SCIENTIFIC SESSIONS 43RD ANNUAL MEETING AMERICAN ASSOCIATION OF VETERINARY LABORATORY DIAGNOSTICIANS

Plenary Scientific Session A Saturday, October 21, 2000 8:00 AM – 11:00 AM Birmingham Ballroom – XI/XII (First Floor)

Graduate Student Award Competition Sponsored by the AAVLD Foundation

Moderators: Drs. David Zeman and Pat Blanchard Page 8:10 Welcome and Opening Remarks

8:15 am AgNOR Score and Ki67 Index as Prognostic Indicators of Cutaneous Mast 1 Cell Tumor in Dogs: Review and Recommendations—K. Boyd* and E. Howerth

8:30 am Production of Clinical Disease and Lesions Typical of Postweaning 2 Multisystemic Wasting Syndrome (PMWS) in Experimentally Inoculated Gnotobiotic Pigs—M. Kiupel* G. Stevenson, J. Choi, K. Latimer, C. Kanitz, and S. Mittal

8:45 am Role of Porcine Circovirus Type 2 in Postweaning Multisystemic Wasting 3 Syndrome of Pigs—R. Pogranichnyy*, P. Harms, S. Sorden, J. Zimmerman, and K. Yoon

9:00 am A Case Report of Suspected Fumonisin B1 Neurotoxicoses in Three Juvenile 4 Equines with Atypical Histopathologic Lesions in the Brain—T. Evans*, S. Turnquist, L. Kellam, A. Templer, N. Messer IV, and S. Casteel

9:15 am Pathological and Epidemiological Investigation of West Nile Virus Disease 5 in Equines in Long Island, New York—A. Wilson, B. Meade, T. Varty, D. Hutto, T. Gidlewski, and D. Gregg

9:30 am Detection of North American West Nile Virus in Animal Tissue by Nested 6 RT-PCR—D. Johnson, E. Ostlund, and B. Schmitt

9:45-10:15: am BREAK

10:15 am Rabbit Calicivirus Disease Confirmed in Iowa in March of 2000—P. Halbur, 7 R. Myers, K. Petersburg, R. Blair, J. Schiltz, K. McKeown, D. Gregg, and L. Thomas

10:30 am After the Outbreak: What Happens After an Exotic Disease is Diagnosed in 8 the USA—K. Petersburg, J. Schiltz, R. Blair, D. Anderson, J. Johnson, B. Bischoff, I. Stewart, J. Annelli, L. Thomas, J. Lubroth, D. Gregg, J. Neilan, S. Shawky, S. Swenson, L. Capucci, and T. McKenna

10:45 am Circoviral Postweaning Multisystemic Wasting Syndrome (PMWS): 9 Retrospective Confirmation of Two Outbreaks in English Herds from 1986—R. Higgins, D. O’Toole, E. Clark, K. West, G. Wells, P. Blackburn, and C. Cundall

11:00-12:00 House of Delegates Meeting

* Graduate student presentation

Pathology Scientific Session A Saturday, October 21, 2000 1:00 PM – 5:00 PM East Meeting Rooms–D/E/F (Third Floor)

Moderators: Drs. Doug Rogers and Jim Turk Page 1:00 pm The Incidence of Clostridium difficile-associated Enteritis in Scouring 10 Piglets and its Association with Specific Gross and Microscopic Lesions— M. Yaeger, N. Funk, and L. Hoffman

1:15 pm Reproduction of Clostridium difficile-associated Enteritis by Experimental 11 Inoculation of Piglets—K. Post, R. Glock, A. Holtcamp, B. Jost, and J. Songer

1:30 pm Evaluation of the Sensitivity of Fetal Serology, IHC and PCR for the 12 Diagnosis of PRRSV in Fetuses—M. Yaeger, J. Benson, J. Christopher-Hennings, and K. Lager

1:45 pm The Sensitivity of Immunohistochemistry for the Detection of Porcine 13 Reproductive and Respiratory Virus (PRRSV) in Vaccinated and Unvaccinated Swine—M. Yaeger

2:00 pm Multiple Abortions in a Herd of Cows Vaccinated while Pregnant with 14 Modified-live Infectious Bovine Rhinotracheitis Virus Vaccine—J. Nietfeld, M. Muenzenberger, S. Kapil, G. Andrews, B. DeBey, and G. Kennedy

2:15 pm Clostridial Abomasitis in Day-old Dairy Calves—J. Songer, C. Reggiardo, 15 J. Biwer, D. Bueschel, and R. Glock

2:30-3:15 pm BREAK AND POSTER SESSION

3:15 pm Fatal Infections in Lambs Associated with a Novel Adenovirus—B. DeBey, 16 H. Lehmkuhl, and K. Ewert

3:30 pm An Outbreak of Ovine Abortions Caused by Coxiella burnetti in Kansas— 17 T. Aboellail and K. West

3:45 pm PrP-Sc Detection in Lymphoid Tissues of Clinically Normal Sheep and 18 Mule Deer Infected with the Agents of Scrapie and Chronic Wasting Disease: Standardized Protocols for Automated Immunostaining— K. O’Rourke, R. Zink, B. Cummings, A. Anderson, and T. Spraker

4:00 pm Comparison of Neuroanatomical Patterns of Spongiform Encephalopathy 19 and Immunohistochemical Staining of Prion Protein in Brain and Lymphoid Tissues of Free-ranging, Research, and Ranch-raised Elk— T. Spraker, R. Zink, B. Cummings, M. Wild, M. Miller, and K. O’Rourke

4:15 pm A Prospective and Retrospective Study on Ovine Herpesvirus-2 20 Malignant Catarrhal Fever in Feedlot Bison—D. O’Toole, H. Li, D. Montgomery, C. Sourk, and T. Crawford

4:30 pm Bovine Tuberculosis in Michigan: An Overview of Five Years 21 Surveillance—S. Fitzgerald, K. Butler, K. Clarke, S. Schmitt, D. O’Brien, T. Cooley, and C. Brunning-Fann

4:45 pm Deer to Deer Transmission of Mycobacterium bovis—M. Palmer and 22 D. Whipple

Microbiology Scientific Session A Saturday, October 21, 2000 1:00 PM – 5:00 PM Birmingham Ballroom III (First Floor)

Moderators: Drs. Kyoung-Jin Yoon and Robert Whitlock Page 1:00 pm Evaluation and Comparison of Veterinary Diagnostic Test Kits— 23 L. Henderson

1:15 pm A New Commercial Competitive Enzyme-linked Immunosorbent Assay 24 for Anaplasma marginale Serum Antibodies—S. Adams, T. McGuire, D. Knowles, and T. McElwain

1:30 pm A Rapid Serologic Test for the Detection of Antibodies to Mycobacterium 25 paratuberculosis with Applications for Bovine Practitioners—T. Jackson

1:45 pm A New Commercial ELISA for Bovine Leukemia Virus Antibody—S. Adams 26 and T. McGuire

2:00 pm Development of an ELISA for the Detection of Antibodies to Mycoplasma 27 hyopneumoniae in Swine Sera—K. Velek, L. Plourde, and H. Liauw

2:15 pm Evaluation of In-house Produced Herrold’s Egg Yolk Agar with 28 Commercially Prepared Formulations With and Without Nalidixic Acid and Vancomycin—B. Love, T. Farrell, B. Byrum, T. Wittum, T. Burns, P. Cullum, and D. Callihan

2:30 pm A New Liquid Culture Method, the Trek ESP Culture System II, for the 29 Rapid Detection of Mycobacterium avium subsp. paratuberculosis in Bovine Clinical Samples—S. Shin, S. Kim, L. Miller, P. Harpending, and D. Lein

2:45-3:15 pm BREAK AND POSTER SESSION

3:15 pm Paratuberculosis in Dairy Cattle: Classification of Cattle by Colony Counts 30 of M. paratuberculosis in Fecal Samples—R. Whitlock, R. Sweeney, T. Fyock, S. Wells, and J. Stabel

3:30 pm If You Use a Different Test, You Will Get a Different Answer— 31 M. Collins and J. Buss

3:45 pm A Fast and Sensitive Diagnostic Assay for the Detection of 32 Mycobacterium paratuberculosis in Bovine Feces—S. Spatz and S. Hogan

4:00 pm Application of Molecular-based Techniques in the Accurate 33 Detection of Mycobacterium avium ssp. paratuberculosis—S. McLellan, H. Pirkov, and R. Lambrecht

4:15 pm Molecular Cloning and Characterization of Mycobacterium avium 34 Subspecies paratuberculosis Antigen 85 Complex Gene Family, 85A, 85B and 85C—Y. Chang, D. Veerabadran, K. Shin, S. Shin, R. Jacobson, and D. Lein

4:30 pm Development of Quantitative PCR Based on the ABI 7700 System 35 (TaqMan) for Mycobacterium avium subsp. paratuberculosis—S. Kim, S. Shin, C. Rossiter, S. Stehman, R. Jacobson, and D. Lein

4:45 pm Comparison of a Novel and Traditional Processing Method with 36 Several Media Formulations to Detect M. paratuberculosis in Bison Tissues—C. Thornton and R. Whitlock

Toxicology Scientific Session Saturday, October 21, 2000 1:00 PM – 5:00 PM East Meeting Rooms–J/K/L (Third Floor)

Moderators: Drs. Catherine Barr and Thomas L. Carson Page 1:00 pm Copper Poisoning in Rabbits: A Case Report—W. Rumbeiha and 37 W. Braselton

1:15 pm Solanum dimidiatum and Crazy Cow Syndrome—A. Barr, B. Abbitt, 38 A. Vardeman, and J. Reagor

1:30 pm The Immunologic and Toxic Changes of Chronic Locoweed Poisoning in 39 Cattle—B. Stegelmeier and P. Snyder

1:45 pm Microcystin-LA Toxicosis Among Cattle in California—B. Puschner, 40 J. Pelton, L. Heath, T. Francis, and D. Holstege

2:00 pm Highly Presumptive Intoxication of Sheep Exposed to Ozark Milkweed 41 (Asclepias viridis Walter)—R. Smith, P. Sharko, D. Bolin, and C. Hong

2:15 pm Facial Eczema in Ruminants – Sporidesmin Toxicosis—C. Hooper 42

2:30 pm Lesions of Experimental Stemodia kingii Intoxication in the Mouse— 43 M. Raisbeck, J. Allen, and S. Colegate

2:45-3:15 pm BREAK AND POSTER SESSION

3:15 pm Nitrate Levels in Cornstalks in Kentucky in the Drought of ’99: 44 Observations of Anomalies that may be Indicative of a Silver Lining— R. Smith

3:30 pm Experiences in Mycotoxin Testing at North Dakota State University— 45 H. Casper, B. Tacke, and D. Iverson

3:45 pm A Sensitive, Automated Method for the Measurement of Total 46 Estrogenic Activity in Feedstuffs—T. Evans, G. Rottinghaus, W. Welshons, B. Judy, and S. Casteel

4:00 pm Effects of Hemolysis on Serum Vitamin E in Horses, Cattle, Sheep, 47 and Cats—S. Hooser, K. Jochim, C. Simaga, C. Wilson, R. Everson, R. Bedel, L. Guptill, and W. Hilton

4:15 pm Acyclovir Toxicoses in Dogs: 10 cases (January 1996-March 2000)— 48 J. Richardson

4:30 pm Acute Renal Failure in a Dog Following the Ingestion of a Chinese 49 Herbal Preparation Containing Indomethacin—R. Poppenga, W. Birdsall, G. Griffin, and M. Cummings

4:45 pm A Case Study Involving Both Acute and Chronic Ibuprofen 50 Poisoning—W. Brewer, P. Parnell, and J. Caver

Avian and Food Safety Scientific Session Saturday, October 21, 2000 1:00 PM – 5:00 PM Medical Forum—G/H/I (Third Floor)

Moderators: Drs. H. L. Shivaprasad and Randy Singer Page 1:00 pm Direct Automated Cycle Sequencing for Diagnosis and Epidemiologic 51 Investigations of Avian Infectious Bronchitis Viruses—S. Hietala, L. Shih, and P. Woolcock

1:15 pm Amyloid Arthropathy Associated with Mycoplasma synoviae in 52 Brown Egg Laying Type Chickens—H. Shivaprasad and B. Daft

1:30 pm Assessing Bias in a Molecular Epidemiologic Study of E. coli in 53 Broiler Chickens—R. Singer, T. Carpenter, J. Jeffrey, C. Cooke, and D. Hirsh

1:45 pm Attaching and Effacing E. coli in Avian Species—H. Shivaprasad, 54 B. Daft, R. Crespo, and D. Read

2:00 pm Anti-microbial Residues Contamination in Shell Eggs: A Pilot Study— 55 G. Scortichini, G. Campana, A. Giovannini, A. Manetta, and A. Simonella

2:15 pm Efficiency of Sampling for the Determination of Colonization Status of Turkey 56 Flocks with Campylobacter spp.—F. Elvinger, S. Spencer, N. Sriranganathan, and F. Pierson

2:30 pm Quality Evaluation of Marketed Eggs at Retail Level in Italy—V. Prencipe, 57 V. Rizzi, A. Giovannini, and G. Migliorati

2:45-3:15 pm BREAK AND POSTER SESSION

3:15 pm Measuring Salmonella Prevalence on Swine Farms and at the Abattoir— 58 H. Hurd, J. McKean, M. Rostagno, R. Griffith, and I. Wesley

3:30 pm Antimicrobial Susceptibility Patterns of Salmonella spp. Isolated from 59 Animals and Animal Environments in Ohio, 1998-1999—B. Love, B. Byrum, A. Berge, E. Dunne, N. Doelling, and F. Angulo

3:45 pm Cost-effectiveness of Serological and Milk Tests for Bovine Brucellosis: 60 Comparison Through a Monte Carlo Simulation Model—A. Giovannini, Conte, and D. Nannini

4:00 pm Screening and Mass Spectral Confirmation of Beta-lactam Antibiotic Residues 61 in Milk Using LC/MS/MS—D. Holstege, G. Whitehead, B. Puschner, and F. Galey

Pathology Scientific Session B Sunday, October 22, 2000 8:00 AM – 11:45 AM Birmingham Ballroom – III (First Floor)

Moderators: Drs. Dave Steffen and Tanya Lemire Page

8:00 am Delayed-onset Papular Dermatitis at Culicoides Feeding Sites as 62 Confounding Factor in Experimental Studies of Arthropod-borne Disease—D. O’Toole, A. Perez de Leon, L. Mei, and L. McHolland

8:15 am Radiculomeningomyelitis due to Halicephalobus gingivalis in an 63 Equine—J. Johnson, C. Hibler, K. Tillotson, and G. Mason

8:30 am Equine Herpesvirus-1 and –4 Differentiation by a PCR Using 64 Formalin-fixed, Paraffin-embedded Tissues—W. Feria, H. Acland, and D. Tewari

8:45 am Possible Mechanisms for Facial Clefts in Wild Northern Leopard Frog 65 Tadpoles—C. Meteyer, D. Green, and K. Converse

9:00 am Novel and Emergent Viral Infections in Wild and Culture (d Sturgeon in 66 North America—S. LaPatra, B. Parker, J. Groff, and R. Munn

9:15 am Is Feline Endomyocarditis Associated with Bartonella Infection?— 67 E. Howerth, G. Jacobs, L. Bauer, D. Perzak, and T. Van Winkle

9:30 am Histologic and Genotypic Characterization of Mycobacteriosis in Two 68 Cats—G. Appleyard and E. Clark

9:45-10:15 am BREAK AND POSTER SESSION

10:15 am Saluki Heart Hemangiosarcomas—T. Bell, M. Sist, and D. Jarman 69

10:30 am Mammary Duct Ectasia in 51 Dogs—M. Miller, S. Kottler, L. Cohn, 70 G. Johnson, J. Kreeger, L. Pace, J. Ramos-Vara, J. Turk, and S. Turnquist

10:45 am Immunohistochemical Detection of Melanocytic Differentiation Antigen 71 Melan A in Canine Steroid-producing Tissues and their Tumors— J. Ramos-Vara, M. Beissenherz, M. Miller, G. Johnson, J. Kreeger, L. Pace, J. Turk, S. Turnquist, G. Watson, and B. Yamini

11:00 am Evaluation of Diagnostic Procedures for Canine Distemper— 72 R. LaRock, S. Mercado, R. Ely, and J. Thilsted

11:15 am Distribution of Lesions in Canine Dysautonomia: Review of 73 35 Cases—G. Johnson, D. O’Brien, K. Bailey, J. Kreeger, M. Miller, L. Pace, J. Ramos-Vara, C. Rosenfeld, A. Schreibman, J. Turk, S. Turnquist, and H. Gosser

11:30 am Interstitial Myocardial Fibrosis and Intramyocardial Coronary Arteriopathy 74 in Azotemic Dogs—J. Turk, S. Stockham, M. Boucher, G. Johnson, M. Miller, J. Ramos-Vara, L. Pace, S. Turnquist, J. Kreeger, C. Loiacono, J. Donald, and M. Scott

11:45 am Pathology Committee Meeting

Microbiology Scientific Session B Sunday, October 22, 2000 8:00 AM – 12:00 NOON Medical Forum – Auditorium (Second Floor)

Moderators: Drs. Lorraine Hoffman and Mitzi Libal Page 8:00 am Comparison of MIC Values for Tetracycline, Chlortetracycline and 75 Oxytetracycline vs. Swine Field Isolates of Actinobacillus pleuropneumoniae and Pasteurella multocida—C. Wu and T. Wolff

8:15 am Multiplex PCR Detection of Swine Respiratory Viral 76 Pathogens—S. Kleiboeker

8:30 am Vesicular Stomatitis in Pigs: Effects of Virus Strain and Serotype 77 on Contact Transmission—D. Stallknecht, L. Bauer, D. Perzak, M. Murphy, and E. Howerth

8:45 am Clostridium difficile as a Cause of Porcine Neonatal Enteritis—J. Songer, 78 K. Post, D. Larson, B. Jost, and R. Glock

9:00 am Molecular Characterization of Chlamydiaceae from Swine—L. Hoelzle, 79 K. Hoelzle, and M. Wittenbrink

9:15 am Improved diagnostic tests for serology and typing of Actinobacillus 80 pleuropneumoniae –T. Inzana, J. Schuchert, and B. Fenwick

9:30 am Assessment of Primers Designed from the Small Ribosomal Subunit RNA 81 for Specific Discrimination between Babesia bigemina and Babesia bovis by PCR—G. Linhares, A. Santana, and L. Lauerman

9:45-10:15 am BREAK AND POSTER SESSION

10:15 am The Use of Porcine Alveolar Macrophages for Detecting Shiga Toxin— 82 W. Mengeling, A. Vorwald, N. Cornick, K. Lager, and H. Moon

10:30 am Characterization of the Interaction of Escherichia coli Heat-stable 83 Enterotoxin (STa) with its Putative Receptor on the Intestinal Tract of Newborn Calves—A. Al-Majali, J. Robinson, E. Asem, M. Freeman, C. Lamar, and A. Saeed

10:45 am Evaluation of the ImmunoCardSTAT Rotavirus (ICS-RV) Assay for 84 On-site Detection of Subgroup A Bovine Rotavirus—C. Han, V. Ciesicki, L. Brown, A. Wise, M. Vickers, C. Kanitz, and R. Maes

11:00 am Simultaneous Detection of Bovine Coronavirus, Bovine Rotavirus and 85 Cryptosporidium parvum in Fecal Samples by Multiplex RT-PCR—A. Wise and R. Maes

11:15 am BVDV Nested RT-PCR Technique to Screen Herds Using Pooled Buffy 86 Coat Samples—L. Braun, D. Peterson, and C. Chase

11:30 am Bovine Viral Diarrhea Virus Infections in Calves from Auction Markets 87 and a Ranch—R. Fulton, J. Saliki, A. Confer, L. Burge, C. Purdy, R. Briggs, G. Duff, and R. Loan

11:45 am Isolation of Bovine Adenovirus Type 7 from Calves with Respiratory 88 Disease—J. Saliki, S. Caseltine, R. Fulton, and H. Lemkuhl

Epidemiology Scientific Session Sunday, October 22, 2000 8:00 AM – 12:00 NOON Birmingham Ballroom – XI (First Floor) Graduate Student Award Competition Sponsored by the AAVLD Foundation

Moderators: Drs. Mark Thurmond and Francois Elvinger Page 8:00 am A Survey of Antimicrobial Susceptibility Testing Practices of 89 Veterinary Diagnostic Laboratories in the U.S.—M. Brooks*, P. Morley, D. Dargatz, and D. Hyatt

8:15 am A Simulation Model to Assess the Effect of Age at Vaccination on 90 Transmission of Bovine Viral Diarrhea Virus in Dairy Heifers— C. Munoz-Zanzi*, M. Thurmond, S. Hietala

8:30 am Prevalence of Johne’s Disease in a Subpopulation of Alabama Beef 91 Cattle—B. Hill*, M. West, and K. Brock

8:45 am Evaluation of PRRS Diagnostics in Detecting Persistently Infected 92 PRRSV Carrier—D. Horter*, R. Pogranichnyy, C. Chang, K. Yoon, and J. Zimmerman

9:00 am The Use of One Tube Nested (OTN) PCR for the Detection of 93 Mycobacterium bovis in Bovine Milk—M. Antognoli*, J. Triantis, J. Hernandez, and M. Salman

9:15 am Epidemiologic Study of Neonatal Equine Clostridial Enterocolitis— 94 D. Hyatt, K. Tillotson, J. Traub-Dargatz, C. Dickinson, R. Ellis, P. Morley, M. Salman, G. Thompson, D. Bolte, and R. Magnuson

9:30 am Molecular Epidemiology of Year 2000 Pseudorabies Outbreaks in 95 Illinois and Tennessee—E. Hahn, B. Paszkiet, R. Weigel, and G. Scherba

9:45-10:15 am BREAK AND POSTER SESSION

10:15 am The Future of Diagnostic Laboratories in Surveillance: More than Just 96 Sentinel Chickens—B. Akey, J. Case, S. Hietala, F. Elvinger, C. Munoz-Zanzi, and M. Thurmond

10:30 am Cloning of Internalin A and Listeriolysin as Antigens in Listeria 97 Serology—P. Boerlin, F. Boerlin-Petzold, and T. Jemmi

10:45 am Outbreak of Multidrug Resistant Salmonella typhimurium (DT104) in Cats in 98 an Animal Shelter with Spread to Humans—R. Frank, J. Bender, K. Culbertson, and K. Smith

11:00 am Molecular Epidemiology of Mycobacterium avium subsp. paratuberculosis— 99 S. Pillai, J. Gummo, E. Hue Jr., D. Tiwari, J. Stabel, R. Whitlock, and B. Jayarao

11:15 am IS900-PCR Assay for Mycobacterium avium subsp. paratuberculosis from 100 Quarter Milk and Bulk Tank Milk Samples Allows Detection of Herds with Johne’s Disease—B. Jayarao, S. Pillai, D. Griswold, D. Wolfgang, L. Hutchinson, C. Burns, and C. Rossiter

11:30 am Geographically Targeted Survey of Cattle in Northeast Colorado for Evidence 101 of Chronic Wasting Disease—D. Gould, J. Voss, K. O’Rourke, M. Miller, B. Cummings, and A. Frank

11:45 am Future Directions in Probabilistic Diagnostic Assessment as Illustrated by Use 102 of Bayes Theorem in Diagnosing Bovine Viral Diarrhea Virus (BVDV) and Neospora caninum Infections—M. Thurmond, C. Munoz-Zanzi, S. Hietala, and W. Johnson * Graduate student presentation

Plenary Scientific Session B Monday, October 23, 2000 8:00 AM – 12:00 NOON Birmingham Ballroom – XI/XII (First Floor)

“Expectations of AAVLD Labs in the Next Century”*

Moderators: Drs. David Zeman and Bruce Akey Page

8:00 am Introduction—D. Zeman 103

8:10 am Future Trends in Food Animal Production in North America—M. Hogberg 104

8:30 am Global Trade Issues and How Science and Politics Shape Animal Health 108 Policies—W. Hueston

8:50 am The Role of the Office International des Epizooties in the Standardization 109 of Diagnostic Testing—J. Pearson

9:10 am Future Expectations of Veterinary Diagnostic Laboratories: Quality Assurance 111 Systems—D. Polson, W. Chittick, and D. Jordan

9:30 am Food Safety—Possible New Role for AAVLD Laboratories—R. Breitmeyer 116

9:50 am BREAK

10:00 am Profiles of Veterinary Practitioners in the Next Century—L. King 118

10:20 am What Future Swine Practitioners Need from Diagnostic Labs—R. Tubbs 120

10:35 am What Future Bovine Practitioners Need from Diagnostic Labs—B. Smith 122

10:50 am Future Needs of Equine Practitioners for Veterinary Diagnostic Laboratory 124 Services—N. Messer

11:05 am Expectations of AAVLD Labs in the Next Century: The Small Animal 125 Practitioner’s Perspective—P. Glouton

11:20 am Maximizing Our Service to the Public by Thinking Out of Our Traditional 129 Boxes—D. Zeman

11:40 am Graduate Student and Pope Awards

11:55 am House of Delegates Meeting

* Financial Support for this Special-Focus Session Provided by: • Pharmacia & Upjohn Co. • Pfizer Animal Health • Boehringer Ingelheim Vetmedica • IDEXX Laboratories, Inc. • Grand Laboratories

Poster Session Saturday, October 21st, 1:00 PM to Monday, October 23rd, 12:00 NOON Birmingham Ballroom I/V/IX (First Floor) Poster No. Page 1. Coronavirus Associated Epizootic Catarrhal Enteritis (ECE) of 134 Ferrets—M. Kiupel*, B. Williams, J. Raymond, C. Grant, and K. West

2. Effect of Transport Enrichment Media, Transport Time, and Growth Media 135 on the Detection of Campylobacter fetus subsp. venerealis—H. Monke*, B. Love, B. Byrum, T. Wittum, and D. Monke

3. Sensitivity and Specificity of Pooled Sample Testing for Diagnosis of 136 Low Prevalence Diseases and Effect on Cost and Predictive Values— C. Munoz-Zanzi*, M. Thurmond, S. Hietala, and W. Johnson

4. Oral and Intravenous Incoulation of Greyhound Dogs with Escherichia coli 137 O157:H7 did not Result in Cutaneous Renal Glomerular Vasculopathy— M. Renninger*, M. White, A. Saeed, C. Wu, J. Christian, and S. Albregts

5. RT-PCR RFLP Patterns of Various PRRSV Isolates Recovered from 138 Ontario Farms, 1998-2000—H. Alexander, S. Carman, D. Lloyd, G. Maxie, G. Josephson, and H. Cai

6. Effect of Iron Dextran on Nested RT-PCR Assay for PRRS Virus— 139 G. Appleyard, J. McMurchy, and B. Yue

7. Design and Evaluation of a Diagnostic PCR Test to Detect Clostridium 140 piliforme—D. Bolin, J. Donahue, M. Hiles, and S. Sells

8. Presence of Salmonella in Pig Ear Dog Treats Obtained from Retail 141 Stores—L. English, S. Ayers, S. Zhao, S. Friedman, D. White, D. Wagner, S. McDermott, and M. Myers

9. Brain Cholinesterase Activities in Exotic/Zoo Species—R. Everson, 142 J. Raymond, R. Bedel, and S. Hooser

10. Evaluation of the Svanovir ELISA for Bovine Leukosis, Bovine Viral Diarrhea 143 and M. paratuberculosis—G. Keefe, J. VanLeeuwen, and S. Hotham

11. Evaluation of IS 900-PCR Assay for Detection of Mycobacterium avium 144 subsp. paratuberculosis Directly from Raw Milk—S. Pillai and B. Jayarao

12. Comparison of Nested PCR, Pepsin/Trypsin Digest and Histology for the 145 Diagnosis of Whirling Disease in Salmonid Fish—T. Qureshi, M. White, and C. Santrich

13. Three Soft Tissue Sarcomas in Two African Hedgehogs (Atelerix albiventris)— 146 J. Ramos-Vara

14. Glucagonoma and Degenerative Joint Disease in a Jaguar (Panthera onca)— 147 J. Ramos-Vara, M. Miller, and D. Preziosi

15. Development of a PCR-based Test for Detection of Leptospira in Bovine 148 Semen—E. Tanaka, S. Druhan, and E. Golsteyn Thomas

16. Melanocytic Schwannoma in a Brown Bullhead Catfish (Ictalurus 149 nebulosus)—M. White and K. Sakamoto

17. Heavy Metal, Organochlorine, and Polyaromatic Hydrocarbon Burdens 150 in Steelhead Trout from Lake Michigan Compared to Rainbow Trout from the Pristine Waters of Northeastern Indiana—C. Wilson, M. White, R. Everson, J. Hall, M. Greeley, R. Bedel, and S. Hooser * Graduate student presentation AMERICAN ASSOCIATION OF VETERINARY LABORATORY DIAGNOSTICIANS (AAVLD)

PURPOSE Dissemination of information relating to the diagnosis of animal diseases. Coordination of diagnostic activities of regulatory, research, and service laboratories. Establishment of uniform diagnostic techniques. Improvement of existing diagnostic techniques. Development of new diagnostic techniques. Establishment of accepted guidelines for the improvement of diagnostic laboratory organizations relative to personnel qualifications and facilities. Consultant to the United States Animal Health Association on uniform diagnostic criteria involved in regulatory animal disease programs.

