. The RT-PCR can be used for diagnosis of NDV and AIV infection. 2. The Kant et al. RT-PCR method can be used for preliminary diagnosis of the NDV virulence (pathotype).

1Department of Avian and Aquatic Animals Disease, Kimron Veterinary Institute, Beit Dagan, Israel 2Department of Clinical Biochemistry, RAMBAM Medical Center, Haifa, Israel

78 Bacteriology Scientific Session Sunday October 24, 2004 8:00 a.m. – 12:00 p.m. Guilford A

Moderators: Brenda Love and Melissa Libal Page 8:00 a.m. Use of a Real-Time Polymerase Chain Reaction Assay for Detection of Mycobacterium avium 81 subsp. paratuberculosis from Bovine Fecal Samples and Confirmatory Cultures - L.A. Estey, P.R. Andersen, A. Asp, T. Bezold, D. Eugene and P. I. Tyrrell

8:15 a.m. Evaluation of Six Decontamination Protocols for the Isolation of M. avium subsp. 82 paratuberculosis from Bovine Feces - K. A. Johansen and J.B. Payeur

8:30 a.m. Development of Real Time Quantitative PCR Technology for the Identification of Mycoplasma 83 bovis in Bovine Milk - B.C. Love, F. Zambito, S.E. Myers, D.P. Shaw

8:45 a.m. Direct Evaluation of Bovine Fecal Samples for Mycobacterium avium subsp. paratuberculosis 84 by a Real-Time PCR Commercial Assay - B.L. Mangold, S.R. Weeks, T.I. Calvin, W.M. Nelson, and J. Christopher-Hennings

9:00 a.m. Optimization of an Algorithm for Laboratory Diagnosis of Mycobacterium avium subsp. 85 paratuberculosis in Bovine Feces Utilizing a Liquid Culture System - S. Rajeev, Y. Zhang, and B. Byrum

9:15 a.m. Culturing Mycobacterium avium spp paratuberculosis from Sheep Feces in the United States - 86 S. Robbe-Austerman, J.R. Stabel, B.N. Harris, K. Johansen, M.V. Palmer, J.B. Payeur

9:30 a.m. Evaluating the Accuracy of the IFN-γ ELISA and the Skin Test to Detect Mycobacterium avium 87 ssp. paratuberculosis (MAP) Infection in Sheep - S. Robbe-Austerman, J.R. Stabel, M.V. Palmer

9:45 a.m. BREAK AND POSTER SESSION

10:15 a.m. Development and Evaluation of a New PCR Test for the Detection - Mycobacterium avium 88 subsp. paratuberculosis - J.R. Stabel, J.P. Bannantine, and M. Paustian

10:30 a.m. A Technique for Enhanced Detection of Acid Fast Bacilli (AFB) (Mycobacterium subsp avium 89 paratuberculosis-MAP) in Liquid Culture - R.H. Whitlock, A. Monson, B. Aksim, S, McAdams, T. Fyock and R. Sweeney

10:45 a.m. Research Challenges for Brucellosis Eradication - P.H. Elzer 90

11:00 a.m. Strain Typing Brucella abortus Isolated from California and Florida Cattle Herds by Using 91 “HOOF-Prints” – a New DNA Fingerprinting Technique for Brucella – B.J. Bricker and D.R. Ewalt

11:15 a.m. Investigation of Reported Resistance of Streptococcus equi ssp. zooepidemicus to 92 Trimethoprim-sulfamethoxazole in Horses - D.J. Feary, J. Traub-Dargatz, S. Roach, D.R. Hyatt, R. Jones, C. C. Wu, and P. S. Morley

79 11:30 a.m. Characterization of a Highly Antimicrobial Resistant Escherichia coli from Newborn Calves 93 with Colibacillosis in North Dakota - P.S. Gibbs, J. Foster, S.R. Petermann, M. Theis, T. Solseng, L.P. Schaan, D.F. Krogh, and G.J.C. Carlson

11:45 a.m. Genetic Variability of Moraxella (subgenus Moraxella) bovis and Moraxella (subgenus 94 Branhamella) ovis Field Strains Recovered from Cattle with Infectious Bovine Keratoconjunctivitis - P.S. Nabity*, H.E. Cerny, P.D. Fey, S.Hinkley

* Graduate Student Competition

80 Use of a Real-Time Polymerase Chain Reaction Assay for Detection of Mycobacterium avium subsp. paratuberculosis from Bovine Fecal Samples and Confirmatory Cultures

L. A. Estey1, P. R. Andersen1, A. Asp2, T. Bezold1, D. Eugene1 and P. I. Tyrrell1

The LightCycler (Roche Applied Science) system has been used to develop a real-time polymerase chain reaction (PCR) assay for detection of Mycobacterium avium subsp. paratuberculosis (M. pt.). The assay uses a hybridization probe detection system that monitors fluorescence energy transfer (FRET) between two adjacent probes during the annealing stage of each PCR cycle. IS900, a sequence shown to be specific to the M. pt. genome (Bull et al., J. Clin. Micro. 2003, 41(7): 2915-2923) and to be present at approximately 10-18 copies per organism, is the genomic sequence targeted in the assay. For these studies, M. pt. genomic DNAs were isolated using either robotic nucleic acid isolation instrumentation (MagNA Pure LC, Roche Applied Science) or a manual DNA purification module (High Pure PCR Template Preparation kit, Roche Applied Science). Assay specificity was demonstrated by the M. pt. reagent set failing to react with other mycobacterial genomic DNAs such as those isolated from Mycobacterium terrae, scrofulaceum, smegmatis, marinum, phlei, flavescens as well as avium subspecies silvaticum. The sensitivity of the reagent set was assessed using an IS900-containing plasmid as an analytical standard. The M. pt. reagent set was able to detect less than 5 copies of purified plasmid- derived IS900 sequence. When the IS900 plasmid was diluted into an M. pt.-negative fecal homogenate and purified using the MagNA Pure instrument, the reagent set was able to detect 1,000 copies of the IS900 sequence. The utility of the reagent set for culture confirmation was evaluated using organisms that were isolated from ileal biopsy tissue, lymph nodes, or fecal material and grown using the TREK system (TREK Diagnostic Systems, Inc.). Of the 152 cultures that were culture-positive or tested positive using a MAV2 primer/probe set, the M. pt. reagent set detected all positives. For the positive population, the mean crossing point was 22.04 ± 3.58, which indicates relatively high organism counts within the cultures. Within the culture-negative, MAV2-negative population (n = 109), the M. pt. reagent set showed a specificity of 92.7%. Interestingly, the eight presumed negatives that tested positive had crossing points > 40; these may represent low levels of poorly growing M. pt. organisms within the culture broth. Ongoing studies evaluating the utility of the M. pt. reagent set for detection of M. pt. from fecal material and peripheral blood lymphocytes will be reported. From these studies, we show that the M. pt. detection reagents developed for the LightCycler provide a rapid and convenient PCR-based method for either culture confirmation or direct detection of DNAs isolated from relevant clinical specimens.

1IDEXX Laboratories, Inc., Westbrook, ME 04092 USA 2IDEXX Scandinavia AB, Osterbybruk, Uppsala S-748 30 Sweden

81 Evaluation of Six Decontamination Protocols for the Isolation of M. avium subsp. paratuberculosis from Bovine Feces

K. A. Johansen1 and J.B. Payeur1

Six decontamination protocols for the isolation of MAP from bovine feces were evaluated as part of the effort to develop a standardized culture procedure. They included four protocols that use the detergent hexadcylpyridium chloride (HPC) as the decontaminating agent, one protocol that uses sodium hydroxide (NaOH) as the decontaminant, and one protocol that enriches for the organism by centrifugation in the detergent CB-18® followed by the use of lytic enzymes as decontaminants. The four protocols that used HPC as a chemical decontaminant differed in the use of sedimentation versus centrifugation for recovery of organisms, as well as the number and speed of the centrifugation steps. They included a “New York” protocol (one centrifugation step at 3000xg), a “Pennsylvania” protocol (one centrifugation step at 900xg) and an “Iowa” protocol (two centrifugation steps at 1700xg). Seventy bovine fecal samples that had been previously identified as sero- and culture-negative for MAP were used in the study. The samples were seeded with MAP ATCC 19698 at levels corresponding to low, moderate and high shedding status, then processed and cultured on Herrold’s Egg Yolk medium with and without mycobactin J. The number of colonies and degree of contamination was recorded at 4, 8, 12 and 16 weeks. The results were analyzed using a mixed models analysis of variance and mean responses for colony counts and contamination were compared. The results indicate that the CB-18®/enzyme protocol gave the highest recovery of MAP for samples seeded with moderate and high numbers of bacteria, while the NaOH method yielded the poorest recovery of organisms overall. Both the NaOH and CB-18®/enzyme protocols had a high degree of contamination. Among the protocols using HPC as the decontaminant, the Iowa and Pennsylvania methods gave good recovery of MAP at low and moderate seeding levels, while the sedimentation protocol yielded poor recovery of the bacteria. The New York protocol had the lowest level of contamination overall, while the Iowa protocol had the highest level of contamination for the procedures using HPC. It was also observed that for all protocols a loss of organisms corresponding to 2 logs was observed. Based on these finding we conclude that the use of chemical decontaminating agents such as HPC and NaOH may impair the growth of MAP, but that the Pennsylvania protocol was the most effective in isolating the organism while controlling contamination for this type of method. However, the use of alternative methods, such as CB-18®/enzyme with improved control of contamination, may lead to increased sensitivity in detection of MAP.

1USDA/VS/APHIS/NVSL/Mycobacteria and Brucella Section, Ames, IA.

82 Development of Real Time Quantitative PCR Technology for the Identification of Mycoplasma bovis in Bovine Milk

B. C. Love, F. Zambito, S. E. Myers, D. P. Shaw

Real-time polymerase chain reaction (RT-PCR) using primers and probe specific for Mycoplasma bovis was compared to standard culture methods to detect M. bovis in milk from cows with mastitis by testing 83 field samples of unknown status using both methods. A standard curve and positive cut-off value was generated with 13 samples (8 negative and 5 positive by culture), and the 83 samples evaluated based on this value. Agreement between the two methods was excellent, with 71 samples negative by both methods, 11 samples positive by both methods, and 1 sample positive by RT-PCR and initially negative by culture. Reculture of this single sample resulted in growth of Mycoplasma spp. Although the method requires further validation, this study demonstrated that RT-PCR has potential usefulness as a diagnostic test for detecting M. bovis in bovine milk, with diagnostic sensitivity and specificity equal to or better than standard culture. In addition, RT-PCR resulted in a quicker turnaround time to diagnosis, and was less labor-intensive than culture.

Department of Veterinary Science, Animal Diagnostic Laboratory, Pennsylvania State University, University Park, PA

83 Direct Evaluation of Bovine Fecal Samples for Mycobacterium avium subsp. paratuberculosis by a Real-Time PCR Commercial Assay

B.L. Mangold1, S.R. Weeks2, T.I. Calvin1, W.M. Nelson1, and J. Christopher-Hennings2

An evaluation of a new real-time PCR commercial kit (VetAlert™ from Tetracore, Inc.) for the direct detection of Mycobacterium avium subsp. paratuberculosis (MAP) from bovine stools was conducted. The VetAlert kit utilizes MAP-specific primers and a MAP-specific FAM-labeled probe, in an optimized, pre-made master mix, for sensitive and specific detection of DNA by real-time fluorescent probe hydrolysis. Utilizing a positive control plasmid containing the cloned amplicon, the VetAlert real-time PCR assay was shown to have sensitivity down to 1 gene copy. Concurrently, a novel stool extraction kit for simplified extraction of DNA from stool compared to conventional procedures was also evaluated. The new extraction procedure combines mechanical disruption with chaotropic solid phase extraction. Under current configurations, 18 stool samples were processed and extracted in 1.5 hours and 50 samples took less than 4 hours.

Twenty-five fecal samples from the National Veterinary Services Laboratories Johne’s Check Test were extracted and evaluated with both the VetAlert test kit and by nested PCR (Collins et al., 1993, Vet Microbiol 36:289). Samples were blinded to the operator, run in duplicate, and, in the case of real-time PCR, evaluated on both the ABI 7700 and Cepheid SmartCycler real-time instruments.

Of the 25 extracted fecal samples, there was 100% correlation between positive and negative results from the VetAlert test and nested PCR. Likewise, results of the real-time VetAlert test were comparable on both the ABI and Cepheid instrument platforms. Real-time PCR and nested PCR were shown to have equally high sensitivity; however, real-time PCR is a very attractive diagnostic test because it can be performed faster than nested PCR, it is less labor intensive, and it is quantitative.

In conclusion, real-time PCR is a sensitive, specific, and rapid diagnostic method which allows for direct evaluation of fecal samples for M. avium subsp. paratuberculosis.

1 Tetracore, Inc., Gaithersburg, MD 2 Animal Disease Research and Diagnostic Laboratory, South Dakota State University, Brookings, SD

84 Optimization of an Algorithm for Laboratory Diagnosis of Mycobacterium avium subsp. paratuberculosis in Bovine Feces Utilizing a Liquid Culture System

S. Rajeev , Y. Zhang, and B. Byrum

Rapid and accurate detection of Mycobacterium avium subsp. paratuberculosis (MAP) infected animals is essential for effective implementation of control programs. Conventional fecal culture using Herrolds egg yolk (HEY) media containing mycobactin J, which is the gold standard for diagnosis of MAP infection, is a labor intensive and time consuming procedure. Recently introduced liquid culture systems permits faster growth and detection of mycobacterium and rely on acid fast staining and PCR for confirmation. However, results based on a single confirmation method may lead to misclassification of animals and may result in economic loss to the producers by culling the uninfected animal or spread of infection. Therefore, this study developed and evaluated a diagnostic algorithm for accurate confirmation of MAP. Since the specificity of IS900 is a concern in PCR diagnosis of MAP, real-time PCR assay was developed and evaluated with a MAP specific target 251. This study evaluated 50 known fecal samples (30 positives and 20 negatives) for the presence of MAP. After cultivation in a liquid culture system, the signal positive samples were acid fast stained with auramineO/ rhodamine fluorochromes and the positive samples were confirmed with IS900 and 251 real-time PCR. Twenty eight out of 30 positive samples were signal positive and acid fast positive. Inclusion of an extra lysis step in DNA extraction protocol resulted in positive PCR results with IS900 and 251 in 24/28 of these samples. PCR negative broth cultures were inoculated into HEY tubes with mycobactin J and were confirmed as MAP. One known positive sample was confirmed as MAP at the end of the incubation period (42 days) based on acid fast staining, PCR and mycobactin dependency. The other known positive sample which was signal negative, was negative on broth and HEY. All the known negative samples tested were negative for the presence of MAP. From these findings, it was concluded that depending on a single test for confirmation of MAP from broth cultures may lead to inaccurate results. Use of the algorithm developed detected the presence of MAP in broth cultures with 100 % specificity when compared with conventional culture method.

Animal Disease Diagnostic Laboratory, Ohio Department of Agriculture 8995 East Main Street, Reynoldsburg OH 43068

85 Culturing Mycobacterium avium spp paratuberculosis from Sheep Feces in the United States

S. Robbe-Austerman1, J.R. Stabel1, B.N. Harris2, K. Johansen2, M.V. Palmer1, J.B. Payeur2

Attempts to culture feces from sheep infected with MAP (write out) using traditional solid media have been unrewarding. Australia has successfully cultured sheep MAP strains using the Bactec™ 460 radiometric culture (Becton Dickinson, Franklin Lakes, NJ USA) by enhancing the media with 18% egg yolk and Panta™ Plus(Becton Dickenson, Franklin Lakes, NJ USA) antibiotic mixture. There is some evidence that U.S. sheep strains differ from the Australian strains, although strains from neither country have been successfully grown on traditional solid media. This study utilized the Australian method to culture fecal material from U.S. sheep known to be infected with MAP.

Two grams of fecal material from sheep, known to be infected with MAP and located in Iowa, Minnesota, South Dakota, and Wisconsin, were decontaminated by the double centrifugation, double incubation method (NADC) or the Cornell method (NVSL) and 100-200 ul was added to a Bactec™ 460 vial containing the following additives, 1 ml of 100% egg yolk, 100 ul of Panta™ plus, and 800 ul of sterile water. Samples were incubated for a total of 12 weeks. All vials having a growth index (GI) reading greater than 10 were sampled for PCR testing and acid fast staining at the end of 12 weeks and earlier if the vials had a GI> 200. Negative samples were retested at the end of 12 weeks before they were considered negative.

Feces from 5 clinical sheep were positive for MAP. Several had a GI > than 200 after 2 weeks of culture. One subclinical animal took 10 weeks before the GI was greater than 10 and never had a GI of over 40. All of these animals were negative on Harold’s egg yolk media.

It appears that the Australian method can be used to grow United States sheep strains, although contamination was a problem. In the U.S., sheep are often fed stored feed rather than grass. Other antifungal agents need to be investigated to decrease the contamination rate.

1 Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Service, USDA 2 National Veterinary Services Laboratory Animal Plant Health Inspection Service, USDA

"Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture."

86 Evaluating the Accuracy of the IFN-γ ELISA and the Skin Test to Detect Mycobacterium avium ssp. Paratuberculosis (MAP) Infection in Sheep

S. Robbe-Austerman, J.R. Stabel, M.V. Palmer

Diagnostic tests for Mycobacterium avium ssp. Paratuberculosis (MAP) in sheep typically identify shedding animals and/or those in the advanced stages of disease. Positive test results usually can be relied upon; however, negative test results tell very little about the true infection status of the animal. None of the current diagnostic tests are accurate for diagnosing Johne’s disease in young replacement lambs or animals in the early stages of infection.

The objective of this study is to evaluate the accuracy of the skin test and IFN-γ ELISA (Bovigam, Biocor Animal Health, Omaha, NE) to detect MAP infected sheep before they become infectious and also to accurately identify uninfected sheep. Tissue culture and/or histology were used as a gold standard.

A cooperative agreement was formed with four flocks with confirmed Johne’s disease. These flocks had annual death loss due to Johne’s disease from 0% to 10%. All breeding and replacement sheep greater than 6 months of age were tested two times a year. Tests included the IFN-γ, skin test, fecal culture, ELISA and AGID. All sheep testing positive by either fecal culture, ELISA or AGID were removed from the flock, euthanized and examined. Sheep testing positive on the INF-γ or the skin test were allowed to stay in the flock until a second positive test. Once an animal tested positive twice, it was removed and euthanized. To evaluate animals testing negative, all cull ewes were euthanized and examined. A total of 80 sheep were submitted and necropsied. ileocecal valve, ileum, distal jejunum, and distal jejunal lymph node, were cultured and a total of 11 tissues were taken for histology.

Histology identified 55% of the tissue culture positive sheep. All sheep that were histology positive were culture positive. Seventy-three percent (16/22) of the sheep removed due to skin test and IFNγ were positive by culture of tissues. Three were fecal culture positive. Over 30% of the sheep that tested positive on the skin test or the IFN-γ are negative on the next test 6 months later. A larger sample size has to be collected to evaluate infection status. Additional results will be presented in the paper.

Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Service, USDA

87 Development and Evaluation of a New PCR Test for the Detection Mycobacterium avium subsp. paratuberculosis

J. R. Stabel1, J. P. Bannantine1, and M. Paustian1

Paratuberculosis has become more widespread in both domestic and wild ruminants in the US and other parts of the world in recent years. Accurate and sensitive ante mortem diagnosis of paratuberculosis is essential for implementation of national control programs. Fecal detection of M. paratuberculosis is the only methodology that can traverse both subclinical and clinical stages of disease. Yet fecal culture requires between 8 and 16 weeks for definitive results causing delays in management decisions to cull infected animals or purchase replacement animals. Nucleic acid detection methods using PCR have made rapid detection of M .avium subsp. paratuberculosis in fecal samples possible, reducing detection time to 2 to 3 days. Application of real-time PCR, nested PCR, and TaqMan PCR to detection assays for M. avium subsp.paratuberculosis have improved the sensitivity of detection and made it possible to perform semi-quantitative analyses. However, these tests have been based upon the amplification of the IS900 (multicopy) gene. This study describes the development of a nested PCR assay utilizing a unique gene for M.avium subsp. paratuberculosis (Map02) that is present in 6 copies in the genome. In addition, the sensitivity of this assay was compared to the detection sensitivity of the IS900 gene in both conventional and real-time PCR assays. Sensitivity of the PCR tests was evaluated using both naturally and experimentally infected samples. In addition, titration curves were generated using pure cultures of M. avium subsp.paratuberculosis and negative fecal samples spiked with known quantities of M. paratuberculosis. Specificity of the Map02 gene was evaluated by PCR of DNA from several isolates of M. avium subsp. paratuberculosis and M. avium subsp. avium, and DNA from M. fortuitum, M. scofulaceum, M. abcessan, M. smegmatis, and M. gordonea. Only M. paratuberculosis DNA was detected by the Map02 gene. Sensitivity of detection for the Map02 gene in either conventional or real-time PCR format determined using pure bacterial DNA was 100 fg DNA, compared to 10 fg DNA detected by the IS900 gene. Experimental spiking of a negative fecal sample followed by Map DNA extraction resulted in detection thresholds of 100 cfu/g for both IS900 and Map02 genes using a real-time PCR format but this sensitivity dropped 10-fold for both genes in a conventional PCR format. Analyses of fecal samples obtained from naturally infected animals demonstrated a higher sensitivity for the detection of Map DNA using the IS900 gene compared to the Map02 gene when it was used in a conventional PCR format. However, the real-time PCR format significantly improved detection levels of the Map02 gene. Using the real-time format detection of fecal samples a higher number of samples containing <10 cfu/g was possible compared to conventional PCR. In conclusion, the Map02 gene provides a very sensitive and specific alternative as a diagnostic reagent for use in PCR assays for the detection of paratuberculosis.

1USDA-ARS, National Animal Disease Center, Bacterial Diseases of Livestock Research Unit, Ames, IA

88 A Technique for Enhanced Detection of Acid Fast Bacilli (AFB) (Mycobacterium subsp avium paratuberculosis-MAP) in Liquid Culture

R. H. Whitlock, A.Monson, B. Aksim, S, McAdams, T. Fyock and R. Sweeney1

During the past three years several diagnostic and research laboratories in the USA have begun the transition from traditional solid media (Herrold’s egg yolk media-HEYM) to liquid culture for the detection of MAP. Liquid culture provides several benefits including shorter incubation time (<45 days) compared to 14 or 16 weeks with HEYM, single tube compared to 4 HEYM tubes, possibly greater sensitivity of MAP detection, requires less laboratory space, repeated assessment of microbial growth at short intervals (hourly), time to detection (TTD) directly correlated to MAP cfu and reduced labor costs. Each liquid culture detection system has computer algorithm that monitors a metabolic by-product on microbial growth (oxygen consumption, C02 production or isotope release). However, liquid culture media, similar to HEYM does not discriminate by allowing only MAP to grow. Once the detector yeilds a positive signal, the resultant microbial growth must be confirmed as MAP. Occasionally, both MAP and other microbial contaminants are present simultaneously. One step in the process is confirming MAP at TTD or at the end of protocol (typically 45 days). This process must be standardized, have built-in quality controls, and be as sensitive as possible. Most liquid culture systems require 1 X 105 cfu/ml of MAP to trigger the mechanical sensor as positive. Therefore the AFB technique needs to be at least this sensitive, but preferably much more sensitive, so as not to miss any potentially MAP positive samples.

Based on the evaluation of several alternative techniques, we recommend that at least 1.0 ml of liquid media be gently aspirated from the bottom of a non-vortexed liquid media tube. MAP settles to the bottom of MGIT-Bactec 960 tubes during the incubation period, thus providing a mechanism of further concentration by collecting MAP in this region of the tube. Vortexing the culture tube results in dispersion of MAP throughout the entire liquid matrix with loss of this natural concentration gradient at the tube bottom. The aspirated 1.0 sample is then centrifuged at 14,000 G in a microfuge for 10 minutes further concentrating MAP in a pellet. Following aspiration of the supernatant, an inoculating loop is used to transfer the pelleted material in the bottom of the microfuge tube to a glass slide. The smear is stained ZN acid-fast stain along with a previously prepared control AFB slide that is also included in the staining process. This method of sample preparation consistently detected 870 to 1600 cfu MAP/ml depending on the volume of egg yolk suspension added to the media tube. Egg yolk seems to trap MAP within the yolk matrix making it more difficult to pellet by centrifugation. Normally 2.5 X 105 cfu/ml of MAP is needed to trigger the computer algorithm. This method of sample preparation for AFB detection consistently detects MAP several days (2-10) prior to the computer algorithm.

In summary, this method of sample aspiration and centrifugation enhances MAP detection thus facilitating determination of positive samples in liquid culture media.

1Department of Clinical Studies-New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA.

89 Research Challenges for Brucellosis Eradication

P.H. Elzer

Brucella species are Gram negative, facultative intracellular pathogens that cause disease in man and animals. The primary hosts for B. abortus are cattle, bison and elk. With the eventual eradication of this disease in the U.S. domestic cattle herds, the bison and elk of the Greater Yellowstone Area (GYA) remain the last natural reservoir of brucellosis. The free ranging and infected wild animals in the GYA migrate from public land onto private lands and may come into contact with cattle. Brucella-induced abortions in both bison and elk have been documented under experimental and field conditions Interagency negotiations culminating in the revised Yellowstone Bison Management Plan and Environmental Impact Statement have identified vaccination as one of the primary means of managing brucellosis in Yellowstone National Park (YNP) bison. Feral swine in the southern United States and caribou in Alaska are infected with B. suis.

There are many challenges which need to be addressed with regards to the eventual eradication of brucellosis the United States. Another challenge is to find an efficacious vaccine which can be used to protect wildlife species. Complicating this issue is the mode of delivery to these animals which have a wide range of habits and habitats. Current commercially available vaccines do not appear to be the answer to these problems. Diagnostic tests and surveillance continue to be important in the eradication effort.

Numerous special interest groups have various stakes and agendas in the GYA with regards to the wildlife in this area. Although not an issue in the GYA, feral swine present problems for regulatory agencies in the United States due to management practices. Caribou in Alaska are also problematic since they can transmit the disease to farmed reindeer.

Current vaccines have been tested in these wild ungulates and have provided no to limited protection against virulent challenge of animals under experimental conditions. New vaccines are being investigated for their safety, pathogenicity, and vaccine efficacy. Once a suitable vaccine is found delivery methods can be tested for wide usage in these wildlife reservoirs.

Louisiana State University, AgCenter and School of Veterinary Medicine, Baton Rouge, LA

90 Strain Typing Brucella abortus Isolated from California and Florida Cattle Herds by Using “HOOF-Prints” – A New DNA Fingerprinting Technique for Brucella

Betsy J. Bricker1 and Darla R. Ewalt2

Techniques for subtyping Brucella abortus strains by conventional means are very limited. Currently, a large panel of metabolic and phenotypic tests is needed to subdivide B. abortus strains into 7 biovars, only three of which occur in the USA. Furthermore, about 85% of B. abortus strains isolated from cattle are categorized as biovar-1, limiting the epidemiological value of subtyping. We have developed a new technique called “HOOF-Prints”, which subtypes Brucella strains on the basis of DNA sequence differences. These differences arise from tandem repeats of an 8-bp sequence. Multiple tandem repeats of short DNA sequences (also known as microsatellites) are known to form hotspots for accelerated mutation. The Brucella genome has eight of these hotspots dispersed throughout its two chromosomes. Analysis of the DNA sequence at each of the eight hotspots produces a very distinctive genetic fingerprint. The procedure is based on PCR technology which is both rapid and very sensitive.

In this study, we examined a collection of 46 Brucella strains cultured from cattle in the early 1990’s. Among these samples were 27 isolates obtained from 5 different infected herds in California, and 19 isolates obtained from 12 different infected herds in Florida. The samples were subtyped by biovar and by DNA fingerprint. Typing by metabolic and phenotypic analyses showed that all three of the U.S. biovars were present in the study group in the following proportions: 89.1% biovar-1, 8.7% biovar-2 and 2.2% biovar-4. Two strains were identified as the vaccine strain S19. DNA analyses of the 46 strain collection revealed 28 different HOOF-Print patterns. Within most herds the fingerprint patterns were identical or very similar, differing at 2 or less of the 8 loci. Comparison of DNA fingerprints across different herds showed that the strains collected from Florida herds were different and probably genetically unrelated. The Florida strains were also different from the California strains. Comparison of the five California herds, however, showed surprisingly similar DNA patterns suggesting a common source of infection.

The data show that HOOF-Print DNA fingerprinting is a highly discriminating method for characterizing Brucella strains, far surpassing the capabilities of biotyping. This technique could be a valuable tool for epidemiologists trying to study the origins and spread of Brucella outbreaks.

1United States Department of Agriculture, Agricultural Research Service, National Animal Disease Center, Ames, IA, USA. 2U.S. Department of Agriculture, Animal and Plant Health and Inspection Service, Veterinary Services, National Veterinary Services Laboratories, Ames, IA, USA.

91 Investigation of Reported Resistance of Streptococcus equi ssp. zooepidemicus to Trimethoprim-sulfamethoxazole in Horses

D.J. Feary,1 J. Traub-Dargatz, 1 S. Roach, 1 D.R. Hyatt2,3, R. Jones,3 C. C. Wu,4 and P. S. Morley1

The objective of this study was to investigate the perceived increase in resistance of Streptococcus equi ssp. zooepidemicus (S. zooepidemicus) isolated from the lower respiratory tract of horses to trimethoprim- sulfamethoxazole (SXT). A retrospective study compared Kirby-Bauer disc diffusion susceptibilities of S. zooepidemicus isolates to SXT in the time periods of 1987-1990 and 1997-2001. The isolates were obtained from the tracheal wash fluid of equine patients examined at Colorado State University Veterinary Teaching Hospital in the 2 time periods. (1987 to 1990 and 1997 to 2001). Results were compared and statistically analyzed using a cross-sectional study design. There was a statistically significant difference between the reported resistance of S. zooepidemicus isolates recovered in the time period from 1987 to 1990 (8%), compared with reported resistance of S. zooepidemicus isolated from 1997 to 2001(42%).

An investigation of laboratory methods for antimicrobial susceptibility testing was conducted following the retrospective study in an effort to determine if the reported discrepancies were due to a true increase in antimicrobial resistance or to problems with test performance and interpretation of results. It was determined that there was inadequate quality control of media and falsely reported resistance of S. zooepidemicus, most likely attributable to antagonism of trimethoprim and sulfamethoxazole in the presence of excessive levels of thymidine found in whole blood-based media. These findings resulted in revision of the Kirby Bauer procedure and enhanced quality control of laboratory media. Strict adherence to laboratory protocols and regular surveillance and monitoring of trends in antimicrobial susceptibility is essential for the detection and correction of the type of problems revealed by this study. In addition, epidemiologists and others collecting data from laboratories should be cautioned to interact with laboratory personnel regarding the interpretation of results of various testing methods so that accurate analysis and conclusions are ensured.

1Department of Clinical Sciences, Colorado State University, Fort Collins, CO 2Veterinary Diagnostic Laboratory, Colorado State University, Fort Collins, CO 3Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 4Indiana Animal Disease Diagnostic Laboratory, Purdue Campus, West Lafeyette, Indiana

92 Characterization of a Highly Antimicrobial Resistant Escherichia coli from Newborn Calves with Colibacillosis in North Dakota

P. S. Gibbs1,2, , J. Foster2, S. R. Petermann1, M. Theis1, T. Solseng1, L. P. Schaan2, D. F. Krogh2, and G. J. C. Carlson3

Colibacillosis is a common problem causing financial losses for many agricultural animal industries. In North Dakota, colibacillosis in newborn calves can be devastating for small farmers. Generally, treatment for colibacillosis is antimicrobial therapy; however, resistance to multiple antimicrobials is becoming a common finding in many cases of calf colibacillosis submitted to the North Dakota Veterinary Diagnostic Laboratory. These highly resistant isolates make treatment in these cases increasingly difficult and often unsuccessful.

In January, 2004, a fecal sample from a calf with scours at 2 days of age was submitted to the NDSU- VDL. At the time of submission, all the calves born on this farm had symptoms of scours by 2 days of age. It was reported that this particular farm had the exact same problem during last year’s calving season. The sample was examined and found negative for Cryptosporidium, Rotavirus, and Corona virus, but was positive on culture for Escherichia coli. A routine antimicrobial sensitivity test was performed for the following antibiotics: Ampicillin, Apramycin, Ceftiofur, Chlortetracycline, Clindamycin, Erythromycin, Florfenicol, Gentamicin, Neomycin, Oxytetracycline, Penicillin, Spectinomycin, Sulphachloropyridazine, Sulphadimethoxime, Sulphathiazole, Tiamulin, Tilmicosin, Trimethroprim/Sulphamethoxazole, and Tylosin. Of all the antimicrobials tested, the only antimicrobial that this E. coli exhibited sensitivity to was apramycin. Using the polymerase chain reaction (PCR) for several potential virulence factors determined this isolate was positive for K99, F41 and Sta.

Since this was an expensive and recurring problem on this farm it was important to determine the source of this E. coli isolate and how it was establishing infections in the calves within 48 hours after birth. The referring veterinarian went to the farm and collected fecal samples from 5 unaffected adult cows and 5 of the affected calves. The feces was diluted and plated for isolation on MacConkey agar. Five suspected E. coli isolates were chosen from each diluted sample for antimicrobial susceptibility testing and PCR for virulence factors.

The results did not indicate that the healthy cows were carrying the highly antimicrobial resistant isolates, nor were they carrying E. coli isolates that contained the K99, F41, and Sta virulence genes. The affected calves, however, were all carrying E. coli isolates similar to the isolate found in the initial submission. Therefore, carriage of the disease-causing isolates in the cows was not established. Currently, the source of this highly resistant and virulent E. coli isolate remains undetermined. Future research will include using these isolates in an animal challenge model to determine differences in the virulence of the isolates found in the cows compared to those found in the calves.

1Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND 2Veterinary Diagnostic Laboratory, North Dakota State University, Fargo, ND 3Southwood Veterinary Clinic, Jamestown, ND

93 Genetic Variability of Moraxella (subgenus Moraxella) bovis and Moraxella (subgenus Branhamella) ovis Field Strains Recovered from Cattle with Infectious Bovine Keratoconjunctivitis

P.S. Nabity*1, H.E. Cerny2, P.D. Fey3, S.Hinkley1

Infectious bovine keratoconjunctivitis (IBK) remains to be an economically significant problem for the cattle industry, affecting beef and dairy cattle alike. Commercial pilus-based vaccines are available but outbreaks of IBK in fully vaccinated herds are not uncommon. Although Moraxella bovis (M. bovis) is thought to be the sole etiologic agent, the Nebraska veterinary diagnostic laboratory has consistently isolated Moraxella (subgenus Branhamella) ovis (B. ovis) from over 75% of the submitted IBK cases and in only 25% of the cases M. bovis was isolated over a period of the last four years. This prompted us to investigate a possible role of B. ovis in the etiology of IBK. Our preliminary data have shown that B. ovis may possess virulence factors similar to M. bovis. The objective of this study was to investigate the genetic diversity of M. bovis and B. ovis field strains. A putative grouping scheme may be able to identify different groups where a representative strain from each group could be chosen as vaccine candidate.

A protocol for pulse field gel electrophoresis for M. bovis and B. ovis was developed based on the official CDC protocol for typing food-borne pathogens, using the same size standard (Salmonella serotype Braenderup) The enzyme used for digestion of both M. bovis and B. ovis was determined to be SmaI which yielded about 15 bands over the size range of 200 kb to 7 kb. The size standard was digested with XbaI. Run time, and initial and final switch time were determined without significantly compromising the expected banding pattern for S. Braenderup. The digested DNA was run on a standard 1% agarose gel, stained with ethidium bromide and then visualized under UV. 110 M. ovis and 35 M. bovis field strains were subjected to this protocol. Images of each gel were stored as tiff files and then subjected to analysis by the BioNumerics software.

Preliminary analysis revealed that within both species, there were several clusters of closely related or identical strains present but the degree of relatedness between clusters was at times very low. Further analysis will determine what factors may be responsible for the clustering. The significant genetic diversity present in field strains may provide an explanation of vaccine failure.

1Dpt. of Veterinary and Biomedical Sciences, Veterinary Diagnostic Center, University of Nebraska- Lincoln, Lincoln, NE 2Williamsburg Veterinary Clinic, Lincoln, NE 3Dpt. of Internal Medicine, Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE

*To be considered for graduate student award.

94 Microbiology Scientific Session Sunday October 24, 2004 8:00 a.m. – 12:00 p.m. Guilford C Moderators: Lindsay Oaks, Jr. and Carole Bolin Page 8:00 a.m. Experimental Infection of Reindeer (Rangifer tarandus) with Mycobacterium bovis: 97 Pathological and Immunological Findings - M.V. Palmer, W.R. Waters, T.C. Thacker, W.C. Stoffregen, R.E. Slaughter, S.L. Jones, J.E. Pitzer, and F.C. Minion

8:15 a.m. Survey of Antibiotic Resistance in Mycobacterium tuberculosis Strains Isolated from 98 Elephants - N.B. Harris, R.A. Osorio, and J.B. Payeur

8:30 a.m. Use of a New Competitive ELISA Procedure for the Serodiagnosis of Glanders 99 (Burkholderia mallei) Infections in Horses - J. B. Katz, T. O. Bunn, D. R. Kinker

8:45 a.m. Competitive ELISA for the Detection of Antibodies Against Foot-and-mouth Disease Virus 100 Using a Biotinylated 3ABC Recombinant Protein - A. Clavijo, E-M. Zhou, P. Kitching

9:00 a.m. Immune Response Differences in Serums from Bovine Herpesvirus-1 Vaccinated Cattle: 101 Dependence of Viral Strain - R. W. Fulton, L. J. Burge, J.M. d’ Offay, R. Funk, G. D. Weaver, H. Van Campen, and B.J. Johnson

9:30 a.m. Use of Antemortem Tests to Identify Cattle with Disseminated Mycobacterium avium ssp. 103 paratuberculosis Infection Detected by Postmortem Culture of 15 Tissues - H.L. Hirst, J.E. Lombard, M.C. Antognoli, M.M. Dennis, M.D.. Salman, , and F.B. Garry

9:45 a.m. BREAK AND POSTER SESSION

10:15 a.m. Apparent Seroprevalence of Anaplasmosis in Wild Ungulates from the Northwestern and 104 Northcentral United States - J. B. Katz

10:30 a.m. Detection of Neospora caninum DNA from Central Nervous System of Deer by Nested 105 PCR - L.G. Corbellini *, L. Xie, J.A. Galeota, B.W. Brodersen, D.J. Steffen

10:45 a.m. Outbreak of West Nile Virus in Farmed Alligators (Alligator mississippiensis) in Southern 106 North Carolina - L.F. Humphries *, S.J. Page, P.G. Moisan, J.M. Law

11:15 a.m. Evaluation of Fluorescent Focus Neutralization Assay in Comparison with SVN, ELISA 108 and IFA for Serological Diagnosis of Porcine Reproductive and Respiratory Syndrome Virus Infection - D. K. Kirby and E.-M. Zhou

95 11:30 a.m. Reconstruction and Analysis of Eradication Efforts During the 2002-03 Outbreak of Exotic 109 Newcastle Disease - R. Speers, M. Webb, B. Howell, M. Grund, C. Hughes, E. Myrus and J. Silverman

11:45 a.m. Patterns of Relationship in Emergency Response: An Exotic Newcastle Disease Case 110 Study - R. Werge, L. Cooper, and C. Cardona

* Graduate Student Competition

96 Experimental Infection of Reindeer (Rangifer tarandus) with Mycobacterium bovis: Pathological and Immunological Findings

M.V. Palmer,1 W.R. Waters,1 T.C. Thacker,1 W.C. Stoffregen,1 R.E. Slaughter,2 S.L. Jones,3 J.E. Pitzer,4 F.C. Minion4

In the United States all species of Cervidae are included in the USDA’s uniform rules and methods for the eradication of bovine tuberculosis and; therefore, are subject to regulations regarding intradermal tuberculin testing. In reindeer, infection with M. bovis is exceedingly rare. The objectives of the present study were to describe the pathologic changes associated with M. bovis infection in reindeer and evaluate the effectiveness of intradermal tuberculin testing and an in vitro blood based assay for interferon-γ (IFN- γ) as means of diagnosis of tuberculosis in reindeer. Eleven reindeer were inoculated intratonsilarly with 2 x 104 CFU of M. bovis while 4 non-inoculated reindeer served as negative controls. The comparative cervical test (CCT) was done on all reindeer 3 and 8 months after inoculation. Blood was collected monthly for IFN- γ analysis. Thirteen months after inoculation, all reindeer were euthanized and examined. Various tissues were collected for bacteriologic culture and microscopic examination. All experimentally inoculated reindeer developed lesions in the medial retropharyngeal lymph nodes. Tracheobronchial, mediastinal and mesenteric lymph nodes, tonsils and lungs were less frequently affected. The CCT accurately identified M. bovis inoculated reindeer, but false positive results were common among negative control reindeer. Modifications in the USDA’s method for interpretation of the CCT decreased false positive results without increasing false negative results. An in vitro blood-based assay to measure IFN-γ production showed that mycobacteria-specific IFN- responses from M. bovis- infected reindeer exceeded those of negative control reindeer. However, positive IFN- responses to M. bovis purified protein derivative (PPDb) were also detected in negative control reindeer. ESAT-6 and CFP-10 are antigens unique to Mycobacteria spp. within the tuberculosis complex. While use of these antigens did not diminish detection of M. bovis-infected reindeer, it did decrease false positive results in negative control reindeer. Reindeer are susceptible to infection with M. bovis; however, lesions are fewer in number, less severe in nature and less widely disseminated than those seen in white-tailed deer similarly inoculated. Comparative cervical skin testing of reindeer can be highly sensitive, but has low specificity. Specificity can be improved by modification of criteria for interpretation of the CCT. A blood-based IFN- assay may prove useful for tuberculosis diagnosis when recombinant CFP-10 or ESAT-6 / CFP-10 antigens are used to enhance the specificity of the IFN- assay.

1Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, USDA, Ames, IA 2Biocor Animal Health, Omaha, NE 3CSL Limited, Victoria, Australia 4Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA

97 Survey of Antibiotic Resistance in Mycobacterium tuberculosis Strains Isolated from Elephants

N. B. Harris1, R. A. Osorio1, and J. B. Payeur1

Since 1996, the National Tuberculosis Working Group for Zoo and Wildlife Species has been monitoring Mycobacterium tuberculosis (TB) in elephants. As part of these guidelines, clinical strains from all culture positive elephants are subject to diagnostic susceptibility testing. Currently, little information exists regarding the effects of chemotherapy on clinical isolates of M. tuberculosis in infected animals. Therefore, a survey of drug resistance in M. tuberculosis isolates from elephants was conducted. For this, 37 clinical strains of M. tuberculosis isolated from elephants were tested against pyrizinamidase (PZA) (100 µg/ml), streptomycin (STR) (2 µg/ml), isoniazid (INH) (0.1 µg/ml), rifampin (RIF) (2 µg/ml) and ethambutol (EMB) (2.5 µg/ml) using the BACTEC 460 TB 460 system. These same isolates were also tested using the BACTEC 960 MGIT system at antibiotic concentrations of 100 µg/ml PZA, 1.0 µg/ml STR, 0.1 µg/ml INH, 1.0 µg/ml RIF, and 5.0 µg/ml EMB.

Overall, eight isolates (21.6%) were resistant to at least one antibiotic. INH resistance was the most prevalent, with 6/37 (16.2%) of the strains demonstrating this phenotype. Resistance to RIF was also noted at a prevalence of 8.1% (3/37), and one isolate (2.7%) was resistant to PZA. All M. tuberculosis isolates were sensitive to EMB and STR at the concentrations tested.

Of the 37 strains of M. tuberculosis surveyed, 18 were isolated from a group of 15 animals. In three animals, two separate isolations were made less than three months apart. Of the 18 strains of M. tuberculosis in this group, two were initially resistant to INH (INHR), but were susceptible to the remaining four antibiotics. In comparison, the other 16 M. tuberculosis strains were sensitive to all antibiotics tested.

The remaining 19 strains represented multiple isolations from six elephants, with isolations being made approximately 6 months to one year apart. Of the six elephants in this category, one had an index case of M. tuberculosis that was resistant to RIF at 2 µg/ml (RIFR), but did not undergo further changes in its susceptibility profile with subsequent isolations. M. tuberculosis strains isolated from another elephant demonstrated a change in antibiotic resistance profile from RIF susceptible (RIFS) to RIFR, whereas isolates from the final elephant demonstrated a similar INHS -to- INHR profile for isoniazid. Strains from a fourth animal showed multiple drug resistance to both RIF and INH in later isolations. Isolates from the remaining two elephants were susceptible to all antibiotics. Since all four animals having isolates that showed changes in antibiotic sensitivity profiles had undergone chemotherapy, restriction fragment length polymorphism (RFLP) profiles were performed on DNA from antibiotic-sensitive and antibiotic-resistant isolates. Interestingly, in one animal the RFLP profiles of the INHR strains were distinct from the INHS strains, suggesting that a re-infection of the animal had occurred with a different, antibiotic resistant strain of M. tuberculosis. For the remaining two elephants, the RFLP profiles were identical for the antibiotic- sensitive and antibiotic-resistant strains, indicating that antibiotic resistance had most likely been acquired during the course of chemotherapy.

Overall, this data demonstrates that, like human strains of M. tuberculosis, INH and RIF resistance are observed most often in clinical isolates from elephants, and that the emergence of drug-resistant strains concurrent with treatment for M. tuberculosis can occur in these animals.

1USDA-APHIS, National Veterinary Services Laboratories, Ames, IA 50010

98 Use of a New Competitive ELISA Procedure for the Serodiagnosis of Glanders (Burkholderia mallei) Infections in Horses

J. B. Katz1, T. O. Bunn1, D. R. Kinker1

Glanders (Burkholderia mallei) is an anciently recognized zoonotic infectious disease of equids that is currently foreign to the United States and excluded by United States Department of Agriculture complement fixation testing (CFT) and rejection of seropositive horses presented for importation. A competitive ELISA (CELISA) has been developed to replace the glanders CFT, in order to reduce problems with anticomplementary sera and to increase the ease, simplicity, and objectivity of the testing program. The CELISA uses all commercially available reagents except a stable, irradiated, whole-cell B. mallei antigen and an anti-B. mallei monoclonal antibody, with results in about 3 hours. Histographic analysis of test results from over 1,300 (presumed) glanders-free, U.S.-origin horses demonstrated that CELISA specificity was 99.5% or greater. Serial endpoint dilutional studies of sera from known B. mallei infected horses proved that the CELISA was 2-4 fold more sensitive to anti-glanders antibodies than the CFT, but 2-4 fold less sensitive than the CFT to sera from animals sensitized to mallein. This enables the CELISA to more readily detect truly infected animals and to differentiate them from uninfected animals which had been previously subjected to intradermal or intrapalpebral mallein testing. The glanders CELISA usually demonstrated seroconversion several days sooner in B. mallei-exposed horses than did the CFT. In a recent serial serologic study of 5 exposed ponies, all 5 were CELISA- positive at 7 days postexposure, while only 2 were CFT-positive by that time. On an intralaboratory basis, the glanders CELISA was highly repeatable and reproducible on a well-to-well, plate-to-plate, and day-to-day basis. A 6-nation, international/interlaboratory, reproducibility study of the glanders CELISA is now in progress. The National Veterinary Services Laboratories is working to replace the glanders CFT with the glanders CELISA and to have the latter method internationally recognized as an accepted serodiagnostic tool for the control of this infectious disease.

1 Diagnostic Bacteriology Laboratory, National Veterinary Services Laboratories, Veterinary Services, Animal and Plant Health Inspection Service, U.S. Department of Agriculture, Ames, Iowa, 50010.

99 Competitive ELISA for the Detection of Antibodies Against Foot-and-mouth Disease Virus Using a Biotinylated 3ABC Recombinant Protein

A. Clavijo1, E-M. Zhou2, P. Kitching1

Foot-and-mouth disease (FMD) is a significant constraint to international trade in live animals and animal products. It can spread rapidly and affect both domesticated and wild ruminants as well as pigs. The FMD virus (FMDV) genome encodes a single polyprotein from which the different viral polypeptides are cleaved by viral proteases, including eight different non-structural proteins (NSPs). Both structural and non-structural antigens induce the production of antibodies in infected animals. Control of the disease in FMD-free countries includes movement restrictions and a slaughter policy. However, elimination of infected and contact animals alone may not be sufficient to eradicate the virus and emergency vaccination may be considered as an additional option. Identifying animals either vaccinated or unvaccinated that have had contact with live virus, from those that have been only vaccinated against the disease is of considerable importance since both groups have neutralizing antibodies in their sera. Using traditional serological techniques it is not possible to distinguish FMD infected animals from vaccinated animals and control authorities have limited possibilities to monitor virus presence or circulation.

We developed a competitive ELISA (cELISA) that uses a recombinant 3ABC protein with a fusion polypeptide containing a site naturally biotinylated by biotin ligase in E. coli. The resulting recombinant protein was expressed and biotinylated in vivo in E. coli and a crude cell lysate applied directly to streptavidin coated plates. This approach provides not only a single step purification of the antigen through its biotinylated moiety, but also a surface with a high binding capacity sufficient to make available an excess of reagent and a negligible nonspecific binding, allowing for the highest possible assay sensitivity and specificity.

Serum samples compete with hyperimmune anti-3ABC guinea pig antisera for specific epitopes on the recombinant protein and the result expressed as a percentage of inhibition. This cELISA was used to investigate whether antibodies to FMDV 3ABC protein could be detected and to explore the possibility of using this test for the detection of antibodies to FMD infection in cattle, sheep and pigs without alterations in the test procedure. We experimentally infected cattle, sheep and pigs with all seven serotypes of FMDV. Serum samples were collected from 1 to 28 days post infection. The cELISA was able to detect anti-3ABC antibodies in FMDV-infected cattle, sheep and pigs as early as 6-7 days post infection. There was no difference in the antibody response of cattle, sheep or pigs to the 3ABC recombinant protein when different serotypes of the virus were used. We are currently in the process of further validating this test for identification and differentiation of vaccinated animals from those naturally infected, in particular in vaccinated animals that have had contact with live virus and become carriers. This perhaps may be the major use of this test.

Currently no NSP test has been fully validated to be used for mass screening. The complexity of FMD and its wide range of hosts make this validation difficult to achieve, but the availability of a competitive ELISA that can be used in all species may facilitate this process.

1National Centre for Foreign Animal Disease,1015 Arlington Street, Winnipeg, Manitoba R3E 3M4, Canada. 2Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011-1250, USA.

100 Immune Response Differences in Serums from Bovine Herpesvirus-1 Vaccinated Cattle: Dependence of Viral Strain

Robert W. Fulton*1, Lurinda J. Burge1, J.M. d’Offay1, Rebecca Funk1, Galen D. Weaver2, Hana Van Campen3, and B.J. Johnson1,4

During fall 2003 and winter 2004 there were reports of bovine herpesvirus-1 (BHV-1) disease in cattle with prior vaccination (often multiple doses) with modified live virus (MLV) vaccines. These disease episodes occurred under feedlot conditions with the respiratory disease 50 or more days after arrival/processing. The disease was not associated with use of any one commercial vaccine. While BHV-1 respiratory disease (infectious bovine rhinotracheitis [IBR]) occurs sometimes in isolated cases several weeks post arrival/vaccination, these 2003-2004 episodes had greater morbidity/mortality than expected

The purpose of the initial phase of this study was to determine if field isolates from these feedlot cases were susceptible to the BHV-1 antibodies in MLV vaccinated calves. Serums were from prior studies and included those from calves receiving each of five commercially available MLV vaccines containing BHV-1. Four of five vaccines contained the Colorado strain and the fifth vaccine the Baker strain. Serums were tested for BHV-1 antibodies in a 24-well plaque reduction assay using the different BHV-1 strains. Monovalent BHV-1 vaccines were obtained representing the five vaccines above. The vaccine vials were reconstituted and assayed for infectivity by both 24-well plaque assay and 96-well plates for TCID50 using MDBK cells. In addition reference viruses included the Colorado 1 strain from USDA NVSL. This BHV-1 (Colorado 1 NVSL) was from Pittman Moore sent to the USDA in 1958. The virus had been passaged 57 times. The second reference was the BHV-1 Cooper strain sent by USDA Center for Veterinary Biologics (CVB) for challenge studies. A BHV-1 isolate was obtained from a diagnostic laboratory and was from a diseased calf with respiratory disease (BHV-1-31751) in the fall 2003.

Results of this study using 5 vaccine strains, a reference BHV-1 Colorado NVSL strain, BHV-1 Cooper challenge strain, and an isolate from a clinic case indicated a wide range of antibody titers when vaccinated calves’ serum were tested against these 8 strains. The titers were highest to the BHV-1 Colorado NVSL and one MLV vaccine virus (Baker strain). The lowest titers were to the BHV-1 Cooper challenge strain and the recent field strain, BHV-1-31751. In almost all serums tested, the differences were 3-10 fold less. Interestingly, the antibody titers in the calves’ serums were quite low to the homologous vaccine (all four BHV-1 vaccines with the Colorado 1) compared to titers to the BHV-1 Colorado NVSL strain and BHV-1 Baker MLV vaccine strain. It appears that most of the vaccines induced serum antibodies that poorly neutralized the BHV-1 Cooper challenge strain and the field strain.

It is possible that high passages in cell culture results in changes in virus neutralization properties of the BHV-1. The implications for differences in efficacy of the vaccines against field strains are unclear. Additional testing is in progress to detect genomic differences among the BHV-1 strains.

1Department of Veterinary Pathobiology, and 4Oklahoma Animal Disease Diagnostic Laboratory, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 2Amarillo, TX 3Veterinary Diagnostic Laboratory, Colorado State University, Fort Collins, CO

101 Sequencing-Based Identification of a Novel Coronavirus in Ferrets with Epizootic Catarrhal Enteritis (ECE) and Development of Molecular Diagnostic Tests for ECE

A.G. Wise1, M. Kiupel1, C. Isenhour1 and R. K. Maes1

Epizootic catarrhal enteritis (ECE) is a relatively new diarrheal disease of domestic ferrets, first described in the spring of 1993 on the East Coast of the U.S. Clinical signs consist of lethargy, anorexia and vomiting, followed by the presence of a foul-smelling bright green diarrhea with high mucus content. The overall mortality rate is low (<5%), but morbidity approaches 100%. The disease in juvenile ferrets is often mild and even subclinical. In older ferrets, the clinical signs are more severe and the mortlity rate is often higher. Earlier investigations on the etiology of the disease implicated a coronavirus, based upon immunohistochemistry and negative staining electron microscopy. In this report, we present the first nucleotide sequence-based evidence substantiating a coronavirus as the etiologic agent of ECE. Using three consensus RT-PCR assays for the genus Coronavirus, a novel coronavirus was detected in diarrheic feces of ferrets exhibiting clinical signs of ECE. These assays made use of highly degenerate primers to amplify short regions of the polymerase (251-bp) and spike (628-bp) genes, and a 735-bp fragment spanning the 3’ end of the membrane glycoprotein gene to the 5’ end of the nucleocapsid gene. The nucleotide sequences of the amplicons were confirmed to be authentic coronaviral sequences by BLAST analysis. The partial polymerase, spike and membrane glycoprotein gene sequences of this novel coronavirus had nucleotide sequence similarities of 76.4%, 60.8% and 66.7%, respectively, to porcine transmissible gastroenteritis virus (TGEV). Subsequently, the entire nucleocapsid gene of this coronavirus was amplified and sequenced using a 3’ RACE method. The 1,125-nucleotide open reading frame of the nucleocapsid gene had sequence similarities of 51.6%, 48.2%, 48.7% and 34.5% to the corresponding genes of canine coronavirus, feline coronavirus, TGEV and a human coronavirus strain, 229E, respectively. Based on these data we designated this novel coronavirus as “ferret enteric coronavirus” (FECV).

Using the nucleocapsid sequence data, a pair of FECV-specific primers which targets a 113-bp region of the gene, was designed to develop a one-step RT-PCR assay for the rapid detection of the virus in fecal or oral swab specimens. This RT-PCR assay was optimized to be run either in a gel-based or real-time format. In addition, an in-situ hybridization (ISH) assay, using an FECV nucleocapsid gene-specific probe, was developed and used to demonstrate the presence of viral RNA in the cytoplasm of apical enterocytes of small intestinal villi from affected ferrets. These tools will be valuable for the rapid and accurate diagnosis of ECE and will facilitate future investigations of the molecular pathogenesis, epidemiology and prevention of FECV infections.

1Diagnostic Center for Population and Animal Health, Michigan State University, East Lansing, MI

102 Use of Antemortem Tests to Identify Cattle with Disseminated Mycobacterium avium ssp. paratuberculosis Infection Detected by Postmortem Culture of 15 Tissues

HL Hirst, DVM, MS1, JE Lombard, DVM, MS2, MC Antognoli, DVM, PhD1, MM Dennis, DVM1, MD Salman, DVM, PhD1, and FB Garry, DVM, MS1

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne’s Disease (JD). MAP infection is a suggested contribution to the cause of Crohn’s disease (CD), a chronic, debilitating enteric disease of humans. Although evidence is not definitive, there is sufficient documentation in the literature to convince some groups that cattle infected with MAP present a risk to human health. If perceived risk escalates in the near future, it may be necessary to exclude cattle infected with MAP from human consumption. A rapid antemortem test would be essential to screen cattle for exclusion prior to slaughter. The objective of this study was to determine the association between antemortem test results and presence of disseminated MAP infection at slaughter.

Seventeen adult lactating Holstein cows were obtained for slaughter from October 2003 to January 2004 from 4 Colorado dairies with a known history of clinical JD. Cows that were purchased had a history of at least one positive serum ELISA result. Physical exam and body condition scoring were used to determine whether clinical JD was present. Prior to euthanasia, serum was collected for ELISA and feces for conventional and BACTEC culture, and liver biopsy was performed for histologic exam. At necropsy the following tissues were collected for conventional culture: skeletal muscle, heart muscle, hepatic lymph node (LN), ileocecal LN, ileum, jejunum, kidney, liver, mesenteric LN, popliteal LN, prescapular LN, medial retropharyngeal LN, supramammary LN, and lung. Ileocecal LN, ileum, and mesenteric LN were submitted for BACTEC culture. Ileum, jejunum, mesenteric LN, liver, hepatic LN, and retropharyngeal LN were submitted for PCR. Ileum, jejunum, ileocecal LN, and mesenteric LN were submitted for histologic exam. Cows with at least one positive tissue culture beyond the intestine and its associated LN were considered to have disseminated infection (DI). Fourteen of 17 cows had confirmed MAP infection via culture or histologic exam. Twelve of these had disseminated infection (DI), but 5 had no evidence of clinical JD, 3 had weight loss without diarrhea, and 4 had obvious clinical JD. The most common tissue found culture positive for MAP in DI cows was the hepatic LN (11 of 12 DI cows). The one cow with DI and negative hepatic LN culture was classified as DI because the retropharyngeal LN was positive for MAP. Fecal culture (FC) with BACTEC and conventional FC detected 8 and 11 of 12 DI cows, respectively. The one cow with DI and negative FC result had negative BACTEC tissue culture results, but the hepatic LN and ileocecal LN were positive on conventional culture. Of 17 antemortem liver biopsies, 4 were reported as having granulomatous hepatitis, but no acid-fast bacteria were seen. Five cows had negative ELISA results at euthanasia, despite previous positive serum ELISA results. Twelve cows had at least one tissue PCR positive for MAP.

Sensitivity (SE) of the conventional FC, PCR, BACTEC FC, liver biopsy, and ELISA to detect DI animals were 92%, 75%, 67%, 67% and 67%, respectively. Specificity (SP) of the conventional FC, PCR, BACTEC FC, liver biopsy and ELISA to detect DI animals were 80%, 40%, 100%, 100%, and 40% respectively. Sensitivity and specificity of the histologic examination of multiple tissues to detect DI animals were 92% and 80%, respectively. If the perceived human health risk leads to mandatory exclusion of cows with disseminated MAP infection from the food supply, serum ELISA may be an option for detecting a large proportion of DI cattle antemortem. Many cattle would be needlessly condemned due to poor specificity of the test in this pilot study. Both FC methods appear to have high SE and SP for detection of DI animals, but would be impractical in most cases due to the time required for culture (6 to 12 weeks).

1Animal Population Health Institute, Colorado State University, Fort Collins, CO. 2Centers for Epidemiology and Animal Health, Fort Collins, CO

103 Apparent Seroprevalence of Anaplasmosis in Wild Ungulates from the Northwestern and Northcentral United States

J. B. Katz

The apparent seroprevalence of anaplasmosis was estimated in a group of 1,332 sera collected from wild ungulates located in the northwestern and northcentral United States. All sera were evaluated, using both an indirect immunofluorescence assay (IFA) and an enzyme-linked competitive immunoassay (CELISA). A 245-serum subset of this group was also evaluated, using the card agglutination test. The IFA was the most analytically sensitive method for detection of anti-A. marginale antibody, using known positive sera. In twofold serial dilution tests, the CELISA was generally 2-4 fold, and the card test 4-8 fold, less sensitive than the IFA. Sera from wild white tailed deer, moose, and pronghorn antelope had 0% IFA seroprevalence in all states where they were found. Bighorn sheep, black tailed deer, mule deer, elk, bison, and mountain goats all had variably higher percentages of seroprevalence (range: 4% - 70%), depending on location. Apparent IFA seroprevalence was very low in the northcentral states and increased generally westward. The CELISA overestimated seroprevalence, relative to the IFA, in nearly all of the ungulate species studied (37.8% vs. 10.2 % overall), especially in moose and elk.

Sera from animals exposed to certain other ehrlichial agents (E. chaffeensis, E. ewingii, A. phagocytophilum) did react positively when using the CELISA, and to a lesser extent - the IFA, but not the card test. Geographic factors, vector-associated factors, species-specific differences, and test method specificity (cross-reactivity with certain other ehrlichial agents) should all influence interpretation in the serodiagnosis of anaplasmosis in wild ungulates from the northcentral and northwestern United States. Wild ungulates may constitute a reservoir of A. marginale infection in these regions. The anaplasmosis CELISA test does not appear to be reliably specific for use with sera from wild ungulate species.

Diagnostic Bacteriology Laboratory, National Veterinary Services Laboratories, Veterinary Services, Animal and Plant Health Inspection Service, U.S. Department of Agriculture, Ames, Iowa, 50010.

104 Detection of Neospora caninum DNA from Central Nervous System of Deer by Nested PCR

L.G. Corbellini *1,2, L. Xie2, J.A. Galeota2, B.W. Brodersen2, D.J. Steffen2

Neospora caninum (NC) is an Apicomplexan protozoa parasite that can infect a number of hosts, mostly cattle and dogs. It is considered one of the most important causes of bovine abortion worldwide. NC has been detected in tissues of naturally infected domestic and wild animals such as bovine, canine, ovine, caprine, equine, deer and rhinoceros. Dogs are a definitive host of NC, since they can eliminate oocysts in the feces. Evidence of the sylvatic cycle of neosporosis is strongly supported by the recent finding that coyotes are also definitive hosts of NC. Moreover, studies on seroprevalence of NC in deer have demonstrated high levels of exposure to this parasite, which could be explained, in part, by environmental contamination from wild canids. Understanding the role of wildlife in the epidemiology of neosporosis is important since there are large populations of deer and wild canids in the USA. The objectives of this study were to develop a nested PCR to detect NC DNA in fresh tissues samples and verify the prevalence of neosporosis in Nebraska deer.

Samples were obtained from 100 deer as part of a study of chronic wasting disease. After overnight incubation with digestion buffer containing proteinase K (100µg/ml), the DNA was extracted with phenol-chloroform (25:25) from approximately 4 g of tissue, which included obex and a small portion of cerebellum and medulla oblongata. A nested PCR was employed to amplify DNA using primers derived from Np21/Np6 (Yamage et al., 1996). Primers were designated 359F and 745R (outer primers) and 424FN and 703RN (nested primers). A reaction mixture containing a volume of 50µl was employed for DNA amplification, which was performed in an automated thermal cycle using 35 cycles with denaturation (95 C; 1 min), annealing (60 C; 30 sec), and primer extension (72 C; 2 min). NC DNA (NC- beef, McAllister et al., 2000) was used as a positive control. The amount of DNA present in the positive control was measured using a spectrophotometer and then, diluted until 10-7 in order to verify the minimal concentration of NC DNA amplified by the nested PCR. Negative control without template was applied in each run. Toxoplasma gondii DNA was also tested.

The NC nested PCR produced a 279-bp product. The minimal amount of DNA amplified was 0.18 pg, corresponding to approximately 2.25 tachyzoites. No T. gondii DNA was amplified. NC DNA was detected in 3% of the samples (3/100). The small area of the central nervous system obtained for this survey could underestimate the prevalence, as the distribution of NC organisms is generally sparse. Neospora abortions occur as occasional epizootics in the study region. NC has been reported in naturally infected deer and transmission between deer and domestic dogs has been demonstrated. Despite the low prevalence, this result suggests that deer might be an important reservoir for NC. As prey for coyotes, deer complete the sylvatic cycle. Deer commonly share habitats with beef cattle and coyotes in Nebraska.

1. Federal University of Rio Grande do Sul, Brazil (CAPES/FAPERGS/Brazil) 2. Veterinary Diagnostic Center, University of Nebraska-Lincoln

*To be considered for graduate student award

105 Outbreak of West Nile Virus in Farmed Alligators (Alligator mississippiensis) in Southern North Carolina

L.F. Humphries1, *, S.J. Page1, P.G. Moisan2, J.M. Law1

West Nile Virus is a Flavivirus of the Family Flaviviridae that has caused disease in birds, mammals, and reptiles of multiple species since its introduction into North America in 1999. West Nile Virus infection and disease was diagnosed at the Rollins Animal Disease Diagnostic Laboratory (RADDL) in 7 juvenile alligators from an alligator farm in North Carolina from 2 separate outbreaks during January and February 2004. Alligators in the first group were approximately 17 months of age and consisted of 1 moribund and 3 dead individuals from a total of 38 dead in the house. Alligators in the second group were approximately 4.5 years old and consisted of 3 dead individuals from 30 dead in the house. The animals were maintained in two separate houses within the facility. Clinical signs included ‘stargazing,’ emaciation, anorexia, and above baseline mortality in the two houses. Necropsy findings included granulomatous hepatitis and splenitis, with caseonecrotizing stomatitis, laryngitis, esophagitis, and enteritis. Histopathology of formalin-fixed tissues revealed meningitis and pyencephaly and confirmed the fibrinonecrotic lesions of the gastrointestinal tract. Reports of encephalitis in alligators infected with West Nile Virus in a previous outbreak prompted testing for WNV in this instance. Bacteria isolated from all animals include: Salmonella sp., Aeromonas hydrophila, nonhemolytic E. coli, Staphlococcus sp., Morganella morganii, Streptococcus sp., Proteus sp., and Edwardsiella tarda. West Nile Virus disease was verified by immunohistochemistry.

To determine the source of infection and to examine the potential risk of spread to humans in the area, the farm was visited and the owner interviewed. The animals at the farm are maintained in the dark at 85- 88oF at all times. Population density in each house is age dependent (300 – 400 small young alligators vs. ~200 larger, near harvest age animals). The animals are fed ground chickens culled from local broiler farms and fortified with vitamins and antibiotics daily in 5-gallon aliquots. Water in each house is changed every other day for larger alligators and once a week for younger, smaller alligators, except during periods of extreme cold when houses may go for up to 10 days between water changes. The water is chlorinated via the addition of 1 quart of bleach per house (800 – 1000 gallons), and 1 quart of table salt (iodized NaCl) and antibiotics are added. Although the animals are maintained in the dark, the houses are not completely isolated from the environment; there are ventilation openings in each house. There were no visible mosquitoes on the initial visit; however traps were deployed on subsequent visits to determine the local mosquito species and to test for captured mosquitoes for WNV.

In summary, West Nile Virus was found to be the causative agent of above average mortality in farmed alligators at a North Carolina alligator farm. Over a 2-3 week period approximately 68 alligators died in two houses at this farm. Investigation of the outbreak indicated that over-wintering mosquitoes are the most likely source of the infection.

References: 1. Brinton MA, 2002. The molecular biology of West Nile Virus: a new invader of the Western Hemisphere. Annu Rev Microbiol 56: 371-401. 2. Miller DL, Mauel MJ, Baldwin C, Burtle G, Ingram D, Hines II MJ, Frazier KS, 2003. West Nile virus in farmed alligators. Emerg Infect Dis 9(7): 794-799.

1 Population Health and Pathobiology Dept. College of Veterinary Medicine, NC State University, Raleigh, NC 2 Rollins Animal Disease Diagnostic Laboratory, North Carolina Department of Agriculture, Raleigh, NC

*To be considered for graduate student award.

106 The Use of Environmental Sampling as a Herd Diagnostic Tool for Detecting Mycobacterium avium subsp paratuberculosis on Minnesota Dairy Farms

Raizman E.A.,*1 S.J Wells1, S.M. Godden1. R.F. Bey2, M.J. Oakes3, D. C. Bentley4, K.E. Olsen4

Paratuberculosis or Johne’s disease is a chronic and progressive intestinal disease in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). The disease becomes manifest in adulthood and results in economic losses. Limited information is available about the distribution of MAP in the environment of dairy farms and its relation to herd infection.

The objective of this study was to characterize the distribution of MAP in the environment of infected and uninfected Minnesota dairy farms. One hundred and eight Minnesota dairy herds were sampled during the summer of 2002, including 80 herds known to be infected based on previous testing in the Johne’s Disease Control Program (JDCP) of the Minnesota Board of Animal Health (MBAH) and 28 herds known to be uninfected based on previous testing in the Voluntary Johne’s Disease Herd Status Program (VJDHSP) of the MBAH. Fecal samples were obtained from up to 100 cows in each herd and were cultured in pools of five cows per pool based on age order. Environmental samples were obtained from each farm, with up to 2 samples collected from various locations. Fecal pools and environmental samples were tested using bacterial culture for Map at the Minnesota Veterinary Diagnostic Laboratory.

Sixty-four of the 80 JDCP herds (80%) had at least one positive pool; 16 of these herds did not have any positive pools. The farm environment was determined to be contaminated on 61 of the 64 (95%) herds with at least one positive pool and in one of the 16 JDCP herds with no positive pools. Twenty-six of the 28 VJDHSP herds (93%) had no positive pools; 2 herds had one positive pool each. The farm environment was determined to be positive in samples from cow alleyways (77% of the herds), manure storage (68%), calving area (21%), sick cow pen (18%), water stream edge and water runoff (6%), and postweaned calves area (3%), but not in preweaned calves or fields near cow area. There was an association between maximum level of colonies per tube from cow alleyways and manure storage and fecal pool prevalence. Herds with both areas cultured negative were estimated to have 0.3-4% fecal pool prevalence. Herds with both areas having a heavy load of bacteria were estimated to have 53-73% fecal pool prevalence. The study results indicate that targeted sampling of cow alleyways and manure storage may be a useful alternative strategy for herd screening and Johne’s infection status assessment and for estimating herd fecal prevalence.

1-Department of Veterinary Population Medicine, University of Minnesota, St Pau,l MN 2- Department of Pathobiology, University of Minnesota, St Pau,l MN 3- School of Public Health, University of Minnesota, Minneapolis, MN 4- Minnesota Veterinary Diagnostic Laboratory, University of Minnesota, St Paul, MN

*To be considered for graduate student award

107 Evaluation of Fluorescent Focus Neutralization Assay in Comparison with SVN, ELISA and IFA for Serological Diagnosis of Porcine Reproductive and Respiratory Syndrome Virus Infection

D. K. Kirby1 and E.-M. Zhou1

Porcine reproductive and respiratory syndrome (PRRS) was first identified in the United States in 1987. Since then its significance in the swine industry has been in the form of reproductive failures, respiratory ailments, and secondary infections. Current common serological diagnostic tests include ELISA (enzyme-linked immunosorbent assay), IFA (indirect immunofluorescence assay) and now the FFN (fluorescent focus neutralization). The FFN was developed as a replacement for the PRRS-SVN (serum virus neutralization), which has shortcomings of lack of replication of test results, diagnostic sensitivity and an incubation time of approximately one week.

With the increased demand on using a neutralization assay for PRRS serological diagnosis, we report here the evaluation results of the fluorescent focus neutralization (FFN) assay in comparison with SVN, ELISA and IFA using experimental serum samples for PRRS serological diagnosis. Throughout this project a protocol adopted from South Dakota State University was used to perform the FFN. The FFN is a neutralization assay using fluorescence-labeled direct anti-PRRS virus nucleocapsid conjugate (SDOW17-F) to detect the degree of neutralization. The assay requires a one hour incubation of serially diluted serum with equal part of standardized virus (100 fluorescent foci units per well). The serum virus mixture is transferred to a plate containing a confluent monolayer of Marc 145 cells and incubated for 48 hours at 37 ْ C with 5% CO2. The plate is then emptied, fixed using 80% acetone and labeled with the conjugate.

Comparing results demonstrated that the FFN was able to detect neutralizing antibodies from 3 groups of 30 pigs experimentally infected with PRRS virus starting at 20 DPI and peaked at about 47 DPI whereas the SVN detected neutralizing antibodies starting at 47 DPI. The ELISA and the IFA detected antibodies starting at 10 DPI and peaked at about 26 DPI. From 54 FFN positive samples, 3, 54 and 52 were detected positives by SVN, IFA and ELISA, respectively.

In conclusion, the FFN is more sensitive than the SVN in detecting neutralizing antibodies against PRRS virus. The FFN demonstrates its appropriateness as a replacement for the SVN with results available within three days instead of seven. It provides an accurate, repeatable, and timely neutralization assay that could be used to determine the presence of neutralizing antibodies in PRRS virus infected as well as vaccinated animals.

1 Serology Section, Veterinary Diagnostic Laboratory, Department of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, Iowa.

108 Reconstruction and Analysis of Eradication Efforts During the 2002-03 Outbreak of Exotic Newcastle Disease

R. Speers, M. Webb, B. Howell, M. Grund, C. Hughes, E. Myrus and J. Silverman1

The Animal and Plant Health Inspection Service, Division of Veterinary Services (APHIS-VS) within the U.S. Department of Agriculture (USDA) asked the CNA Corporation to conduct a reconstruction and analysis of eradication efforts during the 2002-03 outbreak of exotic Newcastle disease (END). Reconstruction of such complex operations can inform APHIS-VS and other organizations about what happened and why, from many different perspectives. During an event, most responders can only see what they directly experience, and it is difficult for even the leading officials to maintain a broad perspective. Unexpected and complex issues often stimulate the most interesting, and most important, decisions and actions. Reconstructing these areas can further understanding of the overall event and enhance preparedness for future disease response operations.

CNA has reconstructed a full timeline of the END eradication efforts at all response levels, by examining the key actions, decisions, events, and how the operations developed over time. To do so, we have drawn evidence from three primary sources: APHIS-VS documents and databases, state and local documentation, and interviews with those involved. Through subsequent analysis, CNA is examining ways to quantify the response and looking for both quantitative and qualitative “models,” or methods of description, that show how the operation was executed and how the response components fit together.

The 2002-03 outbreak of END began in California and later spread to bordering areas of Nevada and Arizona. For the first several months it spread among backyard poultry flocks in southern California, partly due to movements of birds that were bred for cockfighting. It wasn’t until late December 2002, that END was confirmed in a commercial poultry operation. By January, the disease had spread to other states as well as additional counties within California. In April 2003, a different strain of END virus was detected in western Texas and those response operations were folded into the overall Area Command structure. Most quarantined areas were released in May, though several remained until September 2003 when eradication was deemed complete.

Overall, 19 counties were quarantined within five southwestern states (CA, NV, AZ, TX, and NM). A total of 932 infected premises were identified, and over 4 million birds were depopulated. At the peak of response operations, over 1600 personnel were dedicated to the eradication efforts. New processes for laboratory testing, personnel dispatch, risk assessment, response management, data management, and surveillance standards were developed during the eradication efforts.

Differences in authorities, capabilities, and fundamental strategies created differences in response operations among the various Incident Command Posts (ICPs). The Nevada and Texas ICPs established 1 km depopulation zones and 3 km surveillance zones around infected premises. The Forward Command Post located at the Colorado River Indian Tribe reservation established a 1 mi depopulation zone and 5 mi surveillance zone. Within California, depopulation was conducted on infected and adjacent premises, as well as those determined to be dangerous contacts. At times, the definition of a “flock” was extended to include entire neighborhoods with close contact among poultry and other birds. The California ICPs established 1 km quarantine zones around each infected premises. Data sources such as the Emergency Management Response System (EMRS) and situation reports developed by individual Task Forces reflect variations in operational definitions and reporting methods used in the different affected areas.

Careful analysis supported by a complete reconstruction of the eradication efforts will lead to recommendations for improvements in overall preparedness for foreign animal disease outbreaks.

1Operations Evaluation Group, The CNA Corporation, Alexandria, VA

109 Patterns of Relationship in Emergency Response: An Exotic Newcastle Disease Case Study

R. Werge1, L. Cooper2, C. Cardona3

Multijurisdictional animal health emergencies require coordinated response from a wide range of governmental agencies and nongovernmental groups. During the outbreak of Exotic Newcastle Disease (END) in 2002-2003, the USDA and States of California, Nevada, Arizona, and Texas and the California River Indian Tribes worked closely together. They operated in Task Forces under the Incident Command System (ICS) to stop its spread and to eradicate it from the U.S. Besides this level of cooperation, the END response required close collaboration from avian industry groups, county and municipal governments, and local communities.

An END After-Action Review was carried out by a team from USDA/APHIS and the State of California. During that review, over 75 interviews were conducted with Federal, State, Tribal officials as well as representatives from industry and local governments. In addition, ethnographic research was conducted in four affected communities of Southern California.

This paper describes critical factors that determined the patterns of relationship between these agencies and groups. Some of these factors reflect differences in the groups themselves, such as formal status, public visibility, socioeconomic condition, and linkage to the agricultural sector. Other factors, however, reflect the manner in which relationships were formed and evolved after the response to the outbreak began. Two factors were of particular importance in establishing cooperative relations. One is the emergence of “cultural brokers” who maintained duel legitimacy with the ICS Task Force and with their own organization or group. A second is the level of personal or organizational consistency that the ICS Task Force provided to that “cultural broker” and his/her group.

The study of relationships during END also posits the need for a systematic approach to involving non- agriculturally based groups in animal health emergency management. It suggests the concept of “social biosecurity” as framework for establishing those relationships in both preparedness and response actions.

1 US Department of Agriculture, Animal and Plant Health Inspection Service, Policy and Program Development, Fort Collins, CO 2 California Department of Food and Agriculture, retired. 3 University of California, Davis, Veterinary Medicine Extension

110

Molecular Diagnostics Scientific Session Sunday October 24, 2004 8:00 a.m. – 12:00 p.m. Auditorium III

Moderators: Sharon Hietala and Steven Kleiboeker

Page 8:00 a.m. Simultaneous Detection of North American and European Porcine Reproductive and 113 Respiratory Syndrome Virus Using Real-time Quantitative RT-PCR - S.B. Kleiboeker, S.K. Schommer, S-M Lee, S. Watkins, W. Chittick, and D. Polson

8:15 a.m. Quantification of Porcine Reproductive and Respiratory Syndrome Virus in Boar Serum and 114 Semen - J. Christopher-Hennings, J. D. Callahan, Y. Fang, A. Wasilk, M. Dammen , T.A. Gay, M.E. Reos, E.A. Nelson, and W.M. Nelson

8:45 a.m. High Throughput Viral RNA Isolation for Molecular Diagnosis - X. Fang, R. C. Willis, W. 116 Xu, Q. Hoang and M. Bounpheng

9:00 a.m. Fast and Simple Viral RNA Isolation for BVDV Molecular Diagnostics - Q. Hoang, R.C. 117 Willis, W. Xu, M.A. Bounpheng, and X. Fang

9:15 a.m. High Throughput Detection of Bluetongue Virus by a Taqman Real-time RT-PCR - M. A. 118 Jimenez-Clavero, E. San Miguel, M. J. Ruano, M. C. López, E. R. Gómez-Tejedor, C. Gómez- Tejedor

9:30 a.m. Detection of Foot-and-Mouth Disease in Samples from Afghanistan by a Portable Real-Time 119 RT-PCR Assay - K.R. Schumann, T.R. Beckham, L. Jackson, D. Couch, A.J. Eberling, A.C. Giuffre, S.J. Pauszek, J.M. Robida, M.L. Berninger, T.M. Sigafoose, M.A. Kenney, L. Rodriquez, T.S. McKenna, B.M. Martin.

9:45 a.m. BREAK AND POSTER SESSION

10:15 a.m. Phylogeny-Based Multiplex Real-Time RT-PCR for Rapid Detection of Vesicular Stomatitis 120 Viruses NJ and IN-1 from Diverse Geographical Regions - Luis L. Rodriguez, Steve J. Pauszek, George Smoliga and William C. Wilson

111

11:00 a.m. Comparison of Six RNA Extraction Methods for the Detection of Classical Swine Fever Virus by Real-time and Conventional Reverse Transcription-Polymerase Chain Reaction - M.Y. 123Deng, H. Wang, G.B. Ward, T.R. Beckham, and T.S. McKenna 123 11:15 a.m. Development of an Optimized Multiplex PCR to Detect Virulence Genes in Enterotoxigenic 124 Escherichia coli (ETEC) - L.L. Ruesch and D.H. Francis

11:30 a.m. Quantitative Real-Time PCR for the Detection of Mycobacterium avium subsp. 125 paratuberculosis in Bovine Fecal Samples - R.W. Sweeney, B.L. Mangold, S. McAdams, T. Calvin, R.H. Whitlock

11:45 a.m. Identification of Escherichia coli Flagellar Types by Restriction Fragment Analysis of 126 Amplified fliC Gene - C. DebRoy, E. Roberts and M. A. Davis

* Graduate Student Competition

Molecular Diagnostic Session

Sponsored by

CEPHEID

112

Simultaneous Detection of North American and European Porcine Reproductive and Respiratory Syndrome Virus Using Real-time Quantitative RT-PCR

S.B. Kleiboeker1, S.K. Schommer1, S-M Lee1, S. Watkins2, W. Chittick2, and D. Polson2

Porcine reproductive and respiratory syndrome (PRRS) is a one of the most economically important diseases of swine. Detection of the etiologic agent, PRRS virus (PRRSV), represents a diagnostic challenge due to relatively low levels of viremia in infected swine, the heterogeneity of field isolates and the propensity for swine to develop persistent infection in which virus is difficult to detect. Recently European (EU) lineage PRRSV isolates, which are genetically divergent from North American (NA) isolates, have been introduced into NA swine further complicating efforts to diagnose this important disease. In this study, real-time (TaqMan) RT-PCR assays were developed for multiplex detection, differentiation and quantification of NA and EU PRRSV field isolates. Oligonucleotide primers and dual- labeled probes were selected from conserved regions of open reading frame 7 and the 3′ untranslated region. For each oligonucleotide primer and dual-labeled probe, a maximum of a single base mismatch in any individual PRRSV sequence was identified by nucleotide alignment of all available sequences in GenBank. None of these mismatches were within 3 bases of the 3′ end of the oligonucleotide primers. The real-time RT-PCR assays described for the NA or EU genotype of PRRSV detected viral RNA from 74 of 74 NA strains tested and 9 of 9 EU strains tested. These strains were isolated by cell culture between 1992 and 2003. North American PRRSV isolates tested included the prototype strain VR-2332, as well as its attenuated vaccine derivative IngelVac MLV, and the IngelVac ATP vaccine strain. European PRRSV strains tested included the prototype Lelystad isolate plus eight U.S. field isolates of EU PRRSV.