OFFICERS 2000 President ...... Bruce Akey, Richmond, VA President-Elect...... David Zeman, Brookings, SD Vice President...... Patricia Blanchard, Tulare, CA Secretary/Treasurer...... Arthur Bickford, Turlock, CA Immediate Past President...... Doris Miller, Watkinsville, GA

EXECUTIVE BOARD 2000 President ...... Bruce Akey President-Elect...... David Zeman Vice President...... Patricia Blanchard Past President...... Doris Miller Secretary/Treasurer...... Arthur Bickford Northeast...... Beverly Byrum Southeast...... Frederic Hoerr North Central ...... Gary Osweiler South Central ...... Bill Edwards Northwest...... Jerry Heidel Southwest...... Frank Galey Canada Provincial...... Grant Maxie Canada Federal ...... WDG Yates NVSL...... Open

For Membership/Subscription information, contact: AAVLD Secretary/Treasurer PO Box 1522 Turlock, CA 95381 Ph: 209-634-5837 Fax: 209-667-4261 Email: [email protected]

AgNOR Score and Ki67 Index as Prognostic Indicators of Cutaneous Mast Cell Tumor in Dogs: Review and Recommendations.

K. Boyd*, and E. W. Howerth

Cutaneous mast cell tumors are among the most common tumors in dogs. Appearance and biological behavior between individual tumors can be quite variable. Pathologists play a vital role in predicting tumor prognosis by assigning a histologic grade to individual tumors. Histologic grade has historically been one of the most important criteria in predicting tumor behavior. Unfortunately, several pitfalls of assigning a histologic grade remain. Criteria are subjective, pathologists vary in the application of criteria, and a large percentage of mast cell tumors are placed into the intermediate grade. This diminishes the value of current grading systems to clinicians. AgNOR scoring and Ki67 indexing of mast cell tumors have shown recent promise as predictors of aggressive biologic behavior in intermediate grade tumors. It is essential that standardized methods of counting be introduced as these markers become more widely used for MCT evaluation. AgNOR scores are determined by counting the number of AgNORs per nucleus in one hundred cells at 1000X magnification. Ki67 index is determined by counting the number of positive nuclei in 500 or 1000 cells. Performing these counts on MCT that are often edematous and have little to no solid areas can be challenging. Review of the current literature and details for performing these counts, including examples from various tumors ranging from loose edematous tumors to solid tumors will be presented.

Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, GA *To be considered for graduate student award.

Production of Clinical Disease and Lesions Typical of Postweaning Multisystemic Wasting Syndrome (PMWS) in Experimentally Inoculated Gnotobiotic Pigs

M. Kiupel*1, 2, G. W. Stevenson1, 2, J. Choi2, K. S. Latimer3, C. L. Kanitz1, 2, and S. K. Mittal2

Neonatal gnotobiotic pigs were inoculated with either porcine circovirus type 2(PCV2) isolated from a pig with postweaning multisystemic wasting syndrome (PMWS) or with a cell-free filtered tissue homogenate (TH) from a pig with naturally occurring PMWS. Pigs from the PCV and TH group were observed daily for clinical signs and were sacrificed on day 0, 3, 6, 13, 20 and 34 post inoculation (PI). Necropsy examinations were performed and tissues were collected for histopathology, in-situ hybridization and PCR. Only PCV was detected in PCV inoculated pigs, whereas PCV and porcine reproductive and respiratory syndrome virus (PRRSV) were detected in tissues of TH inoculated pigs. Porcine parvovirus (PPV) was not detected in any pig. Pigs in the PCV group were clinically normal until the seventh week PI when pigs occasionally coughed, were mildly dyspneic and one pig developed icterus. Pigs sacrificed on days 20 and 34 PI developed moderate and severe gross and microscopic lesions typical of PMWS, respectively. Lesions included granulomatous inflammation with typical circovirus intracytoplasmic inclusion bodies in macrophages in all lymphoid organs, diffuse hepatocellular necrosis and lympho-histiocytic periportal hepatitis, myocardial necrosis, necrotizing interstitial pneumonia, granulomatous interstitial nephritis and disseminated lympho-histiocytic perivasculitis. PCV nucleic acid was first detected in the nuclei of few macrophages in lymphoid follicles, and later in the cytoplasm of many macrophages in all affected organs as well as in the nuclei of hepatocytes, bronchial epithelial cells, renal tubular epithelial cells, intestinal epithelial cells, smooth muscle cells and cardiomyocytes. Pigs in the TH group developed severe watery diarrhea on day 6 PI and subsequent progressive and severe wasting, icterus and death by day 13 PI. On day 13 PI, gross and microscopic lesions were severe and typical of PMWS. Cells labeled for PCV nucleic acid were more numerous and widespread than in PCV pigs even on day 34 PI. Diarrhea in TH pigs was associated with severe lympho-histiocytic and atrophic enteritis. This paper reports the experimental reproduction of clinical disease and severe multisystemic microscopic lesions of PMWS in gnotobiotic pigs inoculated with only PCV2. Inoculation of gnotobiotic pigs with a cell-free filtered TH containing PCV2 and PRRSV, but not PPV, caused severe wasting disease and severe multisystemic microscopic lesions of PMWS that contained larger numbers of PCV-infected cells than were in pigs inoculated with PCV2 alone.

1Animal Disease Diagnostic Laboratory, Purdue University, West Lafayette, IN 2Department of Veterinary Pathobiology, Purdue University, West Lafayette, IN 3College of Veterinary Medicine, Department of Veterinary Pathology, University of Georgia, Athens, GA *To be considered for graduate student award.

Role of Porcine Circovirus Type 2 in Postweaning Multisystemic Wasting Syndrome of Pigs

R. M. Pogranichnyy*, P. A. Harms, S. D. Sorden, J. J. Zimmerman, and K. J. Yoon

Postweaning multisystemic wasting syndrome (PMWS) has been recognized as an emerging disease of swine in the last several years throughout the world. Porcine circovirus type 2 (PCV2) is the postulated etiologic agent of PMWS. PCV2 is genetically and antigenically different from PCV type 1, which has long been recognized as a noncytopathic contaminant of PK-15 cell lines. Although the virus is infectious for swine, the causal role of PCV2 in PMWS has not been fully elucidated. To assess the strength of association of PCV2 with PMWS, we conducted a field-based case-control study. Cases were defined as individual pigs submitted to the Iowa State University Veterinary Diagnostic Laboratory or collected in the field with a clinical history of progressive weight loss and respiratory signs and which were subsequently diagnosed with PMWS. A diagnosis of PMWS was made on the basis of histological observations, i.e.: 1) some degree of depletion/atrophy of lymphoid tissue and/or 2) granulomatous (macrophage-dominated) inflammation in any tissue, but especially lymphoid tissue, lung, or intestine. Controls were pigs without a diagnosis of PMWS and/or from herds in which PMWS has not been diagnosed and with no clinical signs compatible with PMWS had been reported. Samples from cases and controls were assayed for the presence of specific viral infectious agents and/or antibodies. Serum and various tissues were collected at necropsy and assayed for PCV2 and PCV-specific antibody by virus isolation, immunohistochemistry (IHC), PCR and/or indirect fluorescent antibody (IFA) test. The same clinical specimens were used to determine the presence or absence of other swine viral pathogens, i.e., PRRS virus, parvovirus, enterovirus, SIV, TGE virus, and porcine respiratory coronavirus. Appropriate serologic assays were performed to assess the presence of antibody against these viruses. To date, a total of 31 cases and 56 controls have been identified from diagnostic submissions or farms. The proportions of case and control pigs serologically and/or virologically positive for PCV2 and the other viral agents were determined for case and control groups and statistically compared to determine the strength of the association of each agent with PMWS. In addition, the agreement of various diagnostic tests in detecting PCV2 was evaluated. Six cases and 6 controls in which PCV2 were detected were selected for genetic analysis of PCV2 by direct PCR amplification of PCV DNA and sequencing. Overall, PCR and serology (IFA) showed the best test sensitivity and were in excellent agreement in detecting PCV2 or its infection; however, interpretation of test results in context with diagnosing PMWS was difficult. IHC and virus isolation showed the best performance in detecting PCV2 in association with PMWS. Based on case definition and laboratory test results, PCV2 appeared to be strongly associated with PMWS (odd ratio = 8.7) as compared to other viral agents tested. Risk for PWMS was much higher if animal was co-infected with PRRS virus. However, PCV2 was also found in controls (35/56) and was not detected in 2 of the 31 PMWS pigs. Furthermore, no significant genetic difference was observed among PCV2 isolates from cases and controls. Therefore, further work remains to determine the actual role of PCV2 in PMWS.

Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA *To be considered for graduate student award. A Case Report of Suspected Fumonisin B1 Neurotoxicosis in Three Juvenile Equines with Atypical Histopathologic Lesions in the Brain

T. J. Evans1,*, S. E. Turnquist1, L. Kellam2, A. Templer2, N. T. Messer IV2, and S. W. Casteel1

Three yearling horses, a Standardbred filly and gelding and a Quarter Horse filly were maintained in the same dry lot in Western Illinois and were fed shelled corn, oats, bean meal and an alfalfa/grass hay mixture. There was no history of immunization, and deworming had previously been performed with a cattle preparation of ivermectin. In late February, there was a rapid, sudden onset of neurological signs in all three horses. The Quarter Horse filly exhibited blindness and maniacal behavior and was humanely destroyed at the farm. The Standardbred filly was noticeably depressed and presented recumbent at the University of Missouri Veterinary Medical Teaching Hospital. On physical examination this filly was unaware of her surroundings and exhibited tachycardia, nystagmus and deficits in cranial nerves II, III, IV, VI and VIII. The gelding was depressed and anorexic with tachypnea, tachycardia, decreased intestinal motility, partially weight-bearing, right hind-limb lameness and a diminished menace response. Both horses were afebrile, and the gelding was observed to consistently circle in his stall. The complete blood counts (CBCs) performed at the time of admission were relatively unremarkable, with the exception of high normal and slightly elevated fibrinogen levels in the filly and gelding, respectively. The chemistry panels from both horses demonstrated elevations in total bilirubin (in part due to anorexia), γ-glutamyl transferase (GGT), alkaline phosphatase (ALP), aspartate aminotransferase (AST) and sorbitol dehydrogenase (gelding only), suggesting hepatic involvement. The filly’s normal plasma ammonia level suggested that primary neurologic disease rather than hepatic encephalopathy was the cause of the neurologic signs. Euthanasia was elected on the filly the morning following presentation, but the gelding was hospitalized and treated supportively with fluids, B vitamins and intravenous dimethyl sulfoxide (DMSO). The gelding was discharged from the Veterinary Medical Teaching Hospital after one week, exhibiting no overt clinical signs of neurologic disease at the time of release. Differential diagnoses included mycotoxicoses (aflatoxin B1 and fumonisin B1), equine herpes viral infection, the viral encephalitides (EEE, WEE, VEE and West Nile virus), equine protozoal myeloencephalitis (EPM), rabies and listeriosis. The history of concurrent, common exposure to corn- containing feedstuffs and the simultaneous, clinical manifestation of acute, neurologic and hepatic disease in multiple animals suggested fumonisin B1 (FB1) toxicosis. However, despite gross and histopathologic examination of multiple sections of the cerebrum, cerebellum and brainstem, the only lesion found in the brains of the deceased animals was nonsuppurative encephalitis characterized by lymphocytic and plasmacytic, perivascular cuffing. Leukoencephalomalacia is typically associated with the disruption of normal lipid metabolism in the brain by FB1, but this histopathologic lesion was not observed in either of the brains examined. The portal hepatitis and biliary hyperplasia, although consistent with FB1 toxicosis, were not deemed severe enough to cause hepatoencephalopathy. Low titers in the filly’s cerebrospinal fluid (CSF) ruled out herpes viral infection and the viral encephalitides. Both brains were found negative for rabies and listeriosis. The CSF was positive by Western blot for EPM, but immunohistochemical staining for Sarcocystis neurona and S. falcatula was negative. Aflatoxin B1 was not detected in feed samples, but exposure to FB1 was confirmed by thin layer chromatographic analysis of extracts of the corn and the oats/corn mixture. The levels of FB1 found in the feeds (10 ppm in corn; 5 to 10 ppm in oats/corn mixture) were comparable to FB1 levels demonstrated to cause clinical disease in horses (> 8 ppm of FB1). This case serves to stimulate discussion of the normal lesions associated with FB1 toxicosis and the suspected occurrence of atypical presentations of this condition.

1Veterinary Medical Diagnostic Laboratory, University of Missouri, Columbia, MO 2Department of Veterinary Medicine and Surgery, University of Missouri, Columbia, MO *To be considered for graduate student award.

Pathological and Epidemiological Investigation of West Nile Virus Disease in Equines in Long Island, New York

A. J. Wilson, B. Meade, T. J. Varty, D. Hutto, T. Gidlewski, and D. Gregg

West Nile Virus, (WNV), is an arthropod-borne zoonotic viral disease, caused by a flavivirus first reported in the West Nile district of Uganda in 1937, and is transmitted by mosquitoes with birds as their main host. Infections are most likely in wet, high mosquito and high bird density areas. In infected horses it can cause encephalomyelitis with fever, ataxia and sometimes death. In man it can cause a febrile sometimes-fatal influenza like disease. From August 26,1999 through October 8,1999, a total of 18 cases of equine neurological disease characterized by ataxia, falling down with difficulty standing, hind-limb weakness, head shaking, muscle twitching, hyperexcitability, paresis, depression, convulsions, coma and death, were diagnosed in Suffolk County, New York. Clinically these cases had an acute onset of ataxia with rapidly progressive neuromuscular involvement leading to recumbency. At the request of Dr. John Huntley, New York State Veterinarian, and under the direction of Dr. Jose Diaz, Acting Director of the USDA-APHIS-VS Eastern Regional Hub, members of the Eastern Regional Early Response Team, (ERT), were deployed to investigate the cluster of cases of equine central nervous system disease among horses on Long Island, NY. From October 12-15, 1999, each of the 14 affected premises were visited by members of the ERT and interviews of the owners or managers were conducted to obtain demographic information, symptoms, onset and duration of illness. A survey instrument was used to identify risk factors for equine neurological disease, which included questions related to management practices and exposure to insect and wildlife vectors. Eighty-six horses on the affected premises were sampled for blood and cerebrospinal fluid when possible. Blood samples were also collected from other animals on the premises including cows, swine, sheep, goats, chickens and ducks. All premises surveyed shared a similar ecological environment with cases clustered in a 4-5 mile radius. Standing water, in large pools or in barrel/watering areas was evident on all affected premises. Dense vegetation with unknown mosquito density, sporadic aerial insect spraying and minimal ground fogging on an as-needed basis were also noted. Eighty-three of the 88 horses sampled had adequate diagnostic samples. Of that number, 43% (36/83) were laboratory positive for flavivirus exposure by the serum neutralization test. Eighteen per cent (15/83) had clinical signs of neurological disease and were laboratory positive for flavivirus exposure via the serum neutralization test. Twenty-five per cent (21/83) had no clinical signs but were positive for flavivirus exposure via serology. Three equine tissue specimens collected from horses that died but were not part of the ERT investigation, were tested by the National Veterinary Services Laboratory in Ames and were positive for West Nile Virus by virus isolation (E.N.Ostlund, personal communication). Histopathology test results from two clinically affected dead horses (one before the ERT team arrived and one after it departed Long Island, NY), showed microscopic evidence of moderate to marked, non-suppurative viral encephalomyelitis. Immunohistochemical studies of these tissue specimens are pending. The findings to date, based on additional samples taken for virus isolation and identification and other lab results reported by the Center for Disease Control, as well as NVSL that were not part of the ERT investigation; indicate that a cluster of 25 cases of equine neurological disease, that coincided with several human cases of fatal viral encephalitis in the New York City area, was most likely due to West Nile Virus infection.

United States Department of Agriculture, National Veterinary Services Laboratory, Pathobiology Lab, Ames, IA Detection of North American West Nile Virus in Animal Tissue by Nested RT-PCR

D. J. Johnson, E. N. Ostlund, and B. J. Schmitt

In the fall of 1999, an outbreak of West Nile virus (WNV) occurred in the New York City area. The virus was initially isolated from a crow brain that had been submitted to the National Veterinary Services Laboratories (NVSL) for testing. The initial cell culture isolate was determined to be negative for eastern equine encephalitis, western equine encephalitis, and Venezuelan equine encephalitis by the NVSL, and was subsequently submitted to the Centers for Disease Control (CDC) where it was identified as WNV. At the NVSL, further isolations were made in cell culture from animal samples that had been submitted for testing. These isolates were identified as WNV using a reverse transcriptase polymerase chain reaction (RT-PCR) test that utilized a single set of primers, the sequences provided by the CDC. While this RT-PCR test worked well in identification of cell culture isolates, it was often unsuccessful in detecting WNV directly from tissue. This study describes a nested RT-PCR procedure that has been shown to successfully identify the North American isolates of WNV directly from the tissues of infected horses and birds. Using this procedure, WNV was detected in the brains of three horses and five birds, and from the spleen of one bird. In the fall of 1999, WNV was isolated in cell culture from each of these animals. Sequence analysis was performed on six of the RT-PCR products. Five of the gene sequences were 100% homologous, and one was 99% homologous to that of an isolate recovered from a zoo bird during the 1999 WNV outbreak in the northeastern U.S. (GenBank AF196835).

USDA, APHIS, National Veterinary Services Laboratories, Ames, IA Rabbit Calicivirus Disease Confirmed in Iowa in March of 2000

P. G. Halbur1, R. K. Myers1, K. L. Petersburg2, R. E. Blair2, J. J. Schiltz3, K. McKeown4, D. A. Gregg5, and L. Thomas5

Rabbit calicivirus disease (also known as Viral Hemorrhagic Disease of rabbits) was confirmed in a rabbitry of 27 rabbits in Crawford County, Iowa. The investigation and confirmation of rabbit calicivirus disease in the U.S. is presented as an example of how well the system in place can work to quickly confirm the introduction of a foreign animal disease. The rabbits were Palominos and California Whites. The first rabbit, one allowed to roam near the house, died on March 9, 2000. Rabbits housed in hutches started dying on March 16. Most rabbits were found dead with no prior signs of disease. A private veterinarian forwarded samples to Iowa State University Veterinary Diagnostic Laboratory on March 22. Microscopic examination revealed severe acute periportal to diffuse hepatic necrosis and hemorrhage, pulmonary edema, and fibrin microthrombi in lung and renal glomeruli. Rabbit calicivirus disease or toxic hepatopathy was suspected based on the clinical history and microscopic lesions. Cultures for Pasteurella multocida and other bacteria were negative. A mycotoxin screen of the feed was negative. On March 24, tissues from a second rabbit were submitted and similar lesions were observed. The state and federal officials were notified on March 27, and a foreign animal disease investigation began immediately. Epidemiological information was collected and samples were sent to the USDA’s Foreign Animal Disease Diagnostic Laboratory (FADDL). FADDL diagnosed rabbit calicivirus disease based on characteristic microscopic lesions, positive hemagglutination (HA) tests, and electron microscopy. FADDL forwarded samples to Spain for confirmation; the laboratory in Spain further confirmed the diagnosis by polymerase chain reaction assay. The premises were quarantined as soon as rabbit calicivirus was suspected. By April 6, 25/27 rabbits had died. As a control measure, the remaining 2 rabbits were euthanized and the carcasses incinerated. On April 21, the rabbit hutches and building housing the hutches were burned and the ashes buried. The source of infection has not been determined. There apparently have been no introductions of rabbits onto the premises in the last two years. August 1999 was the last time rabbits left the farm and returned. In January 2000, six rabbits, all healthy and greater than 2 months old, were sold to slaughter. After an extensive epidemiological investigation, Veterinary Services and the Iowa Department Agriculture and Land Stewardship consider the investigation closed; however, rabbit owners and veterinarians and diagnosticians are encouraged to report cases of high morbidity and mortality to state and federal veterinarians. Rabbit calicivirus was first reported in 1984 in the People’s Republic of China. From 1985-1986 it spread through the domestic and wild rabbit populations in continental Europe. The first report of the virus in the Western Hemisphere was in Mexico City in 1988. Mexico was successful in eradicating the virus by 1992. A severe outbreak of rabbit calicivirus disease occurred in Australia in 1995. Rabbit calicivirus disease is a disease of the European rabbit (Oryctolagus cuniculus). This is the species from which all U.S. domestic and commercial rabbits are derived. Rabbits native to North America (cottontail rabbits and jackrabbits) reportedly do not develop clinical disease and are not susceptible to rabbit calicivirus. A rabbit calicivirus Factsheet, an Impact Worksheet, and a Questions and Answers document can be accessed on the APHIS website at http://www.aphis.usda.gov/oa/rcd/rcd/.html.

1Iowa State University College of Veterinary Medicine, Ames, IA 2United States Department of Agriculture, APHIS, VS, Des Moines, IA 3Iowa Department of Agriculture and Land Stewardship, Des Moines, IA 4Twin Valley Veterinary Clinic, Denison, IA 5National Veterinary Services Laboratories, Foreign Animal Disease Diagnostic Laboratory, Greenport, NY

After the Outbreak: What Happens After an Exotic Disease is Diagnosed in the USA

K. L. Petersburg1, J. J. Schiltz2, R. E. Blair1, D. Anderson3, J. L. Johnson2, B. Bischoff4, I. Stewart4, J. Annelli4, L. A. Thomas5, J. Lubroth5, D. Gregg5, J. Neilan6, S. Shawky5, S. Swenson7, L. Capucci8, and T. McKenna5

In March 2000, rabbit calicivirus disease (RCD) was diagnosed in Iowa. This was the first time RCD had been diagnosed in the USA. The initial identification of the presence of RCD in the USA was accomplished through the coordination and cooperation of several different components of the veterinary infrastructure in place in the USA. The initial diagnosis is only the beginning. Once an exotic disease is diagnosed, many things need to be accomplished. The extent of the outbreak must be determined and further spread of the disease limited. This is accomplished through quarantines of affected premises and extensive epidemiological investigation to identify all potentially exposed locations. The source of the outbreak needs to be identified so that we can better understand from where the disease agent was introduced and to adequately identify all potentially exposed premises. In this case, the isolated viral DNA was sequenced and compared to known sequences of RCDV. Monoclonal antibody studies of the isolated virus were used to identify where closely related RCD viruses originated. Additionally, appropriate industry groups, state diagnostic laboratories, private practitioners, state and federal veterinarians, and the international community need to be notified of the presence of a new disease, how to recognize the disease, and how to reduce the spread of the disease. The Emergency Programs staff of the Animal and Plant Health Inspection Service/Veterinary Services spearheaded the notification of appropriate parties and coordinated the disease information dispersal process.

1U.S. Department of Agriculture, APHIS, Veterinary Services, Des Moines, IA 2Iowa Department of Agriculture and Land Stewardship, Bureau of Animal Industry, Des Moines, IA 3Livestock Inspector, Iowa Department of Agriculture and Land Stewardship, Bureau of Animal Industry, Des Moines, IA 4U.S. Department of Agriculture, APHIS, Veterinary Services, Emergency Programs, Riverdale, MD 5U.S. Department of Agriculture, APHIS, Foreign Animal Disease Diagnostic Laboratory, Greenport, NY 6U.S. Department of Agriculture, ARS, Plum Island Animal Disease Center, Greenport, NY 7U.S. Department of Agriculture, APHIS, National Veterinary Services Laboratories, Ames, IA 8Instituto Zooprofilattico Sperimentale Della Lombardia E Dell’Emilia Romagna, Brescia, Italy Circoviral Postweaning Multisystemic Wasting Syndrome (PMWS) - Retrospective Confirmation of Two Outbreaks in English Herds from 1986

R. J. Higgins1, D. O’Toole2, E. Clark3, K. West3, G. A. H. Wells2, P. Blackburn5, and C. J. Cundall5

Postweaning multisystemic wasting syndrome (PMWS) is an apparently new disease of swine and is due to infection with porcine circovirus-2 (PCV-2). The earliest known case of PMWS dates from 1991, when the disease was recognized in a herd of swine in Saskatchewan (Clark EG, Proceedings, American Association of Swine Practitioners 1997: 499 – 501). The present communication documents two earlier outbreaks of PMWS, which occurred in England in the mid-1980s. Details of the outbreaks were reported initially as an idiopathic enteropathy of weaned pigs (Higgins RJ et al.: Clinical, histopathological and ultrastructural observations of a new enteropathy of weaned pigs. Annual Meeting, Association of Veterinary Teachers and Research Workers, Scarborough, Yorkshire, England. April 1987). Virus-like inclusions were noted in macrophages but their etiologic significance was not established at that time. In 1986, an endemic postweaning diarrhea syndrome was recognized in two unrelated herds in Yorkshire, England. The condition presented as acute onset of watery, non-mucoid diarrhea beginning 2–3 weeks after weaning. Most affected pigs responded to treatment with broad-spectrum antibiotics. Some pigs developed chronic ill thrift and few of these animals recovered. The problem was eliminated on one of the premises by destocking and subsequent repopulation with a different bloodline of pigs. Chronically affected pigs were potbellied with an erect haircoat. Gross changes comprised thickened ileal mucosa and colonic dilation with mucosal erosion. The principal microscopic findings were segmental granulomatous ileitis in mucosa-submucosa, villous atrophy, and severe lymphoid depletion in gut-associated lymphoid aggregates. Multinucleated giant cells were numerous in the enteric lamina propria of some animals. Macrophages in depleted Peyer’s patches contained 1 – 7-µm botryoid intracytoplasmic amphophilic inclusions. Ultrastucturally, inclusions contained cellular debris and 10 - 12-nm icosahedral virus-type particles in paracrystalline arrays within autophagolysosomes. Coccidia, cryptosporidia and rotaviruses were present on or in villous enterocytes of some animals. Other lesions present in pigs were superficial colitis, granulomatous lymphadenitis, interstitial nephritis with and without giant cells, interstitial pneumonia, and general lymphoid depletion. Retrospective immunohistochemical staining of selected tissues in 2000 by Prairie Diagnostic Services Inc., Western College of Veterinary Medicine, Saskatchewan established that intracytoplasmic inclusions in mononuclear cells and polykaryons were antigenically positive for PCV using specific monoclonal and polyclonal antibodies. Positive staining was detected primarily in Peyer's patches, and to a lesser extent in scattered cells throughout lamina propria. Most positive staining consisted of variably sized cytoplasmic inclusions in individual and multinucleated giant cells. Cells with large, generally single, intranuclear inclusions were present in most cases. Positive staining of epithelial cells was rare. Age of onset, clinical response to treatment, and gross, microscopic, ultrastructural and immunohistochemical findings were consistent with PWMS. These appear to be the earliest cases of PWMS identified to date.