To assess the sensitivity of the qRT-PCR assays, serial ten-fold dilutions of virus stocks of NA and EU PRRSV were analyzed in parallel by virus isolation, qRT-PCR and nested RT-PCR (nRT-PCR), with nested RT-PCR only performed for NA PRRSV isolates. Virus isolation detected virus at concentrations of 1 and 10 TCID50/0.1 ml for the NA and EU PRRSV viral stocks, respectively. For both strains of PRRSV, qRT-PCR reproducibly detected viral RNA at 100-fold lower concentrations than virus isolation. Nested RT-PCR for NA PRRSV detected viral RNA to the same level as qRT-PCR. To assess assay specificity, RNA or DNA purified from lung and lymphoid tissue of a PRRSV-seronegative pig were amplified in the multiplex qRT-PCR assay and found to be negative. Additionally, RNA and DNA purified from cultured cells and common viral and bacterial pathogens of swine were found to be negative for both assays. When performing multiplex reactions, sensitive detection of both NA and EU stains was possible even when one viral RNA concentration was up to 5,000-fold higher than the second.

In summary, the diagnostic sensitivity and specificity of the multiplex reaction was found to be at a minimum equivalent to that of both nested RT-PCR and virus isolation, and was not significantly reduced for the reactions performed in a multiplex format as compared to results from the single assays. Assays performed in a real-time RT-PCR format have the advantage of higher sample throughput, more rapid turnaround and a lower incidence of false positive due to cross-contamination. It is envisioned that this multiplex assay will allow sensitive, robust detection of diverse NA and EU PRRSV field strains for both experimental and diagnostic applications.

1Veterinary Medical Diagnostic Laboratory, University of Missouri, Columbia, MO 2Boehringer Ingelheim Vetmedica, Inc., Ames, IA

113

Quantification of Porcine Reproductive and Respiratory Syndrome Virus in Boar Serum and Semen

J. Christopher-Hennings1, J. D. Callahan2, Y. Fang1, A. Wasilk1, M. Dammen1 , T.A. Gay2, M.E. Reos2, E.A. Nelson1, Wm. M. Nelson2

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) can be transmitted through boar semen and due to the significant economic and health consequences of this disease, it is important to prevent transmission through this route. The polymerase chain reaction (PCR) has been routinely used for detection of PRRSV in semen, serum and tissues of adult boars. Methods such as real-time PCR have expedited the detection and also allowed for rapid quantitation of virus in these samples. Viral detection and quantitation are useful for pathogenesis, vaccine and treatment studies and may help in answering questions of whether a higher or lower viral load might correlate with virulence, persistence, vaccine efficacy, transmission and identification of reservoir sites.

For this study, a commercially available single-tube, real-time RT-PCR (Tetracore, Inc., Gaithersburg, MD) was used for detecting and quantitating PRRSV in serum and semen of 6 experimentally inoculated boars. Three uninoculated control boars were also used. Serum and semen were collected on the same days, 3 times per week for the first month after inoculation, 2 times per week for the 2nd month, and 1 time per week for the 3rd month, through 96 days post inoculation (DPI). The sensitivity of the real-time RT-PCR was determined using serial cell culture dilutions for the U.S. (SD-23983) isolate. The real-time PCR level of detection matched that of a nested PCR assay, which has been previously reported to detect 10 TCID50/ml and had been compared to the swine bioassay. The minimum level of in-vitro transcript consistently detected by the RT-PCR reaction was 33 copies/ml. This assay did not react with other common swine viruses and inter and intra-assay variability of 2.7-8.8% and 0.1-5.7%, respectively was observed.

For quantitation, a102-bp PCR fragment from the 3’UTR region of the U.S. SD-23983 isolate was cloned into a pGEM®-T Easy Vector System (Promega, Madison, WI) and in- vitro transcribed. The concentration of the transcribed RNA was determined by spectrophotometry. Known amounts of serially diluted in-vitro transcript RNA product (1 x 10 -1 through 1 x 108 copies/µl) were used to generate a standard curve and copy/ml concentrations of the unknown samples were determined by linear extrapolation of the Ct values plotted against the known concentration of the transcript product.

The presence and concentration of PRRSV in serum did not consistently correlate with the presence and concentration of virus in the semen. Viral RNA in serum was consistently detected from 1 through 12 DPI in all PRRSV inoculated boars. For 5 of 6 boars, PRRSV was identified in semen for 0 days (1 boar), 1 day only (1 boar, at 3 DPI), 2 days (2 boars at 8 and 10 DPI) or 4 days total (1 boar at 5, 10, 17 and 19 DPI). The viral load in serum was generally higher than in semen, or PRRSV RNA was not detected in semen even though the boar was viremic. In contrast, in 1 of 6 of the inoculated boars, PRRSV was identified in serum from 1 through 32 DPI and in semen from 5 through 32 DPI. Viral loads in serum from this boar were consistently lower than in the semen. Therefore, this study demonstrated that “compartmentalization” of the virus can occur and the presence and concentration of PRRSV in serum may not necessarily predict or reflect the presence and concentration of virus in semen.

1 Department of Veterinary Science, South Dakota State University, Brookings, SD 2 Tetracore, Inc., 11 Firstfield Road, Gaithersburg, MD

114

Application of Molecular Epidemiological Data to Support Forensic Disease Outbreak Investigations: Evaluation Using the 2002-2003 Exotic Newcastle Disease Outbreak

BM Crossley1, PJ Hullinger2, SL Messenger3, E Skowronski3, Kimothy Smith3, SK Hietala1

Molecular and genetic-based diagnostic tools, including gene sequencing and phylogenetic analysis, are widely used to identify and characterize individual viral and bacterial isolates associated with disease outbreaks, as well as to provide epidemiologic links to the source of those outbreaks. Higher resolution genomic evaluation, including whole genome sequencing and statistical analyses to identify informative nucleic acid and amino acid mutations can also provide a molecular framework for forensic investigation. As part of a diagnostic and epidemiologic investigation, molecular characterization combined with specific temporal and geospatial tracking of individual isolates over the course of an outbreak enables forensic analyses. In-depth forensic investigation supplements the efforts of molecular epidemiology and can potentially be used to identify agent transmission patterns, risk factors, and pathogen evolution, ultimately contributing to enhanced prevention, detection, and disease control.

Whole genome sequences from 12 exotic Newcastle disease virus (ENDV) isolates, and fusion protein sequences from 75 isolates including ENDV, avian paramyxovirus-1, and pigeon paramyxovirus-1 recovered during the 2002-2003 END outbreak were evaluated using phylogenetic analyses. Percent synonymous and non-synonymous mutations, amino acid changes, and predicted antigenicity were evaluated at the individual gene level and over the entire 15224bp genome of the isolates. Whole genome sequences were obtained using a modified shotgun approach and random hexamer amplification to generate sequencing template with approximately 8-fold coverage. A 0.03% sequencing error, based on replicate sequencing of the outbreak index case, was used as a correction factor prior to sequence analysis. An overall 2.4% genomic mutation rate was detected compared to the GenBank reference isolate ZJ1 (AF431744). The greatest phylogenetic divergence over the 8-month course of the outbreak, based on ENDV amino acid sequences evaluated, was found between the index ENDV game bird isolate and a backyard non-commercial chicken isolate obtained 64 days later. Evolutionary distance and mutation differences were additionally documented between host species and/or operation type (pigeons, game birds, backyard chicken, commercial chickens, mixed species premises), clinical presentation, and temporal occurrence (example: first to final commercial premises detected). Geospatial relationships of the isolates using ARCGIS software demonstrated less than 0.1% amino acid change between game bird isolates obtained from Nevada, Arizona, and California during the outbreak. To provide a more cost and time efficient approach to outbreak investigation, additional studies, using the entire ENDV genome with gene chip technology, are currently underway.

Preliminary evaluation of these molecular analyses applied to a veterinary disease outbreak, based on 2002-2003 END isolates, indicates that whole genome and targeted gene sequencing can provide useful documentation and tracking of agent transmission patterns during an outbreak, and serves as a model for the application of forensics to animal disease investigations. The information gained has application in identifying mechanisms for agent introduction and spread, which are critical to focusing control efforts and further understanding of outbreak epidemiology.

1 California Animal Health and Food Safety Laboratory. UC Davis. CA. 95616 2 California Department of Food and Agriculture. Sacramento, CA 95814 3 Lawrence Livermore National Laboratory. Livermore, CA 94550

115

High Throughput Viral RNA Isolation for Molecular Diagnosis

X. Fang, R. C. Willis, W. Xu, Q. Hoang and M Bounpheng

There are many challenges to develop a versatile high throughput method of RNA isolation from various specimens for molecular diagnosis of viral. For example, proteins and lipids rich in plasma, serum and milk can interfere RNA binding and results in very inefficient viral RNA recovery, thus many viral RNA isolation methods have incorporated a proteinase K digestion step, during which RNA will be partially degraded. On the other hand, getting rid of RT-PCR inhibitors is crucial for RNA isolation from cloacal swab samples.

Traditionally, RNA is isolated by phenol/chloroform extraction, which is not amendable to high throughput processes because of the difficulty of extraction with a multi-channel pipette and the high risk of cross contamination. Solid phase binding methods have thus been developed to replace phenol/chloroform extraction for high throughput RNA isolation. Glass fiber filters and beads are among the best choices for the binding matrix. Our bead technology has been successfully used for high throughput Exotic Newcastle disease viral RNA isolation during the 2002/2003 END outbreak in California, which yielded 99.7% sensitivity and 100% specificity based on 1360 virus isolation confirmed samples.

We have further developed this technology into a standard high throughput viral RNA isolation kit. The protocol is thoroughly examined to ensure highest sensitivity and easy to use. We have successful cut down the process while increasing the sensitivity. And the method can be used for many types of specimens, such as oral swab, nasal swab, colacal swab, milk, plasma, serum, and whole blood. The optimized procedure takes about 40 min to process 96 samples in a microtiter plate. Every step is in process is under room temperature, and no special instrument is required. It can be manually performed with multi-channel pipettors, as well as automated on a robotic liquid handler.

Armored RNA and an RNA transcript were used to monitor RNA isolation efficiency. As quantified by real-time RT-PCR, RNA recovery was more than 50% from all type of specimens tested. When the same input was in a whole 96-well plate, the standard deviation of RNA recovery was less than 15% by A260, and the standard deviation of Ct values from real-time RT-PCR is less than 3%. Combining with a well- optimized real-time RT-PCR assay, we can detect as low as 10 copies RNA from original samples. We successfully isolated and detected END virus from as little as 50uL negative swab media where 1 pfu/mL END virus was spiked. This technology has been widely tested for many viral RNA isolation from various specimens, such as BVDV viral RNA from serum and plasma samples; FMDV viral RNA from oral swab, milk and epithelium; CSF viral RNA from nasal swab; and avian leukosis viral RNA from plasma. All the tests done showed that our high throughput method has equivalent or better performance than the gold standard Trizol method.

In conclusion, the magnetic bead-based viral RNA isolation method is very robust, easy to use, and cost- effective. It can be widely used for high throughput viral RNA isolation for molecular diagnosis and surveillance from various specimens.

Ambion Inc., 2130 Woodward St, Austin, TX 78744.

116

Fast and Simple Viral RNA Isolation for BVDV Molecular Diagnostics

Q. Hoang1, R.C. Willis1, W. Xu1, M.A. Bounpheng1, and X. Fang1

Bovine viral diarrhea virus (BVDV) is a small (12.3 kb single-stranded positive RNA) enveloped virus in genus Pestivirus of the Flaviviridae family. BVDV infection is a major problem of worldwide distribution that results in economic losses for the beef and dairy industries. Real-time RT-PCR has become a powerful tool for BVDV diagnosis and surveillance. In the case of an outbreak, numerous samples must be processed within a short time thus requiring the use of automation for high throughput RNA extraction and RT-PCR. Thus the successful high throughput isolation of viral RNA from various biological media and matrixes is an essential component of BVDV infection control.

Milk and blood are ideal sample sources for BVDV RNA isolation due to the simplicity and ease of sample collection. Current methods of BVDV RNA isolation from milk and blood require preprocessing of samples prior to viral RNA isolation. For example, bulk milk must first be cleared of the cream layer by centrifugation while whole blood must be allowed to clot for serum collection before RNA isolation with phenol/chloroform extraction based methods such as TRIzol. The additional processing steps are cumbersome, time consuming and introduce the risk of sample cross contamination due to more hands-on steps. More importantly the preprocessing steps and phenol/chloroform extraction are not amendable for automation and difficult to scale up for high throughput BVDV molecular diagnosis.

We have developed a magnetic bead-based nucleic acid isolation method that is effective for viral RNA isolation from oral and nasal swabs, plasma, serum, whole blood, and milk samples. This method eliminates the clogging problem commonly associated with filter-based methods. In addition, the use of billions of micro spherical beads and agitation can efficiently and effectively capture viral RNA molecules in extremely low concentration samples thus yielding diagnostic assays with high sensitivity and reproducibility. We describe below the successful BVDV viral RNA isolation from whole blood and milk samples, which are easy to collect but difficult to process for RNA isolation.

Our method does not require any preprocessing of milk or blood. The whole milk or blood samples containing viral particles are directly lysed in our proprietary lysis solution, RNA is then captured using paramagnetic beads and subsequently eluted. The isolated RNA is of very high quality and ready for real- time RT-PCR. No inhibition of RT and/or PCR were observed using a volume of eluted RNA up to 60% of the final RT-PCR volume. This method can process 96 samples in less than one hour using a multi- channel pipettor.

A ribonuclease resistant Armored RNA (RNA packaged in MS2 protein) and a 1036 nucleotides artificial mRNA transcript were used to monitor virus lysing and RNA recovery respectively. As determined by quantitative RT-PCR, the overall RNA recovery was >60%, and the detection limit was less than 100 copies of RNA in the processed sample. A BVDV positive serum sample was also spiked into BVDV negative milk and blood samples at 10X serial dilutions, and RNA was isolated using our bead-based method and compared to the gold standard TRIzol method. As quantified by real-time RT-PCR and endpoint gel analysis, our method yielded similar or improved recovery of BVDV viral RNA as evident by the same or earlier Ct values. In conclusion, we have developed a magnetic bead-based high throughput viral RNA isolation method that can be used for BVDV molecular diagnosis. This method clearly offers equivalent sensitivity as the TRIzol method. It is fast and simple, and well suited for automation. This method is also applicable for high throughput RNA isolation of other viruses.

1Ambion, Inc., Austin, TX

117

High Throughput Detection of Bluetongue Virus by a Taqman Real-time RT-PCR

M. A. Jimenez-Clavero1, E. San Miguel1, M. J. Ruano1, M. C. López1, E. R. Gómez-Tejedor1, C. Gómez-Tejedor1

Bluetongue virus (BTV) is an arbovirus within the Orbivirus genus (Reoviridae family) that causes a disease in sheep of economical importance for the international trade of animals and animal products. BTV genome consists of 10 double-stranded RNA segments that encode seven structural (VP1 to VP7) and four non-structural (NS1, NS2, NS3 and NS3A) proteins. There is a marked genetic variation of viruses within the BTV serogroup, with 24 known serotypes. Four of them (2, 4, 9 and 16) have been identified as causing outbreaks in the western mediterranean islands in recent times. Studies on Culicoides vector distribution and abundance, as well as climatic and remote sensing data predict that this area is at high risk of appearance of BTV epizootics in the next years. We have developed a real time-RT-PCR for the detection of BTV that uses a 5’-Taq nuclease-3' minor groove binder DNA probe (TaqMan MGB) and a primer set targeted at a highly conserved region within BTV RNA segment 6. The assay developed has been compared to conventional and nested RT-PCR assays currently in use in BTV diagnosis. The sensitivity of the assay is comparable to that obtained by a highly sensitive nested-RT-PCR assay (0.001 TCID50). As regards to the specificity, the new method can detect all BTV serotypes tested (2, 4, 9, 12 and 16), which include those currently circulating in the Mediterranean area, whereas negative results were obtained when a panel of both related orbiviruses (african horsesickness virus, epizootic hemorrhagic disease virus serotypes 1-9) as well as nonrelated viruses (foot-and-mouth disease virus, ecthima contagiosum virus, bovine viral diarrhea virus, pseudorrabies virus, infectious bovine rhinotracheitis virus, influenza A virus) was analyzed. The method can be performed in 96-well format, which, combined with a nucleic acid extraction protocol that uses 96- well format as well, make possible to process 180 samples in approximately 6 h. This method has become an useful tool in the epidemiological tracking of the recent BTV outbreak occurred in Menorca in 2003, caused by a virus that has been shown to belong to serotype 4 by molecular as well as serological methods. The assay described here is fast, simple, and, performed in 96-well format, is suitable for large- scale epidemiological screening of bluetongue virus in animal populations at risk.

1Emerging Diseases Department, Central Veterinary Laboratory, Algete, (Madrid), Spain

118 Detection of Foot-and-Mouth Disease in Samples from Afghanistan by a Portable Real-Time RT-PCR Assay

K.R. Schumann1, T.R. Beckham1 L. Jackson2, D. Couch2, A. J. Eberling1, A.C Giuffre1, S.J. Pauszek3, J.M. Robida1, M.L. Berninger1, T.M. Sigafoose4, M.A. Kenney1, L. Rodriquez3, T.S. McKenna1, B.M. Martin4 .

Foot-and-mouth disease (FMD) (family Picornaviridae, genus Aphthovirus) is an extremely contagious and economically devastating disease of both domestic and wild cloven-hoofed animals. It is endemic in parts of Asia, Africa, the Middle East, and South America, and is the cause of outbreaks similar to that observed in the United Kingdom in 2001. Rapid and accurate diagnosis of this disease is critical to prevent spread of the virus as well as implement appropriate control measures. Current diagnosis is performed by enzyme-linked immunosorbent assay (ELISA), complement fixation, virus isolation, conventional reverse-transcriptase polymerase chain reaction (RT-PCR), and most recently by real-time RT-PCR methods (rRT-PCR).

In an effort to evaluate a FMDV rRT-PCR assay, field samples were obtained from Afghanistan through a collaboration between the Islamic Transitional Government of Afghanistan, the USDA, and the DOD Coalition Joint Civil Military Operation Task Force (CJCMOTF).

Bovine, ovine, and caprine oral swabs as well as bovine epithelium were collected by members of the CJCMOTF and sent to the Foreign Animal Disease Diagnostic Laboratory for diagnosis and characterization. A portable rRT-PCR assay was used to detect the presence of FMD RNA in each of the samples. The rRT-PCR method was compared to virus isolation and the serotype of each positive sample was further investigated. RNA was extracted from swabs or 30 mg of epithelium according to the manufacturer’s protocol using the RNeasy Mini-KitTM (Qiagen, Valencia, CA). rRT-PCR assays were performed on the Cepheid Smart CyclerTM platform using 2.5 µl of extracted RNA. For virus isolation, the epithelial samples were prepared as 10% homogenates. The oral swabs and epithelial homogenates were filtered through 0.45 micron filters and inoculated onto IBRS-2 cells.

A total of 98 oral swab samples and 12 epithelium samples were tested by rRT-PCR. Twenty (20.2%) of the oral swab samples and nine (75%) of the epithelium samples tested positive for the presence of FMD RNA. A total of four of the samples showed cytopathic effect (CPE) after two passages in IBSRS-2 cells.

Samples positive by virus isolation were serotyped by antigen ELISA. All rRT-PCR positive samples were serotyped by VP1 sequence analysis. ELISA results indicated that 2 of the samples were serotype Asia 1 while two were serotype A. Sequencing and phylogenetic analysis indicated that the FMDV isolates in Afghanistan are closely related to viruses in India.

This work provides valuable information on FMDV serotypes and subtypes circulating in Afghanistan and will aid in choosing of an appropriate vaccine formulation for control programs.

1 United States Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services, National Veterinary Services Laboratories, Foreign Animal Disease Diagnostic Laboratory, Greenport, NY. 2 Department of Defense, Coalition Joint Civil Military Operation Task Force, Combined/Joint Task force-180, Operation Enduring Freedom, Bagram, Afghanistan, APO AE 3 United States Department of Agriculture, Agrucultural Research Service, Plum Island Animal Disease Center, Greenport, NY. 4 United States Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services, National Veterinary Services Laboratories, Diagnostic Bacteriology Laboratory, Ames, IA.

119 Phylogeny-Based Multiplex Real-Time RT-PCR for Rapid Detection of Vesicular Stomatitis Viruses NJ and IN-1 from Diverse Geographical Regions

Luis L. Rodriguez1 Steve J. Pauszek1, George Smoliga1 and William C. Wilson2

Vesicular Stomatitis Virus (VSV) causes clinical disease identical to foot-and-mouth disease (FMD), one of the most devastating diseases of livestock. Vesicular stomatitis (VS) is endemic from Southern Mexico to Northern South America and outbreaks occur sporadically in the United States. It is imperative to rapidly and accurately diagnose VSV in order to avoid costly quarantines and trade restrictions when a case occurs in FMD-free areas. Its great genetic variability and the lack of available genetic sequence have made it difficult to design rapid genetic-based tests capable of detecting viruses from all geographical areas where VS occurs. Nucleotide divergence ranges from 15-20% between serotypes and 8-10% among geographically distant viruses within the same serotype. Third-base mutations are prevalent throughout the genome and long stretches (50 nucleotides or more) of conserved sequence across viruses from various geographical regions are absent. Here we describe a real-time multiplex RT-PCR designed based on the phylogenetic relationship of VSV sequences representing the geographical range of VSNJV and VSIN-1. Full-length sequence of selected viruses identified the nucleocapsid gene (N) as the most conserved gene in the VSV genome. Since N is also the most abundant transcript in VSV infected cells it makes an ideal target for genetic detection tests. Primers and probes were designed to detect conserved target areas in N using 6 VSNJV and 4 VSINV-1, each representing a major phylogenetic lineage comprising the genetic diversity of VSV in North America, northern Central America, southern Central American and South America. Due to lack of conserved areas between serotypes it was necessary to design separate primers and probes for VSNJV and VSINV-1, which were multiplexed and lyophilized into a single-tube test. The multiplex test consists of a mixture of 13 primers and specific probes for VSNJV and VSINV-1 labeled with TET and FAM fluorochromes respectively.

This test was shown specific for VSV and did not detect other vesiculoviruses or viruses causing look- alike diseases such as all FMDV serotypes, swine vesicular disease, vesicular exanthema, bovine viral diarrhea, and bovine herpesviruses among others. When compared to virus isolation in Vero cells, the recognized gold standard test for VS, level of detection ranged from 0.1-2 Log10 TCID50 depending on the viral genetic lineage.

Additional testing of viral samples originating from the various geographical areas representing each major VSV genetic lineage showed that the test detected all strains from the North American and northern-Central American clades, and over 90% of viruses from the southern-Central American and South American clades. Further analyses of non-detectable strains is ongoing. This work demonstrated the utility of phylogenetic analysis in the design of tests for genetic detection of highly variable viral agents.

1Agricultural Research Service, U.S.D.A., Plum Island Animal Disease Center, Orient NY 11957, Tel: 631 323 3364, Fax: 631 323 3006 2Agricultural Research Service, U.S.D.A, Arthropod-Borne Disease Research Laboratory, Laramie, Wyoming.

120 Validation of a Real-Time RT-PCR for Vesicular Stomatitis Virus

G.J. Letchworth1, C. Jiménez2, M. Herrero2, T.E. Cornish3, W.C. Wilson1, G. Smoliga4 S. Pauszek4, C. Dornak, 3 M. George5, L.L. Rodriguez 4

Sporadic outbreaks of vesicular stomatitis (VS) in the United States result in significant economic losses for the US livestock industries because VS clinically mimics foot-and-mouth disease. Control strategies for the two diseases differ radically and thus mandate rapid and accurate differentiation. The objective of this study was to field validate a one-tube multiplexed real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay for the rapid detection of VS-New Jersey virus (VSNJV) and VS-Indiana virus (VSINV) strains occurring from North America to northern South America. This test has undergone bench validation at the Plum Island Animal Disease Center as is described in a separate report.

Vesicular lesion samples obtained from cattle, horses, or swine from throughout Central America were tested at the Laboratorio de Diagnóstico de Enfermedades Vesiculares (LADIVES) in Panama; and samples from Costa Rica were tested at the School of Veterinary Medicine in Heredia Costa Rica. A portion of each sample was prepared for virus isolation in Vero cells and VSNJV and VSINV specific antibodies were used to confirm the presence of each virus. A second portion of each lesion sample was prepared for RT-PCR by RNA extraction using a commercial extraction kit and tested by rRT-PCR. Of approximately 400 samples tested, half yielded a cytopathogenic agent, identified in most cases as VSNJV and occasionally as VSINV by reaction to serotype specific antibodies, reaction with serotype- specific primers and probes, and genomic sequence. Most samples (90%) from which VSV was isolated also reacted in the rRT-PCR but a few specific strains originating from a specific region of Costa Rica were not detected by rRT-PCR. The molecular basis of this lack of detection is being investigated. VSV was not isolated from many RT-PCR positive samples, these samples were confirmed as positive by sequence analysis of N and other viral genes.

We conclude that although rRT-PCR detected the largest number of positives from field samples, there were few specific strains that were not recognized by the current primer/probe combination. Additional primers and probes might be required to identify all of the Central American VSV’s. An important secondary result of this research was the collection of hundreds of new VSV isolates. This benchmark collection constitutes a foundation from which many additional studies can arise.

1 Arthropod-Borne Disease Research Laboratory, USDA, Agricultural Research Service, Laramie, Wyoming. 2 Escuela de Medicina Veterinaria, Universidad Nacional, Heredia, Costa Rica. 3 Department of Veterinary Science, University of Wyoming, Laramie, WY. 4 Plum Island Animal Disease Center, USDA, Agricultural Research Service, Greenport, NY. 5 Laboratorio de Diagnóstico de Enfermedades Vesiculares, Panama City, Panama.

121 Diagnostic Evaluation of a Portable Real-time Reverse Tanscriptase PCR Assay for the Detection of Classical Swine Fever

G. Risatti1, L. G. Holinka1, Z. Lu1, G. Kutish1, J. D. Callahan2, W. M. Nelson1, E. Brea Tió3 and M. V. Borca1

Classical Swine Fever (CSF) is a highly contagious and often fatal disease of swine. Classical Swine Fever Virus (CSFV), the causative agent of CSF, is a member of the genus Pestivirus in the family Flaviviridae. Although not present in the United States, CSF is distributed worldwide and is considered a foreign animal disease (FAD) threat to US swine populations. A rapid, presumptive diagnosis at the site of a suspected disease outbreak would be extremely useful for controlling CSF.

A real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay for classical swine fever virus (CSFV) was developed and evaluated for diagnostic sensitivity and specificity using clinical samples obtained from the Dominican Republic where the disease is enzootic. A total of 449 nasal swab samples taken from both symptomatic and asymptomatic pigs from 9 different farms and were evaluated by the real-time test in parallel with the gold standard method of virus isolation (VI).

Of the 69 samples that were real-time RT-PCR positive, only 50/69 were also VI positive while none were VI positive/ real-time RT-PCR negative. The 19 discordant samples were further tested by a nested RT-PCR that targeted a different genomic area and the nested PCR products were sequenced. Phylogenetic analysis of the sequenced products showed a geographic distribution associated with 4 of 9 farms, highly suggesting that the discordant samples were true positives. There were 4/384 samples that were real-time PCR positive and nested PCR/VI negative and were presumed to be false positives, however these all had Ct values >38 cycles.

The real-time assay exceeded the diagnostic sensitivity of virus isolation (100% vs 72.4%, respectively) with little loss of specificity (98.9% vs 100%, respectively). At the herd level, 3 out of 4 infected farms were detected by virus isolation, while the CSF real time RT-PCR assay identified all four infected premises. This simple and accurate test permits rapid detection of CSFV in affected herds.

1Plum Island Animal Disease Center, Agricultural Research Service, United States Department of Agriculture (USDA), Greenport, New York 11944, United States. 2Tetracore Inc. Gaithersburg, Maryland 20878, United States. 3 Laboratorio Veterinario Central “LAVECEN”, Ave. Monumental Los Girasoles, Santo Domingo, República Dominicana.

122 Comparison of Six RNA Extraction Methods for the Detection of Classical Swine Fever Virus by Real-time and Conventional Reverse Transcription-Polymerase Chain Reaction

M.Y. Deng1, H. Wang1, G.B. Ward1, T.R. Beckham1, and T.S. McKenna1

Reverse transcription-polymerase chain reaction (RT-PCR) is a valuable technique that is increasingly being used for diagnosis of animal diseases caused by RNA viruses including classical swine fever virus (CSFV). RT-PCR required an efficient and simple method for sample preparation. This study compared the efficiency of six RNA extraction methods, and their usefulness for sample preparation for the detection of the CSFV genome by real-time and conventional RT-PCR in swine blood and tissue samples.

All methods used in this study were for recovery and purification of total RNA. The methods were based on the RNAqueous® Kit (Ambion, Austin, TX), Micro-to-Midi Total RNA Purification System (Invitrogen, Carlsbad, CA), NucleoSpin® RNA II (BD Biosciences, Palo Alto, CA), GenElute™ Mammalian Total RNA Kit (Sigma, St. Louis, MO), RNeasy Mini Kit (Qiagen, Germantown, MD), and TRIzol® LS Reagent (Invitrogen). Blood, tonsil, liver, kidney, spleen, bladder, heart, and submandibular lymph node were collected from a CSFV-infected pig. A volume of 200 µl of blood or tissue homogenate was extracted in duplicate by each method. RNA solutions from the duplicated samples were then pooled and the combined RNA obtained by each method had the same total volume of 100 µl. The concentration and the A260/A280 ratio of each RNA solution were determined spectrophotometrically. All RNA solutions and the negative control for each method were subjected to real-time RT-PCR for CSFV using the Tetracore® Dried RT-PCR Detection Assay (Tetracore, Gaithersburg, MD) and to conventional RT-PCR for both CSFV and an internal control gene ACTB coding for β-actin.

The mean concentration of RNA of all samples extracted by the RNAqueous kit, the Micro-to-Midi, the NucleoSpin, the GenElute, the RNeasy, and TRIzol was 306, 224, 138, 175, 102 and 682 ng/µl, respectively, and the mean A260/A280 ratio of these RNA was 2.10, 1.69, 1.68, 1.69, 1.55, and 1.59, respectively. These RNA were all positive for CSFV in the real-time RT-PCR, and all samples except that extracted from blood by TRIzol were positive for CSFV and ACTB in the conventional RT-PCR. The negative control for each extraction method was negative in all the RT-PCR. The RNA extracted from blood by TRIzol became positive for CSFV- and β-actin in the conventional RT-PCR after a simple 1:2 or 1:4 dilution in water. The endpoint of detection (EOD) of the tonsil RNA extracted by each method was determined. Each RNA was diluted in water 1:10 and then two-fold diluted further to 1:20, 1:40, and up to 1:81920. RNA dilutions were tested by real-time and conventional RT-PCR. The highest dilution showing a positive result in a RT-PCR was regarded as the EOD of the PCR. The EOD in the real-time RT-PCR for CSFV on the tonsil RNA extracted by the RNAqueous kit, the Micro-to-Midi, the NucleoSpin, the GenElute, the RNeasy, and TRIzol was 1:5,120, 1:20,480, 1:1,280, 1:640, 1:640, and 1:10,240, respectively. The EOD in the conventional RT-PCR for CSFV on the RNA by these methods was 1:10,240, 1:5,120, 1:10,240, 1:10,240, 1:2,560, and 1:40,960, respectively. The EOD in the RT-PCR for ACTB for these methods was 1:5,120, 1:2,560, 1:10,240, 1:2,560, 1:1,280, and 1:5,120, respectively.

We conclude that all six RNA extraction methods are efficient enough to detect the presence of virus in a clinical sample equally and useful for the detection of the CSFV by real-time and conventional RT-PCR in swine blood and tissue samples although there are differences in quality and yield of RNA and processing time and cost among these methods.

The authors would like to thank Brenda Donahue, Ming-Tsung Yeh, Jessica Dio and Jessica Montez for their assistance in various phases of this work.

1USDA, APHIS, VS, NVSL, Foreign Animal Disease Diagnostic Laboratory, Greenport, NY.

123 Development of an Optimized Multiplex PCR to Detect Virulence Genes in Enterotoxigenic Escherichia coli (ETEC)

L.L. Ruesch, D.H. Francis

A multiplex PCR procedure originally developed a number of years ago at the National Animal Disease Center in Ames, IA, has been widely used among veterinary diagnostic laboratories in the United States and elsewhere. It has been employed for about 5 years at the Animal Disease Research and Diagnostic Laboratory for the characterization of E. coli strains isolated from pigs with diarrhea. Recently, we sought to upgrade the test using currently available primer design software. The new test would potentially increase specificity by increasing the primer melting temperature, thus allowing a higher annealing temperature.

Nine PCR targets were identified: K88 (F4) pilus, K99 (F5) pilus, 987P (F6) pilus, F41 pilus, F18 (F107) pilus, heat labile toxin (LT), heat stable toxin a (STa), heat stable toxin b (STb), and Shiga toxin IIe (Stx2e). Primers of 24-26bp in length were selected within coding-regions of published sequence. A Multiplex Kit from Qiagen Inc. (Valencia, Inc.) containing DNA Polymerase, PCR Buffer and dNTPs was used as the PCR master mix. Optimization assays were performed using a gradient thermal cycler to determine optimal annealing temperature within the multiplex reaction.

One-hundred and fifty-eight E. coli isolates were typed using both old and new PCR tests. As a consequence of increasing the specificity of the primers, atypical genotypes frequently observed with the old test were virtually eliminated. Among the 158 cases used in the test study, the old multiplex PCR resulted in 23 genotypes, while the new multiplex PCR resulted in 14. The eliminated genotypes were atypical, most of which contained two fimbriae, such as: K88+, F18+. Virtually all non-specific PCR banding was eliminated, making gel interpretation less problematic.

Animal Disease Research and Diagnostic Laboratory, South Dakota State University, Brookings, SD 57007

124 Quantitative Real-Time PCR for the Detection of Mycobacterium avium subsp. paratuberculosis in Bovine Fecal Samples

R.W. Sweeney1, B.L. Mangold2, S. McAdams1, T. Calvin2, R.H. Whitlock1

Polymerase chain reaction (PCR) amplification of Mycobacterium avium subsp. paratuberculosis (MAP) DNA represents an attractive alternative to culture, as results may be obtained within hours since cultivation of this slow-growing organism is not required. Additionally, real-time PCR assays offer the capability to quantify the amount of target DNA in the fecal sample, providing an estimate of the severity of infection, and thus culling priority, for an individual infected animal. The purpose of the present study was to evaluate a novel fecal DNA extraction method, combined with a quantitative real-time PCR amplification method (VetAlertTM, Tetracore, Inc.) for the detection of MAP in bovine fecal samples from naturally infected cows.

Fecal samples from 16 cattle were split into 2 to 4 replicates each, for a total of 48 separate samples that were coded so as to blind the operators. The samples were cultured on Herrold's Egg Yolk Medium using a sensitive centrifugation/double incubation method. For quantitative real-time PCR (QRTPCR), the samples were processed with a DNA extraction and purification method which employed bead-beating and chaotropic solid phase extraction. Fecal DNA samples were then subjected to the QRTPCR amplification procedure, which is a real-time fluorescent probe hydrolysis assay. Duplicate sets of samples were tested in two different laboratories using the same procedures.

Of the 48 fecal samples, 4 were culture-negative, 27 were "light shedders" (<10 colonies per culture slant), 11 were "moderate shedders" (10 to 50 colonies per slant), and 6 were "heavy shedders" (> 50 colonies per slant). All 6 of the heavy shedders and all 11 of the moderate shedders were positive on QRTPCR. Of the 26 samples from light shedders, 11 were positive. Seven of the 15 QRTPCR-negative light shedders were samples with <1 colony per culture slant. All of the culture-negative samples were negative on QRTPCR. The number of PCR cycles to reach positive detection threshold, which is related to the amount of DNA in the test sample, was significantly correlated to the number of colonies obtained on culture. For the heavy and moderate shedders, there was 100% agreement between the QRTPCR results obtained in the two different laboratories.

The DNA extraction procedure and quantitative real-time PCR assay show good sensitivity and specificity for the detection and quantification of MAP in bovine fecal samples.

1Department of Clinical Studies-New Bolton Center, University of Pennsylvania School of Veterinary Medicine, Kennett Square, PA 2Tetracore, Inc., Gaithersburg, MD

125 Identification of Escherichia coli Flagellar Types by Restriction Fragment Analysis of Amplified fliC Gene

C. DebRoy1, E. Roberts1 and M. A. Davis1

Escherichia coli are differentiated on the basis of antigenic specificities of the lipopolysaccharide (O- antigen), and flagellar (H antigens) antigens that are present on the surface of the bacteria. The O and H antigens determine serotypes of the E. coli. There are 179 O types that have been classified and 56 H types. H serotyping involves growing the bacteria in special motile media for several days and if found to be motile, collect the freshly growing bacteria and observe agglutination reaction with antisera raised against 56 different standard H types. The serotyping procedure is time consuming and can take more than 7 days to perform. It is not always reliable as the flagellar proteins may not be expressed under certain conditions. Flagellar (H) antigens are mostly encoded by the fliC genes in E. coli. The differences between the fliC sequences in the different H types range between 0.06-3.12% (Wang, et al. 2000) reflecting high degrees of similarity. Amplifying the fliC gene encoding the H antigens by Polymerase Chain Reaction also cannot distinguish the different types. We have developed a method to identify 52 different H types by first amplifying the fliC gene by PCR and further analyzing the restriction fragment lengths by digesting the amplified fragment with restriction enzyme Hha1. The restriction enzyme fragment analysis of amplified fliC gene for different H types reflected that most of them exhibit unique fragment sizes. However, H7 and H23 exhibited similar patterns and H1 and H12 also showed the same pattern. H7 can be distinguished from rest of the H types by PCR (Nagano et al. 1998) and we have developed PCR procedure to distinguish H12 from H1. H4, H17 and H25 could not be amplified using the same primers, therefore we have developed PCR assays using specific primers for H25 and different primer set for H4 and H17. Since H4 and H17 fliC genes are highly homologous, the amplified fragments are distinguished by digesting the amplified DNA with HpaII. This method is being routinely used in our laboratory for detecting H types. We have performed hundreds of assays using the method.

Nagano I, Kunishima M, Itoh, Y, Wu Z and Takahashi Y. 1998. Detection of verotoxin producing Escherichia coli O157:H7 by multiplex Polymerase Chain Reaction. Microbiol. Immunol. 42: 371-376

Wang L, Rothemund D, Curd H and Reeves, PR. 2000. Sequence diversity of the Escherichia coli H7 fliC genes: implication for a DNA based typing scheme for E. coli O157:H7. J. Clin. Microbiol. 38: 1786- 1790

1Gastroenteric Disease Center, The Pennsylvania State University, Wiley Lab, Wiley Lane, University Park, PA 16802

126 Pathology Scientific Session Sunday October 24, 2004 8:00 a.m. – 12:00 p.m. Auditorium IV

Moderators: Michael Yaeger and David Steffen

Page 8:00 a.m. Current Intentional Microbial Threats to Animal Agriculture and Their Relevance to the 129 AAVLD and the Veterinary Pathologist - T.M. Wilson

8:15 a.m. Pathologic and Toxicologic Surveillance of Search & Rescue Dogs Deployed to the World 130 Trade Center & Pentagon: Preliminary Findings - S.D. Fitzgerald, W.K. Rumbeiha, C.M. Otto, and A.B. Downend

8:30 a.m. Canine Coronavirus – Associated Mortality Without Evidence of Concurrent Canine 131 Parvoviral Infection - J.F. Evermann, J.R. Abbott, and S. Han

8:45 a.m. Fatal Mycobacteriosis with Hepatosplenomegaly in a Young Dog Due to Mycobacterium 132 avium - D. O’ Toole, S. Tharp, B.V. Thomsen, E. Tan, and J.B. Payeur

9:00 a.m. New Pathological Observations on Naturally Infected Cats with Physaloptera praeputialis 133 (Nematoda: Spirurida, Physalopteridae) - S. Naem, A.A. Farshid, and V.T. Marand

9:15 a.m. Microscopic Lesions in Cats with Colonic Tritrichomonas Infection - M.J. Yaeger, J.L. 134 Gookin

9:30 a.m. The Role of Immunostimulation on the Pathology of Post-weaning Multisystemic Wasting 135 Syndrome (PMWS) in Pigs Under Field Conditions - J. Haruna*, P. Hanna, D. Hurnik, B. Ikede, L. Miller and C. Yason

9:45 a.m. BREAK AND POSTER SESSION

10:15 a.m. Hemolytic Uremic and Edema Disease-like Syndrome in a Mare and Foal Associated with 136 Escherichia coli 0103 - D.H. Gould, A.H. Davidson, C.E Dickinson, M.E. Legare, D.R. Hyatt and C. DebRoy

10:30 a.m. Gizzard Hemorrhage and Erosion in Broiler Chickens at Hatch - F. J. Hoerr, J. J. 137 Giambrone, L. Li, T. Dormitorio, and D. Poole

10:45 a.m. Utilization of Real-Time PCR for the Detection of Haemophilus parasuis from Swine 138 Tissues - D. Jordan, K. Harmon, and L. Hoffman

11:00 a.m. The Association of Clostridium perfringens alpha and beta2 Toxins with Microscopic 139 Lesions and Diarrhea in Neonatal Swine - M.J. Yaeger and R Ellis

11:15 a.m. Enterotoxemia by Enterotoxin and Beta 2 Toxin Positive Clostridium perfringens Type D in 140 a Goat Kid - F.A. Uzal, D.J. Fisher, S. Sayeed, B.A. McClane, G. Songer, H.T. Trinh and M.E. Fernandez Miyakawa

127 11:30 a.m. Sarcoptic Mange in Raccoons in Michigan - S.D. Fitzgerald, T.M. Cooley, A. Murphy, M.K. Cosgrove, and B.A. King 141

11:45 a.m. Hypocalcemia of Hypovitaminosis D in Mature and Immature Pigs - Y.L. Jones, M. Kiupel 142

* Graduate Student Competition

128 Current Intentional Microbial Threats to Animal Agriculture and Their Relevance to the AAVLD and the Veterinary Pathologist

Terrence M. Wilson

Historical information regarding State sponsored anti livestock and anti plant biowarfare (BW) programs has been well published in the open source literature for the past half century at least. The robust Russian, UK and USA anti agriculture BW programs date back at least 50 years and are well documented. Recently, Post 9/11 there has been an avalanche of information and misinformation regarding non State sponsored agro terrorism (to include criminal) ambitions targeting USA agriculture from the farm to the fork.

Current information regarding the possible intentional introduction of an animal microbial agent into the USA livestock industry will be presented, to include discussions of FMD, END, HPAI, HC and ASF. Suggestions and recommendations will be presented to the AAVLD and veterinary pathologists regarding current methodologies relevant to being prepared to detect these agents and act in a timely and seamless manner to mitigate the consequences of such an introduction.

Using open source information, this presentation will discuss contemporary non-state actor information regarding the intentional introduction of microbial agents into the USA livestock industry. Suggestions and recommendations will be offered to bench pathologists relevant to current methodologies relevant to being prepared to detect these agents in a timely and seamless fashion. Early and rapid diagnosis will assist immeasurably in consequence management.

Armed Forces Medical Intelligence Center, Fort Detrick, MD 21702 - 5004

129 Pathologic and Toxicologic Surveillance of Search & Rescue Dogs Deployed to the World Trade Center & Pentagon: Preliminary Findings

S.D. Fitzgerald1, W.K. Rumbeiha1, C.M. Otto2, A.B. Downend2

Abstract: A prospective surveillance program was undertaken to evaluate the long-term mortality, pathology and toxicology of dogs utilized in search and rescue efforts at the World Trade Center and Pentagon sites immediately following the Sept. 11, 2001, terrorist attacks. A total of 97 dogs deployed to the sites were enrolled in this study, while an additional 55 non-deployed search and rescue dogs that were not deployed to these sites were also enrolled as controls. Standardized protocols for complete necropsies were distributed to participants, and local veterinarians performed the necropsies and forwarded samples for analysis to the Diagnostic Center for Population and Animal Health, Michigan State University. To date, eleven deployed dogs have died (six had complete diagnostic work-ups, five had partial work-ups), while two non-deployed dogs have died. Causes of death in deployed dogs included: one case of cardiomyopathy; six cases of malignant neoplasms; one case of enterotoxemia; one case of non-specified respiratory illness; and one case of euthanasia due to osteomyelitis refractory to treatment. All neoplasms occurred in dogs greater than six years of age. Interestingly, no dogs exhibited nasal, upper airway, or pulmonary tumors; nor did any dog have significant lung lesions other than mild to moderate anthracosis. Toxicologic analyses, including heavy metal screens of livers and kidneys, organic compound screens on livers, and PCB testing of abdominal fat, have failed to reveal any toxicants. This study indicates that mortality in search and rescue dogs appears somewhat higher than in non-deployed dogs, although this is based on early preliminary data. Furthermore, mortality rates in search and rescue dogs were not nearly as high as had been anecdotally reported after the Oklahoma City Federal Building bombing. Based on this study, dogs do not appear to be highly sensitive to develop respiratory tract neoplasms or other chronic inflammatory respiratory conditions, in spite of extended exposure to airborne dusts, smoke and irritants without any respiratory filters or other protective devices. Toxicologic screens have failed to detect any long-term residual toxicants in various tissues.

1Diagnostic Center for Population and Animal Health, Michigan State University, Lansing, MI 48910 . 2Department of Clinical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104.

130 Canine Coronavirus – Associated Mortality Without Evidence of Concurrent Canine Parvoviral Infection

J. F. Evermann1,2, J. R. Abbott 2,3, and S. Han2,3

Canine coronavirus (CCV) has historically been associated with mild enteritis in the dog. However, when concurrent infections occur with canine parvovirus (CPV) there is increased morbidity and mortality. This report presents two cases in which puppy fatalities were associated with CCV, but no evidence of concurrent CPV infection.

Case one involved a 7 week old Chihuahua which had been purchased from a pet store. Within 24 hours the puppy became lethargic and by 72 hours began to vomit and had diarrhea. At that time the pup was admitted to the veterinary clinic where it was placed on IV fluids. A parvovirus Cite® test was run and was negative. The pup died within 12 hours of admission. Gross pathology revealed enteritis with changes suggestive of CPV and associated bacterial septicemia. Histopathology on intestines showed scattered dilated crypts with necrotic cellular debris and neutrophils. There was mild to moderate depletion and necrosis of lymphoid follicles. Electron microscopy (EM) on intestinal contents were positive for coronavirus. Immunohistochemistry (IHC) on selected sections of affected gut for CCV was positive, but negative for CPV.

Case two was an 8 week old Shih Tzu. Two other littermates had exhibited similar clinical signs of severe gastroenteritis, abdominal pain and died. This pup was euthanized by the referring veterinarian and the entire cadaver submitted for diagnostic evaluation. The necropsy revealed a severe ileo-cecal intussuception and segmental necrotic enteritis of the small intestine. Bacteriologic cultures of the intestines and tissue pool were positive for moderate growth of E. coli and Enterobacter sp. Electron microscopy of the intestinal contents was positive for coronavirus. Immunohistochemistry for CCV on selected sections of affected gut was positive, but negative for CPV.

These two cases bring to light the value of pursuing a CCV histologic-IHC diagnosis on puppies (5-10 weeks of age) with severe, fatal enteritis in which CPV was suspected based on clinical signs and gross lesions, but not detected by EM, antigen ELISA, or IHC. These results also suggest that CCV may be involved in more than just a mild self-limiting diarrhea in susceptible puppies. This may be due to unusually virulent strains of CCV, more immunologically vulnerable puppies, highly contaminated kennel environments, or a combination of these factors.

1Department of Veterinary Clinical Sciences, Washington State University, Pullman, WA. 2Washington Animal Disease Diagnostic Lab, Washington State University, Pullman, WA. 3Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA.

131 Fatal Mycobacteriosis with Hepatosplenomegaly in a Young Dog Due to Mycobacterium avium

D. O’Toole,1 S. Tharp,2 B. V. Thomsen,3 E. Tan,4 J. B. Payeur3

Mycobacterial infections with M. avium complex, which includes M. avium and M. intracellulare, and M. tuberculosis complex, which includes M. tuberculosis and M. bovis, are rare in dogs. Dogs are relatively less susceptible to infections with M. avium complex infections than to M. tuberculosis complex. A recent report summarized clinical, microbiological, and morphological features of disseminated mycobacteriosis in 18 dogs (Horn et al., Aust Vet J. 78:320-325) Some breeds appear predisposed to infection, particularly miniature schnauzers and basset hounds. We report generalized granulomatous disease in a young dog infected with M. avium.

A two-year old castrated 5 kg Shih Tzu-poodle-cross developed anemia, abdominal pain, lethargy and splenomegaly. Histological examination of surgically-removed samples of spleen revealed marked granulomatous splenitis with myriad intracytoplasmic acid-fact bacterial rods. Ultrastructual examination demonstrated 3 – 4 µm mycobacteria in phagolysosomes of epithelioid macrophages. Tissue extracts of formalin-fixed spleen was positive by polymerase chain reaction (PCR) testing for M. avium 16S ribosomal RNA. They were negative for M. tuberculosis complex IS6110 DNA. Anemia was associated with the presence of mycobacteria-infected macrophages in bone marrow. The dog’s condition deteriorated and euthanasia was performed after a clinical course of 2 months. Principal morphological finding at necropsy were severe diffuse granulomatous hepatitis, enteric lymphadenomegaly, and segmental granulomatous enteritis with intralesional mycobacteria. M. avium was cultured from enteric lymph nodes sampled at necropsy. The source of infection was not established, but presumed to be environmental with an enteric portal of entry.

Clinical signs and lesions in this dog were fairly characteristic of disseminated mycobacterial infection with M. avium complex. Such dogs are typically young, with a history of malaise, anemia and diarrhea. Lesions characteristically involve the spleen, liver and lymph nodes, which are markedly enlarged due to granulomatous inflammation. The outcome is generally fatal. There is no recognized zoonotic component to the disease. A defect in intracellular killing is presumably involved in dogs that succumb to infection, but the basis for fatal canine M. avium infection remains to be established . 1 Wyoming State Veterinary Laboratory, Laramie, WY 82070 2 Tharp Veterinary Clinic, Worland, WY 82401 3 United States Department of Agriculture, National Veterinary Services Laboratories, Animal and Plant Health Inspection Service, Ames, IA 50010 4 Colorado State University, Fort Collins, CO 80523

132 New Pathological Observations on Naturally Infected Cats with Physaloptera praeputialis (Nematoda: Spirurida, Physalopteridae)

S. Naem1, A. A. Farshid1 and V.T. Marand2

To study the pathological changes in the stomach of cat caused by Physaloptera praeputialis, 12 stray cats were euthanised. At necropsy seven cats were found to be infected with this nematode. Thickening of the stomach, congestion and oedema along with tiny erosions were observed on gross examination. Histopathology observations indicated congestion, oedema, leukocytic infiltration, necrosis, and hyperplasia, cystic changes of glands and hyperplasia of the muscular coat. These changes were indicative of inflammatory and degenerative reaction brought about by the parasitic infection with P. praeputialis and its consequent irritating effects on the stomach. This is the second report of pathological observations of P. praeputialis infection in domestic cats.

1 Department of Pathobiology, Faculty of Veterinary Medicine, Urmia University, P.O.Box 1177,Urmia University, Urmia, Iran. 2 Department of Pathobiology, Faculty of Veterinary Medicine, Islamic Azad University, Urmia, Iran.

133 Microscopic Lesions in Cats with Colonic Tritrichomonas Infection

Michael J. Yaeger1, Jody L. Gookin2

Tritrichomonas foetus has recently been identified as an inhabitant of the large intestine of young domestic cats with chronic diarrhea. This abstract describes the typical clinical presentation and microscopic lesions in 7 naturally infected cats with colonic trichomonads. The clinical signs in these animals were typified by a waxing and waning large bowel diarrhea. Feces were generally semi-formed to cow-pie in consistency, malodorous and occasionally contained fresh blood and mucus. Affected individuals ranged in age from 2.5 – 12 months. The average age of affected individuals was 6.4 months (SD + 3.6). Where tested, cats were FeLV and FIV negative.

The most distinctive microscopic feature in these cases was the presence of numerous trichomonads in sections of colon. The organisms were elongate, tear-drop to crescent-shaped, approximately 7x5 microns with eosinophilic cytoplasm and a faint, hyperchromatic round to oval nucleus approximately 1.5 µm in diameter typically located in the apical portion of the protozoan. Trichomonads were most consistently observed in close proximity to the surface of the colonic mucosa (83%) and to a lesser extent (43%) within the lumen of colonic glands.

All kittens in which trichomonads were identified had mild to moderate expansion of the colonic lamina propria by an infiltrate of plasma cells and lesser numbers of lymphocytes. Significant numbers of neutrophils were observed in the lamina propria in 86% of the cases. Eosinophils were not a prominent feature of the inflammatory infiltrate, were only observed in 43% of the cats, and were generally present in low numbers. Crypt reactivity, loss of goblet cells, crypt microabscesses, and attenuation of the superficial colonic mucosa were consistent microscopic features.

In two of the seven animals trichomonads had infiltrated beneath the colonic epithelium. In one kitten, trichomonads had invaded the colonic lamina propria primarily at the base of the crypts. In the second cat, enteroinvasive trichomonads had resulted in ulceration and intense transmural inflammation with organisms present in all layers of the colonic wall including the subserosal lymphatics. The presence of enteroinvasive trichomonads was associated with more severe and diffuse crypt changes, a lymphoplasmacytic and pyogranulomatous inflammatory response, numerous crypt microabscesses and dramatic loss of goblet cells.

Trichomonads were detected in 56% of the 43 sections of colon from affected individuals. The likelihood of identifying trichomonads in an infected cat would be enhanced by harvesting multiple sections of colon. Based on the above numbers, in order to assure >95% confidence of identifying the organism in an infected animal, 6 separate sections of colon would have to be evaluated.

1Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011 2Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh NC 27606

134 The Role of Immunostimulation on the Pathology of Post-weaning Multisystemic Wasting Syndrome (PMWS) in Pigs Under Field Conditions

J. Haruna*, P. Hanna, D. Hurnika, B. Ikede, L. Miller and C. Yason

PMWS, is a relatively new disease of pigs that was first reported in 1996. The disease is causally associated with infection by porcine circovirus type 2 (PCV-2). Since the initial report, PMWS has now emerged worldwide as an important and devastating disease to the swine industry. Several reports have indicated that immune activation plays a significant role in the development of the disease.

The effects of immunostimulation on the development of PMWS in pigs naturally infected with porcine circovirus type 2 (PCV-2) were evaluated. Nine hundred and thirty 53-54-day-old pigs were allocated to five treatment groups each receiving single IM injection as follows: RespiSure-ONE™, a commercial Mycoplasma hyopneumoniae vaccine for group 1 (n=197), Emulsigen™, an oil based adjuvant for group 2 (n=172), Alhydrogel™, an aluminum hydroxide based adjuvant for group 3 (n=172), physiological saline (injection control) for group 4 (n=218) and no treatment (system control) for group 5 (n=171). Additionally, of the 930 pigs, 417 (category A) received Solutein™ while 513 (category B) did not receive Solutein™ at the source barn in drinking water for 5 days postweaning at 25-26 days old.

PMWS, characterized by the presence of typical gross and microscopic lesions in many tissues, developed in all 5 groups and PCV-2 antigen was detected by immunohistochemistry within lesions in the mesenteric lymph node, spleen, Peyer’s patch and lung of affected pigs. No significant differences in the incidence of PMWS were apparent between the five treatment groups (immunostimulated and non- immunostimulated) suggesting that immunostimulation did not influence the expression of PMWS in this study. There was also no significant difference in the incidence of PMWS between the two categories suggesting that the administration of Solutein™ had no effect in reducing losses due to PMWS in these pigs.

Department of Pathology and Microbiology, and aDepartment of Health Management, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown C1A 4P3 PEI, Canada.

*To be considered for graduate student award

135 Hemolytic Uremic and Edema Disease-like Syndrome in a Mare and Foal Associated with Escherichia coli 0103

D.H. Gould1, A.H. Davidson2, C.E Dickinson2, M.E. Legare1, D.R. Hyatt1 and C. DebRoy3

An 8 year old mare and her 3 day old colt were presented on an emergency basis to the James L. Voss Veterinary Teaching Hospital for evaluation of a 24 hr course of lethargy and fever in the mare. The colt nursed normally following parturition and had no signs of illness on the farm. The mare had a bloody vulvar discharge. On presentation the mare was febrile (102.4°F) and had a fluid-filled, firm uterus. Forty-eight hours after admission, BUN and creatinine were increased, there was thrombocytopenia, and pink serum indicative of gross hemolysis. Acute renal failure was diagnosed based on the presence of azotemia, urinary casts and anuria in the face of fluid therapy. Clinical abnormalities became progressively more severe, and the mare was euthanized. Twelve hours after the mare began to show severe signs, the colt became lethargic, nursed less frequently and, over the course of 12 hr, developed neurological signs progressing to generalized seizures. At this point the foal was euthanized.

At necropsy the mare had reddened, edematous lungs and submucosal edema of the ventral colon and cecum. The kidneys were swollen, pale and wet. Other macroscopic lesions were absent. The colt had no macroscopic lesions. There was no evidence of brain herniation.

The kidneys of the mare had glomerular capillaries filled with, and partially obliterated by, varying proportions of platelets and fibrin (Frazier-Lendrum stain and transmission electron microscopy). Some tubules contained uniformly deposited, finely granular proteinaceous material indicative of hemoglobin. Histologically the endometrium had superficial, light infiltrates of neutrophils along with areas of hemorrhage. Microscopic evaluation of sections of brain revealed well developed perivascular and pericellular spaces, but no evidence of extravasated proteinaceous material. In the foal, microscopic lesions were confined to the brain and consisted of perivascular accumulations of homogeneous, eosinophilic material indicative of extravasated plasma protein in deep gray matter areas of the corpus striatum and thalamus.

Escherichia coli was isolated from necropsy tissue samples of uterus, small intestine and cecum of the mare and the cecum of the foal. Other microorganisms identified in tissues included: Enterococcus. faecalis and Klebsiella pneumoniae from the small intestine of the mare, no growth from kidney of the mare and Enterobacter agglomerans and Pseudomonas from the mare’s uterus. From the foal, Enterobacter cloacae and K. pneumoniae were identified in the cecum, Serratia marcescens in the kidney and Enterococcus and K. pneumoniae in the small intestine. Since the clinical and necropsy findings were consistent with hemolytic uremic syndrome, the E. coli isolates from the mare and the foal were intensively tested. PCR assays for toxins and fimbriae (STb, STa, LT, STX2, CNF1, CNF2, K88, K99, F18, 987P and F41) were performed, and none of the genes were detected. The E. coli from the mare (cecum, small intestine and uterus) and the E. coli isolate from the cecum of the foal belonged to the O103:H2 serogroup and carried the genes for Shiga toxin (stx-I) and hemolysin A (HlyA-EHEC or EhxA).

Based on the microbiological and pathological findings we conclude that these horses had organ-specific, microangiopathic disease due to infection with enterohemorrhagic Escherichia coli.

1Colorado State Veterinary Diagnostic Laboratory, Colo. State University, Fort Collins, CO 80523 USA 2Department of Clinical Sciences, Colorado State University, Fort Collins, CO 80523 USA 3Gastroenteric Disease Center, Department of Veterinary Science, The Pennsylvania State University, University Park, PA, 16802 USA

136 Gizzard Hemorrhage and Erosion in Broiler Chickens at Hatch

F. J. Hoerr1, J. J. Giambrone2, L. Li1, T. Dormitorio2, and D. Poole3

Broiler chickens in the southeastern United States were observed to have focal to confluent hemorrhages and erosions in the mucosal lining (koilin) of the gizzard. The condition spontaneously affects many broiler flocks in a production complex, and involves chicks from multiple breeder flocks of various ages. The lesion occurs in chicks in the hatchery before placement in the broiler house and prior to feed consumption. In the broiler house, stunted broilers are observed with fecal-soiled feathers around the cloaca. At necropsy, affected chicks have focal to confluent or linear erosions and roughening of the gizzard lining, frequently with brown or red discoloration indicative of hemorrhage. The proventriculus may have yellow coagulum adhered to gland openings.

From two broiler complexes experiencing this problem, we examined chicks in the hatchery from 15 broiler breeder flocks, ages 28 to 63 weeks of age. Gizzards showing gross lesions were divided, with proventriculus attached, for viral studies and portions were fixed in neutral buffered formalin for histopathology. Tissues were similarly collected from three-day-old broilers with gizzard lesions in four flocks. At necropsy, gross lesions in the gizzard were as described above.

Histologic examination showed multifocal to confluent hemorrhage in the koilin. Affected regions of koilin had incomplete fusion and were discontinuous. Many sloughed epithelial cells were also present. In some, the hemorrhage was represented by nuclei of degenerated erythrocytes occurring in a laminar pattern with normal koilin forming between the hemorrhage and the mucosa. In others, acute hemorrhage occurred at the interface of the mucosa and the fused koilin layer. Some sections of pancreas had infiltrates of heterophils in periductal interstitial tissue; duodenum and proventriculus had no conclusive findings. In three-day-old broilers, the hemorrhages were less obvious in most gizzards available for examination, however, acute, locally extensive hemorrhage and koilin disruption occurred in some.

The cause of this condition is not currently known, however, both noninfectious and infectious etiologies are under investigation.

1Alabama Department of Agriculture and Industries, C. S. Roberts Veterinary Diagnostic Laboratory, PO Box 2209, Auburn, AL, USA 2Department of Poultry Science, Auburn University, AL 36849 3Degussa Corporation, 1701 Barrett Lakes Blvd Suite 340, Kennesaw GA 30144-3694

137 Utilization of Real-Time PCR for the Detection of Haemophilus parasuis from Swine Tissues

D. Jordan1, K. Harmon1, L. Hoffman1

Systemic disease caused by Haemophilus parasuis in weaned pigs frequently causes significant economic loss for producers. Contributing to the losses from this disease are the inadequacies of diagnostic techniques. Basic bacteriologic isolation from infected tissues is difficult due to prior antimicrobial treatment of the pigs, chronicity of lesions, and the fastidious nature of the bacteria. Experimentally, inoculum strains are only recovered, at best, in 25% of the samples from infected pigs. A diagnosis is presumptive without a bacterial isolate to confirm infection when lesions are present. A real-time PCR assay has been developed, validated for culture isolates and has been utilized on diagnostic tissue specimens. In validation of the test for diagnostic purposes, the real-time PCR platform has been used on over 73 DNA extracts from diagnostic case tissues which encompass normal tissues, pneumonic tissues and tissues representing Glasser’s disease and septicemia. Tissues were classified into three groups of samples: Normal (tissues were within normal limits), Pneumonia only (no pleuritis), Systemic disease typical of H. parasuis infection (polyserositis, meningitis, arthritis). Tissues were cultured for routine bacteriological workup and homogenized for evaluation by real-time PCR. The results are listed in Table 1. The test has had excellent performance on the diagnostic samples. When compared to the lesions of the case, the lesions are consistent with infection by H. parasuis. The table demonstrates the improved sensitivity to detect H. parasuis in the presence of the septicemic disease and the inability to isolate H. parasuis through bacterial culture methods. Furthermore, it is demonstrated that H. parasuis is not commonly detected in normal tissues.

Table 1: Correlation of positive and negative results as determined by bacterial isolation and real-time-PCR Bacterial Isolation/ Real-time PCR Normal Pneumonia Systemic - / - 15 4 9 + / - 0 2 0 - / + 2 2 27 + / + 0 9 3 Normal samples included: lung, brain, liver, spleen, kidney, thoracic fluid, joint swab, and intestinal serosal swab. Pneumonia samples only included lung. Systemic samples included: lung, brain, spleen, heart, and pleural, peritoneal, joint, and heart swab.

Real-time PCR is a technology that can readily provide results of the presence of specific DNA. The use of real-time PCR to detect the presence of H. parasuis from diagnostic specimens will expedite diagnosis of and define infection with H. parasuis.

1 Department of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, Iowa

138 The Association of Clostridium perfringens alpha and beta2 Toxins with Microscopic Lesions and Diarrhea in Neonatal Swine

M. J. Yaeger1; R. Ellis2

A prospective study involving 77 live, piglets less than 1-week of age was undertaken to try and resolve a number of issues concerning C. perfringens infection in neonatal swine, including: a. the detection frequency of C. perfringens alpha and beta2 toxins; b. the correlation between genotyping results and toxin detection; c. the correlation between the presence of C. perfringens toxins and diarrhea; and d. the association between the toxins and specific microscopic lesions.

C. perfringens was isolated from the small intestine of 59 of 77 pigs. Alpha and beta2 toxin were detected in the contents of the intestine or colon in 43 (66.7%) of these animals and the isolate from each of these animals was positive for both the alpha and beta2 toxin genes. C. perfringens was isolated from the small intestine of 19 piglets (21.8%) in which C. perfringens toxins were not detected. Ten of these isolates were positive for alpha and beta2 toxin genes whereas 9 were positive for only the alpha toxin gene.

C. perfringens toxins were detected in the small intestine of 36 of 75 (48%) animals. In all but one of these piglets, both alpha and beta2 toxin were detected. Clostridial toxins, including C. difficile toxins, were detected in 45 of 55 colon samples (82%). Both alpha and beta2 toxin were identified in 36 of 55 colons (65.5%). In 19 of these animals, alpha, beta2 and C. difficile toxins were detected in the colon. In nine (16%) piglets, C. difficile toxins were the only toxins detected.

The presenting complaint in subject pigs was diarrhea. However, when examined grossly, 26 (33.3%) pigs had normal or firm feces and 52 (66.7%) had diarrhea. In 20 pigs, alpha and beta2 toxin were detected in the absence of other enteric pathogens. 19 of these 20 (95%) were considered to have diarrhea. Compared to the overall population, the detection of C. perfringens alpha and beta2 toxin was significantly correlated with gross evidence of diarrhea.

Microscopic changes observed in sections of small intestine in pigs without a viral enteritis or colibacillosis included the presence of numerous large bacilli in the lumen of the small intestine, necrosis of the superficial villus lamina propria, necrosis of superficial villus epithelial cells, an infiltrate of neutrophils into the villus lamina propria, and vacuolar degeneration of superficial villus epithelial cells. The primary microscopic changes observed in the large intestine included mesocolonic edema, a suppurative infiltrate in the lamina propria and focal superficial erosions. There was no correlation between the presence or absence of C. perfringens toxins in the small or large intestine and specific gross or microscopic lesions.

Conclusions: C. perfringens toxins were detected in the intestinal contents of 50 of 77 (65%) study piglets. The presence of C. perfringens alpha and beta2 toxins was positively correlated with diarrhea. Consistent microscopic lesions were not observed in the small or large intestine in cases in which C. perfringens alpha and beta toxins were detected.

1. Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 2. Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine, Colorado State University, Fort Collins CO.

139 Enterotoxemia by Enterotoxin and Beta 2 Toxin Positive Clostridium perfringens Type D in a Goat Kid

F.A. Uzal1, D.J. Fisher2, S. Sayeed2, B.A. McClane2, G. Songer3, H.T. Trinh3 and M.E. Fernandez Miyakawa1

Enterotoxemia by Clostridium perfringens type D in goats is usually believed to be produced by epsilon toxin, a major exotoxin produced by the types B and D of this microorganism. Three forms of caprine enterotoxemia, namely peracute, acute and chronic have been described. The peracute form of the disease is characterized by respiratory and neurological alterations, while the acute and chronic forms are associated with entero-colitis, although neurological alterations can also be seen in the acute form. A condition clinically and pathologically very similar to the peracute form of enterotoxemia has previously been reproduced in goat kids by intravenous inoculation of C. perfringens type D epsilon toxin, which strongly suggests that epsilon toxin is responsible for this form of the disease. The role of epsilon toxin in the intestinal changes of the acute and chronic forms of enterotoxemia in goats, remains controversial and the action of other C. perfringens toxins have been suggested in the past to explain these changes.

A 3 week old alpine doe was found dead in the morning. The kid had appeared normal the night before and had no history of health problems. A post-mortem examination revealed a very good nutritional condition and the perineal area soiled with feces. Multiple strands of fibrin were observed on the small and large intestinal serosas. The mucosas of the rumen, abomasum, ileum and colon were diffusely red and there were multiple shallow, irregular ulcers covered by fibrin in the terminal ileum, more marked around the ileo-cecal valve. The spiral colon was distended with liquid green content and gas, and showed severe serosal edema, which gave the organ a conical shape. The lungs were congested and edematous and there was an excess of pleural fluid. Slight herniation of the cerebellar vermis through the foramen magnum of the skull was also observed. Histologically, there was proteinaceous interstitial and pleural edema of the lungs, perivascular proteinaceous edema of the brain (cerebellar peduncles and cerebellar white matter), fibrino-necrotic ileitis and edema of the colonic serosa.

Alpha and epsilon toxins were detected in ileal and colonic contents and feces by a capture ELISA technique. C. perfringens was isolated from ileal and colonic contents and from feces (16 isolates in total). All these isolates were identified as type D, enterotoxin (CPE) and beta 2 toxin positive by multiplex PCR. Eleven of these isolates showed expression of beta 2 toxin when tested by western blot. Since attempts to induce these isolates to sporulate in vitro were unsuccessful, it was not possible to determine if these isolates produced the sporulation-regulated CPE.

Beta 2 is a novel C. perfringens toxin that has been associated with necrotic enteritis in several animal species, although its role in disease production of animals remains controversial. It is possible that in this case, beta 2 toxin (and/or CPE if present) was at least partially responsible for the necrotic changes observed in the intestine of the goat kid. If this is the case, this finding could help explain the gastrointestinal changes observed in cases of C. perfringens type D enterotoxemia in goats. Possible damage by beta 2 toxin and CPE to the intestinal epithelium would also provide epsilon toxin with an easy route across the intestinal mucosa. The brain and pulmonary lesions observed in this kid are consistent with the action of epsilon toxin reported previously.

1California Animal Health and Food Safety Laboratory System, San Bernardino Branch, University of California Davis, CA. 2Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. 3Department of Veterinary Science and Microbiology, The University of Arizona,Tucson, AZ.

140 Sarcoptic Mange in Raccoons in Michigan

S.D. Fitzgerald1,T.M. Cooley2, A. Murphy1, M.K. Cosgrove2, and B.A. King3

Sarcoptic mange is a cause of pruritic skin disease in domestic dogs and a wide range of wildlife species. This report describes the first reported cases of sarcoptic mange in free-ranging North American raccoons (Procyon lotor). Three adult raccoons from upper Wayne County, Michigan, were captured live by personnel from the Trenton Animal Control, humanely euthanatized by lethal injection, and submitted for diagnostic evaluation. These submissions were part of a much larger clinical outbreak affecting approximately 75% of 300 raccoons handled by animal control personnel in Wayne Co. over a two-year period. All three animals were intensely pruritic, and two had advanced alopecic and crusting lesions covering most of their dorsum and hind limbs. Skin scrapings and skin biopsies revealed a crusting and hyperkeratotic dermatitis with high numbers of Sarcoptes scabiei adults, larvae, nymphs, and eggs. The severe crusting, large body surfaces involved in the lesion, and massive numbers of mites present, are similar to the canine form of sarcoptic mange known as crusted scabies. This condition, first described in humans and known as Norwegian scabies, is characterized by very high numbers of mites and is commonly associated with compromised immune systems. However, these raccoons were not otherwise debilitated, with minimal internal parasites, good body condition, and no evidence of infectious bacterial or viral diseases. Since sarcoptic mange is both highly contagious and zoonotic, it should be included in the differential list for raccoons exhibiting pruritus and alopecia.

1 Diagnostic Center for Population and Animal Health, Michigan State University, East Lansing, MI 2 Rose Lake Wildlife Disease Laboratory, Wildlife Division, Michigan Department of Natural Resources, East Lansing, MI 3 Southeast Michigan Wildlife Rehabilitation, Gross Ile, MI

141 Hypocalcemia of Hypovitaminosis D in Mature and Immature Pigs

Y.L. Jones 1, M. Kiupel1

Dietary levels of vitamin D and calcium are critical for maintaining calcium homeostasis. Rickets, osteomalacia, and osteoporosis are conditions which have been associated with hypovitaminosis D and/or hypocalcemia. Rickets occurs in young animals and is most commonly caused by hypovitaminosis D. Animals typically present with expanded costochondral junctions and enlarged long bones, which appear soft (“rubbery”). Microscopically, the affected bones have thickened and irregular zones of hypertrophic epiphyseal cartilage and excessive amounts of uncalcified osteoid (osteoid seams) in the metaphysis. Osteomalacia occurs in older animals by the same mechanisms as rickets does in young animals, and produces similar gross and microscopic lesions. Low serum levels of ionized calcium or vitamin D3 stimulate the parathyroid gland resulting in secondary hyperparathyroidism. Secondary hyperparathyroidism can eventually cause secondary osteoporosis. In mature and immature calves, experimental Ca deficiency produced osteoporosis without evidence of osteomalacia or rickets. Osteoporosis occurs when bone resorption exceeds bone formation. In these cases, bones are brittle and thin and may fracture spontaneously or following minimal trauma. Microscopically, the bones are thin and porous and the trabeculae are reduced in number and fragmented. Osteoid seams are of normal width and frequency and mineralization typically occurs normally. In osteoporosis hypocalcemia may develop only with severe and prolonged Ca deficiency, because homeostatic mechanisms maximize absorption and resorption of Ca to maintain serum ionized Ca levels. When vitamin D is deficient, the homeostatic mechanisms are impaired and hypocalcemia may develop.

Three pigs (two, 9 week old piglets and one 5 month old hog) were submitted to Michigan State University Diagnostic Center for Population and Animal Health with spontaneous fractures of the rear limbs, which had occurred during transport. All pigs originated from a multiple-site farm that reported a dramatic increase of fractures in the rear tibias of pigs in the postweaning and finisher stages. Fractures occurred mainly during transport, and up to 10 percent of transported animals were affected. However, there had been no changes of transport mechanisms, but there had been dietary changes to compensate for lower feed intakes. Gross lesions in the animals examined were limited to the musculoskeletal system and consisted of comminuted fractures of the right tibia (hog), subcutaneous hemorrhage in the rear limbs (piglet 1), bilateral, open, comminuted tibial fractures (piglet 2). Microscopically, the bones in all three animals had thin cortices with retention of cartilage cores. The trabeculae were fragmented and thin and there was mild periosteal fibrosis. Serum vitamin A and E levels were within normal limits. Tissue levels of Vitamin E and Selenium were within normal limits. Serum ionized calcium levels were marginally decreased in all three animals. There was no evidence of overt rickets based on bone ash contents, which were within reference range. Serum 25-hydroxy vitamin D levels in the two piglets were 14 and 18 nmol/L, respectively. A value of 57 nmol/L is considered normal for a variety of species, however reference values for pigs have not been established by our laboratory. Serum 25-hydroxy vitamin D levels in the hog were 57 nmol/L. These results support a diagnosis of hypovitaminosis D in the piglets. Nutritional analysis performed on a feed sample from the postweaning phase revealed no detectable vitamin D3 or vitamin A.

We concluded that the nutritional data and microscopic lesions supported a diagnosis of osteoporosis. The pathological fractures in these animals were presumed to be due to secondary hyperparathyroidism. Despite a confirmed vitamin D deficiency, there was no evidence of classical lesions of rickets in these pigs. Based on previously described experimental findings in hypocalcemic calves, we surmise that in these rapidly growing pigs, lesions of hypocalcemia of vitamin D deficiency manifested prior to the effects of vitamin D deficiency per se (classical rickets). The marginal decrease in serum ionized calcium levels, in the face of hypovitaminosis D may reflect an underlying dietary calcium deficiency.

1 Department of Pathobiology, Michigan State University, East Lansing, MI

142 Virology Scientific Session Sunday October 24, 2004 8:00 a.m. – 12:00 p.m. Auditorium II

Moderators: K-J Yoon and Edward Dubovi

Page 8:00 a.m. Assessment of the Stability of PRRS Virus RFLP Pattern During In Vivo Replication - S.-H. 145 Cha* and K.-J. Yoon

8:15 a.m. Relative Prevalence of Reassortant H1N1 Swine Influenza Viruses with Avian Polymerase 146 Genes and Classic H1N1 Viruses with Swine Polymerase Genes - B.H. Janke, K.M. Harmon, K-J.Yoon, G.A. Erickson, and R.J. Webby

8:30 a.m. Humoral Immune Responses of Pigs to SIV Infection and Vaccination - W.-I. Kim*, W.-H. 147 Wu, B.H. Janke, K.-J. Yoon

8:45 a.m. Characterization of a Reassortant Human/Swine H1N2 Influenza Virus Isolated from Pigs in a 148 U.S. Swine Herd - M.L. Gramer*, C. Mahlum-Wees, K.D. Rossow, R.J. Webby and S.M. Goyal

9:00 a.m. Nested PCR Detection and Duration of Porcine Circovirus Type 2 in Semen Samples from 149 Naturally Infected Boars - K. A. McIntosh*, J.C.S. Harding, J.A. Ellis and G.D. Appleyard

9:15 a.m. Ulcerative and Hemorrhagic Typhlocolitis in An Angus Heifer Associated with Natural 150 Bovine Enterovirus Type-1 Infection - U. Blas-Machado, M.J Boileau, J.T. Saliki, S.L. Caseltine, S.D. Goens, J.C. Duffy, and R.D. Welsh

9:30 a.m. Bovine Viral Diarrhea Virus Persistent Infection in Two White-Tail Deer in Southeastern 151 South Dakota – C.C.L. Chase, L.J. Braun, P. Leslie-Steen, T. Graham, D. Miskimins and J.F Ridpath

9:45 a.m. BREAK AND POSTER SESSION

10:15 a.m. Bovine Viral Diarrhea Virus (BVDV) Subtypes in Diagnostic Laboratory Accessions from 152 Clinical and Necropsy Cases: Distribution of BVDV1a, 1b, and 2a Subtypes – R.W. Fulton, J. F. Ridpath, S. Ore, J.T. Saliki, L. J. Burge, and A.W. Confer

10:30 a.m. Genetically Identical Bovine Virus Diarrhea Viruses Isolated from an Aborted Ovine Fetus 153 and a Yearling Hereford Heifer on the Same Premises – B. Thompson, E. Dubovi, B. Njaa, J.F. Ridpath

10:45 a.m. Viral Antigen Distribution in the Respiratory Tract of Cattle Persistently Infected with Bovine 154 Viral Diarrhea Virus Subtype 2a - A.W. Confer, R.W. Fulton, D.L. Step, B.J. Johnson, and J.F. Ridpath

11:00 a.m. Comparison and Optimization of High Throughput Viral RNA Isolation Methods For FMDV - 155 A.G. Giuffre, J.M. Robida, X. Fang, M.A. Kenney, T.S. McKenna, B.M. Martin, and T.R. Beckham

143 11:15 a.m. Malignant Catarrhal Fever-like Disease in Sheep Following Intranasal Inoculation with Ovine 156 Herpesvirus 2 - H. Li, D. O'Toole, O. Kim, J. L. , and T.B. Crawford

11:30 a.m. Avian Paramyxovirus Infection -1 in Pigeons – A Retrospective Study - H.L. Shivaprasad, G. 157 Cooper, F. Uzal, P. Woolcock, S. Hietala, B. Crossley, B. Charlton, F. Sommer, R. Crespo, R. Chin, M. McFarland, L. Woods, M. Anderson, H. Kinde, B. Daft, J. Odani, D. Read, A. Bickford, P. Hullinger and B. Barr

11:45 a.m. Isolation of Equine Influenza Virus from Racing Greyhounds with Fatal Hemorrhagic 158 Pneumonia - E.J. Dubovi, P.C. Crawford, R.O. Donis, W.L. Castleman, Iain Stephenson, and E.P.J. Gibbs

* Graduate Student Competition

144 Assessment of the Stability of PRRS Virus RFLP Pattern During In Vivo Replication

S.-H. Cha1* and K.-J. Yoon1,2

Porcine reproductive and respiratory syndrome (PRRS) virus is continuously changing as evidenced by a high degree of genetic and antigenic variability that exists among field isolates. Such a high degree of heterogeneity among PRRS viruses prompted the development of molecular tools for use in characterizing genotypes of the virus and tracing the origin of the virus within a herd or among herds. The most common method is restriction fragment length polymorphism (RFLP) analysis of the virus. The foundation of RFLP analysis is that the pattern of a PRRS virus is relatively stable. However, the emergence of new RFLP patterns and the disappearance of existing RFLP patterns over a period of time have been observed in the field. Furthermore, a high level of genetic variability was shown to exist among PRRS viruses with the same pattern. These observations raised concern on the predictability of the RFLP analysis for genetic relatedness among PRRS viruses. The following study was conducted to evaluate the stability of the RFLP pattern of PRRSV during a long-term in vivo replication.