1Veterinary Investigation Centre, Ministry of Agriculture, Fisheries and Food, Thirsk YO7 1PZ, England 2Central Veterinary Laboratory, Ministry of Agriculture, Fisheries and Food, Addlestone, Surrey KT15 3NB, England 3Prairie Diagnostic Services Inc., University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada 4Cornerways, Sproxton, York YO62 5EF, England 5The Cottage, Sherburn Lodge, Sherburn, Malton, North Yorkshire YO17 8EW, England

The Incidence of Clostridium difficile-associated Enteritis in Scouring Piglets and its Association with Specific Gross and Microscopic Lesions

M. Yaeger1, N. Funk2, and L. Hoffman1

Clostridium difficile has been associated with diarrhea in swine. Lesions reported with enteric C. difficile infection include mesocolonic edema and typhlocolitis. However, the prevalence of enteritis associated with C. difficile infection in young swine has not been defined and the diagnostic significance of mesocolonic edema or colitis in scouring piglets is unknown. This case control study was undertaken to assess the incidence of enteritis associated with the presence of C. difficile toxin in scouring piglets, and to determine if there was an association between the presence of toxin and specific gross and microscopic lesions. The subject population was the first 100, live 1-7 day old piglets submitted to the Iowa State University Veterinary Diagnostic Laboratory with a history of scours. Routine tests were undertaken to assess for E. coli, salmonella, Clostridium perfringens, TGE, rotavirus and coccidiosis. In addition, colonic contents were frozen and analyzed on a weekly basis for C. difficile toxin A and B with a commercially available ELISA (TOX A/B test; TechLab). Incidence of C. difficile-associated enteritis: C. difficile toxin was identified in the colonic contents of 29 scouring piglets. A high level of toxin (+3 or +4) was detected in 19 of the animals in this study. The incidence of enteritis associated with the presence of C. difficile toxin was second only to rotavirus enteritis (36%). Distribution of C. difficile toxin-positive pigs within a group: The pattern of infection was assessed in those cases in which more than one scouring piglet was submitted and at least one animal was positive for difficile toxin. In half of these 16 submissions, 1/3 of the piglets were positive for difficile toxin. An average of 41% of the piglets were positive for difficile toxin in multiple animal submissions with at least one toxin-positive pig. In a single case, all three animals from a group were positive for difficile toxin. The distribution of toxin-positive animals within a group was more consistent with an opportunistic agent than a primary contagious pathogen. The association between the presence of difficile toxin and gross lesions: All of the piglets in which difficile toxin was identified had mesocolonic edema. However, the presence of mesocolonic edema was not specific for difficile-toxin induced diarrhea. Sixty-six piglets had mesocolonic edema to varying degrees, whereas only 29 of these animals were positive for difficile toxin. The positive predictive value of mesocolonic edema was low (0.44). However, the negative predictive value was quite high, as no piglet without mesocolonic edema was positive for difficile toxin. The association between the presence of difficile toxin and microscopic lesions: High levels of difficile toxin (+3 or +4) were detected in 94% of pigs with colitis. Only three pigs negative for difficile toxin had a significant colitis. The positive predictive value of colitis and the presence of high levels of difficile toxin were high (0.84). Colitis was a good indicator of the presence of high levels of difficile toxin. Conclusions: Diarrhea associated with the presence of C. difficile toxin was quite common in scouring piglets less than one week of age. The distribution of toxin-positive animals within a group was more consistent with an opportunistic pathogen than a primary, contagious pathogen. All piglets with difficile toxin had mesocolonic edema; however, mesocolonic edema is not specific for C. difficile infection. The presence of colitis was a good indicator that high levels of difficile toxin would be detected in colonic contents.

1Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Iowa State University, Ames, IA 2Pre-Vet Honors student, Iowa State University, Ames, IA

Reproduction of Clostridium difficile Associated Enteritis by Experimental Inoculation of Piglets

K. W. Post1, R. D. Glock2, A. Holtcamp3, B. H. Jost2, and J. G. Songer2

Diagnostic findings suggest that C. difficile-associated disease in piglets accounts for more than 30% of neonatal pig enteritis accessions in some US laboratories. Experimental reproduction of the infection is an important step in the development of strategies for prevention and control. Ten colostrum-deprived, six-hour-old, crossbred pigs were randomly assigned to 3 groups and given oral passive immunoprophylaxis against Clostridium perfringens Type C and E. coli. Pigs were fed a commercial canine milk substitute. Immediately prior to inoculation, rectal swab samples were collected from all animals for C. difficile culture and enzyme immunoassay (EIA) testing for toxins A & B (Tox A/B Test, TechLab. Blacksburg, VA). Group A (n = 2) were uninoculated controls. Group B pigs (n = 4) were orally inoculated with 4.2 x 1010 CFU of a toxigenic field isolate (Rollins ADDL 10377-00) cultivated in trypticase soy broth incubated anaerobically for 24 h. Group C pigs (n = 4) were inoculated with 1.65 x 106 C. difficile spores (UA strain J9). Rectal swabs were collected at 24, 48 and 72 hours post inoculation for culture and toxin testing. One pig from each group was necropsied after 48 hrs and all other pigs were necropsied at 72 hrs. Tissues were collected for bacteriologic, virologic, parasitologic and histologic examinations. Colon content from each pig was examined for A&B toxins by EIA and cytotoxicity in CHO cells. Cultures and rectal swabs from all piglets prior to infection were negative for C. difficile and A or B toxins. Twenty-four hours PI one pig in Groups B and C had developed pasty, yellow feces. All pigs in Groups B and C had developed scours by 48 hrs PI. All but one of the inoculated pigs (Group B) were culture positive from 24 hours post inoculation. One Group B pig was EIA positive after 24 hours and all but one (Group B) were positive at necropsy. Correlation between EIA results and cytotoxicity for CHO cells was good. Necropsy revealed mesocolonic edema and slight dilation of the small intestine in most piglets. Pasty-to-watery, yellow colon contents were present in most piglets. No viral agents or pathogenic E. coli (genotyping of which did not reveal any genes for known virulence attributes) ere identified. E coli was isolated from 1 group C pig. Moderate numbers of nonenterotoxigenic, β2 toxigenic C. perfringens type A were isolated from both pigs in Group A and 2 pigs each in Groups B and C. The clinical, gross, microscopic lesions, and microbiologic findings consistent with natural C. difficile infection were reproduced with this model.

1Rollins Animal Disease Diagnostic Laboratory, Box 12223 Cameron Village Station, Raleigh, NC 2Department of Veterinary Science and Microbiology, University of Arizona, Tucson, AZ 3Prestage Farms, Clinton, NC

Evaluation of the Sensitivity of Fetal Serology, IHC, and PCR for the Diagnosis of PRRSV in Fetuses

M. Yaeger1, J. Benson2, J. Christopher-Hennings3, K. Lager4

The use of virus isolation (VI) for the diagnosis of PRRS on field cases of porcine abortion has been disappointing. This study was undertaken to compare the sensitivity of VI with fetal serology, IHC and PCR for the diagnosis of PRRSV in experimentally infected fetuses. Twelve, pregnant sows from a PRRSV negative herd that were seronegative at purchase and following two weeks in isolation were challenged with PRRSV (NADC-8) at 90 days gestation. Sows were euthanized 21 days post challenge and fetal tissues and fluids were assessed for PRRSV by virus isolation, fetal serology, PCR and immunohistochemistry (IHC). Serum and fetal thoracic fluid (TF) was collected from 140 fetuses. Fifty-eight (41.4%) fetuses were positive for PRRS by VI. IFA titers were detected in a total of 16, VI-positive fetuses and none of the VI- negative animals. Of the ten litters with PRRSV-infected pigs, fetal serology was positive on at least one fetus from five of these litters. The sensitivity of fetal serology on an individual animal basis was 27.6%. If the entire litter was evaluated, the sensitivity for detecting an infected litter was 50%. Lung, liver, spleen, umbilical cord and thymus from 145 fetuses were placed in a single block for PRRSV IHC. PRRSV-positive cells were detected in 30 of 68 VI positive fetuses and none of the VI negative animals. Overall, PRRSV antigen was identified in 18% of fetuses from PRRSV-infected litters. PRRSV antigen was detected most commonly in the thymus (30/30) followed by liver (19/30), spleen (18/30), lung (8/30) and umbilical cord (5/30). Sensitivity on an individual animal basis was 44%. The sensitivity for detecting an infected litter by IHC (utilizing the procedure outlined above) was 100%. Nine, PRRSV VI-positive fetuses were divided into 3 groups of three pigs each. Individual tissues (brain, liver, spleen, lung, umbilical cord) from each pig were divided evenly into four aliquots and pooled. Group A tissues were stored at 4C, group B at 21C and group C at 37C. Tissues and TF were assessed for PRRSV with a nested RT-PCR immediately after processing (time 0) and following incubation for 24, 48, and 96 hours to mimic postmortem decomposition. All tissue and TF samples from VI positive fetuses were PCR positive at each time period except two tissues (21C day-2, 37C day-2) and two thoracic fluids (37C days 1 & 4). Compared with VI, the overall sensitivity of PCR was 94%, regardless of sample type (tissue or TF) or degree of autolysis. Conditions mimicking autolysis had limited impact on the ability to detect PRRSV by PCR as 21 of 24 (87.5%) samples maintained at 37C for 1-4 days remained positive. Six PCR-positive tissue and thoracic fluid samples were pooled with tissue or thoracic fluid from similarly treated VI/PCR negative controls at 1:1, 1:2, 1:4, or 1:8. Each of the resulting 48 samples was positive for PRRSV by PCR. The pooling of PCR-positive tissue or thoracic fluid with negative samples had no impact on the ability to detect PRRSV by PCR. When compared with fetal serology and IHC, PCR appears to be the best tool for the diagnosis of PRRSV in aborted fetuses. PRRSV PCR was highly sensitive, unaffected by pooling of samples and minimally impacted by autolysis out to 4 days. However, as an average of only 44% of fetuses in each litter were infected, it is important to pool samples from several fetuses to assure that at least one infected fetus is represented in the material assessed by PCR.

1Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Iowa State University, Ames, IA 2Animal Disease Laboratory, Illinois Department of Agriculture, Galesburg, IL 3Animal Disease Research and Diagnostic Laboratory, South Dakota State University, Brookings, SD 4USDA, National Animal Disease Center, Ames, IA

The Sensitivity of Immunohistochemistry for the Detection of Porcine Reproductive and Respiratory Virus (PRRSV) in Vaccinated and Unvaccinated Swine

M. Yaeger

Many variables have the potential to influence the ability to detect PRRSV in the lung of infected swine with immunohistochemistry (IHC). These would include many uncontrolled variables affecting virus load, such as infective dose, stage of infection, PRRSV strain, age of the pig and immune status. Practitioners and diagnosticians can, however, exercise control over the number and location of pulmonary sections collected and assessed. The anterior lung has been documented as the preferred location for the detection of PRRSV by IHC. In one study, PRRSV antigen was detected in the anterior lung approximately twice as often as the caudal lung lobes. In many field cases of PRRSV-induced pneumonia, PRRSV antigen is only detected in one or two of the multiple lung sections evaluated. This observation suggests that the sensitivity of IHC is dependent on the number of sections evaluated. This study was undertaken to assess the impact number of lung sections evaluated has on the sensitivity of IHC to detect PRRSV infection in vaccinated and unvaccinated swine. Fifteen vaccinated and unvaccinated pigs were challenged intranasal with a virulent strain of PRRSV (NADC-20) and necropsied 10 days post challenge. A section of lung taken from each of the five, anterioventral lung regions (right cranial lobe, right middle lobe, the accessory lobe and a section from each portion of the divided left cranial lobe) was evaluated on a single IHC slide. PRRSV was isolated from lung lavage samples and detectable by IHC in all 15, unvaccinated pigs. IHC was positive for PRRSV in 5 of 5 sections of lung in 2 pigs, 4 of 5 sections in 1 pig, 3 of 5 sections in 2 pigs, 2 of 5 sections in 4 pigs, and 1 of 5 sections in 6 unvaccinated pigs. PRRSV was isolated from the lung lavage of 10 of the 15 vaccinated pigs. PRRSV-positive cells were detected by IHC in 6 of VI positive pigs and no VI negative pigs. IHC was positive for PRRSV in 2 of 5 sections in 1 pig, 1 of 5 sections in 5, vaccinated pigs. ______Table 1: The probability (beta binomial) of detecting PRRSV in the lung of vaccinated and unvaccinated swine with IHC as a function of the number of lung sections evaluated.

Number of lung Probability of detecting Probability of detecting Sections evaluated PRRSV with IHC - PRRSV with IHC - Unvaccinated Swine Vaccinated swine

1 0.48 0.14 2 0.70 0.26 3 0.81 0.36 4 0.87 0.45 5 0.91 0.53 6 0.93 0.59 7 0.95 0.65 8 0.96 0.70 9 0.97 0.74

The sensitivity of IHC for the detection of PRRSV depends to a great degree on the number of lung sections evaluated. Factors that decrease virus load in the lung, represented by vaccinated animals in this study, further decrease the sensitivity of PRRSV-IHC.

Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Iowa State University, Ames, IA Multiple Abortions in a Herd of Cows Vaccinated while Pregnant with Modified-Live Infectious Bovine Rhinotracheitis Virus Vaccine

J. C. Nietfeld, M. Muenzenberger, S. Kapil, G. Andrews, B. DeBey, and G. Kennedy

Between approximately January 15 and April 1, 1999, 75 cows in a herd of 125 to 130 head aborted or gave birth to stillborn or weak calves that died during the first postpartum week. Approximately 30 days prior to the first abortion, the herd veterinarian had vaccinated the pregnant cows with a vaccine that contained the following modified live viruses: infectious bovine rhinotracheitis virus, bovine virus diarrhea virus, parainfluenza-3 virus, and bovine respiratory syncytial virus. The label on the vaccine bottle stated in bold print “Do not vaccinate pregnant cows.” Samples from 7 fetuses were submitted for diagnostic evaluation and tissues from 6 of the fetuses were positive for infectious bovine rhinotracheitis (IBR) virus by direct fluorescent antibody (FA) staining. Of the six sets of tissues from which virus isolation was attempted, IBR virus was isolated from 3, including the set that was FA negative. In addition, fetal livers from each of the 7 fetuses contained random foci of necrosis compatible with those commonly caused by fetal IBR virus infection. No other viruses were isolated and tests for bovine virus diarrhea virus, Leptospira spp, Brucella abortus, Campylobacter spp, and Neospora caninum, as well as routine bacterial culture and histologic examination failed to provide evidence of infection by pathogens other than IBR virus. Based on these findings, the abortions were diagnosed as being caused by IBR virus. The herd veterinarian was reluctant to believe that the vaccine IBR virus had caused the abortions because on several previous occasions he had vaccinated groups of pregnant cows with similar modified- live virus vaccines and had not recognized any complications. He requested that we attempt to “fingerprint” the virus to prove that the isolates from the fetuses were different from the vaccine virus. The vaccine manufacturer provided an isolate of the vaccine IBR virus and it was compared to the three IBR isolates from the fetuses. The patterns of the restriction fragments for the 3 fetal isolates and the vaccine strain obtained by 3 restriction endonucleases (HindIII, EcoRI, and BamHI) were identical and the vaccine virus could not be distinguished from the isolates from the fetuses. These findings supported the conclusion that the abortions were most likely the result of fetal infection by the vaccine virus. This case serves as a reminder that many modified-live IBR vaccine viruses are capable of infecting the fetus when given to a naive mother. Ignoring the warning of the vaccine manufacturer not to vaccinate pregnant cows is done with considerable risk.

Veterinary Diagnostic Laboratory, Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS Clostridial Abomasitis in Day-old Dairy Calves

J. G. Songer1, C. Reggiardo1, J. Biwer2, D. M. Bueschel1, and R. D. Glock1

Clostridium perfringens type A is now commonly associated with digestive tract infections in domestic animals. One such syndrome is hemorrhagic, necrotic abomasitis, often with tympany, in neonatal calves. One group (Roeder BL, MM Chengappa, TG Nagaraja, TB Avery, and GA Kennedy. 1987. Isolation of Clostridium perfringens from neonatal calves with ruminal and abomasal tympany abomasitis and abomasal ulceration. J Am Vet Med Assoc 190:1550-1555) reproduced the condition in calves four- to twelve-days-old, but for unknown reasons, attempts to repeat this work have failed. Thus, the etiology has not been solidly confirmed (i.e., Koch’s postulates cannot be reliably fulfilled) and pathogenesis cannot be readily investigated. In addition, lack of a host animal challenge system is a major impediment to development and testing of immunoprophylactic products. A group of eleven calves obtained from various dairies in central Arizona were given pooled colostrum from a single dairy per os approximately twelve hours after birth. Bloating occurred in these calves within six hours, and one died on the day of birth. Additional calves displayed abdominal distension and were treated with antibiotics. Five more had died by the second day, and the remainder died over a three-day period. Post mortem examinations were performed on the farm, and lesions consisted of a hemorrhagic gastroenteritis; abomasums were grossly dilated, with thickened, emphysematous walls and folds. A small amount of fluid was found in the abdomen. Approximately 50-80% of the organisms in stained impression smears of abomasum were Gram positive rods with morphology compatible with C. perfringens. Microscopic lesions included hemorrhage, edema, gas bubbles, and minimal neutrophils in the mucosa and submucosa of the abomasum. Similar lesions were in the duodenum but not in the jejunum. Microcolonies of large rod- shaped bacteria were observed in the mucosa and submucosa. Aerobic bacteriologic culture revealed a moderate population of E. coli and mixed enteric flora. Salmonella spp. were not detected. Anaerobic culture yielded 4+ growth of C. perfringens from abomasum and small intestine. Curds collected from the abomasum also yielded large numbers of C. perfringens. Genotyping revealed that all isolates of C. perfringens were nonenterotoxigenic type A; none contained cpb2, the gene for beta2 toxin. Taken together, these findings suggest a clostridial etiology and accentuate the need for development of prevention and control measures.

1Department of Veterinary Science and Microbiology, University of Arizona, Tucson, AZ 2Sheldon and Associates, Casa Grande, AZ

Fatal Infections in Lambs Associated with a Novel Adenovirus

1 2 3 B. M. DeBey , H. D. Lehmkuhl , and K. M. Ewert

There currently are six recognized species (serotypes) of ovine adenovirus (OAdV) that have been isolated and characterized from sheep. A proposed seventh species is awaiting acceptance by the International Committee on Taxonomy of Viruses. Limited serologic studies in the United States indicate that all six ovine adenovirus serotypes, bovine adenovirus (BAdV) serotypes 2 and 7 are present in the US sheep population. Severe, uncomplicated disease, however, is rarely reported as a result of adenoviral infections in sheep, although ovine adenovirus serotype 6 may be an important contributor to respiratory disease in young lambs. Adenovirus infections were diagnosed in 3 nursing lambs in a group of 130 ewes during the spring lambing season of 2000. Affected lambs ranged from 9 to 25 days of age, and had a short duration of gastrointestinal and systemic clinical signs prior to death. Lesions were found in multiple organs, including multifocal hepatic necrosis, multifocal acute interstitial nephritis, and epithelial necrosis and villus atrophy in the small intestine. Intranuclear basophilic adenoviral inclusions were identified in renal endothelial cells of affected lambs, however not all lambs that died of the adenoviral infection had identifiable intranuclear inclusions. The isolated adenovirus antigenically and molecularly appears to be an OadV 7, and represents the first reported isolation of this adenovirus species in the United States. This virus is genetically unrelated to known ovine adenoviruses, and was neutralized in vitro with antiserum against a newly recognized adenovirus proposed as the prototype strain for goat adenovirus serotype 1.

1Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 2United States Department of Agriculture, Agricultural Research Service, National Animal Disease Center, Ames, IA 3Notkwyta Ranch, McLouth, KS An Outbreak of Ovine Abortions Caused by Coxiella burnetti in Kansas

T. A. Aboellail1, and K. West2

Coxiella burnetti antigen was demonstrated immunocytochemically in the placental tissues of an aborted fetus from a flock of 500 sheep from southeast Kansas. The herd had a history of more than 15 abortions with several other ewes that have not lambed this spring or gave birth to weak lambs. No other bacterial or viral agents were isolated from or detected in the placental tissues from the abortion cases submitted to the Veterinary Diagnostic Laboratory of Kansas State University. Placentas of different animals were examined and had similar lesions. Grossly, the intercotyledonary areas were thickened, leathery, and contained mild to moderate amount of purulent exudate. The placenta showed strong Coxiella-specific staining and histological lesions characterized by moderate to severe, multifocal, necrotizing and purulent placentitis. The antigen was observed in cytoplasmic vacuoles of trophoblasts, especially along the base of chorioallantoic villi. Positive trophoblasts were of normal size or exhibited striking cytoplasmic enlargement. Antigen was also demonstrated in degenerated cells and extracellularly in the intervillous space. Placental vasculitis consisting of infiltrating mononuclear cells and neutrophils was observed. In addition, a few intravascular thrombi were observed. To our knowledge, this report represents the first confirmed diagnosis of Coxiella burnetti abortions in sheep in Kansas.

1Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, KS 2 Prairie Diagnostic Services, Saskatoon, SK, Canada PrP-Sc Detection in Lymphoid Tissues of Clinically Normal Sheep and Mule Deer Infected with the Agents of Scrapie and Chronic Wasting Disease: Standardized Protocols for Automated Immunostaining

K. I. O’Rourke1*, R. Zink2, B. Cummings2, A. K. Anderson1, and T. R. Spraker2, 3

Scrapie of domestic sheep and goats and chronic wasting disease of captive and free ranging mule deer are transmissible spongiform encephalopathies (TSEs). These fatal neurologic disorders are associated with deposition of an abnormal isoform (PrP-Sc) of a normal host protein (PrP-C). Immunohistochemistry assay for PrP-Sc deposition in brain is an approved diagnostic test for sheep scrapie and is used extensively in CWD diagnosis and prevalence surveys. PrP-Sc also accumulates in certain lymphoid tissues of sheep and mule deer well before the appearance of clinical signs. Immunohistochemistry assay of PrP-Sc in lymphoid tissues of tonsil and third eyelid has been proposed as a preclinical test for TSE infection in these species. In this study, optimized conditions for PrP-Sc detection by immunohistochemistry assay of lymphoid tissue from mule deer and sheep were determined. Tonsil, retropharyngeal lymph node, and third eyelids were collected at necropsy from sheep and mule deer, and third eyelid tissue was collected from live sheep. Third eyelid tissues from mule deer rarely contained germinal centers and were not found to be useful in preclinical testing of mule deer. Acid, heat, and protease pretreatments under different conditions were evaluated and two monoclonal antibodies were assessed individually and in combination. Optimal conditions were found to include antigen retrieval with formic acid followed by heat treatment in a modified citrate buffer and protease digestion. A combination of two monoclonal antibodies, F89/160.1.5 and F99/97.6.1, produced sensitive, specific staining in sheep lymphoid tissues. Antibody F89/160.1.5 produced low intensity immunostaining in a small percentage of lymphoid tissues from uninfected mule deer. Antibody F99/97.6.1 produced sensitive, specific staining in mule deer lymphoid tissues. A standard protocol suitable for rapid throughput, high volume automated immunostaining of sheep lymphoid tissues was developed and evaluated in a sample of 50 lymphoid tissues from sheep with scrapie and 50 samples from sheep with no exposure to scrapie. A protocol modified for testing mule deer lymphoid tissues was evaluated in a sample of 100 CWD-positive and 300 CWD-negative mule deer. Concordance between test results and infection status of the animals was greater than 96%. Availability of standard, automated, monoclonal antibody based immunoassays for preclinical and live animal testing of sheep and mule deer for TSEs based on lymphoid tissue immunohistochemistry will facilitate large-scale TSE control programs by state and federal regulatory groups.

1USDA, ARS, Animal Disease Research Unit, 3003 ADBF, Pullman WA 2Colorado State University, Department of Pathology, College of Veterinary Medicine and Biomedical Sciences, Fort Collins, CO 3Colorado State Diagnostic Laboratory, College of Veterinary Medicine and Biomedical Sciences, Fort Collins, CO

Comparison of Neuroanatomical Patterns of Spongiform Encephalopathy and Immunohistochemical Staining of Prion Protein in Brain and Lymphoid Tissues of Free-Ranging, Research and Ranch-Raised Elk

T. Spraker1, R. Zink1, B. Cummings1, M. Wild2, M. Miller2, and K. O’Rourke3

A spongiform encephalopathy compatible with chronic wasting disease has been found in ranch- raised elk in at least 5 states in the US and one province in Canada. There is a tremendous concern in regard to the origin of this disease, is it the same disease as chronic wasting disease of free-ranging elk, and methods to diagnose early cases especially in live animals. A comparison of the histological lesions and immunohistochemical staining of brain and lymphoid tissues (tonsil and retropharyngeal lymph nodes) were examined in 5 free-ranging elk and 10 captive (1 research and 9 ranch-raised) elk with a spongiform encephalopathy compatible with chronic wasting disease and in 95 control elk (5 free-ranging and 90 ranch-raised). The immunohistochemical staining was done with monoclonal 160.1.5 using the Ventana automated system. The neuroanatomical locations of spongiform encephalopathy and immunohistochemical staining of brain in free-ranging elk were identical to those of captive elk. The prevalence of positive immunohistochemical staining of lymphoid tissues was also identical; however, the immunohistochemical staining was not consistently present in the lymphoid tissues of brain positive elk. There were three of 10 brain positive elk that had no lymphoid tissue staining. The demonstration that the spongiform encephalopathy and immunohistochemical staining of brain and lymphoid tissues of free- ranging elk was indistinguishable from those of captive elk may suggest that the spongiform encephalopathy as seen in free-ranging elk of Colorado is the same as observed in these ranch-raised elk. Since the occurrence of positive lymphoid staining was not consistently found in brain positive elk, the use of immunohistochemical staining of lymphoid tissues may be of limited value as a live-animal test in elk.

1College of Veterinary Medicine, Colorado State University, Ft. Collins, CO 2Colorado Division of Wildlife, Ft. Collins, CO 3ARS/USDA, Pullman, WA

A Prospective and Retrospective Study on Ovine Herpesvirus-2 Malignant Catarrhal Fever in Feedlot Bison

D. O'Toole1, H. Li2, D. L. Montgomery3, C. Sourk4, and T. B. Crawford5

Malignant catarrhal fever (MCF) is common in commercially raised Plains bison (Bison bison). We conducted a prospective study of a cohort of 300 healthy yearling male feedlot bison over an 11-month period (September 1999 – July 2000) to define incidence and transmission patterns of malignant catarrhal fever viruses (MCFV). The feedlot was chosen because examination of diagnostic accessions from 1998- 99 revealed that MCF was the leading cause of loss (~150 deaths; Jan 1998 - Dec 1999). The disease has been present in the yard since at least 1994. One group of 300 bison was tested for antibody to MCFV by CI-ELISA at the ranch of origin immediately before shipment to the feedlot. Peripheral blood leukocytes (PBLs) of serologically positive bison were tested for OHV-2 DNA by PCR. Samples from animals that died were tested for evidence of MCF by PCR, CI-ELISA and by histopathology. Bison were followed to slaughter (March - July 2000) to establish whether MCFV infection was associated with subclinical lesions. At entry into the feedyard, 23% (71/300) of bison were seropositive. Of the 71 seropositive bison, 8 (11%) were positive for OHV-2 DNA in PBLs by PCR. None of 40 randomly selected, seronegative bison were PCR-positive for OHV-2 DNA in PBLs. At 6 months post-entry, the overall prevalence of seropositive bison remained essentially the same (23.9%) (68/284). Of 68 seropositive animals, 44 were positive on both occasions (9/99 and 2/00), 26 were positive on the first blood sample and negative on the second, and 24 were negative on the first blood sample and positive on the second. To date, 21/300 bison (7.0 %) died following entry into the yard (9/99 - 4/00). Among the 21 deaths, 15 had gross and microscopic lesions indicative of MCF, 3 had acute pneumonia, 1 had chronic pneumonia and bladder lesions suggestive of chronic MCF, and 2 were not examined post-mortem. Bison with acute MCF died shortly after clinical illness (1 – 5 days). Lesions were characteristic and consisted of hemorrhagic cystitis, ulcerative stomatitis, and hemorrhagic enterocolitis-typhlitis. Arteritis-phlebitis in bison was generally milder and less widespread than in cattle with MCF. No statistically significant patterns of case occurrence were identified that would suggest modes of transmission. Findings to date indicate that: • MCF was the most common cause of fatal losses in a large bison feedlot. • Approximately one in four bison entering the feedlot was seropositive for MCFV. • There was strong agreement between histological findings and the presence of OHV-2 DNA in tissues of bison dying of MCF. • Healthy bison persistently infected with OHV-2 had no detectable lesions in tissues at slaughter. • Persistent infection with OHV-2 of healthy bison (determined by serological status and/or detection of OHV-2 DNA in PBLs) may not be an indicator of increased likelihood of clinical MCF during the subsequent 6 months (final analysis pending). Additional cofactor(s) may be involved in disease expression. • No coherent transmission pattern has yet been identified.