Sequential pig-to-pig passages of the North American PRRS virus prototype, VR-2332, were carried out in 3 independent lines of pigs for a total of 727 days. The experiment began by PRRSV-naïve pigs with a highly homologous inoculum (CC-01) of the VR-2332 prepared through a series of plaque purifications. Three independent lines (A, B, C) of in vivo virus replication were established and monitored over 13 animal passages (P1 to P13). Pigs in a fourth line (D) served both as mock-infected negative controls and environmental sentinels. Each passage took 60 days. Pigs 1A, 1B and 1C were inoculated with CC-01 and P2 to P13 were inoculated with a tissue filtrate from the corresponding pig in the previous passage. Serum samples were collected periodically and used to monitor viremia and antibody response. In addition, 15 viral plaque clones were directly isolated from CC-01 and day 7 serum of each pig of each passage and sequenced for genetic characterization of ORF5. The sequence data was analyzed and compared to CC-01 to evaluate the degree of mutation and RFLP patterns (MluI-HincII-SacII) that occurred during viral replication in pigs.

A total of 495 viral clones were recovered from 33 pigs over 13 passages. Among those, 398 clones (80.4%) had 2-5-2 RFLP pattern which is the same as that of CC-01. However, the remaining 97 viral clones showed RFLP patterns different from the initial inoculum, suggesting that the RFLP pattern can be changed. During the study, 4 different RFLP patterns were observed: 2-6-2 (P2), 1-5-2 (P3), 2-5-4 (P7), and 2-1-2 (P10), indicating that HincII sites are more vulnerable to mutational changes than other sites. More importantly, MluI site (position 196) that was reported to be present only in one of PRRS MLV vaccines (Ingelvac®, Boehringer-Ingelheim Vetmedica) and its parental strain (VR-2332) can disappear during in vivo replication. Among the 4 patterns identified, 2-1-2 pattern became predominant (75 of 97 clones) after long passages of CC-01 in pigs. Nucleotide sequence homology between CC-01 and clones with 2-5-2 RFLP pattern ranged 0.05% up to 1.58%, whereas the identity between CC-01 and clones with RFLP patterns other than 2-5-2 was 0.5% and 1.45%. These observations suggest that RFLP analysis cannot accurately predict genetic similarity/relatedness between or among PRRS viruses.

In conclusion, precaution should be taken when molecular epidemiology of PRRS viruses will be done using only RFLP analysis.

1Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA 2Department of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, IA

* To be considered for graduate student presentation award.

145

Relative Prevalence of Reassortant H1N1 Swine Influenza Viruses with Avian Polymerase Genes and Classic H1N1 Viruses with Swine Polymerase Genes

Janke BH1, Harmon KM1, Yoon K-J1, Erickson GA2, Webby RJ3

In 1998, a new subtype (H3N2) of swine influenza virus (SIV) began to circulate widely in swine populations in the United States. The first H3N2 virus, recovered from swine in North Carolina, was a double reassortant virus with hemagglutinin (HA), neuraminidase (NA) and polymerase PB1 genes from a human influenza virus and the remaining genes from the classic H1N1 SIV. Subsequent H3N2 SIV isolates were triple reassortant viruses with human HA, NA and PB1 genes, classic swine genes for nucleoprotein (NP), matrix protein (M), non-structural proteins (NS), and avian genes for polymerase proteins PA and PB2. Second generation reassortant H1N2 viruses, which were similar to the H3N2 triple reassortants except for reacquisition of the swine H1 gene, were soon discovered. In 2002, new second generation H1N1 reassortants were described which were similar to H3N2 triple reassortants except for reacquisition of both swine H1 and N1 genes. Thus, two different H1N1 viruses appeared to be circulating in U.S. swine populations. Because these new reassortant H1N1 viruses were first identified in herds in which vaccination against the classic H1N1 viruses appeared to have lost its efficacy, concern was expressed about possible antigenic variation in and the prevalence of such new reassortants. To determine the relative prevalence of the new reassortants and gain insight into the appearance of such viruses, H1N1 SIV isolates collected from field cases submitted to the Veterinary Diagnostic Laboratory at Iowa State University from 1997-2003 were characterized using primers for the swine and avian PA and PB2 genes by the SYBR Green RT-PCR technique. Approximately 50 H1N1 isolates from each year, except 1998 (isolates not saved in 1998), were characterized. Year Swine PA, Avian PA, Variable PA, Negative PA, Total PB2 PB2 PB2* PB2** 1997 36 0 8 6 50 1998 0 3 0 0 3 1999 4 33 5 7 49 2000 0 38 6 6 50 2001 0 45 5 1 51 2002 0 56 3 1 60 2003 1 36 9 3 49 *Only PA or PB2 defined as avian or swine, or mixed avian/swine polymerases ** Neither avian nor swine PA or PB2 identified One hundred SIV isolates, 50 from 1999-2000 and 50 from 2003, recovered at the Rollins Animal Disease Diagnostic Laboratory in Raleigh, NC also were characterized. Three of the samples (from 1999- 2000) contained avian PA, PB2 and either swine PA and/or swine PB2 suggesting that more than one virus was in the cell culture supernatant. The other 97 isolates all contained only avian PA and PB2.

These results suggest that the new reassortant H1N1 viruses with avian polymerase genes PA and PB2 have almost completely supplanted the classic H1N1 viruses with all swine genes. Furthermore, this displacement of the classic swine H1N1 by the new reasssortant H1N1 occurred almost immediately after widespread introduction of the H3N2 subtype into U.S. swine populations.

1Veterinary Diagnostic Laboratory, Iowa State University, Ames, IA 2Rollins Animal Disease Diagnostic Laboratory, Raleigh, NC 3Dept of Virology and Molecular Biology, St. Jude’s Children’s Research Hospital, Memphis, TN

146 Humoral Immune Responses of Pigs to SIV Infection and Vaccination

W.-I. Kim1*, W.-H. Wu2, B. H. Janke2, K.-J. Yoon1,2

Swine influenza is one of the economically significant respiratory diseases in swine industry since it was initially recognized in the early 1900’s. Although detection of the virus or viral antigen in clinically affected animals is considered as definitive diagnosis of swine influenza, serological testing is often employed to detect animals that have been exposed to the virus because the disease has a very short course and the causative agent becomes undetectable in infected animals quickly. Serology is also used to assess the immune status of pigs at various stages within an operation so that the level of herd immunity or timing of vaccination can be determined. However, diagnostic value of serologic data is often confounded or diminished by several factors, such as subtype of the infecting virus, presence of vaccine-induced antibody or maternal antibody, and previous exposure. This raises the need for a universal serodiagnosis which can differentiate exposed animals from vaccinated ones. As the first step toward this goal, the following study was conducted to characterize serologic response of young pigs to SIV infection and vaccination.

Known positive serum samples used in the study were collected from several groups of pigs. Ten young crossbred pigs were experimentally inoculated with a H1N1 SIV field isolate (A/Sw/IA/40776/92) at a 10 rate of 1x10 EID50/0.2ml and bled on days 0, 7, 14, and 28 post-inoculation (PI). In addition, 32 serum samples were obtained through sequential bleeding from 12 pigs exposed to a H3N2 SIV isolate 7 (A/Sw/IA/41305/98) at a rate of 1x10 EID50/0.2ml. To obtain vaccine-induced antisera, a group of pigs were vaccinated with one of the commercial bivalent SIV vaccines and sequentially bled over time. Known negative serum samples (n=56) were collected through sequential bleeding from 14 sham- inoculated control pigs in the studies described above. Each sample was tested for its HI activity against H1 or H3 reference strain to assess antibody response of the animals using 1:10 as the cut-off for positive. All sera were also tested using a commercial H1N1 SIV ELISA kit by following the manufacturer’s recommendation. In addition, viral protein specificity of antibody response was determined by Western immunoblotting technique using viral and cellular antigens separated under non-reducing condition. Baculovirus-expressed SIV recombinant proteins were employed as antigen when needed. Viral antigen- serum antibody reactions were assessed using goat anti-swine IgG or IgM antibody.

All inoculated animals had HI antibody to SIV of respective subtype on day 7 PI, ranging from 1:320 to 1:640 and were still seropositive at termination of the study (4 weeks PI) although HI antibody levels of exposed animals tended to gradually decline after 7 days PI. In contrast, ELISA antibody (IgG) against H1N1 SIV was not detectable at days 7 but 14 PI. SIV-specific antibodies detectable on day 7 PI were determined mainly to be IgM antibody, suggesting that IgG-based serologic tests may not be able to detect exposed animals at the early stage of infection.

With respect to the reactivity to viral proteins, both IgM and IgG antibody responses in experimentally infected pigs were initially directed to HA, NP and NS1 proteins of SIV regardless of subtype. Later IgG antibody specific for M protein and NP of SIV became dominant although IgG to HA continued to be detected. In comparison, vaccinated pigs did not develop antibody to NS1 protein during the study period. Collectively, NP appears to be the choice of viral antigen for a non-subtype restricted universal serologic assay. NS1 protein may be the antigenic basis for a serologic assay which can differentiate vaccinated animals from exposed ones.

1Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA 2Department of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, IA

*To be considered for graduate student award

147 Characterization of a Reassortant Human/Swine H1N2 Influenza Virus Isolated from Pigs in a U.S. Swine Herd

ML Gramer1*, CE Wees1, KD Rossow1, RJ Webby2, SM Goyal1

Many swine herds experience severe influenza-induced respiratory disease despite vaccination, the ramifications of which are poor performance in growing swine and herd loss. These continual losses are frustrating to producers and vaccine companies, both of whom need detailed characterization of SIV isolated from pigs that exhibit clinical disease but are well vaccinated. This need has prompted the veterinary diagnostic laboratories to develop molecular diagnostic assays for the detection and characterization of SIV.

Using these molecular tools, we have discovered a new reassortant H1N2 influenza in pigs from the Midwest. An influenza A virus was isolated in early November 2003 from three, 4-week-old pigs with respiratory disease. The virus was initially detected by a TaqMan™ RT-PCR test for the nucleoprotein (NP) gene in late October 2003 in pooled nasal swabs from these pigs received at the Minnesota Veterinary Diagnostic Laboratory (MVDL). The pigs were born to dams that had been vaccinated multiple times at regular intervals (pre-farrowing) with a commercially available, fully licensed, bivalent H1N1/H3N2 vaccine. Serotyping of the virus by hemagglutination inhibition test was non-diagnostic, having no reaction with antisera against A/Sw/IA/1973 H1 or A/Sw/TX/1998 H3N2. The isolate was identified as H1 when the first 900 nucleotides of the HA gene were sequenced and compared to reference H1 sequences in GenBank. There was an amino acid deletion at site 155. Comparison of the first 597 bases of the HA1 gene segment between this isolate and reference strains of classical H1N1 and reassortant H1N2 SIV showed less than 76% similarity. Until this time, most H1 sequences from MVDL SIV isolates were greater than 80% similar to the H1N1 swine reference strains in our analyses. Further phylogenetic analyses of the HA gene demonstrated high similarity (>90%) to H1 human influenza viruses circulating worldwide in 2002/2003. The NA gene was determined to be N2 of swine origin by a multiplex NA typing RT-PCR test conducted at the MVDL and by nucleotide sequencing and phylogenetic analyses at the St. Jude Children’s Research Hospital in Memphis, TN.

This virus is, therefore, considered a human/swine H1N2 reassortant virus. To our knowledge, this is the first description of an H1 human–like reassortant influenza virus to infect and cause clinical disease in swine in the U.S. The impact that this virus may have on the health of U.S. swine is currently unknown. Full genetic characterization of all eight RNA gene segments and serosurveillance of U.S. swine for specific antibodies to this reassortant are ongoing. If this virus persists, it may become yet another subtype of SIV for which there may be no effective commercially-available vaccine. Furthermore, because it possesses a human H1 gene, it may infect humans as well as swine, and the potential zoonotic implications should be considered.

1Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, St. Paul, MN 2St. Jude Research

*To be considered for graduate student award.

148 Nested PCR Detection and Duration of Porcine Circovirus Type 2 in Semen Samples from Naturally Infected Boars

Kathleen A. McIntosh1, John C.S. Harding2, John A. Ellis1 and Greg D. Appleyard1

A nested PCR (nPCR) protocol was applied to porcine semen to demonstrate the porcine circovirus type 2 (PCV2) shedding pattern and duration in naturally infected boars. Boar serum was tested for PCV2 antibodies by competitive ELISA (cELISA) prior to semen collection to confirm previous infection of boars with PCV2. Semen was collected over a period of 6 to 9.5 months from 43 boars representing 6 breeds, aged 8 to 36 months, housed in a commercial artificial insemination (AI) barn. Of the 903 semen samples collected, 30 samples (3.3%) were positive for PCV2 DNA by nPCR from 13 boars (30.3%; 3 Duroc and 10 Landrace). Boars shedding PCV2 DNA in semen ranged between 8 and 16.5 months of age; shedding was not observed in boars older than 16.5 months. The longest duration of PCV2-shedding from a single boar was 5 individual non-sequential collections spanning approximately 6.5 months. The results presented in this study confirm the sporadic and long-term shedding of PCV2 DNA in semen from naturally infected boars and suggest that boar age and breed may contribute to the persistence of PCV2- shedding in semen.

1 Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon. Canada. 2 Harding Swine Veterinary Service Inc, Humboldt. Canada.

* To be considered for graduate student award.

149 Ulcerative and Hemorrhabic Typhlocolitis in an Angus Heifer Associated with Natural Bovine Enterovirus Type-1 Infection

1U. Blas-Machado, 1M.J Boileau, 1J.T. Saliki, 1S.L. Caseltine, 2S.D. Goens, 1J. C. Duffy, 1R.D. Welsh

A 2-year-old, 7 months pregnant Aberdeen-Angus bovine presented with acute, progressive abdominal pain died within 10 hours of hospitalization. Significant post mortem findings were limited to the intestines. The serosa of the distal jejunum, ileum, cecum, and spiral colon were diffusely hyperemic. The contents of the spiral colon were bloody; its mucosal surfaces were dark red, with widely scattered foci of mucosal ulceration. At the ceco-colonic junction, there were multiple, discrete white foci covered by fibrinous exudate mixed with blood. Microscopically, the colon had multiple foci of hemorrhage, necrosis, and ulceration. There was moderate to severe depletion and lymphocyte necrosis of the submucosal lymphoid tissues of the colon and small intestine. The splenic white pulp was moderately depleted. Virus isolation from the intestinal lesions yielded a cytopathic virus, which was classified by electron microscopic examination as a member of the Picornaviridae family. Further characterization classified the virus isolate as bovine enterovirus type 1 (BEV-1). Additional testing of the colonic lesions by PCR was positive for BEV-1. No other significant pathogens (viral or bacterial) were detected. Our results suggest that BEV-1 isolated was associated with the lesions described; however, a direct cause- and-effect relationship was not established. Nevertheless, in the absence of other detectable pathogens, the clinical history, the pathological lesions, and the laboratory results obtained suggest that the BEV-1 isolated from this case could represent a virulent strain. To the best of our knowledge, this is the first report of intestinal lesions and localization in BEV-1 infection, and the first report of BEV-1 from North America.

1Oklahoma Animal Disease Diagnostic Laboratory, Oklahoma State University, Stillwater, OK. 2USDA, ARS, Beltsville, MD.

150 Bovine Viral Diarrhea Virus Persistent Infection in Two White-Tail Deer in Southeastern South Dakota

CCL Chase,1 LJ Braun,1 P Leslie-Steen,1 T Graham,1 D Miskimins1 and JF Ridpath.2

The role of wildlife reservoirs continues to be a major unknown factor in the epidemiology of bovine viral diarrhea virus (BVDV). Serological data indicates that a wide range of wild ruminants have BVDV antibodies. BVDV has been isolated from a mule deer in Wyoming. In this report we examine the gross, histological and virological findings of two cases of BVDV in white-tail deer in south eastern South Dakota.

Two white-tail deer were submitted to the Animal Disease Research and Diagnostic Laboratory in the fall of 2003 by the South Dakota Game Fish and Parks for chronic wasting disease (CWD) testing. Both animals were CWD negative. The first deer was submitted in October and was in good body condition. The second animal was submitted in December and was the past year’s fawn and was in poor body condition. The animals were necropsied and histopathology, viral antigen detection and virus isolation were performed. In addition the BVDV isolates were typed using polymerase chain reaction in both the Erns region and the 5'NCR.

The first deer had no remarkable gross lesions indicative of BVDV. There was an abscess in one lung. The BVDV FA on the lung was negative. A noncytopathic (NCP) BVDV was isolated from the lungs and the intestine. The ear notch was immunohistochemistry positive for BVDV antigen. The isolate was typed in the Erns region and was a BVDV genotype 2. 5'NCR indicated that it was a type 2a. The second animal was unthrifty and stunted. The animal had a gun shot wound and had multifocal mandibular abscesses and diarrhea. Necropsy of this animal revealed multifocal ulcers of the abomasum. Histologically the animal had lymphoid depletion of the spleen. The animal was FA positive for BVDV antigen and a NCP virus was isolated from the kidney, abomasum, and lung. Immunohistochemistry indicated the presence of BVDV antigen in the skin and all tissues tested. The virus was typed in the Erns region and was a BVDV genotype 1. Further genetic in the 5'NCR indicated the virus was a type 1b.

This report is significant because it is the first report of possible persistent infection in deer. This would represent a serious problem for any cattle operation with deer contact. The implications of these BVDV isolations on biosecurity and prevention programs are alarming as the white-tail deer as a reservoir represents a huge risk to cattle. This is the third report of BVDV in deer in North America and the first report of multiple isolations of both genotypes.

1Department of Veterinary Science, South Dakota State University, Brookings SD 57007; 2National Animal Disease Center, Ames Iowa 50010

151 Bovine Viral Diarrhea Virus (BVDV) Subtypes in Diagnostic Laboratory Accessions from Clinical and Necropsy Cases: Distribution of BVDV1a, 1b, and 2a Subtypes

Robert W. Fulton1, Julia F. Ridpath2, Sharon Ore1, J.T. Saliki1,3, Lurinda J. Burge1, and A.W. Confer1

Samples submitted to the Oklahoma Animal Disease Diagnostic Laboratory (OADDL) which were positive for bovine viral diarrhea virus (BVDV) were subtyped by differential PCR and a region of the 5’UTR sequenced. These BVDV samples included supernatants of viral isolation positives from cases representing serums, whole blood with EDTA, nasal swabs and necropsy samples. Submittal forms indicated a wide variety of requests: testing for persistent infections; clinical illness (not specified) yet request for BVDV diagnosis in differential; respiratory disease; digestive tract disease; systemic illness suggestive of Mucosal disease (MD); fetal disease including abortions; and request for BVDV testing from necropsy cases, primarily with respiratory and/digestive tract lesions. These results are in addition to prior studies reported in 1998/1999. There were 63 isolates from 1996-2002 in this study subtyped to date out of 165 positive samples: 14/63 (22.2%) were BVDV1a; 29 (46.0%) BVDV1b; and 20 (31.7%) BVDV2a. Reference and vaccinal strains were included in the assays for comparison.

1Department of Veterinary Pathobiology 2USDA, ARS, NADC, Ames, IA 3Oklahoma Animal Disease Diagnostic Laboratory, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK

152 Genetically Identical Bovine Virus Diarrhea Viruses Isolated from an Aborted Ovine Fetus and a Yearling Hereford Heifer on the Same Premises

B Thompson1, E Dubovi2, B Njaa3, JF Ridpath4

Bovine virus diarrhea virus (BVDV) has been associated with reproductive failure in bovines and caprines. Cattle persistently infected (PI) with BVDV are a source of spread of the virus to susceptible animals. While there has been serological evidence of BVDV infection in ovines, BVDV has not previously been isolated from naturally infected ovine fetuses, nor has the virus been associated with reproductive loss in sheep. In addition, the source of BVDV infection in an outbreak of BVD is often not documented. This report contains evidence of BVDV infection associated with abortion/stillbirth of lambs and supports the suspected epidemiological relationship of a PI heifer with the abortion outbreak.

At the outset of the 2001 spring lambing season, a flock of sheep consisting of 17 adult sheep co-mingled with one goat and two head of beef cattle, experienced an outbreak of near term abortion or stillbirth with losses of 7 fetuses and one mummified fetus over a 3-day period. All of the fetuses and 3 placentas were submitted for diagnostic work-up. The sheep herd was a resident flock, including the goat wether, with a newer ram purchased the previous season. One of the cattle was an older beef cow that had been on the farm for many years. The other was a yearling heifer purchased about 5 1/2 months prior to the fetal losses. She was pastured with one-half of the breeding flock until just prior to expected lambing.

Tissues from two of the fetuses were examined by gross necropsy, histopatholgy, bacteriology, virology and immunohistochemistry. No gross pathological abnormalities were noted for either lamb. One lamb exhibited no significant histopathological lesions. Histopathological lesions in the second lamb consisted of a mild multifocal leukoencephalomalacia with calcification and minimal nonsuppurative encephalitis. The associated placenta exhibited mild placentitis with mild mineralization. BVDV was detected by fluorescent antibody test, immunohistochemistry, and virus isolation from tissues of the latter fetuses. There was no evidence of other pathogens associated with ruminant reproductive loss, including Leptospira spp, Campylobacter fetus, Chlamydia spp, Toxoplasma sp, other aerobic bacteria, or viruses.

Four weeks after the abortions, all adult ruminants were bled and samples were submitted for BVDV serum neutralization (SN) (Types 1&2). All had positive titers to BVDV Type 1, and all but the yearling beef heifer had positive titers to BVDV Type 2. The BVDV serum microplate isolation was positive for the yearling heifer. Her positive test result was confirmed using the BVDV antigen capture ELISA test, and the virus was grown by isolation techniques. The viruses isolated from both the heifer and the lamb were submitted for nucleotide sequencing for further characterization. Results indicated that they were identical BVD Type 1b viruses.

The remainder of the lambs born appeared healthy and thrifty. When the youngest reached 3 months of age, all were bled and their serum tested by BVDV SN and BVDV microplate isolation. All 11 lambs had positive titers to BVDV Type 1, and some had positive titers to BVDV Type 2. None of the lambs had a positive BVDV microplate isolation; persistent infection with BVDV was not demonstrated.

This case demonstrated that BVDV can be an important pathogen associated with sheep reproductive loss, especially if sheep are commingled with cattle that have not been screened for BVDV PI. Epidemiological evidence tying the outbreak to the introduction of a PI heifer is compelling.

1,2,NYS Animal Health Diagnostic Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, NY 3Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 4National Animal Disease Center, Agricultural Research Service, USDA, Ames, IA

153 Viral Antigen Distribution in the Respiratory Tract of Cattle Persistently Infected with Bovine Viral Diarrhea Virus Subtype 2a

A. W. Confer1, R. W. Fulton1, D. L. Step2, B. J. Johnson3, J. F. Ridpath4

Tissues were obtained at necropsy from the nasal vestibule, turbinates, nasopharynx, trachea, tracheobronchial bifurcation and lung from each of ten clinically healthy calves persistently infected (PI) with bovine viral diarrhea virus (BVDV) serotype 2a. Tissues from the nasal vestibule were obtained by biopsy from five additional PI calves. Formalin-fixed tissues were processed for immunohistochemistry to localize the distribution of BVDV throughout the respiratory tract. Antigen distribution and intensity were subjectively evaluated. Throughout the respiratory tract, mononuclear leukocytes, vascular smooth muscle, and endoneural and perineural cells had BVDV immunoreactivity (BVDV-IR). Multifocally, squamous and ciliated columnar epithelium throughout the respiratory tract contained weak to moderate BVDV antigen. Viral antigen was not seen in goblet cells. BVDV-IR in mixed tubuloalveolar glands of the nasal cavity was weak to strong in serous secretory cells and ductular epithelium. Chondrocytes of the concha often diffusely contained BVDV antigen. Nasal mucus secreting and tracheobronchial glands multifocally contained weak viral signal. In all cases, alveolar macrophages had moderate to strong BVDV-IR, whereas BVDV-IR in alveolar epithelial cells was weak to moderate. BVDV was present in interalveolar leukocytes and mesenchymal cells. Results indicate that serous secretions of the nasal cavity, productive viral infection of epithelium, and infected leukocytes in respiratory secretions are likely major sources of infectious BVDV from PI calves. The presence of BVDV antigen in respiratory epithelium is, at least, indirect support for the notion that this virus predisposes PI cattle to secondary microbial infections.

1Department of Veterinary Pathobiology, Oklahoma State University, Stillwater, OK 2Department of Clinical Sciences, Oklahoma State University, Stillwater, OK 3The Oklahoma Animal Disease Diagnostic Laboratory , Stillwater, OK 4The National Animal Disease Center, USDA – ARS, Ames, IA

154 Comparison and Optimization of High Throughput Viral RNA Isolation Methods for FMDV

A.G. Giuffre1, J.M. Robida1, X. Fang2, M.A. Kenney1, T.S. McKenna1, B.M. Martin3, T.R. Beckham1

Foot-and-mouth disease virus (FMDV) is a highly infectious, single-stranded, positive sense picornavirus in the genus Apthovirus which infects cloven-hoofed animals, and leads to significant economic impacts through restricted international trade and production losses. A recent outbreak of FMDV in the United Kingdom resulted in an estimated cost to the UK economy of $15 billion. In the event of an FMDV outbreak in the U.S., a high throughput detection method would be critical for rapid diagnosis, implementation of proper control measures, and recovery from disease. Current diagnostic assays are time consuming and laborious. Recently, real-time reverse transcriptase PCR (rRT-PCR) assays have been developed and validated for FMDV detection. These assays have proven extremely sensitive and specific. In order to adapt these assays to a high throughput format, an efficient 96-well RNA extraction procedure is needed.

Five RNA extraction methods, Trizol (Invitrogen, Carlsbad, CA), Qiagen RNeasy mini kit (Hilden, Germany), Roche Applied Biosystems MagNA Pure Total Nucleic Acid Isolation Kit (Indianapolis, IN), Ambion RNAqueous single tube (Austin, TX), Ambion RNAqueous-96, were evaluated for efficiency, repeatability, ease of use for high throughput applications, and adaptability to automation.

RNA was extracted by all five methods, in duplicate, from 32 oral swabs obtained from FMDV experimentally infected bovine and swine. Extraction efficiency was evaluated by rRT-PCR using the MX 4000 (Stratagene, La Jolla, CA). Comparison of the extraction methods by rRT-PCR indicated that the RNAqueous-96, MagNA Pure, and the RNeasy mini kit were equivalent in extraction efficiency. There was no statistically significant difference among the Ct values from the 32 samples using these three methods (p<0.05). The RNAqueous single tube and Trizol extraction methods were less efficient and consistently gave higher Ct values in the rRT-PCR assay.

Among the extraction methods that performed most efficiently, only the RNAqueous-96 and MagNA Pure were capable of high throughput viral RNA extraction. Upon comparison of these two extraction methods, the MagNA Pure resulted in a higher number of false negative extractions and had more variability among replicates. Of the 32 samples extracted in duplicate, the MagNA Pure kit failed to efficiently extract three known positive samples (each replicate from these samples had no Ct value) and failed to extract one replicate of a duplicate pair.

Because the Ambion RNAqueous-96 assay proved to be a more efficient and reliable extraction method, it was chosen for further optimization. Sample volumes and extraction buffers were optimized for four different sample matrices (oral and nasal swabs, epithelium, and milk). Minor adaptations of the procedure such as initial sample input and the addition of Ca++ chelators were needed depending on the sample matrix.

In conclusion, the Ambion RNAqueous-96 well format proved to be the most efficient, versatile, and reliable method for high-throughput sample extraction. Using this method, a single person, without automation, could extract approximately 1200 samples in one 8 hour shift.

1United States Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services, National Veterinary Services Laboratories, Foreign Animal Disease Diagnostic Laboratory, Greenport, NY. 2Ambion, Austin, TX 3United States Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services, National Veterinary Services Laboratories, Diagnostic Bacteriology Laboratory, Ames, IA.

155 Malignant Catarrhal Fever-like Disease in Sheep Following Intranasal Inoculation with Ovine Herpesvirus 2

H. Li1, D. O'Toole2, O. Kim1, J. L. Oaks3, and T. B. Crawford3

Malignant catarrhal fever (MCF) is a fatal disease syndrome primarily of ruminant species such as cattle, bison and deer, characterized by high fever, inflammation, ulceration, and exudation of the oral and upper respiratory mucous membranes, and sometimes eye lesions and nervous system disturbances. The disease is caused by one of several rhadinoviruses belonging to the gammaherpesvirus subfamily. Two major epidemiological forms of MCF exist, referred as wildebeest-associated MCF and sheep-associated MCF, each defined by the reservoir ruminant species from which the causative virus arises. Majority of MCF cases in the U.S. are sheep-associated MCF caused by ovine herpesvirus 2 (OvHV-2). Virtually all sheep are subclinically infected with the virus under natural flock conditions and serve the source for viral transmission. Spontaneous cases of MCF-like disease in sheep have been suspected but never confirmed. In the course of conducting experimental transmission of OvHV-2 in domestic sheep, an MCF-like syndrome was induced which is reported here.

MCF-like disease was induced experimentally in three sheep following aerosol transmission. Each of three OvHV-2 negative sheep was nebulized with 2 ml of nasal secretions containing approximately 3.7 x 109 OvHV-2 DNA copies from a sheep experiencing an intensive viral shedding episode. OvHV-2 DNA became detectable by PCR in the peripheral blood leukocytes of all sheep within 3 days post- aerosolization (PA), and animals seroconverted between 6 and 8 days PA. The sheep developed clinical signs with severe nasal discharges and fever at 14 days PA. One of three clinically affected sheep was euthanized at 18 day PA. The major lesions at necropsy were multifocal linear erosions and ulcers in mucosa of the cheeks, tongue, pharynx, and proximal esophagus, and mild disseminated pneumonia. Microscopically, there was extensive moderate superficial histiocytic lymphocytic rhinitis, with epithelial hyperplasia, disorganization, and degeneration. Moderate multifocal histiocytic bronchointerstitial pneumonia was associated with loss of terminal bronchiolar epithelium. Vasculitis was present only in the lung. The study revealed that clinical signs and lesions resembling MCF can develop when uninfected sheep are exposed to a high dose of the virus.

1Animal Diseases Research Unit, United States Department of Agriculture-Agricultural Research Service, Washington State University, Pullman, Washington 99164. 2Wyoming State Veterinary Laboratory, 1174 Snowy Range Road, Laramie, Wyoming 82070. 3Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington 99164.

156 Avian Paramyxovirus Infection -1 in Pigeons – A Retrospective Study

H. L. Shivaprasad1, G. Cooper2, F. Uzal3, P. Woolcock1, S. Hietala4, B. Crossley4, B. Charlton2, F. Sommer2, R. Crespo1, R. Chin1, M. McFarland1, L. Woods4, M. Anderson4, H. Kinde3, B. Daft3, J. Odani3, D. Read3, A. Bickfordy2, P. Hullinger5 and B. Barr4

Laboratory records of 100 pigeon submissions to the California Animal Health and Food Safety Laboratory System between 1990 -2004 diagnosed with Avian Paramyxovirus -1 were reviewed. The number of birds examined totalled 277 and was primarily birds raised for squab production and racing but also included feral pigeons and a few show-type pigeons. The data included both males and females of all ages.

Reported clinical signs included torticollis, opisthotonus, paralysis of legs and wings, greenish watery diarrhea, anorexia, ill thrift and increased mortality. Gross lesions included greenish watery contents in the intestine, pale and enlarged kidneys with increased urates, enlarged and mottled pale or red pancreas, and enlarged spleens. Microscopically, the most consistent lesions were non suppurative encephalomyelitis, interstitial nephritis, pancreatitis, enteritis and splenitis. Immunohistochemistry done on a few tissues revealed mild to intense intracytoplasmic staining for Avian Paramyxovirus -1 antigen in the cytoplasm of tubular epithelial cells of the kidney, glial cells, ependymal cells and neurons, including dendrites in the brain, and mononuclear inflammatory cells in the pancreas, intestine, and spleen, myocardium and medullary cells of the adrenal gland.

Avian Paramyxovirus -1 was isolated from brain, intestine or pooled tissues including liver, kidney, spleen and lungs. The fusion protein cleavage site from a few of these isolates and their sequences were similar to those from previously sequenced Pigeon Paramyxovirus isolates. Most of the sera tested were positive for hemagglutination inhibition antibodies to Avian Paramyxovirus -1.

1Fresno, 2Turlock, 3San Bernardino and 4Davis branches. California Animal Health and Food Safety Laboratory System, School of Veterinary Medicine, University of California, Davis. 5California Department of Food and Agriculture, Animal Health Branch, Sacramento, CA.

157 Isolation of Equine Influenza Virus from Racing Greyhounds with Fatal Hemorrhagic Pneumonia

E.J. Dubovi1, P.C. Crawford2, R.O. Donis3, W.L. Castleman4, Iain Stephenson3, and E.P.J. Gibbs4

Recurrent outbreaks of severe respiratory disease characterized by coughing and fever have occurred in greyhounds at racing kennels in Florida in recent years. Most affected dogs recover, but many succumb to a fatal hemorrhagic pneumonia. Investigation of a respiratory disease outbreak in racing kennels in Jacksonville, Florida in January 2004 focused on 22 dogs with clinical signs of cough and high fever. Eight dogs subsequently died from hemorrhagic pneumonia. There are no reports documenting the cause of these acute respiratory disease outbreaks in racing greyhounds. The objective of this work was to isolate and identify the etiological agent(s) responsible for the fatal hemorrhagic pneumonia syndrome in racing greyhounds in the Jacksonville 2004 outbreak.

Lung tissues from 5 of the dogs that died from the hemorrhagic pneumonia syndrome were subjected to virus isolation studies. Lung homogenates were prepared in tissue culture media containing 0.5% bovine serum albumin and antibiotics for inoculation of several cell lines. The cell lines included African green monkey kidney epithelial cells (Vero), Madin-Darby canine kidney epithelial cells (MDCK), primary canine kidney epithelial cells, primary canine lung epithelial cells, primary bovine testicular epithelial cells, canine tumor fibroblasts (A-72), and human colorectal adenocarcinoma epithelial cells (HRT-18). The inoculated MDCK and HRT-18 cells were overlayed with serum-free medium containing 2.5 ug/mL trypsin.

Cytopathology was noted in the MDCK cells on the first passage of lung homogenate from one of the dogs. Loss of cytopathology upon subsequent passage to cells cultured without trypsin coupled with the presence of hemagglutinating activity in culture supernatants suggested the presence of an influenza virus. The virus was initially identified as influenza virus by PCR using primers specific for the matrix gene. Sequence analysis of the viral genome confirmed that the canine isolate is a H3N8 influenza virus closely related to the A/equine/Wisconsin/2003 strain. The hemagglutinin protein of the canine isolate differs from the equine Wisconsin 2003 strain by 6 amino acids. The canine influenza virus has been designated as the A/canine/Florida/43/04 strain.

Immunohistochemistry with a monoclonal antibody against human H3 demonstrated influenza antigen in bronchial gland epithelial cells, bronchial and bronchiolar epithelial cells, and in alveolar macrophages of 4 of the 5 dogs. Seroconversion was demonstrated by hemagglutination inhibition and microneutralization assays using the canine influenza virus and acute and convalescent sera from greyhounds exposed to the dogs that died.

Based on virus isolation from the lungs, the presence of viral antigens in lung tissues by immunohistochemistry, and seroconversion data, we conclude that the novel influenza virus was most likely the etiological agent responsible for the fatal hemorrhagic pneumonia in racing greyhounds during the Jacksonville 2004 outbreak. This represents the first report of an equine influenza virus associated with respiratory disease in dogs.

1 Department of Population Medicine and Diagnostic Science, Cornell University, Ithaca, NY 2 Department of Small Animal Clinical Sciences, University of Florida, Gainesville, FL 3 Influenza Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, GA 4 Department of Pathobiology, University of Florida, Gainesville, FL

158 Joint Plenary Session Monday, October 25, 2004 8:00 a.m. – 11:30 p.m. Guilford B

Animal Disease Surveillance in the 21st Century – Important Tools for Response, Protection of Public Health, and Trade

Moderators: Gary Osweiler and Rick Willer

8:00 a.m. Opening Remarks – Moderators

8:05 a.m. KEYNOTE ADDRESS Global Perspective - the World Organisation for Animal Health (OIE) – 161 Alex Thiermann

8:40 a.m. Importance of Surveillance to North America – Brian Evans 162

9:15 a.m. Emerging and Re-emerging Zoonotic Diseases – Importance of Veterinary 163 Surveillance for Protection of Public Health – Lonnie King

9:45 a.m. Break

10:00 a.m. Surveillance of Disease: Epidemiologic Perspectives - Wayne Martin 164

10:30 a.m. Characterization of the Recent U.S. BSE Case and Methods for Surveillance – 165 Juergen Richt

11:00 a.m. The Value and Risks of Surveillance for Avian Influenza – David Suarez 166

11:30 a.m. AAVLD HOUSE OF DELEGATES

Joint Plenary Session

ANIMAL DISEASE SURVEILLANCE IN THE 21ST CENTURY

Sponsored By

BIO-RAD LABORATORIES RMS, RESEARCH MANAGEMENT SYSTEMS

159

160 Global Perspective - the World Organisation for Animal Health (OIE)

Alex B. Theirmann International Organizations Coordinator USDA, APHIS & Office International des Epizooties Paris, France

161 Importance of Surveillance to North America

Brian Evans Chief Veterinary Officer, Canadian Food Inspection Agency Ottawa, Canada

162 Emerging and Re-emerging Zoonotic Diseases – Importance of Veterinary Surveillance for Protection of Public Health

Lonnie J. King Dean, College of Veterinary Medicine Michigan State University East Lansing, MI

163 Surveillance of Disease: Epidemiologic Perspectives

S. Wayne Martin1

Surveillance is a population-based process and could be viewed as a system with data inputs, internal massaging, organization and analysis of these data, and outputs of information. In simple surveillance systems, the process focuses on data describing the risk of occurrence of a single health outcome, disease, or agent (the “what”), “who” is affected, “where” the events occur, and how the risk varies with time (“when”). More elaborate systems collect additional data on factors potentially affecting these risks and thus allow for a more thorough understanding of the issues (the “why”), including the impacts (real or potential) of the disease/agent on the biologic and economic health of target species and industries. Indeed many surveillance systems are composed of a hierarchy of integrated systems with multiple “surveyed” outcomes, and the effective and efficient design and operation of the system requires knowledge from a variety of disciplines and perspectives.

Laboratory evidence provides crucial information to surveillance systems about the presence or absence of an agent, and an epidemiologic perspective can help decide the optimal allocation of laboratory resources, the best way(s) to obtain representative sample specimens for the laboratory scientist, and the amount of co-variate information needed, consistent with the objectives of the system. Molecular and automated techniques have greatly enhanced the role that laboratories can play in surveillance, and recent advances in epidemiologic techniques have improved the conversion of data to information. This paper discusses issues of importance to achieve maximum value from the integration of laboratory and epidemiologic activities in surveillance systems.

1Department of Population Medicine,Ontario Veterinary College,University of Guelph,Guelph, Ontario, CANADA N1G 2W1

164 Characterization of the Recent U.S. BSE Case and Methods for Surveillance

Jürgen A. Richt1, John P. Kluge2, David P. Alt1, Robert A. Kunkle1, Amirali N. Hamir1, Stefanie Czub3, Arthur J. Davis2, and S. Mark Hall2

Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy of cattle, first detected in 1986 in the U.K. and subsequently in other countries. Here we report on the prion protein polypeptide profile and genotype from the first case of BSE diagnosed in the United States. The six-year old Holstein cow, imported into the State of Washington from Canada in 2001, was nonambulatory at slaughter. The formalin-fixed obex area of the brainstem was found to contain spongiform changes by histopathology and extensive deposition of the abnormal form of the prion protein, PrPres, by immunohistochemistry. Western blot analyses and an enzyme-linked immunosorbent assay using brainstem and cerebellum derived from fresh tissue from the suspect animal revealed positive results. Comparison of the U.S. BSE isolate to the Canadian BSE isolate and European BSE isolates showed similar sized PrPres polypeptide fragments. In addition, the PrP gene from the U.S. BSE case was found to be of bovine origin with a normal cattle PrP sequence. We conclude from these studies that the PrPres profile from the first BSE case diagnosed in the U.S. showed similar molecular properties to the typical PrPres pattern described for the earlier Canadian and European BSE isolates, and that a germline mutation in the bovine PrP gene was not evident. In addition, the sampling and testing methods implemented for the enhanced surveillance program for BSE in the United States will be discussed.

1Virus and Prion Diseases of Livestock Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, Ames, Iowa, 50010, USA; 2Pathobiology Laboratory, National Veterinary Service Laboratory, USDA, Animal and Plant Health Inspection Agency, Ames, Iowa, 50010, USA; 3National BSE Reference Laboratory, Canadian Food Inspection Agency, Winnipeg, Manitoba R3M 3M4, Canada

165 The Value and Risks of Surveillance for Avian Influenza

David Suarez1

Avian influenza virus (AIV) is well known for having a broad host range, including humans, and for its high mutation rate. It also causes a wide range of disease in our domestic poultry species, including asymptomatic infections, mild respiratory disease, serious respiratory disease with some mortality, and systemic disease with high mortality. The reason for this wide range of clinical disease is related to several factors, including how well a virus is adapted to the species in question. Avian influenza is normally an infection of wild birds including ducks, gulls and shorebirds, and from this wild bird reservoir the virus can spread to our domestic poultry species. AIV can also be well adapted for one species and not for another and this can result in a virus causing serious disease for one species and be asymptomatic in another species. For example, highly pathogenic avian influenza causes high mortality in chickens, but in ducks it often causes no clinical disease, although the ducks can be infected and shed virus. This variability in disease expression can complicate disease eradication efforts, since clinical disease can’t always be used to identify infected poultry flocks. In general the methods for determining prevalence of infection for AIV can be summarized for three distinct avian populations, wild birds, large commercial poultry operations, and small commercial poultry operations. The virus is endemic in wild birds, and during parts of the year a high percentage of certain birds will be actively infected, for example, mallard ducks in the fall of the year. For most wild bird surveillance programs, efforts are made to isolate and characterize avian influenza viruses to better understand the ecology of the virus. For our large commercial poultry sector, AIV infections are rare, with relatively few outbreaks occurring each year. However, extensive testing is performed for AIV, including both passive and active surveillance. Most of the active surveillance, primarily using serologic testing, is performed to satisfy requirements for export of poultry or poultry products or through programs like the National Poultry Improvement Plan. During outbreaks of AIV however, direct detection methods such as virus isolation or RRT-PCR are necessary to identify actively infected flocks so that control measures can be used. The most complicated area for surveillance is with the smaller commercial poultry industry, which includes live bird markets (LBMs), gamebird producers, gamefowl owners, hobby farmers, and organic growers. The incidence of AI infection in these groups are higher for several reasons, including the increased opportunity for gallinaceous and waterfowl to be on the same property (ex. LBMs), greater exposure of to wild birds, and often lower biosecurity standards. The many small flocks are also not well documented and the flock owners typically do not have a history of working with state officials in surveillance programs. Efforts to reach out to this population of poultry growers is increasing, since this is considered to be the most likely source of introduction of viruses to the large commercial poultry sector, and active and passive surveillance programs are increasing for this segment of the poultry industry.

1Southeast Poultry Research Laboratory, ARS/USDA Athens, GA 30605

166

Poster Session 47th Annual Meeting American Association of Veterinary Laboratory Diagnosticians Saturday October 23, 12:00 p.m. through Monday, October 25, 1:00 p.m. Guilford Prefunction Area 3rd Floor

No. Poster Title Page

1 Effects of Incubation Temperature, Decontamination Method and Regular Shaking on 171 Diagnostic Performance of a New Liquid Culture Method, the TREK ESP Culture System II and para-JEM Broth, for Detection of Mycobacterium avium subsp. paratuberculosis in Fecal Samples - C. van Maanen, M. G. J. Koene, V. Oosterhuis, T. von Banniseht, S. E. Allen, and N. M. Sullivan

2 In Vitro Evaluation of a Phenolic Disinfectant’s (Environ LpH®) Effect on Chronic Wasting 173 Disease-associated Prion – K.L. Cramer, J. E Jewell, C. T. Larsen, L. A. Baeten, and M. W. Miller

4 Necropsy Findings and Arbovirus Surveillance in Mourning Doves (Zenaida macroura) from 175 the Southeastern United States - R. W. Gerhold, C. M. Tate, S. E. Gibbs, D. G. Mead, A. B. Allison, and J. R. Fischer

5 Use of a Microtiter Serum Neutralization Test to Detect West Nile Virus Antibodies - A.L. 176 Glaser

6 Comparison of the Polymerase Chain Reaction (PCR), Virus Isolation and Electron Microscopy 177 for the Diagnosis of Orf Virus Infections - J. Guo; L.L. Logan and A. de la Concha-Bermejillo

7 Development of a Real-time PCR Panel for Detection of Ruminant Endemic Diseases That 178 Mimic Foot and Mouth Disease - K. Homb, D. Krueger, P. Bochsler, A. Brower, R. Porter, K. Woods and K. Toohey- Kurth

8 Evaluation of the BACTEC® 960 MGIT™ System for Growth of Mycobacterium bovis - N. 179 Hines* and J. Payeur

9 Wasting and Mortality in Beef Cattle Caused by Eurytrema coelomaticum in Southern Brazil 180 M.R.S. Ilha, A.P. Loretti

10 Effect of Hexadecylpyridium Chloride (HPC), Vancomycin and Natamycin on the Growth of 181 Mycobacterium avium subsp. paratuberculosis - K. A. Johansen, E. E. Hugen and J.B. Payeur

11 Diagnosis of Clostridium spiroforme in a Rabbit - D. Jordan, T. Klinefelter, J. Kinyon 182

167 12 Development and Validation of a PCR Test to Detect Lawsonia intracellularis in Tissues and 183 Feces from Swine - G.K.A. Josephson, H.Y. Cai, P. Bell-Rogers, M. Archambault, B. McEwen, M.G. Maxie

13 Antimicrobial Susceptibility Profiles of Recent Isolates of Mycoplasma bovis - Joann M. 184 Kinyon, Ricardo F. Rosenbusch, Lorraine J. Hoffman, and Michael Apley

14 BVDV Infection in Cell Culture-A Laboratory Disaster - J. G. Landgraf, J. V. Warg, L. G. 185 Koster, K. A. Eernisse, M. P. Emery, S. L. Swenson

15 Implementation of Strategy to Control Bovine Viral Diarrhea Virus in the United States - R. L. 186 Larson, D. M. Grotelueschen

16 Implementing the Standard: Personnel – Reading Between the Lines - B. Lawler and M. 187 Pedersen

17 Diagnostic Lab Connectivity and Electronic Health Certificates for Equids - K. Maher and J.A. 188 Facchiano

18 PFGE on Campylobacter jejuni from Animals-Patterns and Clusters - W. Manley, A. Boerger- 189 Fields, K.W. Mills

19 Papillomavirus-associated Basosquamous Carcinoma in an Egyptian Fruit Bat (Rousettus 190

aegyptiacus) - C. McKnight,* M. Kiupel, A. Wise, C. Howe, and R. Maes

22 Aldosterone and Progesterone Producing Cortical Adrenal Tumor in a Cat - I.D.R. Pardo*, 193 G.Johnson, A. Declue, and M. Kiupel

23 Expression of Leptospiral Immunoglobulin-like Protein by Leptospira interrogans and 194 Evaluation of Its Diagnostic Potential in a Kinetic Enzyme Linked Immunosorbent Assay - R.U.M. Palaniappan, Y-F. Chang, F. Hassan, S. P. McDonough, M. Pough, S. C. Barr, K. W. Simpson, H. O. Mohammed, S. Shin, P. McDonough, and R. L. Zuerner

24 Evaluation of lig-based Conventional and Real Time PCR for the Detection of Pathogenic 195 Leptospires – R.U.M. Palaniappan, Y-F. Chang, P. Harpending, S. P. McDonough, E. Dubovi

25 Bacterological Characterization of Respiratory Disease in Calves and Antimicrobial 196 Susceptibility - Y. Pineda, S. Mora, J. Santander

26 The Distribution of Mycobacterium avium subsp paratuberculosis in Minnesota Dairy Farms 197 Using Bacterial Culture of Fecal Pools. Raizman E.A*, S.J. Wells, C. Muñoz Zanzi, S.M. Godden

168

27 Detection of c-Kit (CD 117) on Cutaneous Mast Cell Tumors of Ferrets by 198 Immunohistochemistry - J.A. Ramos-Vara, M.A. Miller, G.C. Johnson, C.M. Loiacono, and M. Kundu

28 Immunohistochemical Detection of Tryptase in Cutaneous Mast Cell Tumors of Ferrets - J.A. 199 Ramos-Vara, M.A. Miller, G.C. Johnson, C.M. Loiacono, and M. Kundu

30 West Nile Virus Outbreak in Horses in North Dakota, 2002: A Characterization of the Equine 201 Cases - L.A. Schuler, M.L. Khaitsa, N. Dyer and C.L. Stoltenow

31 Characterization of a Porcine Teschovirus Isolated During an Emerging Disease Investigation in 202 Imported Pigs - J.V.Warg, J. G. Landgraf L. G. Koster, L. A. Anderson, S. L. Swenson, M. J. Yaeger

33 Disseminated Melanoma and Osteogenic Tumor in a Rabbit - F. Williams III*, C.M. Loiacono, 204 L. Bloomfield

34 Effect of Delayed or Prolonged Fixation on Immunohistochemical Detection of Bovine Viral 205 Diarrhea Virus in Ear-Notch Biopsies - M.A. Miller, .A. Ramos-Vara, S.B. Kleiboeker, R.L. Larson

35 Evaluation of SEM, SE75.3 and SE18.7 in ELISAS for Serum Antibody to Streptococcus equi - 206 J.F. Timoney, A. Graves, S. Muthupalani, and J. Morrow

36 Investigation of Acute Nephrosis in a Herd of Beef Cattle in Nebraska - L.G. Corbellini *, M. 207 Carlson, B. W. Brodersen, A. Doster, D.G. Rogers, D.J. Steffen

37 Neosporosis in Cows Vaccinated with a Neospora caninum Vaccine - L.G.Corbellini, D.G. 208 Rogers, D.J. Steffen, B.W. Brodersen, and D.R. Smith

38 Evaluation of a Multiplex PCR Assay for Identification of Mycobacterium avium subsp. 209 paratuberculosis from Liquid Cultures - K.S. Anklam, E.J.B. Manning, S. Sreevatsan, and M.T. Collins

39 Detection of Mycobacterium avium subsp paratuberculosis in Bovine Feces and Milk Using 210 Adiapure® Extraction Kit and Real Time Adiavet® PCR Kit – B. Blanchard, Y. Versmisse and B. Chevallier

169

42 Transfer and Optimization of a Single-tube CSFV Real-time RT-PCR Assay to a High- 213 throughput 96 Well Format - A.J. Eberling, B.M. Martin, T.S. McKenna , T.R. Beckham

43 Prevalence Study of Coxiella burnetii (Q-fever) in the United States Dairy Herds Based on Bulk 214 Tank Milk Testing by Trans-PCR - S.G. Kim, E.H. Kim, A.E. Cassano, E.J. Dubovi

44 Wildlife Disease Research at the APHIS National Wildlife Research Center - R.G. McLean, L. 215 Clark, M. R. Dunbar, and K. C. Vercauteren

*Graduate Student Competition

POSTER SESSION

Sponsored by

CEPHEID

170

Effects of Incubation Temperature, Decontamination Method and Regular Shaking on Diagnostic Performance of a New Liquid Culture Method, the TREK ESP Culture System II and para-JEM Broth, for Detection of Mycobacterium avium subsp. paratuberculosis in Fecal Samples

C. van Maanen1, M. G. J. Koene1, V. Oosterhuis1, T. von Banniseht1, S. E. Allen2, and N. M. Sullivan2

At the Animal Health Service in the Netherlands fecal culture for M. paratuberculosis is performed on a relatively large scale (600 samples per week). Routinely, fecal samples are decontaminated, and subsequently cultured on solid Löwenstein-Jenssen (LJ) medium in the presence of mycobactin. Cultures are inspected every four weeks up to 16 weeks, if necessary. When suspect growth is observed a Ziehl- Neelsen stain is performed. Ziehl-Neelsen positive cultures are confirmed by IS900 PCR. In previous studies, presented at the AAVLD meeting 2003, we evaluated the performance of a new liquid culture method, the TREK ESP para-JEM Culture System II, as compared with our standard method. In the first experiments, we found equivalent results for both culture systems with a strong reduction in time to detection for ESP. In a subsequent comparison between both culture techniques on 260 bovine fecal samples the LJ system demonstrated a higher sensitivity for detection of low shedders. However, in a subsequent experiment, weekly shaking of liquid cultures enhanced growth, resulting in a better sensitivity and reduction of time to detection by about a week. In the present study effects of incubation temperature and decontamination method with or without shaking were investigated.

Fresh bovine fecal samples were taken from - predominantly light - shedders (n=40) and from cattle originating from herds with a long culture negative history (n=48) and were cultured on LJ agar slants. In parallel samples were also cultured in ESP para-JEM culture bottles with a new poising buffer, para-JEM Blue, after decontamination as prescribed by the manufacturer (Cornell double incubation method), the AHS in-house method (1) or a double incubation method as described by Stabel et al. (2). For the shedder panel for each decontamination method also one ESP series was left undisturbed and one series was shaken vigorously once a week and placed back in the system. Additionally a duplicate ESP series, with and without weekly shaking and after decontamination as prescribed by the manufacturer, was cultured offsite (out of the instrument) at 37o Celsius. All samples detected by the system and all samples not yet detected by the system at the conclusion of the experiment (49 days) were further investigated via ZN and PCR methodologies.

For the shedder panel (n=40) 23 samples were positive on LJ, whereas 21 to 26 samples were detected by the ESP system as confirmed by PCR and ZN with lowest scores for the in-house decontamination method with shaking and highest scores for the prescribed decontamination method with shaking. Additionally a few PCR positive samples were found that were not detected by the system. For the prescribed decontamination method we confirmed in this study the previously observed reduction in Time To Detection (TTD) by weekly shaking. For the in-house and Stabel decontamination methods no difference in TTD was observed between stationary incubation and weekly shaking. Mean TTD was lowest for the in-house decontamination method (22 days), intermediate for the Stabel decontamination method with and without shaking (27 days) and highest for the prescribed decontamination method with stationary incubation (35 days). Offsite incubation at 37oC with and without shaking yielded an equivalent number of positives (n=27). The panel from culture negative herds (n=48) yielded surprising results for the in-house decontamination method. After decontamination according to the prescribed or the Stabel method, in each series six samples were detected by the system, all ZN and PCR negative, and only 2/6 with a suspect graph. However, after decontamination according to the in-house method 26 samples were detected by the system with 19/26 ZN positive and no samples PCR positive. False-positive results clustered strongly in one herd, and the identity of these acid-fast bacteria is still unclear.

171

In this study, the TREK ESP para-JEM Culture System II with the poising buffer, para-JEM Blue, yielded equivalent or slightly better results than the standard LJ system. A reduction in TTD after weekly shaking was confirmed for the prescribed decontamination method. Surprisingly this reduction was not found for two other decontamination methods. The in-house decontamination method yielded unexpectedly high numbers of false-positive results. The Stabel decontamination method combined the advantages of low numbers of false-positives with a relatively low TTD after stationary incubation, which is much more feasible than weekly shaking or any other shaking protocol. Incubation at 37oC did not significantly improve culture results. An extended diagnostic validation of the ESP system using the Stabel decontamination method is currently underway.

1Animal Health Service, The Netherlands 2TREK Diagnostic Systems, USA

172

In Vitro Evaluation of a Phenolic Disinfectant’s (Environ LpH®) Effect on Chronic Wasting Disease-associated Prion

K. L. Cramer1, J. E. Jewell2, C. T. Larsen1, L.A. Baeten1, and M. W. Miller1

We used an in vitro system to evaluate the effects of a phenolic disinfectant (Environ LpH®) on chronic wasting disease-associated prion (PrPCWD) in brain tissue from naturally-infected mule deer (Odocoileus hemionus). A suspension of homogenized brain tissue from CWD-infected mule deer was exposed to a 5% LpH solution for 15, or 30 min, then centrifuged; suspensions of the same homogenized brain tissue without LpH exposure, as well as a suspension of CWD-negative brain tissue, served as controls. After centrifuging, we examined pellets of various exposure groups by Western blot (WB) for evidence of banding typically associated with PrPCWD. LpH treatment did not affect WB performance. After 15 min exposure to 5% LpH, WB banding patterns were partially eliminated as compared to positive controls; after 30 min exposure, PrPCWD-associated bands were completely eliminated. Our findings are consistent with previous in vivo studies of LpH efficacy in inactivating scrapie-associated prion.1 Use of WB may be an efficient alternative to more time-consuming in vivo approaches for evaluating candidate prion inactivating compounds.

Reference: Ernst, D. and Race, R. 1993. Comparative analysis of scrapie agent inactivation methods. Journal of Virological Methods 41: 193-202.

1Colorado Division of Wildlife, Wildlife Research Center, Fort Collins, Colorado USA. 2Department of Veterinary Sciences, University of Wyoming, Laramie, Wyoming USA.

173 Clinical, Virology, Histopathology, Serology, and PCR Findings of a Fatal Eastern Equine Encephalomyelitis Infection in a Puppy

M.D. Farrar1, D.L. Miller1, S.L. Stiver1, C.A. Baldwin1, and C.L. Hall2

Eastern Equine Encephalitis Virus (EEEV) is an Alphavirus in the Togaviridae family and the cause of Eastern Equine Encephalomyelitis (EEE). EEEV is endemic in the Southeastern United States and in sporadic locations along the entire eastern US seaboard. This disease is primarily observed in horses, but has been reported in pigs, cows, goats, humans, numerous avian and selected wildlife species. Herein we describe the clinical and diagnostic findings in 1 of 10 fatal cases of EEE in a puppy.

A male, 17-week-old, Boxer puppy with a 4 day history of depression was presented to the referring veterinarian for clinical evaluation. The puppy was in lateral recumbency with a temperature of 104.1oF, reduced menace response and nystagmus. Supportive care was instituted but the puppy died 3 hours later and a necropsy was performed. Significant gross changes were not noted. Tissues were collected and submitted to the Tifton Veterinary Diagnostic and Investigational Laboratory (Tifton VDIL) for diagnostic examination.

Histopathological examination revealed perivascular infiltrates of lymphocytes, plasma cells, macrophages and neutrophils mainly concentrated in the cortex and midbrain regions. Mild to moderate infiltrates were noted within the meninges. Multiple randomly distributed foci of primarily degenerate neutrophils, occasionally associated with necrosis, were also observed as well as multifocal areas of mild gliosis. Serology tests for EEEV antibodies, utilizing a perimortem serum sample, revealed a 1:8 serum neutralization titer. Virus isolation on fresh brain tissue was performed with two different mammalian cell lines and a virus was recovered that was determined to be EEEV by indirect fluorescent antibody tests. Brain homogenates and virus isolate cell culture was processed for PCR testing and both samples demonstrated bands positive for EEEV.

Over a period of 7 years, 31 cases of clinical neurological disturbances in dogs were examined at the Tifton VDIL. Of these, 10 were diagnosed as fatal EEE and were confirmed by histopathological examination coupled with virus isolation. All cases were puppies less than 6 months of age with no breed predilection. This case indicates the possible susceptibility of puppies to EEEV when exposed to infected mosquitoes in endemic areas of the US. Therefore, EEE infection needs to be added to the list of differential diagnoses for young dogs that present with neurological signs in regions where this virus is known to be indigenous.

1Veterinary Diagnostic and Investigational Laboratory, College of Veterinary Medicine, University of Georgia, Tifton, GA 31793 2Wolfe Animal Hospital, P.C. Jesup, GA 31545

174 Necropsy Findings and Arbovirus Surveillance in Mourning Doves (Zenaida macroura) from the Southeastern United States

R. W. Gerhold1, C. M. Tate1, S. E. Gibbs1, D. G. Mead1, A. B. Allison1, and J. R. Fischer1

Mourning doves (Zenaida macroura) are the most abundant and widespread native member of the columbid family, as well as a major migratory game species, in the United States. However, there is little information on non-hunting mortality factors in mourning doves. Records of necropsy accessions at the Southeastern Cooperative Wildlife Disease Study (SCWDS) from 15 southeastern states between 1971- 2003 were reviewed. In total, 131 mourning doves were submitted from eight states during the thirty-two year period. Trichomoniasis comprised 36.6 % (n=48) of all diagnoses and was the most frequent cause of mortality. Toxicoses and poxvirus infection comprised 26.7 % (n=35) and 15.3% (n=20) of diagnoses, respectively. The remaining diagnoses included trauma, bacterial infection, Ascaridia columbae infestation, and undetermined. Adults were observed more frequently with trichomoniasis (89.5%) and toxicoses (68%) as compared to juveniles, but a gender predisposition was not apparent for either disease. Age and gender predilections were not apparent for diagnoses of poxvirus infection. The majority of the trichomoniasis (72%) and poxvirus infection (90%) cases were observed in the spring and summer, whereas the majority of the toxicoses cases (90%) were observed in the winter and spring. In addition to necropsy accessions, 761 mourning doves were submitted to SCWDS between 2000-2003 from Georgia and West Virginia for West Nile virus surveillance. West Nile virus was isolated from 1.7% (n=13) and Eastern equine encephalitis virus was isolated from 0.26% (n=2) of the submitted birds. The submission of cases was influenced by many known and unknown factors, and the data undoubtedly are biased. As such, these findings may not accurately reflect patterns of these diseases observed in natural conditions.

1Southeastern Cooperative Wildlife Disease Study, College of Veterinary Medicine, The University of Georgia, Athens, GA

175 Use of a Microtiter Serum Neutralization Test to Detect West Nile Virus Antibodies

A.L. Glaser1

Detection of Flavivirus-specific neutralizing antibody in serum has traditionally been done using a plaque reduction neutralization test (PRNT). This assay is time consuming and resource intensive to run as a diagnostic assay and is unpractical when dealing with large numbers of clinical samples. To facilitate higher throughput testing of samples for West Nile virus (WNV) neutralizing antibody, a microtiter serum neutralization test (MTSN) has been developed. The MTSN assay is as sensitive and specific as the PRNT test for the detection of equine serum-neutralizing antibody, but gives two to three fold higher endpoint values. The availability of the WNV MTSN test has allowed more precise quantitation of WNV neutralizing antibody titers in WNV exposed horses. Animals infected with WNV generally have significant WNV SN titers (>1:1024) at the onset of clinical signs. Comparison of WNV-specific and St Louis encephalitis virus (SLE) have to be performed by PRNT as SLE does not exhibit aggressive enough cytopathic effect for the microtiter format.

1Department of Population Medicine and Diagnostic Science, New York State Animal Health Diagnostic Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, NY

176 Comparison of the Polymerase Chain Reaction (PCR), Virus Isolation and Electron Microscopy for the Diagnosis of ORF Virus Infections

J. Guo1,2; L.L. Logan1 and A. de la Concha-Bermejillo1,2

Orf virus (ORFV), the type species of the parapoxvirus genus, is the causal agent of orf, a vesiculo- proliferative dermatitis of sheep, goats, wild ruminants and humans. In the majority of cases, the diagnosis of orf is made based on clinical signs and lesions. Because orf can be confused with other vesicular diseases of livestock, including foot and mouth disease, and other poxvirus infections, such as sheep pox and goat pox, confirmation of the clinical diagnosis by laboratory tests is highly recommended. Electron microscopy (EM) is the test most commonly used for the diagnosis of orf. However, the lack of an electron microscope by many veterinary diagnostic laboratories and the high cost of the test precludes the submission of orf cases for laboratory analysis. Virus isolation is highly specific but lacks sensitivity. The polymerase chain reaction (PCR) is a highly specific and sensitive diagnostic test that has been used for the diagnosis of many infectious diseases of humans and animals. A PCR test for ORFV based on the amplification of a specific DNA fragment of ORFV envelope gene has been developed, but this study did not determine if the PCR test could differentiate between ORFV and other poxviruses of small ruminants. The purpose of the present study was to develop a PCR based on the amplification of ORF virus interferon resistant (VIR) gene, a gene that is highly conserved among parapoxviruses but shares less than 20% nucleotide homology with the VIR of other poxviruses. To determine the sensitivity and specificity of ORF VIR gene PCR, 58 samples with a clinical diagnosis of orf and grounded scab from 5 ORFV vaccines were tested by PCR and the results compared to EM, and virus isolation. All 63 samples (100%) were positive by PCR compared to 62 (98.4%) by EM and 35 (55.5%) by virus isolation. To confirm the accuracy of PCR, the gene product of the PCR-positive, EM-negative case was sequenced and confirmed to correspond to the ORF VIR gene sequence. These results indicate that the sensitivity of the PCR based on the ORF VIR gene was 100% . Currently, goat and sheep poxvirus synthetic VIR genes are being constructed and will be used in the differential diagnosis of ORFV and other small ruminant poxviruses by PCR.

1Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843 2Texas Agricultural Experiment Station, San Angelo, Texas 76901

177 Development of a Real-time PCR Panel for Detection of Ruminant Endemic Diseases That Mimic Foot and Mouth Disease

K. Homb 1 D. Krueger 1, P. Bochsler 1, 2, A. Brower1, 2, R. Porter1, 2, K. Woods1 and K. Toohey- Kurth 1, 2

Real-time PCR has emerged as the assay of choice for rapid detection and concurrent confirmation. This presentation illustrates the progress made on a real-time PCR panel developed for ruminants with endemic diseases that mimic foot and mouth disease (FMD). The following viruses are causative agents of diseases that must be considered in a differential diagnosis: bovine viral diarrhea virus (BVDV), bovine herpesvirus 1 (BHV-1), bovine herpesvirus 2 (BHV-2), bluetongue virus (BTV), malignant catarrhal fever (MCF), orf, and papular stomatitis. Progress on all of the agents will be presented with the major focus of this poster on BHV-1 and BHV-2.

Multiple primer and probe sets were designed for BHV-1 and BHV-2 using Primer Express (Applied Biosystems, Foster City CA). Efficiency of each primer pair was determined and probes were designed for the most efficient primer pairs. Sensitivity of each set was determined using BHV-1 DNA or BHV-2 DNA as appropriate. The specificity of each pair was tested against reference strains of other members of the differential panel and common respiratory organisms.

The real time PCR assay for BHV-1 has a minimum detection level of 10-50 genome equivalents. There was no cross reaction with other members of the differential panel nor common respiratory pathogens. Validation of the assay for BHV-1 thus far includes 49 isolates determined as positive by viral isolation. The real-time PCR assay detected BHV-1 in 48 of the 49 positive clinical isolates. The real time PCR assay for BHV-2 was also sensitive to a level of 10-50 genome equivalents and specific in that there was no cross reaction with other members of the differential panel. Data obtained thus far shows that these primer pairs and probes for BHV-1 and BHV-2 are suitable for further validation with appropriate sample types such as swabs from skin or mucosal membrane lesions.

1Wisconsin Veterinary Diagnostic Laboratory, University of Wisconsin, Madison 53706 2 Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin- Madison 53706.

178 Evaluation of the BACTEC® 960 MGIT™ System for Growth of Mycobacterium bovis

N. Hines¹* and J. Payeur¹

Mycobacterium bovis is a fastidious, acid fast organism that is part of the M. tuberculosis complex. Isolation of Mycobacterium bovis from tissue lesions of animals suspected of having tuberculosis is routinely performed using BACTEC® Middlebrook 12B radiometric media, Middlebrook 7H10 and Middlebrook 7H11 media tubes. However, the BACTEC® 460 culture method is labor intensive and poses a risk of exposure to radiation. When Middlebrook 7H10 (M 7H10) and Middlebrook 7H11 (M 7H11) media are utilized, there is a much longer incubation time (up to 8 weeks), the contamination rate is much higher and fewer isolates are obtained in comparison to the BACTEC® 460 system. The purpose of this study is to compare the sensitivity and growth rate of the BACTEC® MGIT™ 960 liquid culture media, the BACTEC® Middlebrook 12B, M 7H10 and M 7H11 media for the isolation of M. bovis.

The BACTEC® MGIT™ 960 system consists of growth indicator tubes and a BACTEC® 960 instrument. Each tube consists of a fluorescent compound embedded in silicone on the bottom of the tube filled with culture media. Oxygen dissolved in the media quenches the fluorescence until bacterial growth uses up the oxygen and fluorescence is emitted and detected by the instrument. The instrument automatically monitors the fluorescence within each indicator tube every hour to determine the status of the culture.

Bovine lymph node samples were collected at different abattoirs in the United States and Mexico and characterized by histopathology as mycobacteriosis-compatible. The samples were processed using a standard sodium hydroxide-based method. For each processed sample, 0.5ml was inoculated into a MGIT™ tube that was then inserted into the BACTEC® 960 instrument and incubated at 37°C for six weeks. A BACTEC® 12B bottle, 1 tube each of M 7H10 and M 7H11 were also inoculated with each of the treated samples and were used as standards to compare growth sensitivity and rate of M. bovis. BACTEC® 12B bottles were incubated for 6 weeks at 37°C and the solid media was incubated for 8 weeks at 37°C. After six weeks of incubation (or when the BACTEC® 960 or BACTEC® 460 instruments indicate the presence of suspect positives) Ziehl Neelsen slides were prepared for each type of media to check for contaminants and confirm growth of acid fast positive rods. Samples containing acid fast rods were then confirmed as positive using a commercial assay for M. tuberculosis complex organisms (AccuProbe, Gen Probe®Incorporated, San Diego, CA). A niacin-nitrate biochemical test was used to distinguish M. bovis from M. tuberculosis isolates

Preliminary results indicate that there were 59/64 (92% sensitivity) M. bovis cultures isolated using the BACTEC® 960 MGIT™ system, 56/64 (88% sensitivity) M. bovis cultures isolated with the BACTEC® 12B and 49/64 (77% sensitivity) M. bovis cultures isolated using M 7H10 and/or 7H11. Based on these preliminary results, the BACTEC® 960 system using MGIT™ media appears to be over 2% more sensitive than the BACTEC® 460 culture method and over 15% more sensitive than either type of solid media tubes. The average time to detection for the BACTEC® MGIT™ 960 was 14.1 days, 28.5 days for the BACTEC® 12B and 46.4 days for the 7H10 and/or 7H11 solid media tubes. The BACTEC® MGIT™ 960 system has the additional advantage of being non-radioactive and requires a shorter incubation time than solid media. Based on these preliminary findings, the BACTEC® MGIT™ 960 culture system can be used in place of the BACTEC® 460 culture method for isolation of M. bovis.

1USDA/APHIS/VS/NVSL, Mycobacteria and Brucella Section, Ames, IA.

*To be considered for graduate student award

179 Wasting and Mortality in Beef Cattle Caused by Eurytrema coelomaticum in Southern Brazil

M.R.S. Ilha1, A.P. Loretti2

Eurytrema spp. is a trematode that occurs in the pancreatic ducts of cattle, buffaloes, camels, deer, goats, sheep, pigs and human beings. This pancreatic fluke has been found in Europe, Asia and South America. Infestation by Eurytrena spp. leads to chronic interstitial pancreatitis. In Brazil, E. coelomaticum was described for the first time in cattle in 1918. Since then, a few cases of E. coelomaticum parasitism in cattle, sheep, goats and buffaloes have been reported in the central, southern and southeastern regions of the country. A wasting disease has been affecting a small number of cattle in the northern region of Paraná State, southern Brazil, during the last 15 years. This area is endemic for pancreatic eurytrematosis. The disease is characterized by emaciation, recumbency and death despite adequate nutritional management and veterinary care.

The purpose of the present study is to describe the epidemiology, clinical picture, laboratory findings, gross lesions and histological changes of clinical cases of chronic wasting and death associated with pancreatic fibrosis caused by Eurytrema coelomaticum in brazilian beef cattle herds.

Progressive weight loss was the most common clinical sign. Loss of appetite was not observed. Animals were in poor body condition despite plenty of good quality forage was available. Annual losses in the affected herds ranged from 1 to 3%. The clinical course of the disease varied from 2 to 10 months after the onset of the first clinical signs. Sick cattle were humanely euthanatized in extremis. At necropsy, the carcasses were thin. The pancreas was of normal size, slightly enlarged and dark, or white, markedly firm, shrunken and with a shriveled capsular surface. Myriads of leaf-shaped trematodes of the genus Eurytrema were packed inside multiple dilated ducts which had thickened, whitish fibrous walls or were embedded in the pancreatic parenchyma. Microscopic findings included replacement of the glandular pancreatic tissue by extensive fibrosis associated with intralesional flukes and eggs and ductal hyperplasia. Inflammatory reaction varied from absent to severe and consisted of small granulomas composed of lymphocytes, macrophages and giant cells surrounding trematode eggs. One affected animal had high plasma amylase concentration (1580 U/L) suggesting exocrine pancreatic insufficiency. Glucose blood levels were not significantly increased. The diagnosis was supported by different feeding trials with mineral and vitamin supplements which did not improve the body condition of the affected cattle. Other causes of emaciation including gastrintestinal parasitic or infectious diseases, malnutrition due to scarcity of forage or dietary mismanagement, mineral deficiencies, and poisonous plants affecting the alimentary system, were all ruled out.

It is suggested that diffuse, marked fibrosis of the pancreas induced by a large number of pancreatic flukes is the cause of chronic wasting and death in these cattle herds in southern Brazil. The clinical picture and the pathological findings of the cases reported here are similar to those previously described in sheep in Asia also associated with severe parasitism by E. pancreaticum. and characterized by progressive weight loss, weakness, emaciation, recumbency and eventually death. The prevalence of this trematode infestation in cattle in the Paraná, southern Brazil, has been increasing over the last 3 decades. Severe parasitism by pancreatic trematodes in beef cattle in this area could be linked to the specific geographic distribution of the intermediate invertebrate hosts of the pancreatic fluke. The fact that the affected beef herds are kept in pastures suggests that grasses might provide a suitable environment for the development of the invertebrate hosts and maintenance of the trematode in the field.

1Rua Paissandu, 385, Unit 201, Rio de Janeiro, RJ, Brazil, 22210 080 2Department of Veterinary Clinical Pathology, Faculty of Veterinary Medicine, Federal University of Rio Grande do Sul (UFRGS), 91540-000, Porto Alegre, RS, Brazil.

180

Effect of Hexadecylpyridium Chloride (HPC), Vancomycin and Natamycin on the Growth of Mycobacterium avium subsp. paratuberculosis

K. A. Johansen1, E. E. Hugen1 and J.B. Payeur1

Johne’s disease is a chronic enteropathy caused by the intracellular pathogen Mycobacterium avium subsp. paratuberculosis (MAP). Clinical Johne’s disease has been reported worldwide from a wide variety of ruminants, including cattle, sheep, goats, deer, bison, and llamas. Animals are typically infected as neonates, although clinical signs, such as wasting, do not appear until 2-5 years later. This makes early detection of infected animals difficult. Diagnosis of Johne’s disease is also made difficult by intermittent shedding of the organism and a lack of correlation of an antibody response (measured by ELISA) with fecal shedding. USDA regulations state that an organism-based culture test is the official assay for determining the infection status of an animal. Many protocols for the isolation of MAP from feces start with a decontamination step that uses HPC as a chemical decontaminant. However, recent studies from this and other laboratories suggest that the use of HPC may be detrimental to the growth of MAP. Cellular damage sustained by MAP in the presence of HPC may also affect it susceptibility to antibiotics used for additional contamination control during the long culture period required for the isolation of this fastidious organism. We therefore developed a study to examine the effect of HPC on the growth of MAP in the presence of the antibiotics vancomycin and natamycin using a design of experiments approach. Vancomycin was chosen for the study because it is commonly used in the isolation of MAP to inhibit growth of contaminating soil bacteria. Natamycin, a fungicide widely used as a food preservative, was investigated to determine its potential as a tool to control fungal contamination that often occurs with fecal culture.

Stat-Ease® Design-Ease® software was used to design the study. This software was chosen because it randomizes the experimental set-up, thus eliminating user-bias, and allows for statistical analyses that will identify “confounding” interactions among the factors being tested. For this study, 27 different combinations of HPC, vancomycin and natamaycin were tested for their effects on the growth of MAP ATCC 19698 in 7H9 broth. Growth was monitored weekly by determining the absorbance at 600 nm and cell densities were verified by plate counts. Statistical analysis of the results using ANOVA indicate that HPC alone has a significant detrimental effect on the growth of MAP (p< 0.001), while natamycin and vancomycin have no effect. There also appears to be no synergistic effects between natamycin and vancomycin on the growth of MAP. We conclude that natamycin may be useful as an additional fungicide to control contamination during MAP isolation from fecal samples, but efforts to find alternatives for HPC as a chemical decontaminant, such as enrichment procedures, are warranted

1USDA/APHIS/VS/NVSL/Mycobacteria and Brucella Section, Ames, IA.

181 Diagnosis of Clostridium spiroforme in a Rabbit

D. Jordan6, T. Klinefelter1, J. Kinyon1

Clostridium spiroforme is known to be associated with enteric disease in juvenile rabbits. C. spiroforme has been determined to be a sole pathogen in less than 15% of outbreaks of enteric disease of rabbits and a co-pathogen in 33% of cases. A rabbit was presented to the Iowa State University Veterinary Diagnostic Laboratory following sudden onset of diarrhea and death shortly after returning from competition at a show. Several other rabbits in the group had similar clinical signs followed by death. Sections of intestines were submitted to the ISU VDL. Histopathology of the small intestine revealed a fibrinopurulent exudative enteritis and typhlitis. There were numerous coccidia present in the small and large intestinal enterocytes; moderate numbers of Eimeria media and Eimeria magna were detected by fecal floatation. Clostridium spiroforme, a gram-positive loosely coiled rod that favors anaerobic conditions, was recovered from the tissue. Conventional PCR confirmed the identification of C. spiroforme and the associated iota toxin, which is important for the pathogenesis of the disease. It is conjectured that dual infection with coccidia and C. spiroforme, in addition to the stress associated with the show, caused the acute onset of diarrhea and death in this group of rabbits.

1 Department of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, Iowa

182 Development and Validation of a PCR Test to Detect Lawsonia intracellularis in Tissues and Feces from Swine

G.K.A. Josephson, H.Y. Cai, P. Bell-Rogers, M. Archambault, B. McEwen, M.G. Maxie

Lawsonia intracellularis is the causative agent of proliferative enteropathy in a variety of species, including swine, rabbits and horses. This bacterium, widely distributed throughout the world, is an obligate intracellular organism that is extremely difficult to culture. Several serological tests, e.g. indirect immunofluorescence assay (IFAT) and immunoperoxidase monolayer assay, have been developed to detect the presence of antibodies against L. intracellularis, but are limited in that they can only detect previous exposure to the organism. For the detection of L. intracellularis in the live pigs, modified acid fast stain on fecal smear is commonly used but unreliable. A PCR assay has been developed to detect L. intracellularis from fecal samples (Gebhart, et al. 1991, Jones et al. 1993). The PCR assay was shown to be less sensitive than IFAT on faecal samples (Knittel et al. 1998) but more sensitive and specific than either Warthin-Starry silver staining or IFAT when detecting the agents in ileum scrapings (Jordan et al. 1999). This project increased the sensitivity of this PCR assay by optimizing the PCR conditions and improving fecal DNA extraction method. The PCR assay was then validated by testing tissues and feces from clinically affected swine using the combination of histological examination and fecal MAF stain as a comparison standard.

The clinical samples were obtained from routine submissions that were made to the Animal Health Laboratory, Laboratory Services Division, University of Guelph. In addition to PCR testing, samples of ileum and colon were fixed in 10% neutral buffered formalin and further processed to be examined microscopically for lesions compatible with those produced by L. intracellularis. Appropriate sections were stained with Warthin-Starry stain to determine the presence of spriochetal organisms in apical cytoplasm of cryptal epithelium.

DNA was extracted from tissue samples using 2 methods: (1) the Quiagen DNeasy Tissue Kit and (2) the FastPrep FastDNA Kit, and from the fecal samples using the QIAmp DNA stool Kit and the FastPrep FASTDNA Kit.

Based on the results of testing 100 tissue samples and comparing the results to histological evaluations, the sensitivity of the tissue PCR was determined to be 95.5% (95% CI 83.3,99.2) and the specificity was 90.9% (95%CI 79.3,96.6)

Similarly, based on the results of testing 86 fecal samples and comparing to the results mentioned above, the sensitivity of the fecal PCR testing on samples obtained from clinically affected animals was 85.7% (95%CI 69,94.6) and the specificity was 78.4%. (95%CI 64.3,88.2)

We conclude that 1. the PCR test utilized in this study is suitable for identifying the presence of L. intracellularis organisms from either sections of intestine or from feces 2. the fecal test used be used as a herd test, with several samples be submitted from each clinical case.

Animal Health Laboratory, Laboratory Services Division, University of Guelph, Guelph, ON

183 Antimicrobial Susceptibility Profiles of Recent Isolates of Mycoplasma bovis

Joann M. Kinyon1, Ricardo F. Rosenbusch2, Lorraine J. Hoffman1, and Michael Apley1

Mycoplasma bovis is involved in mastitis, pneumonia, and polyarthritis of beef and dairy cattle. Infections can affect all ages and are poorly responsive to antimicrobial therapy. Variation in antimicrobial susceptibility profiles among isolates of M. bovis recovered from various tissues in dairy or beef outbreaks has been suspected but not verified and increasing resistance of isolates has been recently documented. Our objective was to conduct large-scale antimicrobial surveillance to provide current information and to establish a standardized method for in-vitro susceptibility testing. This report contains antimicrobial susceptibilities for 223 isolates obtained from 5 U.S. regions and from 4 tissue sites.

Isolates of M. bovis were obtained from 26 U.S. States as low passage cultures from recent case submissions, along with a report of tissue of origin. Batches of each isolate were prepared in PPLO Broth (REMEL, Lenexa, KS,) supplemented with 5% Alamar Blue (Biosource, Camarillo, CA), and frozen in aliquots at –70C. After titration, antimicrobial susceptibility for the isolate was run on “Sensititer” plates (Trek Lab, Westlake OH) in 200 µl final volume using PPLO Broth with 5% Alamar Blue as redox indicator. Antimicrobials in doubling dilutions were dried onto single well series so that several concentrations were tested. M. bovis was added in the amount of 2 x 103 to 2 x 105 color-changing units/well. Plates were read for color change (blue to red) at 48 hrs. For each antimicrobial, range of results, mode, MIC50 and MIC90 values were obtained globally, as well as by region and tissue site.

The MIC50 for oxytetracycline and chlortetracycline (2 and 4 µg/ml respectively) indicated that these antimicrobials are quite active in-vitro, but the MIC90 values for the drugs (16 µg/ml for both) showed that refractive isolates do exist. Enrofloxacin, florfenicol, and spectinomycin (MIC90 of 0.5, 4, and 4 µg/ml, respectively) were found to be active compounds in-vitro. Erythromycin, and tilmicosin, (MIC50 of 32, >64, and >64 µg/ml, respectively) showed limited in-vitro activity against the 233 isolates.