1Wyoming State Veterinary Laboratory, Department of Veterinary Sciences, University of Wyoming, Laramie, WY 2Animal Diseases Research Unit, USDA Agricultural Research Service, Pullman, WA 3 Texas A&M University, Texas Veterinary Medical Diagnostic Laboratory, Amarillo, TX 4 Sourk Veterinary Clinic, Scott City, KS 5 Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA

This study was supported in part by the National Bison Association. We acknowledge the cooperation of the North American Bison Cooperative in New Rockford, ND. Bovine Tuberculosis in Michigan: An Overview of Five Years Surveillance

S. D. Fitzgerald,1 K. L. Butler1, K. A. Clarke1, S. M. Schmitt,2 D. J. O’Brien,2 T. M. Cooley2 , and C. Bruning-Fann3

This presentation summarizes the spillover of tuberculosis from deer into wild carnivores, domestic deer and cattle, and domestic carnivores observed by our program over the last five years. The management strategies employed to control this tuberculosis outbreak and their effectiveness will be discussed along with changes in the state’s tuberculosis accreditation status. A single hunter-killed white-tailed deer (Odocoileus virginianus) was identified as having Mycobacterium bovis infection during the fall of 1994. Each year since 1995 a survey of hunter-killed deer, elk and wild carnivores has been conducted, which has annually increased in both animal numbers and geographic area. Management changes included increased hunting pressure through extended rifle seasons, increased harvest of antlerless deer, and mandatory bans on both supplemental feeding and baiting of deer. These strategies have resulted in reductions in disease prevalence in deer within the core area of infection. At the same time expanded surveillance has revealed an infected captive deer ranch, multiple infected cattle farms, a dozen infected wild carnivores, and one infected domestic cat. Three positive deer have been found well outside the geographic area of known infection. Genetic analysis indicates that all isolates of M. bovis were closely related. As the geographic area has expanded and numbers of infected animals – both wild and domestic – have increased, Michigan’s status as an accredited TB-free state has changed. Initially, while the geographic area of known infection remained contained in a 5-county area, a plan was developed for “Split State” status. However, as infected animals were found well outside the area, and higher numbers of infected cattle herds were identified. The state was declared modified accredited by the USDA in 2000. Presently, Michigan is involved in statewide deer surveillance, as well as statewide cattle testing, in order to regain its accredited- free status. Human control of wild animal populations may be necessary to prevent concentration and regional overpopulation, which encourages disease transmission. Extensive supplemental feeding encourages high population densities and disease transmission. Limiting domestic and wild animal contact is vital to protect livestock. Continuous surveillance of livestock is needed after obtaining free status to identify transmission from potential wildlife sources. Slaughter check surveillance alone did not detect low level tuberculosis in cattle. Deer can and do serve as reservoirs of bovine tuberculosis.

1Animal Health Diagnostic Laboratory, College of Veterinary Medicine, Michigan State University, East Lansing, MI 2Wildlife Disease Laboratory, Michigan Department of Natural Resources, East Lansing, MI 3United States Department of Agriculture, APHIS, VS, Holt, MI

Deer-to-deer Transmission of Mycobacterium bovis

M. V. Palmer, and D. L. Whipple

In 1994 a free-ranging white-tailed deer (Odocoileus virginianus) in Michigan was diagnosed with tuberculosis due to Mycobacterium bovis. Subsequent surveys in northeast Michigan have identified the first known epidemic of tuberculosis in white-tailed deer. Information is lacking on the pathogenesis and transmissibility of M. bovis infection in white-tailed deer. In order to determine the efficiency with which deer transmit tuberculosis to each other, and the routes by which such transmission occurs we exposed non-inoculated deer to experimentally inoculated deer. Eight deer were experimentally inoculated by intratonsilar instillation of 2x108 colony forming units (CFU) of M. bovis. Eight non-inoculated deer were introduced 21 days after inoculation. Deer were housed in pens 16 sq. meters in size such that 2 in- contact deer were penned with 2 experimentally inoculated deer. Each pen had a single source of water, hay, and pelleted feed. Sixty-nine days after introduction, all in-contact deer developed delayed type hypersensitivity (DTH) reactions to M. bovis PPD as determined by the comparative cervical test. One hundred twenty days after inoculation all experimentally inoculated deer were removed. One hundred fifty nine days after introduction, 4 in-contact deer were euthanized and examined and 4 new non- inoculated deer were housed with the remaining original in-contact deer such that 4 new in-contact deer were housed with 4 original in-contact deer. One hundred days after introduction, all new in-contact deer had developed DTH to M. bovis PPD. At 180 days after introduction of new in-contact deer, all deer were euthanized and examined. All in-contact exposed deer developed tuberculosis with one or more tissues from every deer containing tuberculous lesions and M. bovis. Lesions were most commonly seen in the lung, tracheobronchial and mediastinal lymph nodes. In-contact infected deer shed M. bovis in nasal secretions, saliva, feces, and urine. Hay and pelleted feed contained M. bovis at multiple times throughout the experiment. This study shows that tuberculous deer efficiently transmit M. bovis to other deer in close contact. Lesion distribution in in-contact exposed deer suggests aerosol transmission as a likely means of infection, however, contamination of shared feed must also be considered. Body fluids containing Mycobacterium bovis may become aerosolized or directly contaminate feed, both of which may be sources of infection for other susceptible hosts. Wildlife managers in tuberculosis endemic areas should avoid management practices that encourage unnatural gathering of deer.

Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Service, USDA, 2300 Dayton Avenue, Ames, IA

Evaluation and Comparison of Veterinary Diagnostic Test Kits

L. M. Henderson

Reports which detail the evaluation and comparison of licensed diagnostic test kits provide important information for laboratory diagnosticians. In order to improve communication between investigators and readers, it is important to reach agreement on terminology; this is best accomplished by first understanding how scientists at The Center for Veterinary Biologics evaluate and license diagnostic test kits which are used for the diagnosis of disease and/or vaccination status of animal species. The intended use of a diagnostic test kit impacts the licensing requirements for licensure. In addition, the interpretation of test results plays a critical role in the appropriate uses of the kit. A number of factors are considered and diagnostic test kits are tested and evaluated under routine laboratory conditions prior to licensure. The intended use of the kit plays a critical role in the pre-license evaluation of each kit. Pre-license evaluation varies for kits intended for use in determination of herd vaccination or exposure status as compared to kits intended for use in the diagnosis of individual animal status. Diagnosticians must evaluate the applicability of each kit to the specific needs. The information necessary to do so is found in the product insert, which usually provides sensitivity and specificity data as well as guidance on test result interpretation. During the evaluation of diagnostic test kits, complications introduced by the development of the immune response to the target organism must be considered. For diseases in which infected animals with healthy immune systems develop a strong antibody response within a relatively short defined period of time, sensitivity and specificity determinations are relatively straightforward. For disease circumstances in which the humoral immune response is delayed, such as with Mycobacterium paratuberculosis, the causative agent of Johne's disease, sensitivity varies throughout the lifetime of the animal. Thus, interpretation of kit results is complicated and requires diagnosticians to be aware of other significant factors in addition to test kit results. When investigators compare test kits, it is important to consider the above factors, especially when test kits have different intended uses, and thus have different data supporting licensure. In addition, comparing licensed kits provides important information regarding agreement of test kit results, but may not necessarily indicate test kit specificity and/or sensitivity, which can only be determined when the true infective or vaccination status of the animal from which the serum was obtained is known.

Biotechnology and Diagnostics, Center for Veterinary Biologics-Licensing and Policy Development, USDA, Ames, IA A New Commercial Competitive Enzyme-Linked Immunosorbent Assay for Anaplasma marginale Serum Antibodies

S. Adams1, T. McGuire1, D. Knowles2, and T. McElwain3

Anaplasmosis is an important worldwide disease of cattle and other ruminants caused by a rickettsial hemoparasite, Anaplasma marginale, recently classified among geno group II of Ehrlichaea. Animals suffering from acute anaplasmosis develop severe anemia, abortion, weight loss, jaundice and death. Diagnosis of the acute disease is based upon clinical signs, anemia and finding of Anaplasma inclusion bodies in erythrocytes. Animals surviving the acute phase become lifelong carriers and thus reservoirs of biological transmission by ticks or mechanical transmission by other arthropods. Carriers in various cycles of rickettsemia fluctuate between 102.5 and 107 infected erythrocytes per ml of blood, levels generally undetectable by Giemsa staining. In addition to Giemsa staining, carriers may be identified by detection of serum antibodies to A. marginale. The complement fixation test (CFT) and card agglutination test have been the most frequently used serologic tests for anaplasmosis. Although it lacks sensitivity, the standardized CFT is required by many states and countries for movement of susceptible animals. A cELISA based on recombinant major surface protein 5 (rMSP5) of A. marginale and a monoclonal antibody, ANAF16C1, greatly enhances the ability to diagnose Anaplasma carrier animals by serology because of its significantly increased sensitivity over the CFT. The rMSP5-cELISA showed sensitivity of 96% (145/151) and specificity of 95% (80/84) compared to nPCR coupled with sequence analysis and hybridization to identify A. marginale MSP5 DNA in blood of 235 cattle from an endemic region. A new commercial cELISA has been produced which utilizes rMSP5 as its target antigen and monoclonal antibody ANAF16C1 as the labeled antibody with which Anaplasma-specific antibodies competes for binding. Using 137 samples from the 235 sample set tested by the prototype assay and nPCR, the commercially produced assay had a sensitivity of 96% (92/96 positives) and a specificity of 98% (40/41 negatives). Two diagnostic laboratories certified for export testing by CFT detected 23/96 and 14/96 for 24% and 18% sensitivity, respectively. Both CFTs correctly classified all 41 negatives for 100% specificity. Field trials were performed by personnel in 3 cooperating diagnostic laboratories. They tested 50 interdependency samples assembled by the commercial company and results were identical. They also evaluated 150 field samples that were submitted for routine diagnostic purposes. Of 449 total samples, 131 were positive by the cELISA. Ninety-five (73%) of the 131 cELISA positives were positive by CFT. Of 318 negative by the cELISA, six were positive by the CFT.

1VMRD, Inc., Pullman, WA 2USDA, ARS, Animal Disease Research Unit, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 3Washington Animal Disease Diagnostic Laboratory, Washington State University, Pullman, WA

A Rapid Serologic Test for the Detection of Antibodies to Mycobacterium avium subsp. paratuberculosis with Applications for Bovine Practitioners

T. A. Jackson

A prototype ELISA test has been developed on the IDEXX SNAP ™ device platform to detect antibodies to Mycobacterium avium subsp. paratuberculosis (M.pt.), the causative organism of Johne’s disease in ruminants. Initial validation studies have been completed utilizing bovine serum as the specimen type. This test system has potential applications as an animal-side or in-clinic assay for diagnosing Johne’s disease in symptomatic or suspicious animals. The SNAP ™ test platform has demonstrated efficacious utility as an in-clinic test system for the diagnosis of a variety of infectious diseases in dogs and cats. This new serologic test for Johne’s disease is formatted to provide the same utility to large animal veterinarians who require a rapid test result. Total assay time is seventeen minutes (17’) after a five minute M. phlei pre-treatment. Results are interpreted visually, and may also be quantified with a densitometer. The performance of this new M. pt. SNAP ™ ELISA was evaluated by testing populations of dairy cattle (n = 406) with the prototype test system and with an USDA-licensed microtitre plate antibody test kit. Quantitative data were measured on the SNAP ™ test platform by taking densitometric readings of the diagnostic spot at the completion of the test protocol. These values were compared to the S/P ratios yielded by the microtitre-plate technique. Regression analysis of these data shows a significant correlation of test results between the two techniques utilized, (R2 = 0.82; p < 0.0001; 95% CI). Ninety-six point one percent (96.1%) of the sera tested (390 of 406) yielded an agreement in serologic status as determined by the two test methods evaluated. Specimens tested were collected from seven different dairy herds of varying geographic origin. Herd status was investigated with regard to previous diagnostic test histories, (serology and culture); differential observations of herd veterinarians, (evidence of symptomatic animals); and with regard to the introduction of replacement cattle into the herds, (open vs. closed herd). Three of the seven herds tested (n = 178) had a history of diagnosed M. avium subsp. paratuberculosis cases within each of the herds. Two of the seven herds (n = 133) had no previous evidence of paratuberculosis, and are presumed to be negative for antibodies to M. pt. Each of these two herds did have a prior test history for M.pt. detection by culture and serology. One of the two presumed negative herds was closed to replacement animals. The final two herds tested (n = 95) were of unknown status, and had no previous test history for Johne’s disease. The percent agreement in final test dispositions between the two assay methods utilized (for these three herd groupings) were as follows: known infected herds - 93.3%; presumed negative herds – 99.2%; herds of unknown status – 96.8%. This study has served as a basis in comparing two techniques for the detection of antibodies to M. avium subsp. paratuberculosis. The relative sensitivity and specificity of a new prototype lateral flow test was measured against an established USDA-approved diagnostic product. These data provide evidence that the performance of the prototype M. pt. SNAP ™ test for detecting antibodies to M. avium subsp. paratuberculosis correlates significantly to the assay performance of the USDA-licensed IDEXX M.pt. Antibody Test Kit which is manufactured in a microtitre-plate format. Further, the prototype SNAP ™ test yields specimen dispositions which are consistent with the known source-herd histories for the bovine populations studied.

Production Animal Services Research & Development, IDEXX Laboratories, Westbrook, ME

A New Commercial ELISA for Bovine Leukemia Virus Antibody

D. S. Adams, and T. C. McGuire

A new commercial bovine leukemia virus antibody test kit in ELISA format has been introduced. It detects antibodies to glycoprotein (gp51) in bovine sera. Serum antibodies attach to gp51 molecules bound to plastic wells of a microtiter plate. This complex is then subjected to horseradish peroxidase (HRP)-labeled affinity-purified goat antibodies to bovine immunoglobulins. Attached HRP-labeled antibodies are detected by addition of enzyme substrate and quantitated by subsequent blue color product development. Strong color development indicates the presence of antibody to BLV gp51 in serum. Very weak or no color development indicates the absence of antibody to BLV gp51 in serum. Excluding setup, washing and reading times, total incubation time for the assay is one hour. Solid plate and stripwell formats are available. This assay is unusual in that no verification of positives with non-antigen wells is necessary. Using 476 in-house samples, 205 antibody positive and 271 antibody negative confirmed by another licensed commercial bovine leukemia virus antibody test kit in ELISA format, the new kit showed 100% agreement in results. An additional 237 samples, 150 positive and 87 negative by a licensed agar gel immunodiffusion (AGID) kit, were also tested by the new kit and again showed 100% agreement. Field-testing was set up in 3 animal disease diagnostic laboratories. Laboratory personnel in each of the laboratories tested 50 interdependency samples provided by the manufacturer of the new test, and they also assembled 150 field diagnostic samples for a total of 450 tests. The field diagnostic samples were also tested with another licensed commercial bovine leukemia virus antibody test kit in ELISA format (reference assay). All 3 laboratories and the manufacturer produced identical results on the 50 interdependency samples. Initial analysis of the 450 field samples showed the following: Laboratory 1 had the same results for the 103 negative samples, but 2/47 positive by the reference assay were negative using the new test; Laboratory 2 had identical results for both assays, 106 negative and 44 positive; Laboratory 3 had the same results with both assays on 71 negative samples, but the new assay was negative on 12/79 samples which were positive in the reference assay. Of the 14 samples determined to be positive by the reference assay and negative by the new assay, 12 had sufficient volume for further analysis. At a reference laboratory by AGID and in-house by indirect immunofluorescence (IFA), eight were confirmed as positive and four were determined to be negative. The manufacturer adjusted the positive control of the assay to increase its sensitivity without loss of specificity. After doing this, the eight confirmed positive samples were also positive by the new assay. The remaining 4 samples, which were positive by the commercial reference assay, were negative when re-tested using the new assay, just as they were by AGID (reference lab) and the in-house IFA.

VMRD, Inc., Pullman, WA

Development of an ELISA for the Detection of Antibodies to Mycoplasma hyopneumoniae in Swine Sera

K. Velek, L. Plourde, and H. Liauw

Enzootic pneumonia or Mycoplasma Pneumonia of Swine (MPS), a chronic disease with a high morbidity and a low mortality is caused by Mycoplasma hyopneumoniae. An ELISA has been developed to detect antibody to Mycoplasma hyopneumoniae (Mhyo) present in swine serum. This assay has been designed in the microtiter format, in which purified Mhyo antigen has been coated onto the solid phase and an anti-swine IgG horseradish peroxidase conjugate is used for detection. This Mhyo ELISA was designed as a rapid, standardized, herd-screening method, to be used as an indicator of exposure to the agent. It has been shown to be sensitive, specific, and correlates well with the Tween 20 ELISA. Sensitivity was assessed through the testing of sequential sera from pigs inoculated with a Mhyo bacterin vaccine. Three pigs of known serologic status for Mhyo were inoculated according to the vaccine manufacturer’s recommendation. Serum was collected from each animal at 0, 7, 14, 21, and 28 days post inoculation. Samples were tested by Tween 20 ELISA at the Iowa State Veterinary Diagnostic Laboratory and tested on the experimental Mhyo ELISA at IDEXX Laboratories. The Mhyo ELISA detected seroconversion in 1 pig by day 14, and 2 pigs by day 21; while the Tween 20 ELISA detected seroconversion in 3 pigs by day 21. Additional sensitivity data, demonstrating correlation of the Mhyo ELISA to Tween 20 ELISA was obtained through the testing of field samples from Mhyo infected herds. Samples were obtained from two sites with accompanying Tween 20 ELISA data. From 540 samples tested, 292 samples were considered positive or suspect by Mhyo ELISA, and 255 samples were considered positive or suspect by Tween 20 ELISA. Of these 255 samples, the Mhyo ELISA detected 228 positive or suspect samples. This results in 89.4% sensitivity for the Mhyo ELISA as compared to the Tween 20 ELISA for this population. The overall correlation between the Mhyo ELISA and the Tween 20 ELISA was 77.2%. When all suspect samples for either test were considered positive, the correlation between the two methods was 83.1% and when they were considered negative, the correlation was 82.2%. A set of 190 field samples from Mhyo negative herds, obtained from two sites in the United States, were used to evaluate specificity. Serum samples were assayed on the Mhyo ELISA and results were compared to the Tween 20 ELISA. All of the samples tested were negative (S/P less than 0.30) on the Mhyo ELISA, resulting in 100% specificity for this population. Specific swine serum samples against Mycoplasma flocullare, Mycoplasma hyorhinis, Mycoplasma hyosynoviae, Encephalomyocarditis virus, Hemagglutinating Encephalomyelitis virus, Porcine Adenovirus, Porcine Parvovirus, Porcine Reovirus, Porcine Rotavirus, Swine Influenza virus, and Transmissible Gastroenteritis virus were assayed on Mhyo ELISA, and all of them were negative. The IDEXX Mycoplasma hyopneumoniae Antibody test described above provides good correlation to other serological methods in the detection of seropositive pigs, while maintaining excellent specificity.

IDEXX Laboratories Inc., Westbrook, ME Evaluation of In-house Produced Herrold’s Egg Yolk Agar with Commercially Prepared Formulations with and Without Nalidixic Acid and Vancomycin

B. C. Love1, T. R. Farrell1, B. A. Byrum1, T. E. Wittum2, T. Burns3, P. Cullum3, and D. Callihan3

In February 2000, Becton Dickinson Biosciences initiated a study to evaluate a potential new product, Herrold’s Egg Yolk Agar with Mycobactin J and amphotericin B, nalidixic acid, and vancomycin (BBL HEYA-MANV). The study involved laboratories which produced their own similar product, and compared the ability of the two media to support the growth of Mycobacterium avian subsp. paratuberculosis while suppressing contaminating overgrowth of fungi or bacteria. Fecal samples which the laboratories received for Johne’s culture were processed according to the laboratory’s routine procedure, inoculated onto both media, and evaluated for growth of M. avium subsp. paratuberculosis and contaminants at weekly intervals. Additionally, our laboratory inoculated tubes of BBL’s Herrold’s Egg Yolk Agar with Mycobactin J and amphotericin B(BBL HEYA), for purposes of comparing results to the two media listed above. Our hypothesis was that the additional antibiotics (nalidixic acid and vancomycin) in the media could suppress growth of M. avium subsp. paratuberculosis. The proportion of tubes with contaminating overgrowth was compared among media using the Pearson chi square test. Mean colony counts for the tubes with M.avium subsp. paratuberculosis growth were compared among media using the Kruskal-Wallis test. One hundred sixty two fecal samples were inoculated onto the three types of HEY agar. Growth of M.avium subsp. paratuberculosis was detected on all media as early as 5 weeks. BBL media without amphotericin B and vancomycin had much higher levels of contaminating overgrowth than did the BBL HEYA-MANV or the in-house produced media (31/486 tubes, vs. 0/486 for both in-house and BBL HEYA-MANV; P <0.001). M. avium subsp. paratuberculosis were recovered from 20 (4.1%) of the samples, with growth detected on all 3 media. Thus, media type does not appear to affect the sensitivity or specificity of M.avium subsp. paratuberculosis culture. Mean (SE) colony count among tubes with M. avium subsp. paratuberculosis growth were 22.8 (6.3), 25.4 (6.8), and 31.0 (7.3) for in-house, BBL- HEYA-MANV, and BBL HEYA media, respectively. Differences in mean colony counts were not detected, although statistical power to detect differences was limited.

1 Animal Disease Diagnostic Laboratory, Ohio Department of Agriculture, Reynoldsburg, OH 2 Department of Veterinary Preventive Medicine, The Ohio State University, Columbus, OH 3 Becton-Dickinson Biosciences, Sparks, MD

A New Liquid Culture Method, the Trek ESP Culture System II, for the Rapid Detection of Mycobacterium avium subsp. paratuberculosis in Bovine Fecal Samples

S. J. Shin, S. G. Kim, L. J. Miller, P. R. Harpending, V. H. Patten, and D. H. Lein

Solid medium culture has been the standard procedure for detection of M. avium subsp. paratuberculosis in bovine fecal samples. However, because of the long generation time of the bacterium, it is a slow and labor intensive procedure requiring up to 12 weeks of incubation. The purpose of this study was to develop a rapid detection procedure for M. avium subsp. paratuberculosis in bovine fecal samples using a liquid culture method, the Trek ESP Culture System II (ESP), in conjunction with the Cornell double incubation decontamination process. Bovine fecal samples, a total of 85 including 30 known positive samples and 55 unknown samples, were decontaminated by the Cornell double incubation process prior to culture in ESP MYCO bottles and on Herrold's egg yolk (HEY) agar slants. Of 30 known bovine fecal samples, 10 heavy shedders (>300 CFU/g), 10 medium shedders (31-300 CFU/g) and 10 low shedders (1-30 CFU/g) were included. Of 55 unknown samples, 25 were NVSL were check samples and 30 were field samples. All bottles flagged as positive in ESP and all suspect colonies on HEY agar slants were confirmed as M. avium subsp. paratuberculosis by acid fast staining and PCR. Of 85 bovine fecal samples, 59 (69.4%) were positive for M. avium subsp. paratuberculosis by the ESP method while 51 (60%) were positive by the standard solid medium culture method (HEY). Both systems were able to detect 100% of heavy and medium shedders; however, the ESP detected 8 more low shedders than the standard HEY agar method. Mycobacterium avium subsp. paratuberculosis was detected by the ESP with a mean time to detection of 14.97 days for heavy shedders, 22.79 days for medium shedders, and 34.5 days for low shedders. In contrast, by the standard solid medium culture on HEY agar, M. avium subsp. paratuberculosis was isolated with a mean time to detection of 39.71 days for heavy shedders, 41.9 days for medium shedders, and 48 days for low shedders. Two samples (2.4%) from each method were contaminated with fungus and other microorganisms. In conclusion, the ESP, a liquid medium based detection system, was able to detect M. avium subs. paratuberculosis in bovine feces 2 to 3 weeks earlier than the standard HEY culture method with greater sensitivity and identical contamination rate.

Diagnostic Laboratory, Department of Population Medicine and Diagnostic Science, College of Veterinary Medicine, Cornell University, Ithaca, NY

Paratuberculosis in Dairy Cattle: Classification of Cattle by Colony Counts of Mycobacterium avium subsp. paratuberculosis in Fecal Samples.

R. H. Whitlock1, R. W. Sweeney1, T. Fyock1, S. J. Wells2 and J. R. Stabel3

The extent of fecal shedding by cattle infected with Mycobacterium avium subsp. paratuberculosis is important to determine, since animals shedding more organisms represent the greatest risk to spread the disease and to contaminate the environment. Generally, cattle shedding more organisms (high shedders) represent later stages of infection which also tend to have higher antibody titers and therefore detectable by ELISA. Cattle shedding few detectable organisms (low shedders) by culture represent earlier stages of infection and have lower antibody levels. Awareness of the relative expected proportion of cattle in each shedding category provides more information to the producer, herd veterinarian and regulatory officials about the extent and distribution of infected cattle in herds. The primary objective of this paper is to describe the relative proportion of cattle in one of three categories: high shedders, mid-shedders and low shedders. High shedders are those animals with 70 or more colonies of M. avium subsp. paratuberculosis per culture tube for a total of more than 280 colonies in four tubes. Mid-shedders have less than 70 colonies per tube but more than 10 colonies of M. avium subsp. paratuberculosis per tube with a range of 40 colonies to 279 colonies in four tubes. Low shedders have less than 10 colonies per tube or less than 40 total colonies in four tubes of Herrold's egg yolk media all with mycobactin J. Three populations of Johne's infected cattle including ten dairy herds cultured every six months for four years, twenty-six herds cultured the first time and thirty Johne's infected dairy herds cultured once served as the basis for this study. In the last group of herds, 10 herds had more than 10% fecal culture positive, ten herds had less than 10% culture positive and ten herds were cultured the first time. In each category of infection among the 66 dairy herds in which fecal cultures were done, the relative proportion of infected cattle as detected by fecal culture was similar: 20-30% high shedders, about 10% mid-shedders and 60-70% low shedders. Herds cultured the first time tend to have a higher number of high shedders, while herds tested several times tended to have more low shedders.

1School of Veterinary Medicine, University of Pennsylvania, New Bolton Center, Kennett Square, PA 2College of Veterinary Medicine, University of Minnesota, St Paul, MN 3National Animal Disease Center, USDA-ARS, Ames, IA If You Use a Different Test, You Will Get a Different Answer

M. T. Collins, and J. Buss

The purpose of this study was to test the level of agreement among four serological tests for Johne’s disease. The IDEXX ELISA for antibody to M.avium subsp. paratuberculosis was arbitrarily selected as the basis for comparison. One hundred bovine sera from cattle of unknown infection status submitted to our laboratory for Johne’s disease serology were selected to fulfil the following IDEXX ELISA s/p criteria based on the original test result (IDEXX-1): 15 sera with s/p <0.10 (negative); 10 sera with s/p = 0.10 to 0.24 (suspect); 25 sera with s/p = 0.25 to 0.39 (low positive); 25 sera with s/p = 0.40 to 0.99 (positive); and 25 sera with s/p >1.00 (strong positive). These sera were stored at –20 C, thawed, and tested by four USDA-licensed tests for serum antibody to M. avium subsp. paratuberculosis, including a repeat of the IDEXX ELISA (IDEXX-2) using a different kit lot number from the one used to test the original sample. Other tests used were Parachek® (Biocore Animal Health, Inc.) TipTest® and RJT® (also known as the AGID, ImmuCell, Inc.). All tests were interpreted by manufacturer’s guidelines. For the subjectively interpreted assays, TipTest® and RJT®, all visible reactions, even if weak, were classified as positive. Results: Table 1. Kappa and agreement between pairs of serological tests. Kappa values1 IDEXX-1 IDEXX-2 Parachek® TipTest® RJT®2 IDEXX-1 - .80 .39 .26 .12 IDEXX-2 93% .38 .27 .09 Parachek® 67% 62% .43 .34 TipTest® 65% 62% 71% .25 RJT® 41% 41% 71% 61% Percentage of test results in agreement 1Chi square analysis: all tests except IDEXX-2 had results significantly different from IDEXX-1p<0.01. 2RJT® is only recommended for use on animals with clinical signs of paratuberculosis.

Table 2. Agreement between IDEXX-1 and Parachek® or TipTest® by s/p range Parachek® TipTest® Neg Pos Agreement Neg Pos Agreement Neg+suspect 25 0 100% 17 8 68%

Low-pos 18 7 28% 14 11 44% IDEXX-1 Positive 12 13 52% 11 14 56% Strong-pos 3 22 88% 5 20 80% When analyzed by level of s/p value, highest levels of agreement among tests were found for sera classified at the extremes: negative or strong positive. Five times a serum classified as “suspect” (s/p = 0.10 to 0.24) was positive on retest and one time the reverse occurred. Regression analysis of all 100 s/p results for IDEXX-1 and IDEXX-2 showed a high correlation (r2 = 0.96). For 6% of sera a change in interpretation (pos/neg) occurred between repeated IDEXX ELISAs. For 29%-59% of bovine sera interpretations were different using different commercial kits. This was least common among sera with very low or very high “titers” of serum antibody to M. avium subsp. paratuberculosis. This may be due to differences in antigens used in the kits or other factors. Regardless, the study shows that if laboratories use different commercial kits for Johne’s disease serology, diagnostic results will differ. While the basis for this difference is unknown, it merits detailed investigation.

Department of Pathobiological Sciences, University of Wisconsin, Madison, WI A Fast and Sensitive Diagnostic Assay for the Detection of Mycobacterium paratuberculosis in Bovine Feces

S. J. Spatz, and S. P. Hogan

Johne’s disease is a chronic, debilitating enteritis of cattle, sheep, goats and other ruminants caused by the organism Mycobacterium paratuberculosis. The standard diagnostic assay for Johne’s disease is cultivation of M. paratuberculosis from fecal specimens. This procedure requires a large amount of incubator space and may take months to produce visible colonies. We have developed an assay to determine the presence of M. paratuberculosis in feces that requires no cultivation and can be performed in 2 days. This assay, Mycobacterium paratuberculosis DNA Test Kit, relies on the amplification of M. paratuberculosis from fecal DNA preps. DNA is extensively purified from fecal organisms, amplified using fast-time PCR and detected in a dot-blot format with enzyme conjugates using microarray hybridization conditions. The specificity of the assay was demonstrated using two groups of fecal specimens. One group was comprised of field fecal specimens from two NVSL check sets in which the presence or absence of M. paratuberculosis had previously been determined using cultivation techniques; the other group consisted of fecal samples from a certified negative M. paratuberculosis herd. The results indicated that there was a 100 % correlation between culture negative data and the lack of a signal with the PCR based assay for negative fecal specimens within the NVSL check sets and all the specimens collected from certified M. paratuberculosis negative cattle. With regard to the culture positive specimens, there was a 70.0 % (21/30) correlation between culture positive data and positive PCR results. Since the culture positive specimens that failed to generate a positive PCR signal contained a low number of M. paratuberculosis organisms, the assay’s sensitivity was reexamined in terms of colony forming units (CFU) per sample as originally determined by NVSL. With fecal specimens that contained 0-8 CFU there was a 22.2 % agreement between culture and PCR results. Fecal specimens that contained 9-28 CFU had 81.2% agreement between the two diagnostic methods. Similarly there was a perfect correlation between culture results and PCR results on samples that contained greater than 29 CFU. The low level of agreement with samples that contained 0-8 CFU may represent the detection limit of the PCR assay; however, personnel from many other diagnostic labs were unable to recover M. paratuberculosis from these samples. Therefore, the inconsistent recovery of the organism using these samples most likely represents organisms partitioning in the feces during shedding. This is further supported by a lack of correlation between CFU and PCR signal intensities and the reported clumping of these organisms when propagated in liquid culture.

Production Animal Services, Research and Development, IDEXX Laboratories, Westbrook, ME

Application of Molecular-based Techniques in the Accurate Detection of Mycobacterium avium subsp. paratuberculosis

S. McLellan, H. Pirkov, and R. S. Lambrecht

Mycobacterium avium subspecies paratuberculosis (Map) is the causative agent of paratuberculosis or Johne's disease, a chronic progressive disease affecting the intestinal tract of ruminants. The disease results in a wasting syndrome of infected dairy and beef cattle with tremendous economic loss to the agricultural industry. The diagnosis of Johne’s disease and the identification of Map are extremely difficult due to the slow growth of Map in the laboratory and the poor sensitivity of laboratory tests, especially during the subclinical stages of disease. Prompt and accurate diagnosis of infection is central to effective management of livestock enterprises. Although Map shares 99-99.5% DNA homology with other related mycobacteria belonging to the Mycobacterium avium complex (MAC), the insertion sequence IS900 differentiates Map from these close phylogenetic neighbors and has been used for the basis of molecular diagnostic testing. Insertion sequences similar to the IS900 in other MAC strains have been a source of false-positives in commercial PCR-based assays that target IS900. Analysis of the alignment of these similar IS elements reveal a 4 base pair deletion in each element (at bases 121 and 125) which is not present in the IS900 sequence. We have used this unique region to design primers that have an increased specificity for the IS900 sequence, resulting in a decrease in the rate of false positives. Our testing strategy also relies on size characterization of the amplification product providing an additional level of confidence for specificity. PCR detection methods which employ primers that do not take into consideration the potential for cross- amplification of portions of other insertion sequences, or that are unable to distinguish nonspecific amplification products of related sequences, have suffered from high rates of false-positives. Primer design as well as size characterization of the PCR product are both critical in discerning IS900 from other related IS elements in mycobacteria, and greatly increase the specificity of the diagnostic probe for IS900. Another insertion sequence designated IS1311 has been identified in Map and Mycobacterium avium subs. avium and Mycobacterium avium subsp. silvaticum. According to restriction enzyme digestion patterns of IS1311, it has been recently reported that Map can be further classified into “sheep” and “cattle” strains, and can be differentiated from Mycobacterium avium-avium and Mycobacterium avium- silvaticum. Five point mutations differentiate the IS1311 sequence of Map from M. avium-silvaticum and M. avium-avium. The IS1311 test has a sensitivity equal to that of the IS900 test and has been shown to be useful on a range of crude and purified DNA preparations from a variety of sources. We have used the IS1311 test to evaluate Map strains isolated from a wide range of hosts besides cattle and sheep. Most of these strains have resulted in a pattern similar to cattle strains; however, Map strains isolated from bison gave a unique restriction pattern, suggesting evolutionary divergence of Map in unique hosts. Testing for IS900 and IS1311 is simple and rapid, and when performed together can provide accurate and confirming diagnosis of Map infection, thereby offering herd managers and animal health programs useful information for the control and management of paratuberculosis.

Department of Health Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI Molecular Cloning and Characterization of Mycobacterium avium subspecies paratuberculosis Antigen 85 Complex Gene Family, 85A, 85B and 85C

Y-F. Chang, D. Veerabadran, K-S. Shin, S. Shin, R. H. Jacobson, and D. H. Lein

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the etiologic agent of paratuberculosis (Johne’s disease), a chronic granulomatous enteritis in cattle, sheep and goats. Currently, there is an urgent need for an effective recombinant vaccine to control the disease and an improved diagnostic test for reliable detection of M. paratuberculosis infection. The dominant exported proteins and protective antigens of other Mycobacterium spp. have been identified to be the antigen (Ag85) complex. In this study, we have cloned and sequenced the genes (85A, B and C) from M. paratuberculosis and expressed the proteins in E. coli. Ag85A, B, and C have been implicated in disease pathogenesis through its fibronectin-binding capacities. A carboxylesterase domain was found within the amino acid sequences of these proteins. The function of 85A, B and C is to act as a mycolyltranferase involved in the final stages of mycobacterial cell wall assembly. These secreted proteins or genes may be useful as antigens for serologic diagnosis and/or recombinant and DNA vaccine development.

Department of Population Medicine and Diagnostic Science, College of Veterinary Medicine, Cornell University, Ithaca, NY

Development of Quantitative PCR-based on the ABI 7700 System (TaqMan) for Mycobacterium avium subsp. paratuberculosis

S. G. Kim, S. J. Shin, C. A. Rossiter, S. M. Stehman, R. H. Jacobson, and D. H. Lein

There have been numerous reports for PCR-based diagnostic methods to detect Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne’s disease. The result of conventional PCR tests has been only qualitative, either positive or negative; therefore, the result does not present any quantitative information about the number of the agents in the specimen. We have developed a quantitative PCR method using the ABI system (TaqMan) to measure the number of M.avium subsp. paratuberculosis present in test samples. The sensitivity of the method was 10 CFU for M. avium subsp. paratuberculosis ATCC 19698. The specificity of the method was tested for 14 Mycobacterial species (M. abscessus, M. asiaticum, M. avium subsp. avium, M. bovis, M. fortuitum subsp. fortuitum, M. intracellulare, M. kansasii, M. marinum, M. phlei, M. scrofulaceum, M. simiae, M. smegatis, M. terrae, M. ulcerans) and 9 non-Mycobacterial species (Borrelia burgdorferi, Chlamydia psituicci, Ehrlichia canis, E. equi, E. risticii, Escherichia coli, E. coli O157:H7, Streprococcus equi, S. zooepidemicus). Even at high level of cell numbers (105 CFU), most of the organisms tested negative except M. marinum and M. scrofulaceum. The finding with M. scrofulaceum was consistent with a recent report by Austrian investigators who found some isolates closely related to M. scrofulaceum carry 70% to 79% homology with M. paratuberculosis in the region of IS900. Using this TaqMan-based quantitative PCR method with the Trek ESP System II for bovine clinical fecal samples, we were able to confirm that most of the positive samples contained 105 to 106 CFU/ml of M.avium subsp. paratuberculosis. The quantitative PCR was also useful in the study of growth characteristics of three groups of M. avium subsp. paratuberculosis strains classified by shedding levels, heavy, medium, and low based on CFUs on HEY slants.

Diagnostic Laboratory, Department of Population Medicine and Diagnostic Science, NYS College of Veterinary Medicine, Cornell University, Ithaca, NY

Comparison of a Novel and Traditional Processing Method with Several Media Formulations to Detect Mycobacterium avium subsp. paratuberculosis in Bison Tissues

C. G. Thornton1, and R. H. Whitlock2

Isolation of Mycobacterium avium subsp. paratuberculosis (Mpa), the causative agent of Johne’s disease in ruminants remains the gold standard for diagnosis of infection. Traditional culture methods have relied on decontamination with hexadecylpridium chloride (HPC) and Herrold’s egg yolk media (HEYM). This study evaluated a novel decontamination method that relies on the combination of the zwitterionic detergent C18-carboxpropylbetaine (CB-18) and lytic enzymes (CB-18/LE) and compared the results with the traditional method using a variety of Bison tissues. Tissues harvested from 59 Bison at slaughter included ileum, one section of mesenteric lymph node from each of three levels (upper, middle and lower) of the mesenteric lymph node chain and the ileo- cecal-colic lymph node in most animals. In total, 270 Bison tissues suspected of being infected with Mpa were processed using both decontamination methods. HPC processed specimens were inoculated onto four different media formulations: HEYM with pyruvate (HEYMP) HEYM without pyruvate (HEYMNP), Lowenstein-Jensen (L-J) media and Middlebrook 7H10 media supplemented with egg yolk (7H10EY). CB-18/LE processed specimens were inoculated into BACTEC 12B liquid media supplemented with 1% egg yolk and PANTA, as well as inoculated in duplicate onto 7H10EY and HEYMNP media slants (both solid media formulations used in conjunction with CB-18/LE processing included PANTA to control contamination). Preliminary results indicated tissues from 46 (88%) of the 59 Bison and 149 of 270 (55%) tissues were classified as positive. Of the 149 tissues processed by CB-18/LE on three media formulations (12B, 7H10EY and HEYMNP) identified 98% (142) of the tissues positive compared to 57% (85) when processed by HPC and four media formulations (HEYMP, HEYMNP, L-J and 7H10EY). Similar results were obtained when Bison were classified as positive according to processing method, 44 (95.7%) were positive with CB-18/LE while only 28 (60.9%) were positive when processed by HPC. These results indicate both tissue processing method and media formulation greatly influence the recovery of M. avium subsp. paratuberculosis from Bison suspected of Johne’s Disease. Overall, Bison isolates of Mpa grew better when processed with CB-18/LE. The most sensitive detection method was the combination of CB-18/LE processing and BACTEC 12B liquid culture compared to the traditional method of HPC/HEYM analysis.

1Integrated Research Technology, 1901 Sulfur Springs Road, Baltimore, MD 2School of Veterinary Medicine, University of Pennsylvania, New Bolton Center, Kennett Square, PA

Copper Poisoning in Rabbits: A Case Report

W. K. Rumbeiha, and W. E. Braselton

Copper is an essential element in domesticated species including rabbits. It is a constituent of metalloenzymes such as cytochrome oxidase, lysyl oxidase and tyrosinase. Copper is also a component of ceruloplasmin, which plays a role in iron absorption. Copper is usually added to diets of rabbits to improve weight gain, control gastrointestinal infectious diseases and to improve hair pigmentation. However, dietary copper levels recommended for rabbit feeds are not clearly defined and potential for copper toxicosis exists. However, the authors are only aware of two published reports on copper poisoning in rabbits. In December 1999, at least 2 separate farms in Michigan reported increased mortality and weight loss in rabbits. One farm had changed feed source 2 months before problems were first noticed. This particular producer lost 35/55 rabbits. Dwarf breeds such as Jersey Woolys and Mini Rexs were the most affected, while Hollands and Dutch breeds were not or only minimally affected. The most prominent clinical finding was emaciation. Histopathology indicated that the rabbits had mild to moderate hepatic coccidiosis. Liver copper concentration was 1,490 ppm dry weight. Normal liver copper concentration is 32-200 ppm dry weight. Mean copper concentration in feed samples was 279 ppm (recommended is 20- 100 ppm). Feed tested negative for mycotoxins, polychlorinated biphenyls and polybrominated biphenyls. Tissue samples also tested negative for bacterial pathogens. The disease stopped when a new low copper diet was introduced. Rabbits on the second farm had used the same feed for a shorter period of time and experienced similar but milder clinical signs. On the basis of circumstantial and clinical evidence, and on the presence of toxic liver concentrations, we conclude that copper poisoning was responsible for toxicosis in these cases.

Animal Health Diagnostic Laboratory, CVM, Michigan State University, East Lansing, MI

Solanum dimidiatum and Crazy Cow Syndrome

A. C. Barr1, B. Abbitt1, A. Vardeman2, and J. C. Reagor1

During the recent drought in Texas, a herd of cattle was rotated into a large pasture for the breeding season and was maintained there for five months. During this time, observers found the animals to be in good flesh with no obvious problems. When the cattle were gathered in the spring for health evaluation, some animals began to stagger. Paresis progressed to uncoordinated collapse if the cattle were pressured. The down animals recovered spontaneously – a hallmark of Crazy Cow Syndrome. Four cows and a calf were presented to the veterinary clinic for evaluation. Blood counts and clinical chemistry profiles gave no indications of an etiology. None of the animals appeared responsive to thiamine therapy. Solanum dimidiatum (potatoweed, western horsenettle), associated with Crazy Cow Syndrome, was known to grow in the pasture, so one animal was euthanized and the brain was submitted to Texas Veterinary Medical Diagnostic Laboratory (TVMDL) for evaluation. Microscopic examination revealed a marked paucity of cerebellar purkinje cells with many of those remaining exhibiting hypereosinophilic cytoplasm and clear cytoplasmic vacuoles. This specific lesion is postulated to be a result of consumption of the calystegin B2-related alkaloids, which are potent inhibitors of beta- glucosidase and beta-galactosidase; these have the potential to produce a lysosomal storage disease similar to that seen in locoism. The effects of S. dimidiatum poisoning are irreversible, but deaths directly attributed to the plant toxicosis are rare. Deaths are more often the result of uncoordinated movement in hazardous surroundings (drowning in the process of drinking, falling off steep ledges). Affected cattle live a normal life span and continue to be productive when allowances are made for the cerebellar impairment during gathering and examination.

1Texas Veterinary Medical Diagnostic Laboratory, 1 Sippel Road, College Station, TX 2Mitchell County Veterinary Clinic, 2145 South Highway 208, Colorado City, TX

The Immunologic and Toxic Changes of Chronic Locoweed Poisoning in Cattle

B. L. Stegelmeier1 , and P. D. Snyder2

Locoweed (Astragalus and Oxytropis spp.) poisoning has an insidious onset, with signs of poisoning not becoming apparent until animals have grazed the plant for several weeks. Poisoning results in non- specific clinical signs of anorexia, lethargy, muscular weakness, intention tremors, proprioceptive deficits, and emaciation. Although the microscopic vacuolar microscopic lesions are characteristic of intoxication, these quickly resolve leaving subtle permanent neurologic changes. The locoweed toxin has been identified as swainsonine, an indolizidine alkaloid that inhibits lysosomal að-mannosidase and Golgi mannosidase II. Poisoned animals develop both lysosomal storage disease and altered glycoprotein processing. Serum biochemical changes including alterations in aspartate aminotransferase, alkaline phosphatase, and að-ðmannosidase are often remarkable, but these also return to normal soon after animals stop ingesting locoweed. Hematologic changes of poisoning, including vacuolation of lymphocytes and monocytes has been used as indicators of poisoning, but little known of their progression or fate. The purpose of this study is to document the hematologic, biochemical and immunologic changes of locoweed poisoning. Four groups of 6 crossbred heifers were dosed with locoweed to obtain swainsonine dosages of 0.0, 0.25, 0.75, and 2.25 mg swainsonine/kg body weight/day for 45 days. After dosing, half of the animals were allowed to recover for 45 days after which all animals were killed and necropsied. Throughout the dosing and recovery period blood, serum, lymph node and liver biopsies were collected. Hematologic and serum biochemical changes included a normocytic, normochromic mild anemia that was seen in all treated groups. The leukogram had no significant changes in cell populations; however, lymphocyte and monocyte vacuolation was apparent 4 to 7 days after dosing began. During recovery, both the anemia and leukocyte vacuolation returned to normal within 7-9 days. There were no apparent permanent hematologic or serum biochemical changes. Immunologic studies found mild irregularity in CD4 and CD8 lymphocyte numbers during late locoweed dosing and early recovery. Initially, low dose locoweed treatments had a mild stimulative effect on lymphocyte blastogenesis that tended to be depressed during recovery. No differences were detected in ability to react to injected antigens or in cell mediated response, but locoweed treated animals had significantly increased IgA secretion. All changes rapidly returned to normal during the recovery period. Additional work is needed to determine the importance of these changes on the immunocompetence of clinically poisoned livestock.

1 USDA/ARS Poisonous Plant Research Laboratory, Logan, UT 2 Purdue University, Dept. of Veterinary Pathobiology, West Lafayette, IN

Microcystin-LA Toxicosis among Cattle in California

B. Puschner1, J. Pelton2, L. Heath1, T. Francis1, and D. M. Holstege1

In October 1999, 6 of a heard of 45 Holstein heifers died acutely over a period of 3 weeks, near Petaluma, CA. The cattle were found dead without a history of clinical signs prior to death. The remaining heifers had signs of weakness, depression, anorexia and rough hair coat. One fourth of the surviving animals had signs of photosensitization with lesions on noses and vulva. Serum was taken from 2 heifers and both sera showed high elevations of (-GT and AP. A complete necropsy was performed on 2 heifers that died. The liver had acute massive hepatic necrosis, with individualization of hepatocytes and marked centrilobular congestion. Although the history of ill thrift suggested a chronic condition, histopathological findings were supportive of acute hepatotoxicosis. Possible sources of toxin included blue-green algae, cocklebur, mycotoxins, hepatotoxic mushrooms, clay pigeons and tar. Water samples from 5 ponds were collected. Algae were identified by means of light microscopic examination of structural characteristics of the bloom. Granular, nonfilamentous clumps of algae surrounded by a clear calyx were observed, and were consistent with the morphology of organisms of the genus Microcystis. High-performance liquid chromatography was used to determine whether water from the ponds and animal specimens contained microcystin. The specimens were sonicated; frozen and thawed three times to disrupt any algae cells present. After centrifuging, the extract was removed and cleaned-up using a C18 solid-phase-extraction cartridge. The extract was then injected onto a Waters 2690 HPLC with photodiode array detection, monitoring 238nm. Injections were made on a Spherisorb ODS2 4.6 x 250mm column with a mobile phase consisting of a 55:45 mixture of methanol:aqueous sodium sulfate (0.7%, wt/vol). The HPLC conditions allowed quantitation of commercially available standards of microcystin-LR, YR, RR and LA. Microcystin-LA was present in 1 pond water sample at a concentration of 64 ppm. Both rumen contents contained microcystin-LA at concentrations of 5.7 and 23 ppm, respectively. Water and rumen contents contained none of the other listed microcystins. In this case report the combination of histologic lesions and detection of microcystin-LA in pond water and rumen contents confirmed the exposure of cattle to the hepatotoxic blue-green algae that had bloomed in the pond. Microcystin-LA is one of the less commonly encountered toxins produced by Microcystis spp. Recent surveys have demonstrated that microcystin-LR constitutes more than 70% of the microcystins in any sample, and is found worldwide. Thus, the detection of microcystin-LA in pond water and rumen contents is an unusual finding. Based on these findings, initial diagnostic screening of animals suspected to have blue-green algae toxicosis should include microcystin-LR, YR, RR and LA as markers.

1California Animal and Food Safety Laboratory System–Toxicology Laboratory, University of California, Davis, CA 2Sonoma-Marin Veterinary Service, Large Animal Practice, Petaluma, CA Highly Presumptive Intoxication of Sheep Exposed to Ozark Milkweed (Asclepias viridis Walter)

R. A. Smith, P. Sharko, D. Bolin, and C. B. Hong

In an attempt to control the weeds on a farm overgrown with them, a producer in Fleming County Kentucky, pastured a large flock of sheep on the farm. leming County is situated on the Eastern Knobs, a hilly and heavily forested region underlain by calcareous rocks. The sheep began to die one by one starting in late July 1999. Altogether some 20 head died prompting a field investigation by Diagnostic Center staff on August 27. Marked posterior paresis was also observed in one animal, a condition often observed in animals suffering from this disease. Animal poisonings by plants containing cardiac glycosides often occurs in stock recently introduced from areas of the country which are not infested. The flock in question had been moved about a year previously from a state well north of Kentucky and outside the range of Asclepias. viridis. That plant was found in abundance on the farm and showed evidence of having been grazed. The symptoms reported by the owner included repeated tetanic seizures following a period of depression, weakness and staggering. The summer had been characterized by drought, a condition known to in increase the risk of exposure to other species of Asclepias. Three animals were submitted for necropsy during the course of the disease. Animal #1 showed all lung lobes red, wet and heavy with white froth in the airways. Petechial and ecchymotic hemorrhages covered the epicardial surface of the heart. The distal portion of the rectal mucosa was dark red and hemorrhagic. Mild multifocal nonsuppurative myocarditis was noted on re-examination. Cardiorespiratory failure was suspected. Animal #2 had wet and heavy lungs. Froth was present in the trachea and bronchi. The liver was tan colored. The diagnosis was mild multifocal nonsuppurative myocarditis. Cardiorespiratory failure was suspected in this animal also. Animal #3 was presented in poor post-mortem condition. The lungs appeared pale with small areas of consolidation in the anterior portion. The diagnosis was bacterial bronchopneumonia. We feel that this case history strongly implicates Asclepias viridis Walter as an additional toxic member of the genus

Livestock Disease Diagnostic Center, Veterinary Science Department, University of Kentucky, Lexington, KY

Facial Eczema in Ruminants - Sporidesmin Toxicosis

C. C. Hooper

Facial eczema is a common disease in ruminants in the North Island of New Zealand. It is the most important mycotoxicosis in production animal medicine in New Zealand. The mycotoxin sporidesmin is produced by the saprophytic fungus Pithomyces chartarum. The ingested mycotoxin is excreted in bile where it is concentrated 10-100 fold from the serum. It produces free radicals causing damage to the biliary epithelium, impairing the excretion of phylloerythrin in bile. Warm wet weather in late summer and autumn is associated with outbreaks of the disease. In other countries, P.chartarum often does not produce sporidesmin, unlike in New Zealand where 95% of the isolates of the fungus produce the mycotoxin. Clinical signs: The main signs are photosensitization and weight loss with icterus, dysuria, diarrhoea and death also occurring in some stock. Gross lesions: The carcass is in poor condition and icteric. Non-haired skin and unpigmented areas of the carcass have areas of edema and necrosis. The liver is pale tan with patchy firmness and distorted lobulation. The left lobe of the liver is the most severely effected. Severe fibrosis of the intra and extra hepatic bile ducts and gall bladder is present. Plugs of bile stained material can be seen in bile ducts. Petechial haemorrhage and fibrosis can be seen in the urinary bladder and ureters. Histopathology: The large portal tracts contain the typical lesions. The large intrahepatic bile ducts are severely damaged with necrosis and sloughing of the lining epithelium and severe fibrosis of periductular connective tissue. Veins and arteries in these large tracts have proliferation of the fibroblasts of the tunica intima causing occlusion of the lumen. A striking lesion is the abundant intimal fibrosis occurring on the vessel side nearest the damaged bile duct (causing a crescent shaped fibrous occlusion). In the smaller portal tracts are severe fibrosis, biliary hyperplasia and moderate chronic inflammation. The extrahepatic bile ducts and gall bladder have multifocal necrosis of the lining epithelium and severe fibrosis. Haemorrhage and fibrosis can be seen in the urinary bladder and ureters. Clinical Pathology: Serum gamma glutamyltransferase (GGT) levels correlate well with the hepatobiliary damage and correlate well to spore counts in pasture and feces. Serum GGT has a fairly narrow normal range (5-30 IU/l at 37 degrees Celsius in our laboratory). Proportionately large increases in serum GGT occur with even mild sporidesmin induced damage. In most cases, the serum GGT will return to normal 3-6 months after exposure to sporidesmin.

Auckland Animal Health Laboratory, Agriquality NZ Ltd, Auckland, New Zealand Lesions of Experimental Stemodia kingii Intoxication in the Mouse

M. F. Raisbeck1, J. G. Allen2, and S. W. Colegate3

Stemodia kingii is a shrub native to the Pilbara region of northern Western Australia, which has been implicated in sheep poisonings. Recent feeding trials reported by Agriculture Western Australia confirmed its toxicity in sheep and suggested that the active toxin might be a cardiac glycoside. As a suitable bioassay is a prerequisite for chemical isolation and structure elucidation of the toxin, this presentation describes validation of a murine bioassay of Stemodia activity. As in sheep, the principal organ systems affected by Stemodia intoxication were the gastrointestinal tract, the heart and the liver. Within 4-12 hours of a toxic dose, mice exhibited diarrhea and a decreased level of physical activity. Some individuals recovered uneventfully with 24 hours. Lethally poisoned mice rapidly became moribund, cyanotic and exhibited signs of circulatory shock prior to death within a few hours. Histologic examination revealed vacuolar degeneration and necrosis of the villi in the small intestine and sloughing of the mucosa in the colon. There were scattered foci of degenerating and necrotic myocytes in the myocardium. The livers of a few mice exhibited mild degenerative centrilobular changes. The lesions seen in this experiment are similar to those reported with spontaneous and experimental Stemodia intoxication in sheep.

1 Department of Veterinary Sciences, University of Wyoming, Laramie, WY 2Agriculture Western Australia, South Perth, Western Australia 3CSIRO Division of Animal Health, Australian Animal Health Laboratory, Geelong, Victoria

Nitrate Levels in Cornstalks in Kentucky in the Drought of ’99: Observations of Anomalies that may be Indicative of a Silver Lining

R. A. Smith

In August 1999, drought plagued much of Kentucky and resulted in very high nitrate levels in corn stalks. Not only did local producers fail in many cases to produce any grain-bearing cobs at all, they were unable to cut their losses and put their cattle out to graze the failed crop for fear of nitrate poisoning. Many were prevented by financial constraints from harvesting and ensiling the crop, despite assurances that this could reduce the nitrate concentration to a safe level. The producers simply sold their cattle at very depressed prices since they had nothing to feed to them. Many hundreds of analyses were done during the year. Analytical methodology involved sampling, pooling of stalks, oven drying them, grinding them and extraction of the ground samples in boiling water with subsequent analyses of the extracts by ion chromatography. The nitrate content in the stalks of several corn hybrids, all being grown at an experimental farm, was determined, resulting in a serendipitous discovery. The stalks of some hybrids contained very high nitrate levels while others grown side by side with the toxic cornstalks contained only innocuous levels of nitrate ion. The potential now exists to utilize existing hybrids and even to produce new hybrids of corn that show little propensity to accumulate nitrate ion. The use of these hybrids in soils with low moisture retention characteristics may result in crops that can still be grazed in situ despite severe drought in subsequent years. The following nitrate levels were found in corn plots growing side by side under identical conditions. Hybrid C was being grown in every other plot and hence appears several times in the list below.

Nitrate Level, Corn Hybrid Nitrate Level, Corn Hybrid ppm ppm 50 A 7,300 H 880 B 9,700 I 2,600 C 11,000 J 2,900 C 11,000 K 3,200 C 13,000 L 3,400 C 13,000 M 3,800 C 13,000 N 5,600 D 14,000 O 5,700 C 16,000 P 6,300 E 17,000 Q 6,400 F 18,000 R 7,200 G -- --

Livestock Disease Diagnostic Center, Veterinary Science Department, University of Kentucky, Lexington, KY

Experiences in Mycotoxin Testing at North Dakota State University.