Table 1. Antimicrobial activity against 233 isolates of Mycoplasma bovis. Range* Mode** MIC50*** MIC90**** Chlortetracycline 0.25 to >32 4 4 16 Enrofloxacin 0.03 to 4 0.215 0.25 0.5 Erythromycin 4 to >32 32 32 >32 Florfenicol 0.06 to 8 1 1 4 Oxytetracycline 0.125 to >32 2 2 16 Spectinomycin 1 to >16 2 2 4 Tilmicosin 0.5 to >128 64 64 >128 Ampicillin >32 >32 >32 >32 Ceftiofur 64 to >64 >64 >64 >64 * Range of results for 223 isolates; ** Mode- the most frequent result; *** MIC50- the central result; ****MIC 90 – 90% of the isolates with MIC below the value. All values are expressed as µg/ml.

1Department of Veterinary Diagnostic and Production Animal Medicine, 2Department of Veterinary Microbiology and Preventive Medicine Iowa State University, Ames, IA 50011

184 BVDV Infection in Cell Culture-A Laboratory Disaster

J. G. Landgraf1, J. V. Warg1, L. G. Koster1, K. A. Eernisse2, M. P. Emery1, S. L. Swenson1

Since the late 1970’s, the Diagnostic Virology Laboratory (DVL) of the National Veterinary Services Laboratories (NVSL) has used fetal bovine serum (FBS) enriched growth and maintenance media in all primary cell cultures and continuous cell lines. The serum has been collected under contract and then raw serum tested and processed by United States Department of Agriculture laboratory personnel. Serum meeting laboratory requirements is irradiated by a commercial company prior to use in the DVL.

In late November 2002, cell cultures used in the Bovine and Porcine Viruses Section of the DVL became infected with bovine viral diarrhea virus, apparently contaminated from the FBS that was being used to propagate cells. This report will describe the testing done to identify and confirm the source of the contamination and the difficulty encountered finding a source of “clean” FBS. Results of the extensive testing performed and implications of the results will be discussed.

1 USDA-APHIS-VS, National Veterinary Services Laboratories, Diagnostic Virology Laboratory, Ames, IA 2 USDA-APHIS-VS, Center for Veterinary Biologics Laboratory, Ames, IA

185 Implementation of Strategy to Control Bovine Viral Diarrhea Virus in the United States

R.L.Larson1, D. M. Grotelueschen2

The cattle industry has recognized the need for increased levels of control for bovine viral diarrhea virus (BVDV). Organizations including the Academy of Veterinary Consultants, American Association of Bovine Practitioners, and the National Cattleman’s Association (Cattle Health and Well-Being Committee) have endorsed the need for effective BVDV control. Discussions have involved various aspects of control as well as eradication, with some resistance to targeting BVDV eradication. BVDV control can be defined as the implementation of planned strategies to maintain negative status, reduce incidence or eliminate BVDV from a unit of interest, including documentation and/or monitoring of progress. BVDV eradication can be defined as the implementation of planned strategies to eliminate BVDV from a unit of interest, including documentation of that status.

A control strategy that is embraced by all interests, including scientific disciplines, veterinary practitioners, and cattle producers is needed. Education at all levels is critical as the strategy is conceptualized and implemented. Surveillance, biosecurity and biocontainment are critical components that require input and adoption across multiple scientific disciplines as well as by those implementing the plan. Diagnostic laboratory leadership, innovation and participation is a key component for success. Use of scientifically valid, cost effective surveillance is needed for better detection of BVDV-infected herds. Biosecurity plans include methods to prevent entry of BVDV into herds, monitoring for BVDV and persistently infected (PI) reservoirs, and vaccination to control losses if exposure occurs. Biocontainment plans focus first on elimination of PI BVDV animals and include biosecurity, monitoring and vaccination to control losses incurred by exposure.

Setting goals and objectives is critical to successful BVD control within individual herds or larger subsets of the cattle population. Goals may or may not include elimination of BVDV from a particular herd or unit of interest. There is great diversity among beef operations that are exploring and/or implementing BVD control strategies. Control plans must be effective and economically beneficial within that diversity.

Clearly, more effective strategies are needed if the cattle industry expects to achieve better control of BVDV. A comprehensive strategy for BVDV biocontainment and biosecurity is proposed.

1Department of Veterinary Medicine and Surgery, University of Missouri, Columbia, Missouri 2Pfizer Animal Health, Gering, NE

186 Implementing the Standard: Personnel – Reading Between the Lines

B. Lawler1 and M. Pedersen2

Of all the “P” categories in the AAVLD Essential Requirements Standard (personnel, places, procedures, processes, papers), personnel is arguably the most important. After all, it is the Laboratory’s personnel who handle all the other P’s. The Standard’s section on ‘Personnel’ is concise (5.2.1 – 5.2.3), but related requirements are scattered throughout most sections of the document. While the personnel requirements seem obvious, some ‘between the lines’ elements may be overlooked in implementation.

One tool used in implementation is GAP analysis, which is the comparison of Laboratory processes and procedures required by the new Standard versus those currently in place. The comparisons are straightforward for facilities, equipment, procedures, processes and paperwork. However, GAP analysis comparing aspects of the Standard regarding personnel does not seem so inherent.

Can personnel be, in a sense, standardized or calibrated? Should they be? There are times when this would be important, such as when an assay is routinely performed by multiple people. It is important that such procedures be done consistently, by qualified staff, according to approved procedures. An effective training program will ensure that staff is properly trained to perform the elements detailed in their job description and encourage them to continuously build upon their skills and knowledge. Additionally, documenting who is trained and authorized to perform certain tests and tasks will allow substitutes to fill in as required, without compromising the quality of testing results.

Through Management’s responsibilities under the Standard, they authorize, and can energize, the entire staff in their job duties. It is vital for management to: specify responsibility, authority and inter- relationships; provide resources (tangible and intangible); give feedback and recognition; promote and reward teamwork as well as individual ideas and actions. Quality is Management driven!

1Veterinary Diagnostic Laboratory, Iowa State University, Ames, IA 2Veterinary Diagnostic Center, University of Nebraska, Lincoln, NE

187 Diagnostic Lab Connectivity and Electronic Health Certificates for Equids

K. Maher and J.A. Facchiano

Diagnostic Laboratory connectivity with electronic health certificates provide laboratories and private practitioners with real-time record keeping, accurate epidemiology data queries for the dissemination of information relating to the diagnosis of animal diseases, animal movement tracking and trace back reports necessary for regulatory surveillance, monitoring, and control of existing, emerging and/or foreign animal diseases.

The rationale for this abstract is built upon significant accomplishments in the development and implementation of electronic health certificates and diagnostic lab connectivity since 1999.

The objective of this abstract is to present the epidemiology results with electronic health certificates with diagnostic laboratory connectivity for Equine Infectious Anemia (EIA) between September 2001 and September 2003.

In 1999, the Florida Department of Agriculture and Consumer Services (FDACS), contracted with GlobalVetLink, LC of Ames, Iowa, for a project encompassing Internet applications for all species and diagnostic lab connectivity for Equine Infectious Anemia (EIA) applications necessary for animal health regulatory management.

From a time period of September 2001 through September 2003, the State of Florida produced a total of 50,114 certificates for 19,701,679 total animals. Of the total queried, the numbers represent 19,437 EIA/Coggins and 2,681 Official Certificates of Veterinary Inspection that include diagnostic lab test results. The system is used in the export of horses to more than 47 states and 3 US territories.

Tests for the 20,200 EIA/Coggins application were submitted to one (1) state and two (2) private diagnostic labs. All tests were reported Negative. None were positive, suspect or needed retest. Ninety percent were run by AGID and 10% ELISA. The reasons for testing were: 18,834 Annual, 9 Breeding, 49 Change of Ownership, 31 Export, 274 First Test, 87 Market, 123 Other, and 51 Show.

Digital images, an additional method of identification, replace the hand drawings, are provided on the lab submittal form and are available in the lab applications should a Certified Copy be requested by a veterinarian and/or client. With diagnostic lab results and vaccination records readily available on OCVI’s, the electronic health certificates provide immediate ability to verify tests results and vaccines requires for the movement of animals.

We conclude that electronic health certificates offer the practitioners and diagnostic labs the ability to create complete and legible documents, incorporate digital images and signatures of practitioners and lab technicians, compile real time data, allow for ease of data analysis, and disseminate documents to the appropriate animal health officials with the same ease as sending e-mail. Reduction of paper work and time/cost benefits to administrative staff accomplishes the goals supported by USAHA, which are now in national implementation stages by USDA / APHIS / VS. This project compliments the goals of the National Animal Health Lab Network (NAHLN) and their partnership with state and federal agencies to safeguard animal health and fully coincides with the National ID Plan and US Animal ID Plan (USAIP).

GlobalVetLink, LC., Iowa State University Research Park, Ames, IA

188 PFGE on Campylobacter jejuni from Animals-patterns and Clusters

W. Manley1, A. Boerger-Fields2, K.W. Mills2

Pulsed-field gel electrophoresis (PFGE) using Sma 1 was applied to Campylobacter jejuni isolates from animals. Bovine isolates (n=66), bison isolates (n=4), ovine isolates (n=3), canine isolates (n=2, feline isolates (n=2) as well as one equine isolate and one from a lynx yielded 43 different patterns. Isolates represented 19 of 23 Wyoming counties and three surrounding states. The three patterns containing the largest number of isolates were made up of 15, 12, and 6 isolates. These three groups were bovine isolates except for one ovine. Canine, feline and lynx isolates did not match isolates from another species. Isolates from an equine, bovine and a bison yielded the same pattern. Two isolates from a single bison yielded two patterns. Patterns identified in this study also matched human C. jejuni isolated from diarrhea cases. Because C. jejuni is the leading cause of human bacterial enteritis in developed countries, it is important to investigate techniques that will aid in the determination of the source of infection and PFGE analysis of animal isolates encountered in a veterinary diagnostic laboratory can provide useful information.

1Wyoming Public Health Laboratory, Cheyenne, WY 2Departrment of Veterinary Sciences, University of Wyoming, Laramie, WY

189 Papillomavirus-associated Basosquamous Carcinoma in an Egyptian Fruit Bat (Rousettus aegyptiacus)

2 3 C. McKnight,*1 M. Kiupel,1 A. Wise, C. Howe, and R. Maes,2

An approximately 5-year-old, female Egyptian Fruit bat (Rousettus aegyptiacus) from The Organization of Bat Conservation, in Bloomfield Hills, MI, had a small raised pigmented mass located at the lateral canthus of the left eye. The mass progressively increased in size over six months, and was surgically removed in January, 2004. At the time of surgery, the mass was 12mm in diameter, and extended 6mm into the underlying subcutaneous tissue. Further examination identified multiple (approximately 6) variably sized, raised, smooth to cauliflower-like skin masses randomly distributed throughout the left wing membranes. An additional three masses were removed in February, 2004. All excised tissues were submitted to the Diagnostic Center for Population and Animal Health at Michigan State University for microscopic examination.

All four masses appeared microscopically similar and were characterized by elongate to polygonal neoplastic cells with prominent intracellular bridging that were arranged in lobules and thick pegs extending from the overlying hyperplastic epithelium. Neoplastic cells had variable amounts of eosinophilic cytoplasm and large vesiculate nuclei with prominent, sometimes multiple nucleoli and surrounded central areas of ketatinization with numerous intermixed dyskeratotic cells. There were 1-3 mitoses per high power field (HPF). All masses were diagnosed as basosquamous carcinomas. Immunohistochemistry for papillomavirus on the four examined masses detected positive intranuclear staining in all tumors. DNA extracts from formalin fixed-paraffin embedded tumor tissue were tested by PCR, using degenerate primers designed to amplify a 450 bp segment of the L1 region of the human papilloma virus genome. A 450-bp product was obtained and directly sequenced. A BLAST analysis of the sequence data showed that there was 42.9% sequence identity with the L1 region of human papillomavirus.

This is the first report of a papillomavirus-associated carcinoma in a bat. Papillomaviruses have been associated with a number of hyperplastic and neoplastic lesions in a wide variety of vertebrate species, including humans.2 Papillomavirus in bovines most commonly results in benign lesions, while papillomavirus-induced lesions in humans may progress to squamous cell carcinomas.2 Other species- specific papillomaviruses may have a similar carcinogenic progression, including canine oral papillomavirus, cottontail rabbit cutaneous papillomavirus (Shope papillomavirus), and rodent (Mastomys natalensis) papillomavirus . Interestingly, bovine papillomaviruses (BPV-1, BPV-2) have been associated with neoplastic lesions in other species, such as cutaneous sarcoids in horses.1 In this case, papillomavirus antigen was detected in all 4 examined carcinomas, strongly suggesting a role of this virus in tumorigenesis. It is uncertain whether the papillomavirus identified in this bat represents a novel species– specific bat papillomavirus, or cross-infection of a known papillomavirus from another species.

1. Fenner, F. J., E.P.J. Gibbs, F.A. Murphy, R. Rott, M.J. Studdert and D.O. White (eds). 1993. Papoviridae. In: Veterinary Virology 2nd ed. Academic Press, Inc., San Diego, California. Pp. 321-328. 2. Sundberg, J.P. (1987). Papillomavirus infections in animals. In: Syrjanen, K., L. Gissmann, and L.G. Koss (eds). Papillomaviruses and Human Disease. Springer-Verlag, Berlin. Pp. 40-103.

1Diagnostic Center for Population and Animal Health, Michigan State University, E. Lansing, MI. 2Department of Veterinary Microbiology, Michigan State University, E. Lansing, MI 3Crossroads Animal Hospital, 3232 N. Dettman Road, Jackson, MI 49201, USA

*To be considered for resident award

190 Correlation of Leptospira interrogans Fluorescent Antibody Test with Histology and Immunohistochemistry in Aborted and Abattoir-collected Bovine Fetuses

E. K. Meseck*1, B. Njaa1, B. Thompson2, L. Warnick2 and P. McDonough2

Leptospirosis causes a variety of reproductive problems in dairy cattle. Bovine abortions submitted to the New York State Animal Health Diagnostic Laboratory (NYSAHDL) are routinely examined by a Leptospira interrogans fluorescent antibody (FA) test; however, a suspect or even positive result is often of uncertain significance to the diagnostic pathologist, because few or no histologic lesions and no spirochete organisms are present on histologic examination of the fetal tissues and membranes.

To better define the significance of a positive or suspect Leptospira interrogans FA in bovine abortion submissions, a population of 56 randomly selected, ostensibly healthy, fetuses with a median estimated gestational age of 145 days were collected from an abattoir. Fresh placentome, kidney, lung, liver and abomasal fluid were submitted for Leptospira interrogans FA, and identical tissues plus thymus were fixed in 10% neutral buffered formalin, embedded in paraffin-wax, sectioned 5 micrometers thick and stained with hematoxylin and eosin. Immunohistochemistry with a monoclonal antibody for Leptospira interrogans and Modified Steiner’s silver stain were applied to all sections. Neither lesions nor spirochete organisms were identified in the 56 bovine fetuses and FA results were negative for all 56 cases. From this preliminary study, we conclude that the FA test for Leptospira interrogans is not commonly positive in a randomly selected, healthy population of bovine fetuses.

In order to infer that the converse is true, we reviewed all 166 bovine abortions submitted to the NYSAHDL in 2002, which included six positive and three suspect positive Leptospira interrogans FA tests. In the six positive cases, fetal membranes (n=1), kidney (n=4) and/or abomasal contents (n=4) were positive. The three suspect positive cases were based on examination of kidney. Within this pool of nine positive and suspect positive cases, no specific diagnostic lesions were identified. Four of the 9 cases had no significant lesions, whilst the remaining cases had suppurative placentitis (n=1), karyorrhexic debris in the placenta (n=1), multifocal gliosis in the brain (n=2), thymic lympholysis (n=1) and nonsuppurative myocarditis (n=1). Citrobacter amalonaticus was cultured from the lung and abomasal fluid of one positive case, and Escherichia coli was cultured from the lung of one suspect positive case. No other infectious agent was identified in the remaining positive and suspect positive cases. Although a pattern of diagnostic lesions specific for Leptospira interrogans abortion was not identified within this small pool of positive and suspect positive cases, we conclude that a positive FA test for Leptospira interrogans has diagnostic significance in bovine abortion submissions, based on the abattoir-collected population results.

1Department of Biomedical Sciences, Section of Pathology (EKM and BN) and 2Department of Population Medicine and Diagnostic Sciences (BT, LW and PM); College of Veterinary Medicine, Cornell University, Ithaca, NY

* To be considered for graduate student award

191 Postmortem Evaluation of Thirty-one Brucella-Reactor Cattle from Wyoming and Evidence of Apparent Transmission from Elk

D. Montgomery1, K. Mills1, D. O’Toole1, H. Edwards2, W. Cook2, S. Olsen3, B. Bricker3, D. Ewalt4, A. Jensen3, P. Yaeger1, J. Logan5, B. Combs6

Transmission of Brucella abortus between domestic livestock and free-ranging ungulates threatens eradication efforts in the United States. Six episodes of B. abortus infection of domestic cattle herds in Wyoming have been associated with potential exposure to Brucella-infected wildlife since 1969. Incrimination of elk or bison as sources of infection for cattle has been based largely on epidemiological data rather than on similarities between strains of B. abortus cultured from cattle and wildlife.

Following the identification of a B. abortus seropositive cattle herd in Wyoming during November 2003, 31 reactor cows were examined postmortem. Purposes of the study were to confirm active infection, to compare various techniques for the diagnosis of brucellosis in reactor cattle, and to compare B. abortus isolates with those of elk recovered from the Muddy Creek feedground for elk in northwest Wyoming. Historically, cattle from the index herd had intermingled with elk (approx. 29% seroprevalence) on the feedground. B. abortus was cultured from an aborted elk fetus on the feedground in 2002. Timing of infection in the index cattle herd is undetermined but infection is believed to have possibly occurred the preceding year. Epidemiological investigation found no evidence of a cattle source for the infection.

Of the 31 reactor cows, 7 were open at the time of postmortem examination. In the remaining pregnant cows, gestational age of fetuses ranged from 4 to 7 months, the majority being in the 6th or 7th month of gestation. Active infection was confirmed in several cows based on positive culture for B. abortus. Positive cultures were obtained most commonly from mammary gland and regional (supramammary and iliac) lymph nodes. The WSVL also cultured B. abortus from one of the fetuses.

Nonspecific lesions, compatible with but not diagnostic for infection, were found in the majority of cows. In lymph nodes, these included reactive lymphoid hyperplasia with plasmacytosis, sinusal neutrophilia and histiocytosis. Lesions in mammary glands included stromal/intraductal accumulations of macrophages, lymphocytes, and plasma cells. Lesions regarded as more specific for infection consisted of granulomatous inflammation with discrete accumulation of macrophages and multinucleated giant cells in lymph nodes and mammary stroma and ducts. Immunohistochemical (IHC) staining for B. abortus was definitively positive only in mammary gland from one cow. Compared to bacterial culture, IHC is an insensitive and inefficient method to confirm a diagnosis of brucellosis in serologically positive cattle.

Three seropositive elk were subsequently euthanized and necropsied at the Muddy Creek feed ground. B. abortus was cultured from one elk. Comparisons of the isolates from the index cattle herd and the elk are ongoing. Comparison is based on analysis of the variable number tandem repeats in the Brucella genome, a technique termed HOOF-Prints [Bricker, BJ et al: Brucella ‘HOOF-Prints’: strain typing by multi-locus analysis of variable number tandem repeats (VNTRs). BMC Microbiol 3:15-27, 2003]. Initial comparison of B. abortus cultured from one cow with the cervid isolate revealed a close match, suggesting infection by a similar strain. The close match between these isolates, the proximity between infected cattle and elk, and the lack of a known bovine source, suggest that brucellosis was transmitted from elk to cattle.

1Wyoming State Veterinary Lab (WSVL), Dept. of Vet Science, University of Wyoming, Laramie, WY 2Wyoming Game & Fish Dept, Wildlife Disease Laboratory, Laramie, WY 3USDA-ARS, National Animal Disease Center, Ames, IA 4USDA-APHIS, National Veterinary Services Laboratory, Ames, IA 5State Veterinarian, Cheyenne, WY 6AVIC, USDA-APHIS, Cheyenne, WY

192 Aldosterone and Progesterone Producing Cortical Adrenal Tumor in a Cat

I.D.R. Pardo*1, G.Johnson1, A. Declue2, and M. Kiupel3

Formalin fixed tissues from a left adrenal mass and a liver biopsy originating from a 13 years old, neutered male, domestic long haired feline were submitted to the Veterinary Medical Diagnostic Laboratory of the University of Missouri. This cat was presented to the Veterinary Teaching Hospital at MU for evaluation of diabetes mellitus, weight loss and a possible renal mass. The physical findings at presentation included a thin, greasy haircoat, thin skin, moderate dental tartar, a III/VI left sternal systolic heart murmur, and a mass cranial to the left kidney. The mass was identified as the left adrenal gland (2 cm by 2.5 cm) based on radiographs and ultrasound. Hyperglycemia, azotemia, hypokalemia, hypochloremia, hyperphosphatemia, elevated total CO2, mild corrected hypernatremia and glycosuria were noted with serum chemistry and urinalysis tests. Blood pressure and low-dose dexamethasone suppression test were within the normal limits. Aldosterone was significantly elevated (>3329 pmol/L, reference range 194-388 pmol/L). Progesterone was elevated both before ACTH administration, and at 30 and 60 minutes after administration. Other androgens and cortisol concentrations were within the reference range.

Grossly, the left adrenal mass was diffusely yellow, firm, and multilobulated. Histologically, the cortex of the gland was markedly expanded by cords and sheets of dense neoplastic growth, which compressed the adjacent parenchyma. Tumor cells had round central, stippled to hyperchromatic nuclei with occasional single prominent nucleolus. The cytoplasm contained single to multiple, well demarcated vacuoles. There were two mitotic figures in ten 400X fields. Multifocally, packets of tumor cells infiltrated the adrenal capsule. In the liver, there was diffuse hepatocellular swelling, with single to multiple, well demarcated to feathery intracytoplamic vacuoles. The tumor cells were immunochemically positive for melan A and vimentin, but were negative for aldosterone.

Cortical adrenal gland neoplasms in the cat are uncommon malignant tumors that frequently invade adjacent structures such as the vena cava, descending aorta, lymph nodes, and kidneys. They may also metastasize to distant organs such as the lung. Cats with these tumors have high levels of aldosterone, progesterone and testosterone. Melan A and vimentin are two immunohistochemical stains that are commonly used to confirm the diagnosis of adrenal cortical tumors in humans and dogs; however, there are no publications utilizing the technique in cats. Although the levels of aldosterone were high in this animal, the tumor cells could not be confirmed as the source of this hormone by immunohistochemistry. The result may be due to the production of this hormone by the non-neoplastic right adrenal gland. Further immunohistochemical studies of feline adrenal cortical tumors may be useful in developing additional date for the diagnosis of this condition. The liver lesions are most likely associated with long periods of anorexia.

1Veterinary Medicine Diagnostic Laboratory, University Missouri Columbia 2Veterinary Teaching Hospital UMC 3Department of Pathobiology and Investigation, Michigan State University.

*To be considered for graduate student award

193 Expression of Leptospiral Imunoglobulin-like Protein by Leptospira interrogans and Evaluation of Its Diagnostic Potential in a Kinetic Enzyme Linked Immunosorbent Assay

Raghavan U.M. Palaniappan1, Yung-Fu Chang1, Fahad Hassan1, Sean P. McDonough1, Margaret Pough1, Stephen C. Barr1, Kenneth W. Simpson1, Hussni O. Mohammed1, Sang Shin1, Patrick McDonough1, and Richard L. Zuerner2

The search for novel antigens suitable for improved vaccines and diagnostic reagents against leptospirosis led to the identification of LigA and B. LigA and B expression were not detectable at the translation level but were detectable at the transcription level in leptospires grown in vitro. Lig genes were present in pathogenic serovars of Leptospira, but not in non-pathogenic L. biflexa. The conserved and variable regions of LigA and B (Con, VarA and VarB) were cloned, expressed, and purified as GST fusion proteins. These purified recombinant proteins of LigA and B were evaluated for their diagnostic potential in a KELA assay using sera from vaccinated and MAT positive dogs. Sera from vaccinated dogs showed reactivity to whole cell antigens of leptospires but did not show reactivity in the KELA assay with recombinant antigens suggesting the lack of antibodies to Lig proteins in the vaccinated animals. The diagnostic potential of recombinant Lig antigens in the KELA assay was evaluated by using 67 serum samples with MAT ≥ 1600, which showed reactivity of 76, 41 and 35% to rConA, rVarA and rVarB, respectively. These findings suggest that recombinant antigen to the conserved region of LigA and B can differentiate between vaccinated and naturally infected animals.

1College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 2Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Ames, Iowa 50010.

194 Evaluation of lig-based Conventional and Real Time PCR for the Detection of Pathogenic Leptospires

R.U.M. Palaniappan, Y.-F. Chang, P. Harpending, S.P. McDonough, and E. Dubovi

Leptospirosis is an important disease affecting wild and domestic animals as well as humans. Pathogenic leptospires encode immunoglobulin-like protein (Lig) that is found to expressed only during infection. We report the development of conventional and real time PCR assays targeting lig genes of leptospires for the early diagnosis of leptospirosis. Sensitivity of the newly designed Lig1/Lig2 primers for conventional PCR was compared with previously published primers LP1/LP2 and G1/G2. G1/G2 primers amplified the target DNA from all the serovars including non-pathogenic L. biflexa whereas LP1/LP2 and Lig1/Lig2 primers amplified only pathogenic leptospires. The diagnostic PCR assay was also developed for the detection of pathogenic L. interrogans in urine samples. We obtained the highest sensitivity in PCR using our Lig1/Lig2 primers with a detection limit of 6 leptospires. A lig-based real time PCR assay was also developed with a detection range of 10-107 gene copies. To evaluate the early diagnosis of leptospirosis, we compared culture with conventional and real time PCR for the detection of spirochetes in experimentally infected hamsters during a time-course study. Culture of infected hamster tissues detected the presence of leptospires from day 2 of infection but not on the day of infection or day 1, whereas conventional PCR and real time PCR detected the leptospires from the day of infection. Hence, conventional and real-time PCR with lig primers would be a sensitive and rapid tool for early diagnosis of leptospirosis.

College of Veterinary Medicine, Cornell University, Ithaca, New York 14853

195 Bacterological Characterization of Respiratory Disease in Calves and Antimicrobial Susceptibility

Y. Pineda1, S. Mora2, J. Santander1

Respiratory disease (RD) in calves is a complex disease syndrome caused by many bacterial and viral agents, influenced by management and environment. This disease affected 58% of fatal cases in a study of calves in Venezuela. The most frequent etiologic bacterial agents from fatal cases of RD in calves are pasteurellae. Antimicrobial therapy is the most effective method for treatment of RD and antimicrobial susceptibility patterns are important in determining appropriate therapy against bacterial pathogens. The objective of this study was to determine the bacteriological characterization from RD of calves in two farms of Guárico state, Venezuela. And the antimicrobial susceptibility for the bacteria most commonly isolated.

A total of 50 samples from nasal swabs of dairy calves, males and female, between 1 and 4 months of age with RD, were carried out for bacteriological studies. The isolates were identified using standard bacteriological methods and for antimicrobial susceptibility by means of antimicrobial test for bacteria isolated from animal according to NCCLS. The antimicrobial agents used were amikacin, ampicillin, cephalotin, colistin, enrofloxacin, streptomycin, florfenicol, kanamycin, neomycin, tetracycline, trimethoprim/sulfamethoxazole, triple sulpha and trimethoprin.

The bacterial agents most frequently isolated were Pasturella multocida (28%), Streptococcus spp. Β hemolytic (22%), Klebsiella pneumoniae (18%), Corynebacterium spp. (14%), Staphylococcus epidermidis (6%), Bacillus spp. (4%), E. coli and Pseudomonas aeruginosa (2%). It was found that isolates in mixed cultures were predominant over pure cultures. Antimicrobial susceptibility was determined for Pasteurella multocida against 13 antimicrobial agents of use common in veterinary medicine. All Pasteurella multocida isolates were susceptible to enrofloxacin and florfenicol. High frequency of resistance was found to triple sulfa and trimetoprin-sulfametoxazol in both farms.

These in vitro observations should be used only as a guide for the clinician in the selection of the most appropriate antimicrobial agent, Therapeutic approaches to bacterial calf pneumonia in the area under study, should be modified according to the isolated bacterial population and the observed antimicrobial resistance.

1 Laboratory of Bacteriology, Animal Health, Instituto Nacional de Investigaciones Agrícolas (INIA- CENIAP) Maracay- Estado Aragua, Venezuela. 2 Department of Animal Health, Instituto Universitario de Tecnología de los Llanos, Valle de la Pascua- Estado Guárico, Venezuela.

196 The Distribution of Mycobacterium avium paratuberculosis in Minnesota Dairy Farms Using Bacterial Culture of Fecal Pools

Raizman E.A.*, S. J. Wells, C. Muñoz Zanzi, and S M. Godden

Paratuberculosis or Johne’s disease is a chronic and progressive intestinal disease in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). The disease becomes manifest in adulthood and results in economic losses. Studies have validated the use of bacterial culture of pooled fecal samples as a herd screening strategy for detection of MAP. The objective of this study was to characterize the distribution of MAP on Minnesota dairy farms, using pooled fecal samples, and to compare it to serum ELISA test results obtained in each herd.

One hundred and eight Minnesota dairy herds were sampled during the summer of 2002, including 80 herds known to be infected from previous testing, which participated in the Johne’s Disease Control Program (JDCP) of the Minnesota Board of Animal Health (MBAH), and 28 herds known to be uninfected based on previous testing, which participated in the Voluntary Johne’s Disease Herd Status Program (HSP) of the MBAH. Fecal samples were obtained from up to 100 cows in each herd and were cultured in pools of five cows based on age order. Fecal samples were tested using bacterial culture for MAP at the Minnesota Veterinary Diagnostic Laboratory. In addition, results from serum ELISA test obtained up to 9 month prior and subsequent to the cow fecal sampling were used to compare with the fecal pool results.

Of the 28 HSP herds 93% (26) had no positive pools; 7% (2) had one positive pool each. Of the 80 JDCP herds 80% (64) had a least one pool positive and 20% (16) did not have any positive pools. In small fecal pool positive herds (<100 cows), 30% had >30% fecal pool positive, whereas fecal pool prevalence was >30% in 43% of fecal pool positive herds with 100≤ cows. In 74% of the herds with positive pools, the maximum level of shedding was >50 colonies per tube. Within each herd, on average, 21% of positive pools were heavy shedders. The overall average pool age among all fecal pool positive herds (n=1180 pools) was 4 years (min.=2, max. =11). Overall Chi-square results indicate no significant difference between all age groups (2, 3 ,4, 5, and 6≤ years) in terms of pools status (p=0.19) or level of shedding (p=0.257). There was a positive correlation between fecal pool prevalence and ELISA prevalence (г = 0.54, p<0.0001). Positive correlation was also found between maximum level of shedding and maximum ELISA result (г=0.60, p<0.0001). The within-herd prevalence estimated using pool fecal culture was on average 15% (95%CI= 9.7-50% min. 2% max. 143%).

Conclusions: Larger herds have higher fecal pool prevalence. Of all fecal positive herds, 75% had at least one pool with >50 colonies per tube. Since age was not found to be a cluster factor in fecal pool status and level of shedding, resources may be saved if cows would be randomly allocated to each pool. There was a correlation between ELISA within herd prevalence and fecal pool within herd prevalence.

Department of Clinical and Population Sciences, College of Veterinary Medicine, University of Minnesota, 1970 Commonwealth Av St Paul MN 55108 USA. Phone: 612-624-3766.

*To be considered for graduate student award

197 Detection of c-Kit (CD 117) on Cutaneous Mast Cell Tumors of Ferrets by Immunohistochemistry

J.A. Ramos-Vara1, M.A. Miller1, G.C. Johnson2, C.M. Loiacono2, and M. Kundu1

Mast cell tumors are the second most common tumor of the skin in the ferret. In this species, cutaneous mast cell tumors are generally benign and may be single or multiple. Histologically mast cell tumors are well demarcated, unencapsulated dermal masses of monotonous round cells with hyperchromatic central nuclei. Metachromatic stains reveal only few cytoplasmic granules.

The KIT protein is a tyrosine kinase receptor that is a product of the c-kit proto-oncogene. This protein is expressed in numerous tissues including glioblastoma cells, gastrointestinal stromal tumors, term placenta, brain, erythroid precursors, melanocytes, basophils and mast cells. The ligand for the KIT receptor, stem cell factor, also called mast cell growth factor is involved in mast cell proliferation and maturation. The purpose of this work was to detect KIT in ferret mast cell tumors by immunohistochemistry.

Twenty-eight cutaneous mast cell tumors were used. Samples had been fixed in 10% formaldehyde and routine processed. For IHC, heat antigen retrieval, immersing slides in citrate buffer (pH 6.0) at 90-95 C for 20 min., was used. A rabbit polyclonal antibody (DakoCytomation) to CD 117 (KIT) at a dilution of 1/200 was incubated for 60 minutes at room temperature. A peroxidase-DAB, polymer-based detection system (EnVision+) was used to demonstrate the immune reaction.

All tumors were positive for KIT. This protein was detected in the cytoplasmic membrane or/and the cytoplasm of the majority (>80%) of cells in all tumors examined. The staining was granular and diffuse in most cases but in 10 cases there was a concurrent paranuclear polar staining, interpreted as the Golgi area. In two cases there was a ring of DAB precipitate around the nucleus, in addition to the diffuse cytoplasmic staining. Plasma membrane staining was observed in 23 cases. Nuclear staining was not apparent.

Different patterns of KIT expression have been described in normal and in neoplastic mast cells. In dogs, normal mast cells and grade 1 mast cell tumors express KIT mainly on the cell membrane, whereas in grade 2 and 3 mast cell tumors, KIT accumulates in the cytoplasm (Reguera et al., 2000). In our ferret series of mast cell tumors, the staining was frequently observed on the cell membrane and the cytoplasm which is somewhat different from that observed in grade 1 canine mast cell tumors. Although we did not examine other cutaneous tumors in ferrets, we conclude that IHC for KIT is a valuable method to confirm mast cell tumors, particularly with the difficulty in detecting metachromatic granules in mast cells in this species.

Reguera MJ, Rabanal RM, Puigdemont A, Ferrer L: Canine mast cell tumors express stem cell factor receptor. Am J Dermatopathol 22:49-54, 2000

1ADDL and Department of Veterinary Pathobiology, Purdue University, West Lafayette, IN 2VMDL and Department of Veterinary Pathobiology, University of Missouri, Columbia, MO

198 Immunohistochemical Detection of Tryptase in Cutaneous Mast Cell Tumors of Ferrets

J.A. Ramos-Vara1, M.A. Miller1, G.C. Johnson2, C.M. Loiacono2, and M. Kundu1

Tryptase is a major neutral protease of mast cells in many species. Immunohistochemistry for tryptase is a very specific and sensitive method to identify mast cells in humans and dogs. We report the detection of tryptase by immunohistochemistry in cutaneous mast cell tumors of ferrets.

Twenty-seven cutaneous mast cell tumors were used. Samples had been routinely processed (fixed in 10% formaldehyde and embedded in paraffin). We used as primary antibody a mouse monoclonal antibody to tryptase (DakoCytomation) at 1/200 dilution, incubated for 30 min. Tissue sections were heated at 90-95 C in citrate buffer, pH 6.0, for 20 min for antigen retrieval. A peroxidase-DAB, polymer- based detection system (EnVision+) was used to demonstrate the immune reaction. The reagent negative control was substituting the primary antibody for normal mouse immunoglobulins

All tumors were strongly positive for tryptase. The reaction was detected only in the cytoplasm, usually in a diffuse and granular pattern. The percent positive cells were usually higher than 80%, but in 4 cases, fewer than 50% were positive. The distribution of staining was usually heterogeneous, with strongly labeled cells admixed with phenotypically similar but weakly stained or negative cells. In five tumors, neoplastic cells in the middle or deeper portions of the mass were weakly stained or unstained whereas the superficial tumor cells were strongly labeled.

Cutaneous mast cell tumors of ferrets are considered benign until proven otherwise. In our series, these tumors had a benign morphology. Therefore, variations in the staining of tryptase among cells within the same tumor might not correlate with differentiation; however, in several cases, distance from the epidermis was inversely proportional to immunohistochemical reactivity. Fixation may have contributed to this heterogeneous staining, but the small size of these masses (usually < 2 cm diameter) makes this unlikely.

1ADDL and Department of Veterinary Pathobiology, Purdue University, West Lafayette, IN 2VMDL and Department of Veterinary Pathobiology, University of Missouri, Columbia, MO

199 Isolation of Persistently Infected P388D, a Mouse Macrophage Cell Line, with Ehrlichia risticii, the Causative Agent of Potomac Horse Fever

S. Sahu1, D.D. Pedersen1, J. Stasko2, and E. N. Ostlund1

Potomac Horse Fever (PHF) is a disease of horses characterized by fever, anorexia, leucopenia, and diarrhea. The mortality rate in infected, untreated horses is 15 to 30%. The causative agent, Ehrlichia risticii, belongs to the genus Ehrlichia, in the family Neorickettsiaceae. Indirect fluorescent antibody (IFA) and ELISA tests have been developed to monitor antibodies in equine sera resulting from infection with E. risticii. The IFA test is commonly used in veterinary diagnostic laboratories for detection of E. risticii antibodies. Evidence of false positive results with the IFA test for E risticii antibody in horses has been reported (Madigan et al. J Am. Vet. Med. Assoc. 207:1448: 1995) and have been observed at the National Veterinary Services Laboratories. To produce antigens for the E. risticii IFA test which do not produce false positive reactions, the murine macrophage cell line P388D and E. risticii were obtained from Dr. S. K. Dutta, University of Maryland, Collage Park, M.D. During the process of antigen production, a clone of infected P388D cells infected with E. risticii was observed which did not require the addition of fresh cells or E. risticii. The infected clone demonstrates cell proliferation in culture media supplemented with 6% fetal bovine serum. The clone was passaged for 8 times and the presence of E. risticii was detected by IFA and electron microscopy. The new clone is being evaluated for its utility in IFA tests for detecting equine serum antibody to E. risticii.

1Diagnostic Virology Laboratory, National Veterinary Services Laboratories, VS/APHIS/USDA, Ames, IA 2National Animal Disease Center, Agricultural Research Service, USDA, Ames, IA

200 West Nile Virus Outbreak in Horses in North Dakota, 2002: A Characterization of the Equine Cases

L.A. Schuler1, M.L. Khaitsa2, N. Dyer3 and C.L. Stoltenow4

West Nile virus (WNV) is an arbovirus of the genus Flavivirus, family Flaviviridae. The virus was first isolated from a febrile woman in Uganda in 1937 in the district of West Nile. Since then, WNV encephalomyelitis has been reported in humans in Africa, Europe, the middle East, West and Central Asia, and Oceania. WNV is maintained in nature by a bird-mosquito cycle, and birds are the only animals in which a viraemia develops that are sufficient to infect mosquitoes and propagate the infective cycle. Equine encephalomyelitis caused by WNV has been reported in Egypt, Morocco, Portugal France and Italy. Unlike WNV in various bird species, the virus does not amplify sufficiently in infected horses and humans to infect mosquitoes and allow spread to other susceptible hosts. WNV was first reported in the United States in 1999 in New York, and caused disease in birds, horses and humans. The virus has been isolated from a large number of avian and mammalian species but causes disease (encephalomyelitis) mainly in birds, equine animals and humans. The objective of this work was to describe the initial 2002 West Nile virus (WNV) outbreak in horses in North Dakota; document the characteristics of the disease in horses, evaluate the effectiveness of the available vaccine, and determine which horse characteristics and WNV clinical signs were associated with the outcome to WNV infection.

The study sample comprised horses from North Dakota that tested positive to WNV by IgM ELISA in 2002 at the North Dakota State University Veterinary Diagnostic Laboratory (VDL), Fargo. A questionnaire was developed and mailed to veterinarians of the 569 horse cases from ND that tested positive to WNV by VDL. The cases were distributed in 52 out of the 53 counties, with the majority originating from the central region, whereas the highest incidence was in the East and North East. Among the cases, 27% (152) were vaccinated against WNV while 54% (309) were not, and 19% (108) were of unknown WNV vaccination status. Also, 61% (345) of the horse cases recovered, 22% (126) died, and the outcome of 17% (98) was unknown. It was observed that the odds of death among unvaccinated horse cases were 3 times (OR = 3.16) and 16 times (OR = 16.13) more than among those that received only one dose or 2 doses of vaccine not given per the manufacturer’s recommendation and those that were vaccinated as per manufacturer’s recommendations, respectively. Horses exhibiting recumbency, posterior paresis and increased age were associated more strongly with death, whereas those with incoordination appeared to be associated with lower odds of death.

We concluded that among this group of WNV horse cases, those that were not vaccinated with WNV vaccine showed higher odds of death than those that were vaccinated. Horses exhibiting incoordination were less likely to die than those that did not show incoordination as a clinical sign. Older horses and those that were recumbent and/or had posterior paresis were more likely to die than those without these clinical signs.

1North Dakota State Board of Animal Health, 600 E. Boulevard Avenue, Dept.602 Bismarck, ND 58505-0020 2Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND 3Department of Veterinary Diagnostic Services, North Dakota State University, Fargo, ND 4Department of Animal and Range Sciences, North Dakota State University, Fargo, ND

201 Characterization of a Porcine Teschovirus Isolated During an Emerging Disease Investigation in Imported Pigs

J.V. Warg1, J.G. Landgraf1 L.G. Koster1, L.A. Anderson2, S.L. Swenson1, and M.J. Yaeger3

A finishing operation experienced sudden deaths (greater than 12 %) in apparently healthy pigs. The pigs were from a group of 1200 swine that were imported. Four sets of diagnostic samples were submitted to the Iowa State University Veterinary Diagnostic Laboratory (VDL) with no consistent microorganisms isolated. Potential pathogens isolated by VDL included PRRS, circovirus, P. multocida, B. bronchiseptica, S. suis, and an unidentified virus. Microorganisms isolated were not consistent with the clinical signs.

Two additional sets of diagnostic samples were collected and evaluated at the National Veterinary Services Laboratories. Porcine reovirus was isolated from two different pigs. Porcine enterovirus (PEV) was isolated from an intestinal tissue suspension from a single pig. This virus was identified as belonging to PEV serogroups 1-7 by indirect fluorescent antibody assay. Follow-up testing showed strong neutralization with PEV-1 antiserum, and some neutralization with PEV-5 antiserum.

Some strains of porcine enterovirus serotype 1 cause a virulent, highly fatal nonsuppurative encephalomyelitis called enterovirus encephalomyelitis. Other strains of PEV serotype 1 cause less severe forms of disease or no disease at all. Enterovirus encephalomyelitis (previously Teschen/Talfan disease) is listed on the Office International des Epizooties (OIE), World Organization for Animal Health List B, a list of reportable diseases that affect international trade of animal and animal products. Isolation and identification of a PEV serotype 1 virus requires expensive and time consuming pig inoculation studies to rule out enterovirus encephalomyelitis. Often farms are held under quarantine until a definitive diagnosis can be made. Molecular assays have been developed that differentiate porcine enteroviruses into their respective CPE groups I - III and serotypes. Both methods were used in this investigation to rule out the possibility of an emerging or foreign animal disease.

Five pigs were inoculated intranasally and orally with the enterovirus isolate. One control pig was inoculated with the cell culture medium. Pigs were monitored for 28 days. No signs of illness were observed during the course of study. All six pigs were seronegative for PEV-1 antibodies on day of inoculation. All 5 inoculated pigs developed neutralizing antibodies to PEV-1, while the control pig remained negative.

RT-PCR and sequence data were utilized to further characterize the enterovirus isolate. Two different PCR assays were utilized. The first assay differentiated the porcine enterovirus into porcine enterovirus CPE group I, and the second assay identified the virus as a porcine enterovirus serotype 1. Phylogenetic analysis of sequence data for this isolate suggests that it belongs to the new picornavirus genus Teschovirus. Further molecular characterization is underway.

1 USDA-APHIS-VS, National Veterinary Services Laboratories, Diagnostic Virology Laboratory, Ames, IA 2 USDA-APHIS- Veterinary Services, Des Moines, IA 3Veterinary Diagnostic Laboratory, Iowa State University, Ames, IA 50011

202 Differentiation of Mycobacterium bovis Infection of Cattle from M. avium subsp. avium and M. avium subsp. paratuberculosis Infection Using a Recombinant ESAT-6:CFP-10 Fusion Protein

W.R. Waters1, M.V. Palmer1, B.J. Nonnecke2, and F.C. Minion3

Immunological diagnosis of Mycobacterium bovis infection of cattle often is confounded by cross- reactive responses resulting from exposure to other mycobacterial species, especially Mycobacterium avium. Early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) are dominant interferon (IFN)-gamma-inducing antigens of tuberculous mycobacteria; and, they are absent from many environmental non-tuberculous mycobacteria. Because M. avium exposure is the primary confounding factor in the diagnosis of M. bovis-infected animals, in vitro responses to a recombinant ESAT-6:CFP-10 fusion protein by blood leukocytes from cattle naturally-exposed to M. avium or experimentally- challenged with M. avium subsp. avium or M. avium subsp. paratuberculosis (Map) were compared to responses by M. bovis-infected cattle. Responses to heterogeneous mycobacterial antigens [i.e., purified protein derivatives (PPD) and whole cell sonicates (WCS)] were also evaluated. Tumor necrosis factor (TNF)-alpha, IFN-gamma, and nitric oxide responses by M. bovis-infected cattle to rESAT-6:CFP-10 exceeded (P<0.05) corresponding responses by cattle sensitized naturally to M. avium. Experimental infection with M. bovis, M. avium, or Map induced significant (P<0.05) IFN-gamma and nitric oxide production to WCS and PPD antigens, regardless of the mycobacterial species used for the preparation of the antigen. Responses to homologous crude antigens generally exceeded responses to heterologous antigens. Nitric oxide and IFN-gamma responses to rESAT-6:CFP-10 by blood leukocytes from M. bovis- infected calves exceeded (P<0.05) corresponding responses of non-infected, M. avium-infected, and Map- infected calves. Despite the reported potential for secretion of immunogenic ESAT-6 and CFP-10 proteins by M. avium and Map, it appears that use of the rESAT-6:CFP-10 fusion protein will be useful for the detection of tuberculous cattle in herds with pre-existing sensitization to M. avium and/or Map.

1Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, ARS, USDA, Ames, IA 2Periparturient Diseases of Cattle Research Unit, NADC, ARS, USDA, Ames, IA 3Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA\

203 Disseminated Melanoma and Osteogenic Tumor in a Rabbit

F. Williams III*,1 C.M. Loiacono1, L. Bloomfield2

Melanomas are very uncommon spontaneous tumors of rabbits. Generally, these tumors present little diagnostic challenge, with behavior similar to that observed in other domestic species. Osteosarcomas are extremely rare tumors of rabbits, and generally occur in the long bones. There are extremely rare reports of amelanotic melanomas occurring in the same sites as osteosarcomas in humans. To the submitter’s knowledge, this is the first case of a simultaneous cutaneous melanoma and cutaneous osteosarcoma in the rabbit.

A subcutaneous biopsy specimen from the flank of a 9-year-old, neutered male, rabbit was presented for biopsy following identification of multiple raised nodules. Findings included an aggressive, malignant melanoma. In one specimen, there are also multiple spindloid cells associated with osteoid development, often within lacunae separated by compact bone, consistent with osteosarcoma, closely associated with neoplastic melanocytes.

The same rabbit subsequently presented with multiple additional subcutaneous masses as well as lameness and anorexia. The subject was euthanized and submitted to the Veterinary Medical Diagnostic Laboratory for necropsy. On gross examination, there are multifocal to coalescing nodular, diffusely black masses throughout the subcutaneous tissues. Numerous tissues including the marrow spaces of long bones were infiltrated by black nodular tumors.

Microscopically, the tissues are infiltrated by multifocal to coalescing, polygonal to spindloid cells consistent with malignant melanoma. In long bones, there is effacement of the marrow parenchyma with morphologically similar neoplastic cells.

1 Veterinary Medical Diagnostic Lab, University of Missouri, Columbia, MO 65205, USA 2 Animal Care Hospital 1146 Blairs Ferry Rd NE, Cedar Rapids, ID 52402

*To be considered for the resident award

204 Effect of Delayed or Prolonged Fixation on Immunohistochemical Detection of Bovine Viral Diarrhea Virus in Ear-Notch Biopsies

Margaret A. Miller1, José A. Ramos-Vara1, Steven B. Kleiboeker2, Robert L. Larson3

Bovine viral diarrhea virus (BVDV) causes reproductive losses, immunosuppression, diarrhea, hemorrhagic disease, and mucosal disease. Elimination of persistently infected (PI) cattle, the major viral reservoir, is essential in disease control. Immunohistochemistry (IHC) on ear-notch biopsies is frequently employed as a one-time test for persistent BVDV infection, but little is known of the effects of biopsy handling on the sensitivity of IHC results. If formalin is unavailable at the time of biopsy, practitioners need to know the best way to handle biopsies until they can be immersed in fixative; practitioners who are screening an entire calf crop for PI animals may wish to hold samples in formalin until all or at least multiple animals have been biopsied. This study examined the effects of delayed formalin fixation and prolonged formalin fixation on the sensitivity of IHC detection of BVDV persistent infection.

Multiple full-thickness specimens of the pinna were procured immediately after euthanasia of two calves, each known to be persistently infected by ante mortem and post mortem IHC and reverse-transcriptase polymerase chain reaction tests. Specimens were handled in one of three ways: (1) immersion within 5 minutes in 10% buffered formalin at room temperature (22 – 24 C), (2) sealed in plastic bags and refrigerated (3 – 4 C) within 30 minutes of procurement, or (3) exposed to room air (22 – 24 C) on a Styrofoam tray. Specimens that had been promptly fixed were removed from formalin and processed into paraffin blocks after 3, 5, 10, 15, 22, 29, 36, or 176 days. Specimens in plastic bags were refrigerated for 1, 3, 5, or 10 days, and then placed in formalin for 2 – 5 days before processing. Those exposed to room air were allowed to desiccate for 1, 3, or 5 days, then placed in formalin for 2 – 5 days before processing. Paraffin blocks were sectioned and stained by immunohistochemistry with antibody to BVDV on the same day. A serial section of each block was reacted with unrelated antibody as a negative control. Microscopic slides were randomly numbered and evaluated without knowledge of fixation procedure. Intensity of immunohistochemical staining in epidermis and hair follicles (follicular epithelium and dermal papilla) was graded as 0 = no apparent reaction, 1 = weak, 2 = moderate, or 3 = strong.

Immunohistochemical staining of specimens that had been promptly fixed in formalin was moderately to strongly positive for BVDV antigen at all fixation periods through 36 days. However, after 176 days in formalin, no reaction was detected in one calf, whereas the other calf had no epidermal reaction and only weak reaction in occasional follicles. Specimens that had been refrigerated in sealed bags for 1 – 10 days before fixation became gelatinous after 3 days, with separation of skin from cartilage, but generally had moderate to strong IHC staining. Occasional weak follicular reactions were noted in samples refrigerated for 3 or 5 days, but even with 10 days refrigeration, no negative reactions were recorded. Specimens exposed to room air desiccated, became brittle, and were difficult to section by 24 hours. Furthermore, no immunohistochemical reaction was detected in either calf at any exposure period.

Prompt formalin fixation is optimal for BVDV IHC; samples can be held in formalin at least 36 days without loss of reactivity. A 24-hour delay in fixation causes no apparent loss of reactivity in regrigerated specimens protected from desiccation. However, exposure to room air for even 24 hours caused false- negative IHC results.

1Department of Veterinary Pathobiology, Animal Disease Diagnostic Laboratory, Purdue University, West Lafayette, IN 2Department of Veterinary Pathobiology, Veterinary Medical Diagnostic Laboratory, University of Missouri, Columbia, MO 3Department of Veterinary Medicine and Surgery, Veterinary Medicine Extension, University of Missouri, Columbia, MO

205

Evaluation of SeM, Se75.3 and Se18.7 in ELISAs for Serum Antibody to Streptococcus equi J.F. Timoney1 , A. Graves 2 , S. Muthupalani1 , and J. Morrow 2

Streptococcus equi of Lancefield group C causes equine strangles, an economically important and widespread disease of horses. The causative organism appears to be a clonal descendent of S. zooepidemicus with which it shares extensive DNA homology and expresses an almost identical array of proteins. Immunogenic proteins uniquely expressed by S. equi include the antiphagocytic SeM, the pyrogenic mitogens SePE-I and H, Se75.3 and the antiphagocytic Se18.7. SeM has been widely used in studies of equine immune response to S. equi and in diagnosis of post strangles sequelae, including bastard strangles and vasculitis. The aim of this work was to compare SeM with Se75.3 and Se18.7 as antigens for assay of S. equi specific antibody in sera of horses. Categories of sera included pre-exposure, convalescent, post-vaccination, purpuric, bastard strangles and post strangles myopathy. All 3 antigens were associated with strong antibody responses in all categories except post-vaccination. Post- vaccination sera showed no reactivity with Se75.3 so this protein should be useful distinguishing vaccine from infection responses. Pre-exposure sera generally had very low antibody levels to each antigen confirming that exposure to S. zooepidemicus as a mucosal/tonsillar commensal did not trigger responses to unique proteins of S. equi. Purpuric, bastard strangles, and myopathic sera usually had very high levels of antibody to all 3 proteins. SeM was generally the most useful antigen although the antibody responses of some horses were partially cross-reactive with S. zooepidemicus.

1 Gluck Equine Research Center, University of Kentucky, Lexington, KY 2 IDEXX Equine Biodiagnostics, Coldstream Research Campus, University of Kentucky, Lexington, KY

206

Investigation of Acute Nephrosis in a Herd of Beef Cattle in Nebraska

L.G. Corbellini1,2*, M. Carlson1, B. W. Brodersen1, A. Doster1, D.G. Rogers1, D.J. Steffen1

Numerous toxic plants, metals, antimicrobials, and infections have been associated with cases of nephrosis in cattle in US. Ingestion of oxalate-bearing plants such as the genus Rumex sp. (dock), Kochia sp. (fireweed), Amaranthus sp. (redroot pigweed) and Halogeton sp. can cause severe renal damage. The diagnosis of oxalate poisoning is based on observing microscopic lesions in the kidneys which are characterized by nephrosis with oxalate crystals within renal tubules. This study describes cases of acute nephrosis in cattle in which rare oxalate crystals were observed in the renal tubules.

Three hundred and fifty yearling Red Angus heifers had been estrous synchronized, mated and then, transported to a pasture on June 8th, 2002. On June 22nd all heifers looked normal; however, a few days later five animals were found dead in the pasture. Overall, 42 heifers were found ill of which 12 died during a period of three weeks. The affected animals were dehydrated, anorectic and depressed. Two heifers examined by the veterinarian had protein (+++) and hemoglobin in the urine. A total of four animals were necropsied by the veterinarian practitioner and the gross findings reported were markedly perirenal edema and hemorrhage within the retroperitoneal space and in two animals mucosal sloughing was observed in the esophagus. Specimens from five affected animals including liver, lung, kidney, esophagus, intestine, lymph node, serum and rumen content were analyzed. Tissues from the esophagus were tested for the presence of BVDV by immunohistochemistry (IHC). The toxicological exams included multi-elemental analysis (Zn, Fe, P, Mn, Mg, Ca, Cu, and Na) and detection of heavy metals (Hg, As, Cd, Pb and Cr) from tissues samples (kidney, liver), and evaluation of nitrate/nitrite levels from serum samples. Rumen content from one case was submitted for microscopic examination and plant identification. Creatinine levels were measured in the serum from 45 affected animals. Histologic examination revealed marked acute tubular nephrosis in all cases analyzed. Fine granular casts were seen within proximal convoluted tubules. The epithelial cells had sloughed and many tubules were lined only by a basement membrane and surrounding connective tissue. Rare oxalate crystals were observed only after careful examination of kidney sections. Sections of esophagus were characterized by ulceration of the mucosa and replacement by a fibrinonecrotic membrane. Multi-elemental analysis did not detect deficiency or excess of minerals in the tissues and no detectable amounts of heavy metals was found. IHC and virus isolation were negative. Analysis of rumen content from one animal revealed significant amount of Rumex sp. seed and Kochia schoparia leaf material. Of 45 animals tested for creatinine, 31 (68.9%) had levels ≥2mg/dl (2-33.7mg/dl).

The severe nephrosis with the presence of oxalate crystals and the lack of other diagnostic findings suggests that the mortality observed in this herd occurred due to renal failure after ingestion of oxalate- containing plants. The significant amount of Rumex sp. and Kochia scoparia material detected in the rumen support those findings, since both plants contain oxalate. It was not possible to determine which plant was responsible for the toxicosis, however, the possibility of concurrent poisoning should be considered. Some studies report minimal amounts of oxalate crystals in the renal tubules after ingestion of Rumex sp., Kochia sp. In this report, rare oxalate crystals were found only after careful examination, denoting that the scarcity or even absence of oxalate crystals could preclude the diagnosis of some oxalate-containing plants.

1Federal University of Rio Grande do Sul, Brazil (CAPES/FAPERGS/Brazil) 2Veterinary Diagnostic Center, University of Nebraska-Lincoln

*To be considered for graduate student award

207 Neosporosis in Cows Vaccinated with a Neospora caninum Vaccine

L.G.Corbellini1,2, D.G. Rogers2, D.J. Steffen2, B.W. Brodersen2, and D.R. Smith2

The intracellular protozoan Neospora caninum (NC) is an important cause of bovine abortion worldwide. This parasite is transmitted horizontally to cattle via canids and vertically from dam to fetus. Protection against this infection depends on cell-mediated immunity (CMI). The objective of this investigation was to study the role of NC-infection among vaccinated dairy cows within a herd experiencing abortions.

Eight lactating cows, 3-8 years of age, in a 280-cow dairy located in Eastern Nebraska aborted during March through July, 2003. The 30-day cumulative incidence of observed abortions among lactating cows was 5.8% (1/17), 3.7% (1/27), 5.5% (2/36), 11.1% (3/27) and 2.9% (1/34) in March, April, May, June and July respectively. Based on Dairy Herd Improvement Association (DHIA) records only two observed abortion occurred in 2002. Six of those aborted fetuses, 3-7 months of gestation, were submitted for laboratory examination. Three fetuses had lesions consistent with neosporosis, and NC tachyzoites were detected by immunohistochemistry in tissues from two of these fetuses. Those NC abortions occurred in May, June and July. Mycotic placentitis, placentitis of unknown cause and fetal mummification with no discernible lesion were observed in the remaining cases. All fetuses tested negative for BVDV by immunohistochemistry and virus isolation and the results of bacteriologic culture were negative.

Based on an investigation visit to the farm, lactating cows were housed in a free stall barn and replacement heifers were raised on-pasture at a separate farm. No information about abortion rates in the heifers was available. No cattle additions had occurred in the last several months; however, the owner had introduced a puppy in February, 2003, which had been observed feeding on placenta, and had access to the free stall barn and feed storage areas. Neosporosis had been diagnosed in this herd years earlier, and a vaccination program using a commercial killed NC tachyzoite vaccine was implemented in 1998. Adult cattle (3 to 11 years of age) received two doses of vaccine annually in early gestation (during routine pregnancy testing). All post-parturient cows were routinely vaccinated with Leptospira spp. and modified-live IBR and BVD vaccines. Replacement heifers were not vaccinated for NC. Serum samples from unvaccinated heifers and vaccinated lactating cows were collected. Seroprevalence (ELISA-Idexx) among vaccinated cows and unvaccinated 1 and 2 year-old heifers was 84% and 36%, respectively.

Some, but not all, abortions in this herd could be attributed to NC infection. The rate of NC seroprevalence found among unvaccinated heifers is probably explained by vertical transmission and the relatively higher seroprevalence rate in the cow herd might be explained by vaccination. However, it is also possible that previously uninfected adult cattle had recently become infected by horizontal transmission coincident with the puppy being introduced in February. Point source exposure in endemically infected herds related to introduction of naïve dog has been described. Unfortunately, it was not possible to determine if the aborting cows were recently or chronically NC-infected. Vaccination also impaired interpretation of the NC serology results. We could not determine if NC vaccine failed to prevent abortion in this herd because there was not an unvaccinated comparison group. It is possible that the adverse consequences of NC infection might have been greater if the herd was not vaccinated. Little is known about NC vaccine efficacy in cattle infected either by ooccyst infection or transplacental transmission but this case-study demonstrates that NC vaccine efficacy is not 100%. Mid-gestation transmission from dam to calve has been observed in chronically infected cattle, probably due to downregulation of specific cell proliferation, which is crucial to protect against intracellular parasite infection. Killed vaccines usually do not stimulate CMI mechanisms. Further study of NC vaccine efficacy is needed in naturally-infected cattle populations.

1Federal University of Rio Grande do Sul, Brazil (CAPES/FAPERGS/Brazil) 2Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln

208 Evaluation of a Multiplex PCR Assay for Identification of Mycobacterium avium subsp. paratuberculosis from Liquid Cultures

K.S. Anklam1, E.J.B. Manning1, S. Sreevatsan2, and M.T. Collins1

Paratuberculosis (Johne’s disease), caused by Mycobacterium avium subsp. paratuberculosis (Mptb), is an untreatable chronic granulomatous enteritis of ruminants. Economic losses attributable to paratuberculosis are reported in agricultural enterprises in many countries; US dairy industry losses are estimated to be $200 to $250 million annually. Control of the spread of this infection is based on hygienic measures and removal of animals shedding Mptb in their feces and milk. These efforts are hampered by the lack of accurate and rapid diagnostic tests. Culture of Mptb from feces has been the most reliable method for identifying infected animals but it is very slow and complicated by the presence of closely related mycobacteria. Various mycobacterial insertion sequences (IS) have been used to differentiate among the different species and subspecies of the M. avium-M. intracellulare complex (MAIC), of which Mptb is a member. This study evaluates a multiplex polymerase chain reaction (PCR) assay developed for clinical sample testing by the University of Wisconsin-Milwaukee with molecular methods developed for research purposes by The Ohio State University (OSU). The goal of the methods is to reliably differentiate Mptb from other closely related mycobacteria found in primary liquid culture by targeting specific insertion sequences.

The multiplex PCR assay uses five sets of primers designed from published sequences of mycobacterial DNA. Resultant PCR products include a 484bp product from the 16S ribosomal DNA gene found in all mycobacteria, a 398bp product from IS900 a target consistently found in Mptb, a 753bp product from IS901 specific to M. avium subsp. avium (MAA), plus a 1291bp and a 608bp product from IS1245 and IS1311 respectively found in subspecies or subtypes of M. avium and M. intracellulare and possibly associated with virulence. To increase the sensitivity of the multiplex PCR assay the DNA extraction procedure was optimized to ensure an adequate amount of DNA was efficiently extracted from BACTEC liquid culture media supplemented with egg yolk, antibiotics and mycobactin-J.

The multiplex PCR assay was evaluated with acid-fast bacterial isolates obtained from 114 different clinical samples. Identification of these isolates by the OSU molecular methods was considered the reference method for this study. All isolates were positive for the 16S element by the multiplex PCR assay. A 92% concordance was demonstrated between the multiplex PCR assay and the OSU molecular methods; that is, 105 of 114 isolate identities were in agreement (69 Mptb, 10 MAIC non-Mptb and 26 outside MAIC). Nine isolates proved discrepant between the methodologies. Six of the nine discrepancies were reported as Mptb by OSU testing while the multiplex PCR assay was unable to demonstrate either IS900 or IS1311 PCR products. Two other discrepant strains were identified as MAIC, non-Mptb by the multiplex assay. One of these strains was reported as outside MAIC by OSU methods and the other was reported as Mptb. The remaining discrepant strain demonstrated IS901 and IS1311 PCR products identifying this strain as MAA by the multiplex assay. The OSU methods identified this strain as outside MAIC. Possible causes under investigation for the discrepant results provided by the multiplex PCR include genomic DNA preparation methods, amplification inhibition by elements in the initial sample or amplification interference due to multiple elements being amplified in a single reaction.

The results indicate that the multiplex PCR assay is an effective and reliable confirmatory assay for the identification of Mptb and other mycobacteria from primary liquid cultures.

1Department of Pathobiological Sciences, University of Wisconsin, Madison, WI 2Food Animal Health Research Program, Ohio Agricultural Research and Development Center, and Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, OH

209 Detection of Mycobacterium avium subsp paratuberculosis in Bovine Feces and Milk Using Adiapure® Extraction Kit and Real Time Adiavet® PCR Kit

B. Blanchard, Y. Versmisse and B. Chevallier

M. avium subsp. Paratuberculosis causes Johne’s disease in ruminants. This disease is economically significant in the cattle industry but its control is hampered by the lack of sensitive and rapid detection test. We have developed two commercial kits: Adiapure® for DNA extraction from milk and feces and Adiavet® ParaTB for real-time IS900-based PCR. Adiapure extraction kit combines bead brinding of feces with an ultrafiltration system. This new system allows to extract the DNA of 96 feces in 1 hour. For milk DNA extraction immunomagnetic beads separation coupled with bead brinding is used. Adiavet ® ParaTB PCR kit was found to be very effective and allows the detection of 5-10 organisms in 1g of faecal samples. This new system was compared with classical DNA extraction without brinding. 94 faeces stored at -20°C from 2 infected herds were analyzed, 40% more of positive animals were found with the new protocol of DNA extraction.

For initial development of milk DNA extraction, 10 ml of healthy milk were infected with a suspension of faecal sample containing 5.106 organisms/ gram. Serial dilutions were prepared. M. paratuberculosis were captured with magnetic beads and disrupted with glass beads in a mixer mill. The disrupted solution was then purified by ultrafiltration. The collected DNA was then tested for the presence of M. Paratuberculosis with a real time PCR test. Using this protocol, we can detect less than 80 organisms in 1 ml of milk.

The new commercialized Adiavet® ParaTB and Adiapure® extraction kits are ready to use and results can be obtained under 4 hours. This improved method for detection and identification of M. paratuberculosis is a powerful and helpful tool for veterinary laboratory involved in Johne’s disease programs.

Adiagène SA, 38 rue de Paris. 22000 Saint-Brieuc. France.

210 Development of Multiplex Real-Time RT PCR for the Detection of Akabane and Aino Viruses and Molecular Characterization of Israeli Isolates of Akabane Virus

Y. Stram1,A. Levine2, L. Kuznetzova1, J. Brenner1, Y. Braverman1, M. Guinni1.

In the last few decades the ruminant population of Middle Eastern countries including Israel was considered to be endemically exposed to akabane virus (AKAV). More recently, outbreaks of newborn calf teratogenic malformations in Israel have appeared. Surviving calves were found to have high titers of AKAV and in some cases aino virus (AINV) neutralizing antibodies indicating exposure to those viruses.

AKAV and AINV belong to the Simbu serogroup of the arthropod-born Bunyaviridae which consists of 24 antigenically different viruses that are related serologically. They can cause severe teratogenic malformations when susceptible pregnant ruminants are infected. Infection of susceptible cows usually causes subclinical viremia of short duration and the virus is cleared rapidly from the blood. In pregnant cows the virus can invade the central nerve system and/or the skeletal tissues of the fetus and may cause arthrogryposis (AG) or hydranencephaly/hydrocephaly/microencephaly (HE/ME) encephalomyelitis. Blood-sucking insects such as biting midges and mosquitoes serve as vectors and transmit the viruses to vertebrates. AKAV and AINV were identified serologically or by virus isolation in Japan, Korea, Taiwan, Israel, Turkey, Saudi Arabia and Australia.

The virion is enveloped and the genome consists of three segments of ss (-) RNA. The L segment RNA carries the polymerase gene, the M segment RNA encodes the two G1, G2 glycoproteins, and the 858 base S segment RNA and encodes the nucleocapsid (N) and nonstructural (NSs) proteins. To detect the AKAV and AINV genomes in affected calves and arthropod vectors, a multiplex quantitive reverse- transcriptase real-time PCR was developed using MGB TaqMan chemistry. Each specific probe was labeled with a different fluorescent dye - VICR for detecting AKAV and 6-carboxy-fluorescein (FAM) for detecting AINV. Using the developed real-time RT PCR, AKAV was identified in Calicoidies imicola trapped at the Volcanic Center. It was calculated that the insect extract contained 1.5x105 copies of the genome segment S. Following amplification of the entire S genome segment, its nucleotide sequence was determined and found to have over 93.4% identity with the S segment of other AKAV isolates. The deduced amino acid (aa) sequence of the combined nucleocapsid and the non structural protein showed more than 96.6% identity. Phylogentic trees constructed using the combined deduced nucleocapsid and the nonstructural protein aa sequences and the nucleotide sequences showed that the Israeli isolate forms a fourth cluster of AKAV indicating a separate virus lineage. AKAV genome was also identified in the brain of a calf with typical Simbu related teratogenic malformations. When trying to amplify the entire viral S segment it was revealed the viral genome is truncated representing only 430 bases from the 5' end of the genome.

1. Kimron Veterinary Institute, P.O. Box 12 Beit Dagan, 50250. 2. Molecular Virology Department, The Faculty of Medicine, The Hebrew University of Jerusalem, P.O. Box 12272 Jerusalem, 91120.

211 Detection of Brachyspira hyodysenteriae and pilosicoli in Porcine Feces Using Real-Time PCR

P. L. Bell-Rogers1, M. Archambault1, G. Josephson1, B. McEwan1, R. Friendship2, J. Prescott3, G. Maxie1 and H.Y. Cai1

The spirochete Brachyspira hyodysenteriae is the etiological agent of swine dysentery (SD), a severe mucohemorrhagic diarrheal disease of pigs. Brachyspira pilosicoli causes the less severe swine intestinal spirochetosis (SIS). Detection and diagnosis of SD and SIS have traditionally been based on culture from fecal samples, however this method is unreliable due to the fastidious nature and slow growth of these spirochetes. In this study, quantitative real-time PCR assays were developed for the Roche LightCycler™ (LC) using primers and hybridization probes specific to the NADH oxidase gene (nox) of B. hyodysenteriae and the 23S rRNA gene of B. pilosicoli. Specificity of the real-time PCR was confirmed using nine ATCC strains of B. hyodysenteriae, B. pilosicoli and B. innocens, and a panel of DNA isolated from twenty-six common porcine intestinal pathogens. The sensitivity of the real-time PCR was determined using both pure cultures and fecal samples spiked with serial dilutions of culture. Several commercial DNA extraction kits were used to compare the sensitivity of real-time and block thermocycler PCR using DNA extracted from B. hyodysenteriae-spiked fecal samples. The PCR assays were evaluated by testing field samples.

The B. hyodysenteriae LC PCR was able to detect 0.1-10 cells per PCR reaction from pure cultures compared to a detection limit of 10 – 1000 cells per PCR reaction for conventional block thermocycler PCR. Similarly, the real-time PCR was 100-1000-fold more sensitive than block themocycler PCR for the detection of B. hyodysenteriae in porcine fecal samples in spiking experiments with a detection limit of 1000 cells/200mg feces.

The B. pilosicoli LC PCR was able to detect 10 cells per PCR reaction from pure cultures, compared to 100 cells per PCR reaction for block thermocyler PCR. B. pilosicoli LC PCR detected 10,000 cells/g feces and was 10-fold more sensitive than the conventional block thermocycler PCR.

Both real-time PCR assays were evaluated by testing 124 sentinel herd fecal samples collected from Ontario farms, as well as clinical cases submitted to the Animal Health Laboratory. Among the sentinel herd samples, one (0.8%) was positive for B. hyodysenteriae, and none were positive for B. pilosicoli. Among 23 enteric diseased porcine fecal or intestinal tissue samples submitted to the Animal Health Laboratory, 2 cases were determined to contain 105 – 107 cells of B. hyodysenteriae/200mg feces and 4 cases were found containing 104 – 107cells of B. pilosicoli/200mg feces. In addition, intestinal tissue from 2 cases was positive for B.hyodysenteriae, and tissue from 2 cases was positive for B. pilosicoli. We conclude that: 1. The real-time PCR assays for both B. hyodysenteriae and B. pilosicoli provide a sensitive and specific method for the detection of these difficult-to-culture organisms. In addition, real-time PCR permits the quantification of bacterial load in fecal samples. 2. The real-time PCR on pure cultures was at least a hundred-fold more sensitive than traditional PCR (B. hyodysenteriae) and ten-fold more sensitive for B. pilosicoli. 3. Field validation of both assays using sentinel herd fecal samples showed a low incidence of both organisms.

1Animal Health Laboratory, Laboratory Services Division, University of Guelph, Guelph, ON 2Department of Population Medicine, University of Guelph, Guelph, ON 3Department of Pathobiology, University of Guelph, Guelph, ON

212 Transfer and Optimization of a Single-tube CSFV Real-time RT-PCR Assay to a High-throughput 96 Well Format A.J. Eberling1, B.M. Martin2, T.S. McKenna1 ,T.R. Beckham1.

Classical Swine Fever virus (CSFV) is a highly contagious disease that affects both domestic and wild pigs. The etiological agent of CSFV is a small, enveloped, single stranded RNA virus belonging to the Flaviviridae family in the genus Pestivirus. Due to its potential for rapid spread and high morbidity, CSFV outbreaks can have serious consequences on international trade of swine and swine products. Although the United States is considered “free” of CSFV, numerous outbreaks in neighboring countries such as Mexico, Cuba and the Dominican Republic continue to threaten the pork industry. Current assays use to diagnose CSFV are time consuming and laborious. A single-tube “dried-down” real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) assay (Tetracore, Inc., Gaithersburg, MD) for the detection of CSFV is currently being validated on the SmartCycler™ II (Cepheid, Sunnyvale, CA). This rapid diagnostic test can detect and identify CSFV infection early during the course of the disease. This assay should be available in a high-throughput format for use in surveillance and/or an outbreak situation. The SmartCycler™ II has the capacity to run 96 samples at once when 6 processing blocks are serially connected. However, this option is expensive and cumbersome. Other commercially available rapid nucleic analysis platforms allow for 96 sample capacity and are easily transitioned to automation using a standard liquid handling system. The present work describes the transfer and optimization of a “dried-down” single-tube rRT-PCR assay for CSFV to a 96 well platform (MX4000, Stratagene, La Jolla, CA) using a wet chemistry.

RNAs from viral isolates and field samples were extracted using the RNeasy Mini-Kit as per the manufacturer’s instructions (Qiagen, Valencia, CA). All PCR reactions, regardless of platform were performed in a final volume of 25 µl using 2.5 µl of extracted RNA as template. Primer and probe concentrations as well as thermocycling conditions were optimized for the 96 well platform. Due to the slower ramping rate, thermocycling conditions on the 96 well platform required longer denaturation and extension times than used on the SmartCycler™ II. Denaturation time was extended from 2 sec to 30 sec and the extension time was extended from 30 sec to 1 min.

A total of 101 viral isolates from the Foreign Animal Disease Diagnostic Laboratory repository, the EU Reference Laboratory in Hannover, Germany, and field samples (nasal swabs) from the Dominican Republic were used to demonstrate equivalency between the wet and dry chemistries and across platforms. Limit of detection (LOD) assays indicated that the SmartCycler™ II and the MX4000 detected equivalent amounts of CSFV regardless of chemistry (wet or dry). In addition, the MX4000 detected 96 out of 101 samples at a Ct equivalent to or lower than that reported on the SmartCycler™ II. Eighteen of the samples were within 1.5 Ct of the value obtained on the SmartCycler™ II. The remaining 78 samples had a mean Ct value of 4.0 cycles less than that observed on the SmartCycler™ II.

In conclusion, a single-tube “dried-down” CSFV assay for use on the SmartCycler™ II platform has been transferred to and optimized on the MX4000. A demonstration of equivalency on the MX4000 to the SmartCycler™ II platform has been shown using a CSFV rRT-PCR assay. This assay can now be used for high-throughput sample analysis in a national surveillance program or in an outbreak situation.

1United States Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services, National Veterinary Services Laboratories, Foreign Animal Disease Diagnostic Laboratory, Greenport, NY. 2United States Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services, National Veterinary Services Laboratories, Diagnostic Bacteriology Laboratory, Ames, IA.

213 Prevalence Study of Coxiella burnetii (Q-fever) in the United States Dairy Herds Based on Bulk Tank Milk Testing by Trans-PCR

S.G. Kim, E.H. Kim, A.E. Cassano and E.J. Dubovi

Bulk tank milk samples from dairy herds across the United States were tested to determine the prevalence of Coxiella burnetii (Q-fever) at the herd level over a 3-year period. Conventional and real-time Trans- PCR assays, targeting the unique repetitive transposon-like element (IS1111) of C. burnetii, were used in the study.

Of the 316 bulk tank milk samples, 233 (71.5%) were strongly positive by Trans-PCR. Thirty-four samples (19.3%) produced weak bands after PCR. Combining these results, the overall prevalence of C. burnetii at the herd level is greater than 90% in the United States. Positive results were confirmed by nested PCR and DNA sequencing. The sequencing results were consistent with the published sequence of IS1111with 100% homology. Individual milk samples from the entire cows on a Q-fever PCR positive dairy were tested two times over a two-month period. Of a total of 53, 28 (52.8%) were either positive or weak positive. Daily shedding levels of C. burnetii by individual cows were assayed by real-time Trans- PCR, which was developed in this study. The shedding numbers in milk by each cow stayed relatively steady over a 7-day period. Quarter milk samples from 5 cows were tested weekly for 6 weeks to examine shedding patterns between quarters. Though shedding levels were consistent in general, intermittent shedding and variations between quarters of the same cow were observed.

Commercial pasteurized milk samples also tested positive by Trans-PCR with a titer ranging from 102 to 103 genome-equivalents/ml. The public health implications of this finding needs further studies though it is known that C. burnetii cells in milk are killed by the commercial pasteurization process.

New York State Animal Health Diagnostic Laboratory, Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853

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Wildlife Disease Research at the APHIS National Wildlife Research Center

R.G. McLean1, L. Clark, M. R. Dunbar, and K. C. Vercauteren

Research on wildlife diseases at the newly formed Wildlife Disease Program at the National Wildlife Research Center, Wildlife Services, APHIS, concentrates on diseases of importance to domestic animal and human health and include multiply studies on wildlife rabies, bovine TB, chronic wasting disease (CWD), West Nile virus (WNV), pathogenic bacteria of birds, pseudorabies (PR), and avian influenza (AI). The goal of the research is to develop innovative methods for surveillance, intervention, prevention, and control of these diseases.

Rabies research is conducted to support the national management of wildlife rabies that utilizes an oral rabies vaccine baiting program to combat rabies transmission in raccoon populations in the eastern United States, and in coyote and gray fox populations in Texas. Field and laboratory research is aimed at improving the effectiveness of the baiting strategies and developing new baits and baiting methods for the target species and for additional host species. Research on identifying potential wildlife reservoirs or carriers of Bovine TB besides white-tailed deer is being conducted in Michigan. Investigations of wildlife-cattle interactions are being conducted to develop methods to reduce or eliminate deer and cattle contact that is conducive to TB transmission. Research on chronic wasting disease is concentrating on measuring the interaction of free-ranging and captive cervids and developing affordable methods to prevent that contact, on investigating the population biology, dynamics, and movement patterns of deer in relation to the transmission and geographical expansion of CWD, and in developing potential vaccines and decontamination procedures. Investigations of WNV are designed to develop and test new surveillance methods, to better understand the ecology of WNV in vertebrate hosts, to develop epidemiological models, and to test and apply prevention strategies guided and evaluated by the model. Birds as hosts for and carriers of pathogenic bacteria were identified in urban goose populations and will be investigated in dairy and confined feeding operations. Studies on the potential threat of PR in feral pigs to domestic swine herds have begun at our new field station in south Texas and AI studies in wild birds associated with poultry will be initiated in northeastern states.

National Wildlife Research Center, Wildlife Services, APHIS, USDA, Fort Collins, CO

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ACKNOWLEDGEMENTS

On behalf of the Program Committee, THANK YOU to all who have contributed ideas, information, presentations, posters and the hard work and detail needed to support a national meeting. This involves many individuals and groups including:

Our loyal and continuing members and guests of AAVLD who shared and interacted to provide the basis for our scientific program

Our invited speakers for the First Plenary Session on Transmissible Spongiform Encephalopathies, as well as David Benfield and James Evermann, who organized the session.

Invited speakers for the Joint Plenary Session

The moderators for the scientific sessions who keep to program moving and lend their expertise to keeping our sessions dynamic and on time

The program committee who evaluated and edited nearly 160 abstracts for inclusion in the program

Kathy Berrett for assistance in editing and compiling the proceedings book

Special thanks to Donna Dare, Linda Ragland, Allison Reitz, Pat Blanchard and others who provide the knowledge and innumerable details that turn ideas and abstracts into the reality of a meeting

Our Plenary Session sponsors, Patrons and Exhibitors named on the following page, whose presence and financial support helps to expand and enhance the quality and scope of our meeting

Program Committee 2004

Gary Osweiler, Chair John Adaska Catherine Barr Tim Baszler Carole Bolin Francois Elvinger Scott Fitzgerald Sharon Hietala Lorraine Hoffman Mitzi Libal Brenda Love Terry McElwain Carlos Reggiardo H.L. Shivaprasad David Steffen Trish Talcott Melissa Behr Tom Besser Mark Thurmond

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The AAVLD Scientific Program and related activities are sponsored in part by generous support from the following companies or organizations

First Plenary Session

Bio-Rad Laboratories

Joint Plenary Session

Bio-Rad Laboratories & RMS, Research Management Systems, Inc

Molecular Diagnostics Session & Poster Session

Cepheid

JVDI Patrons Addison Biological Laboratories Bio-Rad Laboratories Grand Laboratories Trek Diagnostics Veterinary Diagnostic Technology, Inc Viral Antigens, Inc

AAVLD Meeting Exhibitors

• BD Diagnostic Systems • Prionics AG • Bio-Rad Laboratories • Quality Systems Integrators • Biovet, Inc. • Synbiotics Corporation • Centaur, Inc • Tetracore, Inc. • Cepheid • Trek Diagnostic Systems • Crawford Industrial Group • Ventana Medical Systems, Inc. • Global VetLink, LC • Vetstar • IDEXX Laboratories, Inc. • Viral Antigens, Inc. • Key Scientific Products • VMRD, Inc. • National Institute for Animal • Waste Reduction by Waste Agriculture (NIAA) Reduction, Inc. (WR2)

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