H. H. Casper, B. K. Tacke, and D. M. Iverson

Trichothecene mycotoxins are numerous (>170) and North Dakota State University has provided an assay for 15 non-macrocyclic trichothecenes (DON, SCRIP, T-2 TET, NIVAL, FUS-X, 15-ASCIRP, 3- ADON, 15-ADON, DAS, NEOSOL, T-2 TRI, HT-2, T-2, AC T-2 plus zearalenol and zearalenone (ZEAR) since 1989. Mycotoxins in feeds are extracted with acetonitrile:water (84 + 16) and derivatized for analysis by GC/MS. The detection limit for the above 17 mycotoxins was ~0.5 ppm. Quality assurance is maintained by a corn pool with 5.2 + 0.3 ppm of DON, 0.8 + 0.01 ppm of 15-ADON, 1.7 + 0.1 ppm of HT-2, 1.7 + 0.1 ppm of T-2 toxin and 1.6 + 0.2 ppm ZEAR. Details on the mycotoxin procedures are available at: www.ndsu.edu/mycotoxins. Barley heads were inoculated with 3 Fusarium species, collected from field samples of barley. Intact heads infected with F. graminearum contained ~28 ppm DON; those with F. sporotrichioides contained ~2 ppm T-2, ~9 ppm HT-2 and ~4 ppm T-2 TET; and those with F. poae contained ~3 ppm NIVAL. This data demonstrates that potential does exist for mycotoxins other than DON and ZEAR. From 1990 to 1999, ~8,000 diagnostic samples were assayed for 17 mycotoxins and positive (>0.5 ppm) results were recorded. In the 8,000 diagnostic samples 3,123 (39%) were positive for DON, 398 (13%) were positive for ZEAR, 43 (0.6%) were positive for HT-2, 36 (0.4%) were positive for T-2 and 15 (0.2%) were positive for NIVAL. Co-occurrence of HT-2 and T-2 was found in 25 (0.3% of the samples. Co-occurrence of HT-2 (>0.5 ppm) and DON (>5.0 ppm) was found in 26 (0.3%) of the samples. The mycotoxin levels are shown in Table 1:

Table 1: Mycotoxin Levels (ppm)

0.5-1 1-2 2-3 3-4 4-5 5-10 >10 DON 1195 914 362 181 121 182 150 HT-2 30 2 4 2 3 1 - T-2 21 11 2 - - 1 - ZEAR 183 103 31 16 8 10 9

Unusual samples include; a) corn from Manitoba with 10 ppm DON, 2 ppm HT-2, and 1 ppm T-2; b) horse feed from Virginia with 3 ppm DON, 9 ppm T-2 TET, 2 ppm HT-2, 1 ppm T-2 and 26 ppm ZEAR; c) moldy noodles from Virginia with 2 ppm NEOSOL, 7 ppm HT-2, and 15 ppm T-2 toxin. Feeding guidelines for mycotoxins vary widely, depending on source, and can be lower than values from controlled research. In example, North Carolina State University recommends a maximum of 0.5 ppm DON for cattle. North Dakota State University, in contrast, found no toxic effects from 8 to 12 ppm DON in the diet of heifers and steers.

Veterinary Diagnostic Laboratory, North Dakota State University, Fargo, ND

A Sensitive, Automated Method for the Measurement of Total Estrogenic Activity in Feedstuffs

T. J. Evans1, G. E. Rottinghaus1, W. V. Welshons2, B. M. Judy2, and S. W. Casteel1

Dietary estrogens of fungal and plant origin have been associated with hyperestrogenism in swine (zearalenone), “clover disease” in sheep (genistein and formononetin), and “bulling” in heifers (coumestans). Other, less well-defined syndromes presenting clinically as infertility, cyclic irregularity, edema of the female genitalia and mammary glands, abortion, and other reproductive problems in animals may also be caused by the ingestion of natural or synthetic estrogenic compounds in feedstuffs. However, the investigation and diagnosis of suspected xenoestrogenic exposure in animals has been hampered by the lack of readily available, reliable, and sensitive methods for measuring total estrogenic activity in feeds. Estrogen-responsive proliferation of MCF-7 breast cancer cells, as measured by an increase in DNA, has been used as a bioassay for the detection of dietary estrogens. Robotic instrumentation for making serial dilutions has facilitated the automated screening of feed samples for estrogenic activity in cases where hyperestrogenism or endocrine disruption has been suspected. The half-maximal response for estradiol occurs at 2 pM or 0.54 ng/ml, and zearalenone, a weaker estrogenic compound, produces a half- maximal response at 200 pM or 64 ng/ml. This bioassay has been calibrated against a number of known estrogenic compounds (estradiol, zearalenone, zearalenol, zearalanol, formononetin, genistein, daidzein, biochanin A, and coumesterol), and the estrogenic activity of feed extracts is expressed as ppm equivalents of zearalenone required to induce an equal amount of stimulation of MCF-7 cellular proliferation. The detection limit of this assay has been determined to be 0.05 to 0.1 ppm equivalents of zearalenone in the feed. Competitive inhibition of this bioassay with an anti-estrogen is used to confirm an estrogen receptor-mediated mechanism for the measured estrogenic activity. This automated assay for total estrogenic activity shows promise as a reliable and sensitive method for the determination of the role of dietary estrogens in the etiology of behavioral, structural, and/or functional reproductive abnormalities in animals.

1 Veterinary Medical Diagnostic Laboratory, University of Missouri, Columbia, MO 2 Veterinary Biomedical Sciences, University of Missouri, Columbia, MO

Effects of Hemolysis on Serum Vitamin E in Horses, Cattle, Sheep, and Cats

S. B. Hooser 1,2, K. B. Jochim 1, C. Simaga 1, C. R. Wilson 1, R. J. Everson 1, R. R. Bedel 1, L. Guptill 3, and W. M. Hilton 3

Vitamin E is a fat-soluble vitamin, important as an antioxidant and in the functioning of the reproductive and skeletomuscular systems. As a free radical scavenger, it helps to quench lipid peroxidation in cell membranes, resulting in its degradation. The Toxicology Section of the Animal Disease Diagnostic Laboratory of Purdue University is frequently asked to analyze serum samples for vitamin E concentrations to ascertain the health of individual animals and the effectiveness of vitamin E addition to feed. Our laboratory previously reported studies utilizing porcine blood which demonstrated that if serum samples were noticeably pink with hemolyzed red blood cells, then the serum vitamin E concentrations were reduced or below our limits of detection. Further studies demonstrated that red blood cell hemolysis in porcine blood results in increased lipid peroxidation which correlates with decreased serum vitamin E concentrations. However, one additional study has been published which indicated that hemolysis in equine blood resulted in only a partial decrease (to approximately 70% of normal) of serum vitamin E. Therefore, because of this apparent species difference, we began a study to determine the effects of RBC hemolysis on serum vitamin E concentration in a variety of species of livestock. Whole blood was obtained from normal horses (n=6), cattle (n=7), sheep (n=6), and cats (n=5), carefully placed in red top serum tubes, allowed to clot, and centrifuged to separate serum from RBCs. No visible hemolysis was present in any of the serum samples that were used. Sera were analyzed for vitamin E the day of collection via HPLC with fluorescence detection. Additional blood from each animal was collected, frozen and subjected to three cycles of freeze-thaw prior to analysis for vitamin E. In each species tested, severe hemolysis resulted in a dramatic decrease of serum vitamin E concentrations to less than 10% of the value of the non-hemolyzed serum sample in each animal. The mean % of non-hemolyzed value and standard error for different species were: horses, 5% + 1.8; cattle, 4% + 0.9; sheep, 7% + 0.8; cats, 7% + 2.6. These results are comparable to previous reports using porcine blood and indicate that RBC hemolysis results in nearly complete degradation of serum vitamin E in horses, cattle, sheep, cats, and pigs. Based on these results, it may be assumed that RBC hemolysis results in vitamin E degradation in all species. Therefore, care must be taken to avoid RBC hemolysis when taking serum samples for vitamin E analysis in any species. Future mechanistic studies will evaluate the links between RBC hemolysis, lipid peroxidation, and vitamin E degradation.

1 Animal Disease Diagnostic Laboratory, Purdue University, West Lafayette, IN 2 Department of Veterinary Pathobiology, Purdue University, West Lafayette, IN 3 Department of Veterinary Clinical Sciences, Purdue University, West Lafayette, IN

Acyclovir Toxicoses in Dogs: 10 cases (January 1996- March 2000)

J. A. Richardson

Acyclovir is an antiviral agent that is used to treat herpes simplex, cytomegalovirus, Epstein-Barr syndrome, and varicella-Zoster. Acyclovir terminates viral DNA synthesis by inhibiting viral reverse transcriptase. Acyclovir is considered to have low toxicity. However, acyclovir can cause renal injury from accumulation of crystals in renal tissue, causing an obstructive nephropathy. A retrospective study (January 1996-March 2000) was conducted of acyclovir toxicoses in dogs that were reported to the ASPCA National Animal Poison Control Center. Data analysis included amount ingested, clinical effects, and time of onset of signs. Records from 10 dogs were retrieved. Ingested dosages ranged from 40-2195 mg/kg. The most common signs seen included vomiting, diarrhea, anorexia, and lethargy. Other signs reported include polydipsia, polyuria, tachycardia and mydriasis. In 60 percent of cases, clinical signs developed within three hours. Because of the potential development of renal failure, a large ingestion of acyclovir should be considered dangerous and should be treated aggressively. Treatment of overdoses would include standard decontamination procedures, such as emesis and activated charcoal, in combination with fluid diuresis and supportive care.

ASPCA National Animal Poison Control Center, Urbana, IL

Acute Renal Failure in a Dog Following the Ingestion of a Chinese Herbal Preparation Containing Indomethacin

R. H. Poppenga1, W. J. Birdsall1, G. M. Griffin2, and M. R. Cummings1

A five-year-old, 17.3 kg, neutered, male Cocker Spaniel was referred to the Emergency Service of the School of Veterinary Medicine at the University of Pennsylvania in acute renal failure one week following the ingestion of a number of Chinese herbal “balls” prescribed to the owner of the dog for “arthritis.” It was estimated that the dog ingested between 20 and 30 of the balls, although the exact number was unknown. The weight of individual balls varied but approximated 6 grams each. The dog was initially presented to a local veterinary clinic five days after the ingestion because of anorexia, emesis and diarrhea and referred to Penn two days later. Abnormal serum chemistry results included elevated creatinine (14.1 mg/dl), urea nitrogen (220 mg/dl), phosphorus (16.7 mg/dl) and potassium (8.1 mmol/L). Initial treatment included administration of a balanced electrolyte solution, furosemide, sucralfate, cimetidine, doxycycline and enrofloxicin. A diagnosis of acute renal failure was made based upon urinalysis, serum chemistry and kidney ultrasonographic results. Ethylene glycol and aminoglycoside antibiotic toxicoses, leptospirosis and neoplasia were systematically excluded from the differential list based upon history and laboratory results. The herbal preparation was submitted to the PADLS/Penn Toxicology Laboratory for analysis. A metal screen by ICPAES and an organic chemical screen by GC/MS detected the presence of lead at 4.48 ppm, mercury at 3.05 ppm and caffeine at 3300 ppm (all reported on an as received basis). No NSAIDs were detected by our routine GC/MS screen protocol. The dog slowly improved over the next ten days with symptomatic and supportive care. Nine days after admission to the hospital, serum creatinine, urea nitrogen, phosphorus and potassium values were all within expected ranges. A conservative estimate of caffeine ingestion was ~ 500 mg (based upon the concentration of caffeine in the preparation and ingestion of 25 balls with an average weight of 6 g). This was felt to be below a lethal dose, but above that generally associated with the occurrence of clinical symptoms. However, with the exception of emesis, no clinical signs exhibited by this dog at the time of admission to Penn could be attributed to caffeine ingestion. Estimated ingestion of lead and mercury were not believed to be clinically significant. The dog was released from the hospital ten days after admission and a follow-up examination one-week later was unremarkable. Subsequent to this case, the toxicology laboratory developed a NSAID screen using LC/MS. The original herbal balls had been retained and were re-analyzed for the presence of NSAIDs using the LC/MS screen; indomethacin was detected in the herbal preparation at 3,200 ppm. Again, assuming the ingestion of 25 balls weighing ~ 6 g each, a total dose of indomethacin was calculated to be 480 mg. There is limited information available regarding the acute toxicity of indomethacin. The oral LD50 of indomethacin, based upon 14-d mortality, is 12 mg/kg and 50 mg/kg in rats and mice, respectively. Thus, the ingested dose of ~ 28 mg/kg could reasonably be expected to be toxic. Therefore, based upon all of the information available for this case, NSAID-induced acute renal failure was likely. Adulteration of Chinese patent medicines is common and appropriate laboratory analyses for detection of adulterants is recommended following their accidental ingestion by or intentional administration to animals.

1Pennsylvania Diagnostic Laboratory System, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA 2Veterinary Hospital of the University of Pennsylvania, Philadelphia, PA A Case Study Involving both Acute and Chronic Ibuprofen Poisoning

W. E. Brewer, P. G. Parnell, and J. A. Caver

Extreme weight loss, vomiting and tarry stools were reported in a 10-year-old dog. BUN and creatinine levels were elevated. The dog was treated with I.V. fluids and Tagamet. Several days later, another dog from the same owner was moribund and unresponsive. This 5-year-old dog died within an hour of noting these symptoms. Subsequently, the 10-year-old dog died 3 weeks after the initial symptoms of vomiting. Gross examination of the liver, gastrointestinal tract and kidneys was rather unremarkable in the 5-year-old dog, but mild erosion of the gastric mucosa was noted. The liver in the 10-year-old dog was diffusely pale with a slight lobular pattern, and renal papillae were diffusely necrotic bilaterally. Additionally, a large perforating ulcer in the proximal duodenum was associated with large amounts of luminal hemorrhage. Thrombosis was present in the pulmonary arteries of the caudal lung lobes. The liver in the 5- and 10-year-old dogs contained 81 and 0.12-ppm ibuprofen, respectively. These results indicate that the younger dog died acutely from ibuprofen poisoning, and the older dog died from renal failure due to chronic ibuprofen toxicity. Apparently, a neighbor had threatened to harm these dogs with Advil. An empty bottle of Advil was found just outside the fence of the two dogs.

Clemson Veterinary Diagnostic Center, Clemson University, Columbia, SC

Direct Automated Cycle Sequencing for Diagnosis and Epidemiologic Investigations of Avian Infectious Bronchitis Viruses

S. K. Hietala1, L. M. Shih1, P. Woolcock2

Avian infectious bronchitis virus (IBV) isolated over the past several years from clinically affected chicken flocks in California include several variant strains that cannot be typed by routine monoclonal antibody-based dot blot or fluorescent antibody techniques. In addition, molecular-based techniques such as restriction fragment length polymorphism (RFLP) and serotype-specific PCR typing have provided variable results. In efforts to improve diagnostic identification and allow for epidemiologic tracking of IBV isolates, direct sequence analysis was evaluated as an approach to virus identification and typing. IBV was isolated from clinical specimens using standard egg inoculation techniques, viral RNA was recovered by single-step surfactant-based extraction, and pre-selected sequences were amplified by RT- PCR using degenerate primers complementary to conserved regions of the genome. The nucleic acid sequence of the amplified fragments was determined by automated direct cycle sequencing. An approximate 620 nucleic acid sequence, encoding the immunologically important S1 spike glycoprotein, and composed of two hypervariable regions separated and flanked by conservative regions, was selected as the target sequence. The S1 subunit of the IBV protein is involved in viral infectivity and contains epitopes recognized by neutralizing antibody, making it a diagnostically and functionally significant target. The S1 sequence patterns and predicted amino acid sequences obtained from the 60 IBV isolates evaluated were compared to IBV vaccine strains, IBV reference strains, and previously published gene sequences of IBV field variants. Phylogenetic analysis of the IBV isolates characterized resulted in 8 distinct subgroups, with S1 genetic homology ranging from 78% to 100%. The phylogenetic relationships indicate that IBV variants continue to emerge in vaccinated flocks, and that circulation of variants may be limited by geographic isolation. Direct cycle sequencing shows promise for both virus identification, and as a diagnostic tool to support management and biosecurity decisions based on molecular epidemiologic tracking of IBV variants in flocks.

1California Animal Health and Food Safety Laboratory - Davis, UC Davis, Davis CA 2California Animal Health and Food Safety Laboratory - Fresno, UC Davis, Davis CA Amyloid Arthropathy Associated with Mycoplasma synoviae in Brown Egg Laying Type Chickens

H. L. Shivaprasad1, and B. Daft2

Amyloid arthropathy was diagnosed in a flock of 20-week-old, brown-egg laying chickens. Approximately 15% of the birds in a flock of 94,000 had locomotor problems due to swollen joints. The birds had been vaccinated for Marek’s disease, infectious bursal disease, Newcastle disease, infectious bronchitis, pox, and avian encephalomyelitis. Several birds submitted for laboratory evaluation revealed mild to moderately swollen hock joints. Most of the birds had orange exudate in and around the synovium. A few birds had erosions of the articular surfaces. Histopathology revealed a proliferative synovium with the accumulation of large amount of homogenous eosinophilic material and infiltration by lymphocytes. The homogenous eosinophilic material was positive on Congo Red staining and exhibited apple green birefringence when viewed with polarized light. There was an accumulation of similar homogenous eosinophilic material to a milder degree in the interstitium of the liver, adrenal, in and around the blood vessels of the spleen and other organs. All the birds were serologically positive with high titers for Mycoplasma synoviae by plate agglutination, hemagglutination-inhibition, and ELISA tests. Serological titers for other common pathogens were within the normal range for vaccinated birds. Mycoplasma was not cultured from the joints or tracheas of these birds. No other bacteria were isolated from the joints. The flock had a Mycoplasma synoviae outbreak when the birds were 10-weeks of age. The birds had swollen joints with cloudy exudate, and Mycoplasma synoviae was isolated from the joints. It is probable that the chronic stage of the disease resulted in the failure to culture Mycoplasma synoviae at 20-weeks of age.

1California Animal Health and Food Safety Laboratory System, Fresno Branch, University of California, Davis, CA 2California Animal Health and Food Safety Laboratory System, San Bernardino Branch, University of California, Davis, CA Assessing Bias in a Molecular Epidemiologic Study of E. coli in Broiler Chickens

R. S. Singer1, T. E. Carpenter2, J. S. Jeffrey3, C. L. Cooke4, and D. C. Hirsh4

In infectious disease epidemiologic studies, researchers often rely on specific cues of the host, such as clinical signs, as surrogate indicators of pathogen presence. A selection bias would manifest if the specific visual cues used in sampling for the pathogen were not representative of the full range of signs caused by the strains of that pathogen. In addition, in many molecular epidemiologic studies, isolates are collected over extended periods of time. Inferences are then made about isolates collected during this time interval. If a clone of an organism were to be sampled at the beginning and end of this time interval, random genetic drift may result in different DNA fingerprints between the isolates. Consequently, a misclassification bias due to random genetic drift would cause the isolates to be considered different even though they were derived from the same clone. We assessed for the presence of these biases during a one-year molecular epidemiologic investigation of Escherichia coli associated with avian cellulitis in broiler chickens. This condition is characterized by a diffuse inflammatory reaction in the subcutaneous tissue that results in the complete or partial condemnation of the carcass at processing. Numerous investigators have causally linked the presence of E. coli with cellulitis. The lesions induced by these E. coli can vary considerably in their morphological appearance as well as overlying skin involvement. In our studies, carcasses were collected at the processing plant prior to evisceration so that the skin of the bird was still intact and the lesion was not contaminated. We used visual cues, such as skin discoloration, to select birds that we suspected to have cellulitis lesions, and this method may have resulted in a selection bias. Therefore, we utilized a validation protocol to assess the potential for selection bias in our molecular epidemiologic studies of E. coli and avian cellulitis. In two different trials, E. coli DNA fingerprints were compared between birds that our observers collected and the birds that the observers missed. Using Fisher's exact tests and simulation models, we determined that the isolates collected by the observers were not significantly different from the isolates missed by the observers (P > 0.60 in both trials). Our method of selecting birds suspected of having cellulitis did not significantly bias our inferences about the population of E. coli associated with cellulitis in the flock. Because the carcasses were collected over a one-year period, we also evaluated the potential influence of misclassification bias due to random genetic drift. We designed an experiment in which antibiotic resistant mutants of four broiler-derived isolates of E. coli were inoculated onto the litter of an artificial broiler environment. Forty 14-day-old broilers were living on the litter when the isolates were inoculated. Litter was sampled by drag swab weekly for five weeks, and a minimum of 24 colonies of each antibiotic-resistant isolate were DNA fingerprinted using pulsed-field gel electrophoresis at each sampling interval. We observed no genetic drift during this study. Consequently, the genetic structure of E. coli isolates in the broiler chicken environment is likely to be stable over time allowing isolates to be collected reliably over extended periods.

1Department of Veterinary Pathobiology, University of Illinois, Urbana, IL 2Department of Medicine and Epidemiology, University of California, Davis, CA 3Department of Population Health and Reproduction, University of California, Davis, CA 4Department of Pathology, Microbiology and Immunology, University of California, Davis, CA

Attaching and Effacing E. coli in Avian Species

H. L. Shivaprasad1, B. Daft2, R. Crespo1, and D. Read2

Escherichia coli can cause various syndromes in poultry including colisepticemia, coligranuloma, salpingitis, omphalitis, osteomyelitis, synovitis and cellulitis. However, the literature on E. coli as a cause of enteritis in poultry as well as in other species of birds is limited. Between 1989 and 2000, numerous cases of enteritis associated with attaching and effacing E. coli were diagnosed most often in turkeys, but also in chickens, pigeons, quail, partridges, pheasants, ducks, ostriches, a parakeet, and a bullfinch. Most of these birds were young and had a history of diarrhea and increased mortality in the flock. Grossly, the intestines, including the ceca, were distended with watery frothy contents in most birds. E. coli was cultured from the intestine of many of these birds, and eae gene was demonstrated by PCR. All of the E. coli isolated were negative for heat labile and heat stable toxins, as well as Shiga-like toxin and cytonecrotizing factors. Histopathology of the intestine revealed the attachment of Gram negative bacilli to the tips of the villi, and transmission electron microscopy revealed intimate attachment of the bacteria to the enterocytes with effacement of the microvilli and formation of cup-like pedestals. Attaching and effacing E. coli should be considered as one of the causes of diarrhea and enteritis in avian species, especially in turkey poults.

1California Animal Health and Food Safety Laboratory System, Fresno Branch, University of California, Davis, CA 2California Animal Health and Food Safety Laboratory System, San Bernardino Branch, University of California, Davis, CA

Anti-microbial Residue Contamination in Shell Eggs: A Pilot Study

G. Scortichini, G. Campana, A. Giovannini, A. Manetta, and A. Simonella

Council Directive 96/23/EC lays down the criteria for the surveillance of residues of unauthorized substances or products and veterinary drugs and contaminants in live animals and in food of animal origin. Since the Directive applies to the whole European Union, the sampling plan to be carried out in all European States may be unsuitable to evaluate the risk for consumers of single member Countries. According to the Directive's provisions on anti-microbial drugs in shell-eggs, in Italy in 1998, 434 samples were analyzed for tetracyclines, 8 samples were examined for sulfonamides, 496 samples were examined for anticoccidials, none were examined for quinolones, nitrofurans, macrolides. In order to evaluate the need for an assessment of the risk for the Italian consumer to assume anti- microbial drugs with shell-eggs, a pilot study was carried out in the two largest Italian cities (Rome and Milan). Thirty-five boxes containing 4 to 10 eggs were examined to search for oxytetracycline, tetracycline, enrofloxacin, flumequine, oxolinic acid, sulfadiazine, sulfaquinoxaline, sulfadimethoxine, sulfamerazine, sulfamethazine, sulfamethoxazole, sulfathiazole, sulfamethoxypiridazine, nitrofurazone, nihydrazone, furazolidone, furaltadone, carbadox, olaquindox, nicarbazin, tylosin, spiramycin. Four eggs per box were homogenized, extracted using a solvents mixture, purified through Solid Phase Extraction and analytes were identified by HPLC. Twenty-six out of 35 samples (74%) resulted negative to all substances, 2 samples (6%) were positive for enrofloxacin (limit of detection 1,6 ppb), 1 sample (3%) was positive for oxolinic acid (limit of detection 3,0 ppb), 8 samples (23%) were sulfadiazine positive (limit of detection 13,4 ppb). The results obtained confirmed the need for a wider scale assessment of the risk for the Italian consumer to assume anti-microbial drugs through shell-eggs consumption.

Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise, Teramo, Italy

Efficiency of Sampling for the Determination of Colonization Status of Turkey Flocks with Campylobacter spp.

F. Elvinger, S. J. Spencer, N. Sriranganathan, and F. W. Pierson

Determination of the infection / colonization status of a flock or herd with any pathogen or contaminant depends on the proportion of animals that are infected / colonized, the number of samples that are collected, and the characteristics of the diagnostic tests used. We collected 127 cloacal swabs, 121 cecal drop swabs and 24 drag swabs in 8 commercial turkey flocks of 2 to 11 weeks of age for detection of Campylobacter spp. An additional 127 cecal drop swabs were collected in 22 flocks. Swabs were stored refrigerated overnight in peptone water supplemented with Cefoperazone. Specimens were plated on modified charcoal deoxycholate agar and incubated for 36-48 hours under microaerobic conditions at 42oC. In the first 8 flocks, 110 cloacal swabs (86.6%; range on farm: 76 to 100 %), 113 cecal drop swabs (93.4%; range on farm: 83 to 100 %) and 20 drag swabs (83.3%; range on 7 farms: none to all) were positive for Campylobacter spp. Cloacal and cecal drop swabs from the one farm with 0 of 3 negative drag swabs were Campylobacter spp. positive. Campylobacter spp. was isolated from 99 of the additional 127 cecal drop swabs (77.9%). Assuming 100% sensitivity and specificity of our diagnostic test, and applying the binomial distribution function we can, based on all cecal drop swab results (212/248=85.5% samples positive), establish that collection of 3 cecal drop swabs provides a >99% probability of detecting one or more positive samples in a colonized flock, and thus diagnose a flock as either colonized or free of Campylobacter spp. A greater number of swabs are necessary to determine colonization status of younger flocks when proportions of colonized birds are lower. Reduced test sensitivity necessitates an increased number of samples to be collected such that the probability of detecting at least one positive sample in a colonized flock will be maintained at 99% or above. With reduced specificity of the diagnostic test, the number of positive samples necessary to establish the true infection status needs to exceed 1, i.e. be 2 or more, thus requiring the calculation of the probability of detecting 2 or more positive samples to avoid misclassification as positive of a negative flock. The cost of misclassification, ultimately, has to guide us in determining the level of confidence that needs to be maintained, and thus the number of samples / animals to be tested, when classifying a flock as positive or negative for colonization with a foodborne or other pathogen.

Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA Quality Evaluation of Marketed Eggs at Retail Level in Italy

V. Prencipe, V. Rizzi, A. Giovannini, and G. Migliorati

Commission Regulation (EEC) No 1274/91 lays down the criteria for inspection of marketed shell eggs. In Italy, inspections are carried out to evaluate the compliance with the legislation in force and data collected within control activities have never been used as surveillance data nor statistically analyzed. Therefore, a pilot study was carried out to evaluate the need of an assessment of marketed egg quality, from the point of view of sanitary aspects and freshness. Thirty-five samples of grade ‘A’ and grade ‘Extra’ were collected in the two largest Italian cities (Rome and Milan). Each sample was made up of one or more boxes containing 4 to 10 eggs. In order to mimic consumers’ behavior, samples were kept at refrigeration temperature up to the date of expiry and then examined. Examination included the following: external inspection (to evaluate integrity and cleanliness of shell), candling (to evaluate the depth of air cell and the presence of microscopic checks in the shell), determination of the Haugh unit value. Analysis of collected data was performed using SPSS for Windows, release 9.0.1 (χ2 test, Fisher exact probability, Spearman correlation coefficient), and MS- Excel ’97 to estimate the defects frequency in the egg lot from which the sample was drawn through the application of Bayes’ theorem. Forty-three percent of samples had at least 1/6 cracked eggs, 31% of samples had at least 1/6 dirty eggs, 69% of samples had at least 1/6 eggs with microscopic checks, 11% of samples had a mean air cell depth greater than 6 mm (the legal threshold according to European legislation), 71% of samples had a Haugh unit value (calculated as the average of 10 eggs) lesser than 60. No statistically significant correlation was detected between the air cell depth (the European indicator of freshness) and the Haugh unit value (ρ=0,104, p=0,554). Application of Bayes’ theorem showed that, even when the number of defects involves 1 egg out of 6, the probability that the lot of origin of the sample complies with the tolerance foreseen by the European legislation (7% of faults) is lesser than 0.01. The results obtained confirmed the need for a wider scale assessment of the quality of shell eggs marketed in Italy.

Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise, Teramo, Italy

Measuring Salmonella Prevalence on Swine Farms and at the Abattoir

H. S. Hurd1, J. D. McKean2, M. Rostagno2, R. Griffith2, and I. Wesley1

Measurement of Salmonella prevalence in food animals can serve multiple purposes: 1) to estimate the on-farm prevalence so that diagnosticians will have data for risk factor analysis, intervention assessment and producer feedback, 2) to predict the food safety risk of products, or 3) to evaluate status of a given animal or load of animals. The objective of this report is to discuss the Salmonella measurement tools of fecal culture; culture of abattoir collected tissues, and serum/meat juice ELISA in market swine. In a study of market pigs from one commercial producer, fecal (1g) samples collected 24 hours before slaughter revealed an average herd prevalence of 3.5% (10/290). However, after slaughter 67.4% of these same pigs were Salmonella positive on at least one abattoir collected tissue (cecal contents, ileocecal lnn, colon contents). Salmonella was isolated from only three of these pigs, when testing samples that might reflect a food safety risk, such as carcass swabs, ventral thoracic and subiliac lnn. Based on the Danish serum ELISA, 1.7% (5/293) of pigs showed evidence of historical Salmonella infection (OD%≥ 40). At an OD% ≥ 20, 6.9% (20/291) were positive. These prevalence estimates were not significantly different (p< 0.05) from the 3.5% estimated by fecal culture. Of the 10 pigs that were culture positive by on-farm fecal, one was positive at the OD%≥ 40 cutoff; 2 at the OD%≥ 20 cutoff. Using six herds depopulated during the Accelerated Pseudorabies Eradication Program (APEP), we tested and necropsied almost 600 market pigs. Three days before depopulation, 100 finish pigs on each farm were snared, ear tagged, and 1g feces was collected for culture. On the day of depopulation, one half of the group was sent to slaughter at a commercial abattoir. After 2-3 hours holding, these pigs were stunned, exsanguinated, and necropsied. The remaining 50 pigs were necropsied on the farm, the next day. The same tissues were collected at the abattoir and on the farm (ileocecal lnn, cecal contents, fecal loops, superficial inguinal lnn). The following results are based on presumptive biochemical tests for Salmonella and serogrouping. Culture methods included preenrichment in GN Hajna and Tetrathionate, enrichment in Rappaport- Vassiliadis followed by streaking onto brilliant-green sulfa and XLT-7 plates. Suspect colonies were inoculated into triple sugar iron and lysine iron agar. The estimated average prevalence for all six herds was 3.2% (9/281) using the 1g antemortem fecal. The estimates from on-farm collected ileocecal lnn and cecal contents were 3.9% and 2.8%, respectively. The average herd prevalence estimate was 8.5%, when combining cecal contents, ileocecal lnn, and postmortem fecal samples. This increase in prevalence is to be expected when an insensitive test is repeated on the same population. When comparing the fecal samples collected at necropsy with cecal contents and ileocecal lnn, there was no agreement on positive samples; kappa statistics were less than one. For pigs necropsied at the abattoir, the estimated average herd prevalence was 40.8% (117/287). These studies suggest that antemortem fecal culture severely underestimates the true Salmonella prevalence. They also demonstrate the confounding effects that transport and holding have on Salmonella prevalence estimation. Fecal culture and serum ELISA do not appear efficient for evaluating the status of a given pig or load of pigs.

1National Animal Disease Center USDA, Ames, IA 2Iowa State University, Ames, IA

Antimicrobial Susceptibility Patterns of Salmonella spp. Isolated from Animal and Animal Environments in Ohio, 1998-1999

B. C. Love1, B. A. Byrum2, A. C. B. Berge2, E. F. Dunne3, N. Doelling3, and F. J. Angulo3

The Animal Disease Diagnostic Laboratory of the Ohio Department of Agriculture isolates Salmonella spp. from around the state of Ohio from clinically affected animals, as well as surveillance samples from healthy animals and animal environments. Antimicrobial resistance patterns (antibiograms) are generated on a subset of these isolates, using an automated microdilution technique (Sensititre, Trek, Westlake, OH). A study of these antibiograms has been performed in order to provide information regarding the different Salmonella species and their antibiotic resistance patterns in the state. This data does not provide information regarding prevalence of different serotypes or resistance patterns in the state, but it may be of value to monitor yearly trends and to provide information to be used in animal and public health settings. In 1998, there were 1,066 isolations that resulted in 187 antibiograms. Commercially available MIC panels are routinely used in the laboratory. Antimicrobial concentration (in µg/ml) at which Salmonella spp. are considered resistant are included parenthetically after each drug. The antibiotic panel in 1998 consisted of amikacin (32), apramycin (16), ampicillin (16), cephalothin (16), tilmicosin (16), clindamycin (5), oxacillin-2% NaCl (2), penicillin (0.03), spectinomycin (16), novobiocin (4), sulfachloropyridazine (256), tetracycline (8), tiamulin (8), trimethoprim/sulfamethoxazole (2), tylosin (10), ceftiofur (4), neomycin (8), and erythromycin (4). In addition, enrofloxacin (1) and sarafloxacin (0.12) results are available for some poultry isolates. Antibiograms from all Salmonella isolates were evaluated by serotype, by species of animal from which the isolate was recovered, and by sample type (surveillance vs. clinical). Serotypes for which antibiograms were generated frequently enough to yield significant information were analyzed independently. These serotypes included typhimurium, kentucky, heidelberg, and cerro. All serotypes show 100% resistance to tilmicosin and tylosin. Thirty-five of 88 (40%) S. typhimurium isolates were resistant to ampicillin, sulfonamides, and tetracycline. Two of these isolates were additionally resistant to cephalothin. Of 15 S. heidelberg strains, 100% were resistant to clindamycin, erythromycin, florfenicol, novobiocin, oxacillin, penicillin, spectinomycin, tiamulin, and tylosin. Between 13-38% of S. heidelberg isolates were resistant to ampicillin, ceftiofur, cephalothin, gentamicin, neomycin, sulfonamides, tetracycline, and trimethoprim/sulfa. S. cerro isolates exhibited a lower level of antibiotic resistance than did the above two serotypes. In 1999, there were 1,003 isolations that resulted in 257 antibiograms. In 1999 the panel changed slightly by replacing tetracycline with chlortetracycline (8) and oxytetracycline (8), removing amikacin, cephalothin, oxacillin-2% NaCl, and novobiocin and adding florfenicol (1), sulphadimethoxine (40), sulphathiazole (100), and gentamicin (8). These data are currently being analyzed. Analysis of antibiogram information that is routinely generated at veterinary diagnostic laboratories can be extremely valuable, both for practitioners who are treating clinically ill animals, and for monitoring/surveillance of trends of resistance development over time. As organisms become more resistant, it is important for clinicians to use antimicrobials which will be effective against the true pathogen. Monitoring of resistance trends in veterinary pathogens is becoming more important also, as this has become a matter of public health significance. Veterinary diagnostic laboratories harbor a wealth of information that can be analyzed and utilized for both of these purposes.

1Animal Disease Diagnostic Laboratory, Ohio Department of Agriculture, Reynoldsburg, OH 2University of California-Davis, Veterinary Medical Teaching and Research Center, Tulare, CA 3Centers for Disease Control and Prevention, Foodborne and Diarrheal Disease Branch, Atlanta, GA Cost-effectiveness of Serological and Milk Tests for Bovine Brucellosis: Comparison Through a Monte Carlo Simulation Model

A. Giovannini, A. Conte, and D. Nannini

The qualification for Brucellosis-free status of cattle herds, according to EC Council Directive 97/12/EC, can be based either on two serological tests (Rose-Bengal Test and Complement fixation test) of the whole herd at an interval of more than three months and less than 12 months (regimen a) or on three milk ring tests at three-monthly intervals followed by a serological test at least six weeks later (regimen b). A herd will retain official brucellosis-free status if it is negative either to two serological tests carried out at an interval of at least three months and not more than six months (regimen a) or to three milk ring tests (regimen c) or milk-ELISA tests (regimen d) carried out at intervals of at least three months. In the case of positive results in testing regimens (b), (c) and (d), individual serological testing is required to detect the infected animals. The aim of this study as to compare the effectiveness of testing regimens foreseen by Council Directive 97/12/EC and of some alternative testing strategies, including an evaluation of costs. The effectiveness of testing regimens was evaluated through a Monte Carlo simulation model, running 10,000 iterations. Variables used in the model were (1) sensitivity and specificity of tests employed, both on individual basis and on bulk milk, derived from the results of previous researches, (2) number of herds and herd size from census data on cattle herds of a Southern Italian region, (3) prevalence of infection for categories of herd size, obtained from routine serological testing of cattle herds in the same region, (4) fertility data and duration of milking, obtained from breeders’ association data of a Southern Italian province, (5) veterinarians’ fees for field activities, stated by Italian legislation and (6) cost of laboratory testing. Estimated sensitivities to detect an infected herd were 97% for regimen (a), 95% for regimen (b), 66% for regimen (c), and 98% for regimen (d). Regimens (b), (c) and (d) were affected by the proportion of milking cows in the herd, but their relative effectiveness remained unchanged. Costs estimated for the testing of a population of 42,314 animals in 2,347 herds of a Southern Italian region (including serological confirmation costs and extra costs for serological testing in case of false positive results for bulk milk tests) were: 73,019 Euro for regimen (a), 94,245 Euro for regimen (b), 57,227 Euro for regimen (c) and 36,248 Euro for regimen (d). Results demonstrate both the considerable variation in the ability of the regimes to detect an infected herd, ranging in Se as 98% to as low as 66%, and the wide difference in costs of regimes, ranging from 36,248 Euro to 94,245 Euro. These results suggest that when developing legislation that affects continental areas, efforts should aim at establishing minimal levels of effectiveness of the testing procedures (i.e. the probability to detect an infected herd) and at considering the costs associated with proposed procedures.

Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise, via Campo Boario, 64100 Teramo, Italy

Screening and Mass Spectral Confirmation of Beta-lactam Antibiotic Residues in Milk Using LC/MS/MS

D. M. Holstege1, G. Whitehead1, B. Puschner1, and F. D. Galey2

Milk is typically screened for beta-lactam antibiotics by non-specific methods. While these methods are rapid and sensitive, they are not quantitative, and can yield false positive findings. A sensitive and specific method for the quantitation and mass spectral confirmation of five $-lactam and two cephalosporin antibiotics commonly used in the dairy industry is described using HPLC with tandem mass spectrometry. The antibiotics studied were ampicillin, amoxicillin, penicillin G, penicillin V, cloxacillin, cephapirin and ceftiofur. The antibiotics were extracted from milk using precipitation with acetonitrile, followed by reverse phase solid phase column cleanup. The extract was analyzed by liquid chromatography coupled with a mass spectrometer, using a water-methanol gradient containing 1% acetic acid on a C-18 reverse phase column. The antibiotics were detected by positive ion electrospray ion trap MS/MS, with quantitation based on the most abundant fragment ions of each compound. The method was validated at the tolerance level for these compounds in milk. The method had a limit of quantitation of 5 ng/mL for each compound using a nominal sample size of 5 mL.

1California Animal Health and Food Safety Laboratory System, Toxicology Laboratory University of California, Davis, CA 2Wyoming State Veterinary Laboratory, Department of Veterinary Sciences, University of Wyoming, Laramie, WY

Delayed-onset Papular Dermatitis at Culicoides Feeding Sites as Confounding Factor in Experimental Studies of Arthropod-borne Disease

D. O'Toole1, A. A. Perez de Leon2, L. Mei2, and L. McHolland2

The USDA’s Arthropod-borne Animal Diseases Laboratory uses known or suspected insect vectors to transmit various viral diseases and to this end maintains closed colonies of the biting midge Culicoides variipennis. In the course of experiments using midges inoculated intrathoracically with vesicular stomatitis virus (VSV), we recognized that a delayed onset papular dermatitis occurred following feeding, regardless of insects’ inoculation status. This communication describes the clinical and morphological features of the dermatitis. Its clinical course and histological features were distinct from recognized cutaneous syndromes that are associated with Culicoides feeding, such as acute ventral midline dermatitis and hypersensitivity dermatitis (sweet itch or Queensland itch in horses). Two studies were performed using guinea pigs never exposed previously to biting insects. Midges in feeding chambers fed for 30 minutes on freshly shaved abdominal skin of anesthetized adult male guinea pigs. In the first study, 19 adult male guinea pigs were used. Animals (n = 9) were injected intradermally with VSV (New Jersey strain), exposed to VSV-inoculated C. variipennis (n = 6), injected with cell- culture medium (n = 3), or exposed to uninoculated C. variipennis (n = 1). Guinea pigs were euthanized and examined post-mortem 9 days post-exposure (DPE) to C. variipennis. In a second study, guinea pigs (n = 6) were exposed to 200 – 300 uninoculated C. variipennis. Animals were examined post-mortem in pairs at 6, 9 and 13 DPI. Unexposed guinea pigs (n = 2) served as negative controls. Following exposure to midges, petechial hemorrhages developed at C. variipennis feeding sites and disappeared within 9 – 24 hours. Most animals then developed 3 – 40 papular erythematous < 1 mm non- pruritic lesions at feeding sites between 7 – 10 DPE. None of the guinea pigs became ill, and lesions were absent in other parts of the integument or in internal organs. The earliest histological change, which developed at 4 DPE, was mild multifocal hyperplastic eosinophilic dermatitis with degeneration of basilar keratinocytes. This progressed to mild or moderate papular dermatitis, eosinophilic micropustules, and sparse intra-epithelial and intradermal multinucleated cells at 9 and 13 DPE. Vesiculobullous change was absent. Ultrastructurally, the principal change was intense inflammation and degeneration of keratinocytes in germinal and spinous layers. Attempts were uniformly negative to demonstrate infectious agents by virus isolation, transmission and negative-stain electron microscopy, and immunocytochemistry. Insects examined immediately after feeding had intact mouthparts and no mouthparts were found in cutaneous lesions. This excludes a foreign body reaction as the basis for dermatitis. We are uncertain of the basis for delayed onset papular dermatitis in guinea pigs exposed to C. variipennis. Similar gross and histological lesions develop in cattle exposed to uninoculated midges (Perez and O’Toole, unpublished). Likely possibilities are that they are a reaction to chemical components in insect saliva, or are due to an unidentified infectious agent endemic to the USDA’s C. variipennis colony. The development of this lesion complicates studies in which midges are used to transmit infectious agents, particularly dermatotropic viruses.

1 Wyoming State Veterinary Laboratory, University of Wyoming, Laramie, WY 2 ABADRL-USDA-ARS, Laramie, WY Radiculomeningomyelitis Due to Halicephalobus gingivalis in a Horse

J. Johnson1, C. Hibler2, K. Tillotson3, and G. Mason1

A 14-year-old gelded Paint horse was presented to the Colorado State University Veterinary Teaching Hospital with a complaint of progressive hind-limb paresis, incontinence and ataxia. A clinical diagnosis of cauda equina neuritis was made and the horse was humanely destroyed. Significant gross post-mortem and histopathologic findings were limited to the sacral spinal cord and cauda equina. The meninges of the caudal sacral spinal cord and connective tissues of the spinal rootlets of the cauda equina were thickened. Microscopic examination revealed sclerosing pyogranulomatous inflammation with necrosis involving the rootlets, epineurium, sacral spinal cord and meninges. The lesion contained numerous approximately 15 µm diameter by 350 µm long parasites with a reflexed ovary and rhabditiform esophagus with a corpus, isthmus and valved bulb. Morphologic features lead to identification of the parasite as Halicephalobus gingivalis. H. gingivalis is a sporadic cause of granulomatous gingivitis, nephritis, and meningoencephalitis in the equine, however no reports were found that describes disease limited to the caudal spinal cord and rootlets.

1Department of Pathology and Veterinary Diagnostic Laboratory, Colorado State University, Fort Collins, CO 2Department of Pathology (Ret.), Colorado State University, Fort Collins, CO 3Department of Clinical Sciences, Colorado State University, Fort Collins, CO Equine Herpesvirus-1 and -4 Differentiation by a PCR Using Formalin-fixed, Paraffin-embedded Tissues

W. Feria, H. Acland, and D. Tewari

Equine herpesvirus (EHV) -1 and -4 are viral agents of horses that cause economically important disease. EHV-1 infection results mainly in abortions, respiratory infections and paresis. EHV-4, on the other hand, causes mainly respiratory infections but has also been associated with ataxia (Verheven et al. 1998) and abortions. Differentiation of EHV-1 from 4 in diagnostic specimens is important for evaluating the effectiveness of the vaccine strategies because both EHV-1 and 4 may result in similar disease conditions. We describe here a technique for diagnosing EHV-1 infection in formalin-fixed, paraffin-embedded tissues. DNA obtained from paraffin embedded tissues of 16 equine abortion cases was subjected to a glycoprotein B gene based multiplex PCR for diagnosing EHV infection. The test detected presence of viral DNA in tissue sections and also allowed differentiation between EHV-1 and 4.

Pennsylvania Veterinary Laboratory, 2305 N Cameron Street, Harrisburg, PA Possible Mechanisms for Facial Clefts in Wild Northern Leopard Frog Tadpoles

C. U. Meteyer1, D. E. Green1, and K. A. Converse1

Early in our investigation of malformations in the northern leopard frog it became clear that the malformations were the result of teratogenic events occurring during early development. With this insight, we began an investigation in 1998 examining tadpoles at sensitive stages of development. As part of this two-year investigation, tadpoles just beginning metamorphosis (Gosner stages 41,42) were collected and examined. Of the 28 tadpoles collected from one Wisconsin site over ten days, 15 had facial clefts involving degeneration and loss of the suprarostral cartilage. Approximately 2,200 additional tadpoles have been examined from 15 sites in 4 states and this malformation has not been seen again. Microscopic examinations of the affected tissues showed degenerative changes consistent with apoptosis. Results of application of a commercial kit for Tunnel (transferase mediated dUTP nick end labeling) procedure, which amplifies and labels specific breakpoints in DNA, were also consistent with Apoptosis. Apoptosis is genetically programmed cell death critical to normal remodeling of tissues during development. Facial clefts have been documented in Xenopus laevis tadpoles and Rana temporaria experimentally exposed to DDT as well as Xenopus laevis tadpoles exposed to corticosteroids. Experimental studies in mammals have also produced facial clefts (including cleft lip and cleft palate) by exposing the developing fetus to vitamin A and jervine, a steroid alkaloid from the toxic plant Veratrum californicum. These chemicals probably mediate facial cleft through apoptosis. Toxic levels of ethanol; methyl mercury; and hyperthermia also cause facial clefts but these factors more likely mediate their effect through necrosis not apoptosis. This is the first reported case of facial clefts occurring spontaneously in free-living tadpoles.

1USGS-BRD National Wildlife Health Center, 6006 Schroeder Rd, Madison, WI Novel and Emergent Viral Infections in Wild and Cultured Sturgeon in North America

S. E. LaPatra1*, B. L. Parker2, J. M. Groff3, and R. J. Munn4

Sturgeons are susceptible to a variety of viral, bacterial, and fungal diseases. The viruses that have currently been reported include the white sturgeon iridovirus (WSIV), white sturgeon herpesvirus-1 (WSHV-1), white sturgeon herpesvirus-2 (WSHV-2), and the white sturgeon adenovirus (WSAV). Previously, these viruses have only been identified in cultured populations. However, these cultured fish were all spawned from wild white sturgeon brood stock. A survey of lower Columbia River juvenile (<1-year old) wild white sturgeon Acipenser transmontanus was conducted in the fall of 1994, 1996, and 1997. Fish were collected by standard methods and either sampled and released or held in captivity using virus-free water supplies. Captive sturgeon with morbidity and mortality were sampled for virus isolation and histological examination. In 1994, a virus identical to WSHV-2 was isolated and two novel viruses were detected in 1996. Additionally, a virus similar to the enteric adenovirus previously described in white sturgeon from California was isolated in cell culture. A WSIV infection in wild white sturgeon from the Columbia River was also diagnosed in 1997. These observations provide evidence that viral pathogens are normally present in wild stocks and that new viruses exist and need to be considered in the development of management strategies for free-ranging and cultured populations of sturgeon. Additionally, the potential broad distribution of these emerging diseases is supported by the recent report of an iridovirus detected in Russian sturgeon A. guldenstadi in northern Europe (Adkison et al. 1998); as well as the presumptive viral etiology of acute mortalities that have occurred in pallid Scaphirhynchus albus, shovelnose S. platorynchus, and shortnose A. brevirostrum sturgeon in North America.

1Clear Springs Foods, Inc., Research Division, P.O. Box 712, Buhl, ID 2Columbia River Inter-Tribal Fish Commission, 729 N.E Oregon, Suite 200, Portland, OR 3Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA 4Department of Pathology, School of Medicine, University of California, Davis, CA

Is Feline Endomyocarditis Associated with Bartonella Infection?

E. W. Howerth1, G. Jacobs2, L. Bauer2, D. Perzak2, and T. Van Winkle3

Endomyocarditis is an important cause of death in cats, but the cause of this disease is unknown. The domestic cat is the principle reservoir of Bartonella henselae. This bacterium is transmitted by fleas and can cause persistent bacteremia in cats. Clinical disease due to this organism is uncommon. However, chronic experimental infection of cats has been associated with cardiac inflammation similar to that seen in endomyocarditis. Our hypothesis is that chronic Bartonella infection is the cause of endomyocarditis. The objective of this study is to determine the prevalence of Bartonella in the heart of cats with endomyocarditis as compared with other forms of cardiomyopathy and in cats without heart disease. We examined paraffin embedded cardiac tissue from these cats for the presence of Bartonella using silver stains, immunohistochemistry, and PCR. Hearts of 9 of 25 cats with endomyocarditis had organisms suggestive of Bartonella in tissue sections stained by either a silver stain or by immunohistochemistry. Twelve of 12 hearts from cats with endomyocarditis were positive for the Bartonella citrate synthetase gene by PCR and restriction fragment length polymorphism suggests that the amplified DNA is from B. henselae. Three of the PCR positive hearts had organisms compatible with Bartonella as visualized by silver staining or immunohistochemistry. Although preliminary, these results suggest the Bartonella is associated with endomyocarditis in cats. Further work will be needed to definitively state that Bartonella is the cause of the disease.

1Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, GA 2Department of Small Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 3Laboratory of Pathology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA Histologic and Genotypic Characterization of Mycobacteriosis in Two Cats

G. D. Appleyard, and E. G. Clark

Two cases of feline atypical mycobacteriosis, one from 1994 and one from 1999, were identified as being similar on the basis of two unusual histological features: there were massive numbers of macrophages in all affected tissues, including skin, containing large numbers of filamentous organisms and these organisms were visible on Hematoxylin and Eosin stained slides. PCR amplification, using two sets of Mycobacterium-specific primers targeting different genes, demonstrated the presence of Mycobacterium nucleic acid in the samples of skin lesions from both cases. This was consistent with the presence of gram negative, acid-fast bacteria observed histologically. Bacterial culture, special stains and immunohistochemistry showed no evidence of mixed infection with other microorganisms such as Nocardia. PCR assisted cloning and DNA sequence analysis of a 600bp section of the Mycobacterium 16S RNA gene from both cases generated DNA sequences which were 99% identical suggesting that the two isolates were of the same species. Alignment with comparable 16S RNA gene sequences from 65 other Mycobacterium species highlighted a 93-95% similarity to M. bohemicum, M. bovis, M. leprae and M. ulcerans as the closest matches. These significant molecular genetic and histological differences between the two new isolates and other known species of Mycobacterium suggested to us that the organism may not have been previously described.

University of Saskatchewan, Dept. Veterinary Pathology, Saskatoon, Saskatchewan, Canada

Saluki Heart Hemangiosarcomas

T. G. Bell1, M. D. Sist2, and D. W. Jarman3

An investigation of Saluki heart pathologic changes found a high site incidence (32.5%) of cardiac hemangiosarcoma (CHSA). This study was undertaken because of the frequent clinical diagnoses of “heart disease” and some sudden deaths among Saluki dogs. Samples (38) were received from 1991 to 1999 from Saluki owners aware of the concerns for cardiovascular health in the breed and this allowed us to accumulate a “Select Saluki Group” (SSG) of hearts. Veterinarians have judged Saluki hearts to be enlarged on sonograms but few histologic lesions in this study were consistent with hypertrophic or dilatative cardiomyopathy. In the last ten years, the Animal Health Diagnostic Laboratory received specimens from 73 Salukis, including the SSG hearts. During that same period 77,883 biopsy/necropsy samples were examined from dogs of all breeds resulting in over 99,000 individual diagnoses. Fatal hemangiosarcomas occurred in 659 of those canine cases (0.86%), 108 of 17,556 feline cases (0.6%), and 13 of 7,400 equine cases (0.18%). We also looked for trends in hemangiosarcoma (HSA) occurrence when compared to lymphomas. Dog lymphosarcomas have remained fairly constant at about 1.5% of diagnoses, while canine HSA have increased from less than 1% to approximately 1.5%. The location of canine HSA was found to be in the skin of 32.8% of all HSA cases, in the spleen of 28.8% and in the heart of only 7.1%. The tumors were diagnosed with a 41% : 56% female to male ratio. The breeds of highest incidence were the Saluki (32/73), Golden Retriever (119/5196), German Shepherd (37/2796), Labrador Retriever (47/5159), and Boxer (16/2033), respectively. The average age of all HSA cases was 9 years, while it was roughly 11 years for the Saluki. The extraordinarily high incidence of CHSA in the SSG hearts is consistent with a 1999 review of cardiac tumors at Iowa State / Purdue from 1992-1995. In that study, the Saluki breed was also found to have the highest incidence of CHSA of any breed (0.75%). For all breeds, the frequency of CHSA at Michigan State University (0.073%) is not substantially different from the frequency found in that study (0.087%). The Saluki hearts from SSG were evaluated utilizing standardized pathology procedures, including gross, macro- and microscopic examinations; of the 32.5% of the SSG hearts with CHSA of the right atrium, the majority of the CHSA were primary; however, in two cases, noncardiac hemangiosarcomas appeared to be primary in the spleen. A heart tumor of an unrelated type, a carotid body tumor, was also diagnosed in a Saluki. The vast majority of SSG hearts had an array other lesions because the average age at death was 9.7 years. Most of the lesions were acquired, most frequently myxomatous degeneration of the endocardial surface of the mitral valve. This verrucous valvular disease occurred in 42% of the SSG hearts with severe changes occurring in 12%. Among other heart lesions were congenital insufficiency of the tricuspid valve (5%), and congenital insufficiency of the mitral value (2%). There were vascular changes including mineralization of the aorta (5%), arteriosclerosis, and a single case of atherosclerosis. Other lesions included myocardial degeneration, pericarditis and conduction system abnormalities. By actively soliciting for submissions of hearts from Saluki owners, it is apparent that we have uncovered a CHSA problem in the Saluki dog, the cause of which must be explored to insure the future health of the breed.

1Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, Lansing, MI 2Animal Care Clinic, Williamston, MI 3DVM Class of 2000 Mammary Duct Ectasia in 51 Dogs

M. A. Miller1, S. J. Kottler1, L. A. Cohn2, G. C. Johnson1, J. M. Kreeger1, L. W. Pace1, J. A. Ramos-Vara1, J. R. Turk1, and S. E. Turnquist1

Canine mammary duct ectasia is reportedly inducible with progestagens, curable by ovariectomy, and common, yet veterinary practitioners seldom include it in the differential diagnosis for mammary enlargement. From 1992 through 1999, it was the major lesion in 51 (2.8%) of 1,825 canine mammary biopsy submissions. Another 18 cases with a diagnosis of duct ectasia were excluded because of concurrent neoplasia (which was not always apparent in each histologic section). Duct ectasia comprised 48% of nonneoplastic canine mammary diseases. The mean age at diagnosis was 6.1 + 3.1 years; cases were evenly distributed over a range of 1-13 years of age. This distinguished duct ectasia from mammary neoplasia cases in which dogs averaged 9.2 + 2.8 years and prevalence increased with age. Great Danes, Chihuahuas, English Springer Spaniels, and Miniature Pinschers were over-represented. Chihuahuas and English Springer Spaniels were also over- represented among dogs with mammary neoplasia. All dogs with duct ectasia were female; 20 had been spayed before diagnosis. The date of ovariohysterectomy was known for 13 of these: 5 were spayed during the last 6 months before diagnosis; 8 had been spayed >1 year. The mean age at ovariohysterectomy was 5.1 + 3.1 years (range 0.5 – 10 years); 11/13 were spayed after 4 years of age. Of 26 dogs with reported reproductive history, 16 were nulliparous; 10 had whelped at least once. The dates of the most recent whelping and weaning were known for 8 dogs: 3 had last whelped >1 year before diagnosis; 2 had whelped 6-8 months before diagnosis; 3 had whelped 2-3 months and weaned pups 1-6 weeks before diagnosis. No dog had been treated with progestagens or other sex hormones. Referring veterinarians usually reported nodules or masses (26 cases), but recognized the cystic nature of duct ectasia in 13 cases. In 36 of 47 dogs, only one gland was reportedly affected. Caudal glands (31 dogs) were affected more frequently than cranial (14) or middle glands (10). Upon dissection, if grossly apparent, ectatic ducts were cylindrical or saccular and filled with yellow or blue inspissated secretion. Histologically, major ducts were dilated from 500 µm to 2.5 cm in diameter and usually lined by 1-2 layers of cuboidal epithelial cells, but 34 dogs had focal epithelial stratification (>2 cell layers); 37 dogs had areas of papillary proliferation; and 11 had solid proliferation. Duct epithelial erosion was associated with localized inflammation, cholesterol granulomas, or squamous epithelial metaplasia. Mammary acini were considered atrophied in 24 dogs, hyperplastic in 23, and secretory in 4. Thirty-six dogs had mastitis. All dogs were treated by surgical excision; 11 were also spayed at the time of mastectomy (these were counted as sexually intact for this study). Outcome was reported for 38 cases. Three dogs subsequently developed similar mammary lesions that were excised but not examined histologically; another developed mammary carcinoma. No recurrence was reported during a 3-month to 7-year follow- up period in the remaining 34 dogs. Duct ectasia apparently may develop independently of ovarian or exogenous progestagens and should be considered as a cause of mammary enlargement in spayed or intact bitches of any age, especially when its cystic nature is apparent. Concurrent neoplasia should be ruled out by careful histologic examination.

1Veterinary Medical Diagnostic Laboratory, University of Missouri, Columbia, MO 2Veterinary Teaching Hospital, University of Missouri, Columbia, MO Immunohistochemical Detection of Melanocytic Differentiation Antigen Melan A in Canine Steroid-Producing Tissues and Their Tumors

J. A. Ramos-Vara,1 M. E. Beissenherz,1 M. A. Miller,1 G. C. Johnson,1 J. M. Kreeger,1 L. W. Pace,1 J. R. Turk,1 S. E.Turnquist,1 G. L. Watson,2 and B. Yamini2

Melan A is a melanocytic differentiation antigen used in human and canine immunohistochemical diagnosis of melanomas. Antibodies to Melan A also stain steroid producing cells in human ovary, adrenal gland and testicle. Here, we report immunoreactivity of Melan A in canine steroid-producing tissues (testicle, ovary and adrenal gland) and their tumors. A total of 197 normal, hyperplastic or neoplastic tissues including testicular tumors (Leydig cell tumor, Sertoli cell tumor, and seminoma), ovarian tumors (granulosa-theca, luteoma, dysgerminoma, carcinoma), and adrenal lesions (adrenal cortical hyperplasia, adenoma, adenocarcinoma and pheochromocytoma) were stained with a monoclonal antibody to Melan A. Results are presented in the following Table.

ORGAN TUMOR POSITIVEa NEGATIVEb TOTAL Testicle Normal Leydig cells 42 (100%) 0 (0 %) 42 Leydig tumor 23 (100%) 0 (0 %) 27 Sertoli cell tumor 12 (80%) 3 (20 %) 15 Seminoma 0 (0 %) 26 (100 %) 26 Ovary Normal ovarian follicles 1 (50 %) 1 (50 %) 2 Granulosa-Theca 11 (58 %) 8 (42 %) 19 Dysgerminoma 0 (0 %) 1 (100 %) 1 Carcinoma 0 (0 %) 1 (100 %) 1 Luteoma 1 (100 %) 0 (0 %) 1 Adrenal gland Normal cortical cells 3 (100 %) 0 (0 %) 3 Cortical hyperplasia 2 (100 %) 0 (0 %) 2 Cortical adenoma 13 (87 %) 2 (13 %) 15 Cortical carcinoma 22 (67 %) 11 (33 %) 33 Pheochromocytoma 0 (0 %) 10 (100 %) 10 aPercentage of positive cases in parentheses; bPercentage of negative cases in parentheses.

Leydig cell tumors were 100% positive for Melan A. Seminomas were consistently negative. In the adrenal tissues, no pheochromocytomas were positive, whereas 100 %, 87 %, and 67 % of cortical hyperplasia, cortical adenomas and cortical carcinomas were positive, respectively. Ovarian granulosa- theca tumors yielded 58 % positive cases but usually the reaction was focal or weak. The staining was usually diffuse cytoplasmic; however, Leydig cells (normal and neoplastic) and occasionally Sertoli cell tumors had also nuclear staining. In humans, the majority of Leydig cell and adrenal cortical neoplasms are positive for Melan A whereas granulosa-theca tumors are also usually positive but the staining is focal, similar to that seen in our canine samples. It is concluded that Melan A can help in the definitive diagnosis of canine testicular and ovarian steroid-producing tumors and may help to distinguish between adrenal cortical and medullary neoplasms.

1Veterinary Medical Diagnostic Laboratory, University of Missouri, PO Box 6023, Columbia, MO 2Animal Health Diagnostic Laboratory, Michigan State University, East Lansing, MI Evaluation of Diagnostic Procedures for Canine Distemper

R. La Rock1, S. Mercado1, R. Ely2, and J. Thilsted1

There are multiple methods used to diagnose canine distemper in dogs. Traditionally, histopathology has been done to determine whether typical histologic lesions are present in lung and brain, and whether typical intracytoplasmic and/or intranuclear inclusion bodies are present in lung, brain, stomach, urinary bladder, etc. Diagnosis is made by finding typical lesions and characteristic inclusion bodies. More recently, immunologic and molecular methods, which demonstrate viral antigen or viral genes in tissues, have been used as an aid to diagnosis. Serology for canine distemper virus antibodies is used to diagnose distemper antemortem, although the prevalence of the virus in dogs and the prevalence of vaccination complicate interpretation of titers. We have observed a lack of correlation of various test results for distemper in some individual cases, which may be due to false positives, lack of sensitivity of some tests, and/or a high prevalence of subclinical canine distemper virus infection in dogs. Retrospective and prospective studies were undertaken to evaluate several immunologic methods of diagnosing canine distemper in dogs at postmortem. Results of the immunologic tests were compared to clinical findings and to pathology findings. The immunologic tests evaluated included 1) direct fluorescent antibody staining (DFA) for viral antigen on brain and conjunctival impression smears, 2) Immunoperoxidase staining (IHC) for viral antigen on sections of formalin fixed brain, lung, and skin, and 3) indirect fluorescent antibody staining (IFA) for canine distemper virus antibodies on CSF. The animals studied were dog cadavers submitted for necropsy to the NMDA-Veterinary Diagnostic Services. Sixteen dogs were included in the retrospective study. Four of the 16 dogs had histologic lesions compatible with canine distemper in brain and/or lung and had typical inclusion bodies in one or more tissues. In all 4 of these dogs, both the DFA on brain impression smears, and the IHC on brain sections were positive. Six dogs had no brain/lung lesions typical of canine distemper and no inclusion bodies in tissues examined histologically. In five of these 6 dogs, the DFA on brain impression smears was negative, and in the 6th the DFA was positive. The immunohistochemistry (IHC) on brain sections was negative on all six. The prospective study is in progress. At this point, lack of correlation has been noted between pathology findings and immunologic findings in 2 dogs. One of these had typical brain/lung lesions and inclusion bodies, but negative DFA on brain and conjunctival smears and negative IFA on CSF. The other had no brain/lung lesions and no inclusion bodies, but positive DFA on brain and conjunctival smears. Additional dogs are being added to this study and immunohistochemistry is in progress.

1New Mexico Department of Agriculture-Veterinary Diagnostic Services, Albuquerque, NM 2Oklahoma Animal Disease Diagnostic Laboratory, Stillwater, OK Distribution of Lesions in Canine Dysautonomia: Review of 35 Cases

G. C. Johnson1, D. P. O’Brien2, K. L. Bailey1, J. M. Kreeger1, M. A. Miller1, L. W. Pace1, J. A. Ramos-Vara1, C. Rosenfeld1, A. Schreibman1, J. R. Turk1, S. E. Turnquist1 , and H. S. Gosser1

Gross and histologic findings were reviewed in 35 cases of canine dysautonomia, an idiopathic disease of young, predominantly rural dogs. Of these, thirty accessions were dead animals submitted for necropsy, and 5 were fixed tissues from necropsies done by referring veterinarians. Clinical signs, diagnostic response to pharmacological agents, and the presence of neuronal degeneration in at least one autonomic ganglion were criteria used for diagnosis. The most common gross finding was megaesophagus, which was noted in 17 necropsy accessions. Five of these patients had gross or histologic lesions of aspiration pneumonia, as did 3 additional dogs in which there was no mention of esophageal dilation. Histologic changes of neuronal degeneration and loss were most consistently observed in sympathetic postganglionic prevertebral and (when sampled) paravertebral ganglia; multiple ganglia were usually involved. Parasympathetic pelvic and ciliary ganglia were less consistently affected. Additional lesions were found in lateral and ventral horn preganglionic neurons along the length of the spinal cord. Degenerative lesions were found in 20/23 patients in thoracolumbar cord, which contains preganglionic sympathetic neurons, but in only 7/22 samples taken from the parasympathetic cervical or sacral spinal cord. Preganglionic parasympathetic nuclei of the X and III cranial nerves were affected in about half the cases sampled. Examination of the enteric nervous system revealed eosinophilic neuronal degeneration in the submucosal and myenteric ganglionated plexes of large and small intestine. The myenteric neurons were more frequently involved at both sites, with 64-90% of patients having lesions. In 3 animals, the submucosal ganglia were not affected in any specimen, although myenteric lesions were present. In two instances, the enteric nervous system was not involved at all, although prevertebral ganglia had degenerated. These findings emphasize that involvement of the autonomic nervous system may be inconsistent in canine dysautonomia, indicating a need for sampling multiple organs in obtaining a diagnosis. The somewhat preferential involvement of sympathetic pathways could reflect factors having to do with the susceptibility of the postganglionic sympathetic fibers by virtue of their longer axons, or may reflect some interaction between the patient and the unknown etiology of this condition.

1Veterinary Medical Diagnostic Laboratory, University of Missouri, Columbia, MO 2Department of Veterinary Medicine & Surgery, University of Missouri, Columbia, MO Interstitial Myocardial Fibrosis and Intramyocardial Coronary Arteriopathy in Azotemic Dogs

J. R. Turk, S. L. Stockham, M. Boucher, G. C. Johnson, M. A. Miller, J. A. Ramos-Vara, L. W. Pace, S. E. Turnquist, J. M. Kreeger, C. M. Loiacono, J. Donald, and M. A. Scott

A specific cardiomyopathy characterized by impaired diastolic relaxation in association with interstitial myocardial fibrosis and coronary arteriopathy occurs in human uremia. We performed retrospective examination of formalin-fixed paraffin-embedded sections of left ventricle from the hearts of 16 adult dogs with antemortem azotemia and a postmortem diagnosis of glomerulonephritis or renal amyloidosis. Sections of left ventricle from 7 non-azotemic adult dogs of similar age with no gross or histopathologic evidence of pulmonary, cardiac or renal disease were used as control tissues. Interstitial and perivascular myocardial collagen content was assessed by computerized image analysis of polarized sections that had been stained with Picrosirius red. The thicknesses of the intima and media as a percent of external diameter of intramyocardial coronary arteries were calculated in sections stained with Verhoeff’s method for elastin. There was significant increase in anisotropic interstitial myocardial collagen in azotemic as compared to non-azotemic dogs (P<0.01). There were insufficient cross sections of intramyocardial arteries within sections from every dog to permit statistical comparison, however, the arteries of 7 azotemic dogs had intimal fibrosis, splitting of the internal elastic lamina, and/or fibrinoid necrosis that were not observed in the non-azotemic dogs. These findings are similar to those reported in human uremia and suggest the need for further characterization of uremic cardiomyopathy in dogs.

Veterinary Medical Diagnostic Laboratory, P.O. Box 6023, University of Missouri, Columbia, MO Comparison of MIC Values for Tetracycline, Chlortetracycline, and Oxytetracycline vs. Swine Field Isolates of Actinobacillus pleuropneumoniae and Pasteurella multocida

C. C. Wu1, and T. Wolff2

The tetracycline family of antimicrobials is commonly used in pig production to prevent or treat swine respiratory disease. Veterinarians rely on antimicrobial susceptibility testing to guide them in appropriate medication selection. Traditionally, the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) have recommended that tetracycline be used in laboratory susceptibility testing as the class representative for the tetracycline family. This study compared in vitro susceptibility patterns for tetracycline (TC), chlortetracycline (CTC) and oxytetracycline (OTC) vs. two key swine respiratory pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, and demonstrated that TC, while a fairly good predictor of OTC susceptibility, is a poor predictor of CTC susceptibility. The Sensititre (Sensititre Ltd., East Grinstead, Sussex, England) plate broth microdilution technique was used to compare the susceptibility of TC, CTC and OTC vs. 100 field isolates each of A. pleuropneumoniae and P. multocida. The antimicrobial agents were incorporated into the wells of the plate in a doubling dilution pattern over a range of 8 concentrations (0.5, 1, 2, 4, 8, 16, 32, and 64 µg/mL). Each well of the Sensititre plate was inoculated with 50 µL of inoculum containing 105 colony forming units (CFU) of A. pleuropneumoniae or P. multocida per ml, prepared from an inoculum of bacteria with adjusted turbidity visibly comparable to that of a 0.5 McFarland turbidity standard. Test plates were incubated at 35±1°C for 18-20 hours. The minimum inhibitory concentration (MIC) was recorded as the lowest concentration of each antimicrobial to inhibit growth in a well. Each batch of isolates was run together with reference strains ATCC 29213 (S. aureus), ATCC 25922 (E. coli) and ATCC 27090 (A. pleuropneumoniae). The traditional tetracycline susceptibility interpretations of sensitive (≤ 4 µg/mL), intermediately sensitive (= 8 µg/mL) and resistant (> 8 µg/mL) were used to compare the MIC results for TC, CTC and OTC. Sixty eight percent and 84% of the 100 A. pleuropneumoniae isolates were resistant to TC and OTC, respectively, but none of the isolates was resistant to CTC. Seventy one percent of the A. pleuropneumoniae isolates were sensitive to CTC, while only 10% of the isolates were sensitive to TC or OTC. Twenty eight percent, 34% and 3% of the 100 P. multocida isolates were resistant to TC, OTC and CTC, respectively. Eighty eight percent of the P. multocida isolates were sensitive to CTC, while 61% and 58% of the isolates were sensitive to TC or OTC, respectively. When susceptibility patterns for the individual isolates were examined, all 68 (100%) of the TC-resistant A. pleuropneumoniae isolates were sensitive or intermediately sensitive to CTC, but only 5 of 68 (7%) of the TC-resistant A. pleuropneumoniae isolates were sensitive or intermediately sensitive to OTC. Of the 28 P. multocida isolates that were resistant to TC, 25 (89%) were sensitive or intermediately sensitive to CTC. Eight of the 28 TC-resistant P. multocida isolates (29%) were sensitive or intermediately sensitive to OTC. Practitioners who make their antimicrobial choices based on tetracycline susceptibility results would likely have discounted CTC as a medicinal option, choosing instead a less effective and/or more costly therapeutic. On the basis of this and other study results, several veterinary diagnostic laboratories in the U.S. are using a panel which as been redesigned to inlcude both CTC and OTC instead of tetracycline.

1Animal Disease Diagnostic Laboratory, Purdue University, West Layfayette, IN 2Alpharma Animal Health, Kansas City, MO

Multiplex PCR Detection of Swine Respiratory Viral Pathogens

S. B. Kleiboeker

Respiratory diseases of swine are one of the most costly disease complexes facing producers. Viral pathogens are often implicated as the inciting cause and swine influenza (SIV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV-2), and porcine respiratory coronavirus (PRCV) are among the main viral pathogens considered in the differential diagnosis of swine respiratory disease. Numerous separate diagnostic approaches have been described for each of these pathogens. Here a single multiplex PCR/RT-PCR assay, which can detect each of these pathogens following a single amplification reaction, is described. Total cellular RNA and DNA were extracted from tissue samples or infected cell culture homogenates using Trizol LS (GibcoBRL) according to the manufacturer’s instructions. Both PCR and RT-PCR were performed in a one-tube format with the Qiagen One Step RT-PCR reagents for a total of 50 amplification cycles. Oligonucleotide primers were selected from published sequence information and were designed to recognize conserved domains within the genome of the target pathogens. For SIV, primers which recognize the nucleoprotein (segment 5) gene generated a 659-bp product. For PRRSV, primers were selected from ORFs 4 and 5 and generated a 489 bp fragment. For PCV-2, primers were selected from the 3’ end of ORF 2 and generated a 289 bp product. For PRCV, primers which recognize the 5’ end of the S protein gene were modified from a previous report (J Virol Methods 66:303-309) and generated a 204 or 215 bp product. The sensitivity of the multiplex assay for each target pathogen was determined by amplification of RNA or DNA purified from serial ten-fold dilutions of titered reference stocks. The multiplex assay was capable of routinely detecting a minimum of 4 x 10-5 HAD units of SIV, 0.1 TCID50 of PRRSV, 1 TCID50 of PRCV, and 1 FFU50 of PCV-2. The specificity of amplification was determined by nucleotide sequencing of amplicons from known positive viral stocks followed by Southern blot hybridization of products from amplification of diagnostic laboratory specimens. Specificity was further demonstrated by a negative reaction when using template RNA and/or DNA purified from: 1) noninfected cell cultures of MARC-145, MA-104, ST, PK15, or Vero cells; 2) grossly and histopathologically normal pig lung tissue; and 3) viral cultures of porcine circovirus type 1, porcine adenovirus type 3, porcine enterovirus, porcine parvovirus, encephalomyocarditis virus, bovine viral diarrhea virus, and parainfluenza virus type 3. Based upon the sensitivity and ability to detect both RNA and DNA virus, it is anticipated that this assay will assist in the rapid and cost-effective diagnosis of this economically important disease complex of swine.

Veterinary Medical Diagnostic Laboratory and the Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, 1600 E. Rollins, Columbia, MO

Vesicular Stomatitis in Pigs: Effects of Virus Strain and Serotype on Contact Transmission

D. E. Stallknecht1,2,L. D. Bauer1, D. E. Perzak1, M. D. Murphy3, and E. W. Howerth3

Previous experimental infections of pigs with vesicular stomatitis virus New Jersey serotype (VSV- NJ) isolated from a sand fly (Lutzomyia shannoni) from Ossabaw Island, Georgia suggest that pigs represent an ideal model system to investigate pathogenesis and transmission of this virus. Pigs can be infected by various routes that are compatible with biological vector, mechanical vector, and contact transmission and these infections result in a full spectrum of clinical disease ranging from subclinical infection to the development of primary and secondary vesicles. Evidence of viremia in pigs has never been detected but contact transmission has been demonstrated. Recent vesicular stomatitis outbreaks in the United States have involved horses and cattle rather than pigs and have been restricted to the western states. In addition, two serotypes have been associated with these outbreaks; VSV-NJ and vesicular stomatitis virus Indiana (VSV-I). In order to test the application of our model system to other strains and serotypes of vesicular stomatitis virus, the objective of this study was to determine if infection of pigs with recent western isolates of VSV-NJ and VSV-I would result in a similar outcome to that observed with the Ossabaw strain of VSV-NJ. Groups of three pigs (20-30 pounds) were infected with VSV-NJ (Ossabaw), VSV-NJ (Colorado 95) and VSV-I (New Mexico 97) by intradermal inoculation of the snout, application of virus to scarified areas of oral mucosa, and application of virus to intact oral mucosa. For 6 all three routes of inoculation approximately 10 TCID50 of each virus was used. With the intradermal 4 2 route, doses of 10 and 10 TCID50 also were evaluated. Pigs were examined for vesicular lesions and swabs of the nasal planum, nasal cavity, saliva, tonsil and feces were collected for virus isolation for 12 days following inoculation. Serum samples were collected pre and post-infection for serologic testing for neutralizing antibodies. Infection, as detected by virus isolation and/or seroconversion, was observed with all virus/route treatments, and with one exception (VSV-I at 102), at all doses used for intradermal inoculation. Although the duration of viral shedding varied with virus and route of inoculation, all three viruses were isolated from swab samples of nasal planum, nasal cavity, saliva, and tonsil. A single isolate of VSV-NJ (Colorado 95) was made from a fecal swab collected from a pig with oral vesicular lesions. Positive fecal swabs have been previously observed from pigs infected with VSV-NJ (Ossabaw). The development of vesicular lesions and the duration and extent of viral shedding were similar between the two VSV-NJ strains. Although vesicular lesions developed, the duration and extent of viral shedding was reduced in the VSV-I infected pigs with detectable viral shedding lasting for a maximum of 3 days and <2.3 3.6 viral titers ranging from 10 to 10 TCID50 /swab. Results suggest that contact and mechanical vector transmission may have more relevance in epizootics of VSV-NJ than VSV-I. Actual contact trials with these viruses, however, and validation of results in horses and cattle are needed.

1Southeastern Cooperative Wildlife Disease Study, The University of Georgia, Athens, GA 2Department of Medical Microbiology and Parasitology, The University of Georgia, Athens, GA 3Department of Pathology, College of Veterinary Medicine, The University of Georgia, Athens, GA Clostridium difficile as a Cause of Porcine Neonatal Enteritis J. G. Songer1, K. W. Post2, D. J. Larson3, B. H. Jost1, and R. D. Glock1

Clostridium difficile is found widely in soil, water, and the intestinal tract of various mammals, birds, and reptiles. Spores are ingested and germinate in the large intestine, and if the normal flora is not intact, vegetative cells may multiply in open niches and produce toxins. This organism is an established cause of antibiotic-associated diarrhea and pseudomembranous colitis in humans, enterocolitis in foals, nosocomial diarrhea and typhlocolitis in adult horses, and typhlitis in adult hamsters. Diagnostic findings from several US laboratories suggest that C. difficile-associated disease in pigs is widespread, and data from North Carolina suggest a peak incidence in January through March. More than 50% of neonatal pig enteritis accessions involve C. difficile as an etiologic agent, and it is the sole pathogen identified in more than 36% of the total cases. This may place the importance of C. difficile-associated disease above that of E. coli, C. perfringens type A, and viral agents in at least some parts of the US. Most cases manifest as scours beginning soon after birth. Lesions include moderate-to-severe mesocolonic edema with scattered foci of suppuration in the colonic lamina propria, accumulation of neutrophils in the mesocolon, and exudation of neutrophils into the lumen. Enteritis is sometimes accompanied by hydrothorax or ascites, with respiratory signs. Cultures of affected tissues commonly yield heavy growth of C. difficile, but toxin detection is the gold standard for diagnosis; the correlation between clinical, pathologic, and bacteriologic diagnosis, on the one hand, and toxin detection on the other is 93.7%. Treatment with antimicrobials and use of probiotics has yielded mainly unsatisfactory results, but anecdotal evidence indicates that bacitracin methylene disalicylate may be effective against C. difficile infection in some herds. The lack of commercially available immunoprophylactic products has led some producers to the use of autogenous bacterin:toxoids. Penicillins or cephalosporins administered to neonatal pigs may be potentiating factors in the incidence of C. difficile-associated enteritis. Extensive knowledge of C. difficile and the pathogenesis of infections by this organism, accumulated as a result of its importance as a human pathogen, may have positive impact on attempts to develop prevention and control strategies for use in pigs.

1Department of Veterinary Science and Microbiology, University of Arizona, Tucson, AZ 2Rollins Animal Disease Diagnostic Laboratory, Box 12223 Cameron Village Station, Raleigh, NC 3Iowa Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Iowa State University, Ames, IA Molecular Characterization of Chlamydiaceae from Swine

L. E. Hoelzle, K. Hoelzle, and M. M. Wittenbrink

In our report, lung and intestine of 49 pigs with respiratory diseases and endocervical swabs from 205 sows with reproductive disorders were investigated for Chlamydiaceae infection by polymerase chain reaction. Samples from 49 healthy slaughter pigs and endocervical swabs from 30 fertile sows served as controls. PCR primers targeted DNA sequences flanking almost the entire chlamydial omp1 gene and sequences flanking a 590-bp fragment of the chlamydial omp2 gene. PCR amplicons of the expected size were generated from 49.0% of pigs with respiratory disease and 60.0% of sows with reproductive disorders. Corresponding values for the respiratory healthy controls were significantly lower (24.5%; p < 0.05). No PCR amplicons were obtained from endocervical swabs of fertile sows. By DNA hybridization of PCR amplicons a high prevalence of mixed infections with Chlamydophila (Chl.) abortus and Chlamydia (C.) suis in the porcine lung and intestine was found and further confirmed by restriction fragment length polymorphism analysis and nucleotide analysis of the omp1-gene-PCR amplicons. 81.3% of the PCR amplicons from endocervical swabs were identified as Chl. abortus, indicating an association of this known genitopathogenic chlamydial species with reproductive disorders in sows. Nucleotide sequence analysis of omp1 gene amplicons identified as C. suis shared maximum 82.7% homology with the reference strain S45.

Institute of Veterinary Bacteriology, University of Zürich, Switzerland Improved Diagnostic Tests for Serology and Typing of Actinobacillus pleuropneumoniae

T. J. Inzana1, J. Schuchert1, and B. Fenwick2

Actinobacillus pleuropneumoniae (Ap) is the etiologic agent of swine pleuropneumonia. This pathogen is responsible for high morbidity and mortality in swine producing areas throughout the world. There are 12 serotypes of Ap that are recognized, and different serotypes predominate in different geographic regions. In the United States, for instance, serotypes 1, 5 and 7 predominate. Current vaccines are partially protective, but are serotype specific. Therefore, accurate knowledge of infecting serotypes is important for prevention as well as epidemiology. Determining exposure of swine to Ap by serology, and serotyping Ap isolates has been problematic due to antigenic cross-reactivity of Ap toxins, lipopolysaccharide, and outer membrane proteins between species and Ap serotypes. To minimize these problems we have developed a serologic assay that utilizes highly purified capsular polysaccharide (CP) conjugated to biotin bound to streptavidin-coated ELISA plates. The assay could detect antibodies to the CP of Ap with high sensitivity and specificity, indicating the serotypes pigs were exposed to. We have previously reported the high sensitivity and specificity of a field latex agglutination assay, utilizing IgG to Ap CP, for serotyping isolates (T. Inzana, J. Clin. Microbiol. 33:2297, 1995). The ELISA and latex agglutination assays are now available for use by veterinarians and diagnostic laboratories. However, development of latex reagents for all 12 serotypes and identification of new or untypable isolates would be difficult. Therefore, a multiplex PCR assay was developed to simultaneously identify Ap and serotypes 1,2, and 5. The assay is based on amplification of specific DNA fragments using one set of primers to the conserved CP export region and a second set of primers to the serotype-specific CP biosynthesis region of serotypes 1, 2, or 5. The capsule biosynthesis regions of additional Ap serotypes are being sequenced to expand the capability of the multiplex PCR assay to identify additional serotypes.

1College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 2College of Veterinary Medicine, Kansas State University Manhattan, KS

Assessment of Primers Designed from the Small Ribosomal Subunit RNA for Specific Discrimination between Babesia bigemina and Babesia bovis by PCR

G. F. C. Linhares1, Â. P. Santana1 , and L. H. Lauerman2

Six pairs of species-specific primers were designed from the alignment of the sequences of the SS rRNA gene obtained from the GenBank database for Babesia bigemina (accession number X59604) and for B. bovis (accession number U06105). Three pair of primers were designed specifically for B. bigemina and another 3 primer sets for B. bovis. All oligonucleotide sequences selected as primers were examined for similarities to other organisms through the GenBank Blast procedure and these 6 sets of primers demonstrated a high level of specificity. The synthetic oligonucleotides were further tested for specificity using genomic DNA extracted from blood of clinically ill animals showing specific parasitemias detected by blood smear examinations from 6 different states of Brazil. All 6 sets of primers were validated as 100% specific for the respective parasite using DNA amplification by PCR assay. The PCR amplified the expected fragments for each set of primers, as follows: a) B. bigemina: primers GAU5 forward/GAU6 reverse – fragment of 1,124 bp; primers GAU5 forward/GAU8 reverse – fragment of 458 bp; primers GAU7 forward/GAU6 reverse – fragment of 685 bp; b) B. bovis: primers GAU9 forward/GAU10 reverse – fragment of 541 bp; primers GAU9 forward/GAU 13 reverse – fragment of 883 bp; primers GAU 14 forward/GAU13 reverse – fragment of 358 bp. The variation in size of the amplicons produced by the different combinations of primers for each species of Babesia allowed the procedure of Multiplex PCR assay as well as the use of internal controls for PCR to be applied for enhanced reliability.

1 College of Veterinary Medicine, Federal University of Goias, Goiania, Brazil 2 Avian Health Laboratory, Washington State University, Puyallup, WA

The Use of Porcine Alveolar Macrophages for Detecting Shiga Toxin

W. L. Mengeling1, A. C. Vorwald1, N. A. Cornick2, K. M. Lager1, and H. W. Moon2

Earlier this year we investigated an epidemic of clinical illness among a large group (3600) of approximately 50 lb. pigs that had recently been moved from nurseries to grow/finish facilities. The most prominent clinical signs were profuse, sometime fatal, diarrhea and incoordination. Blood (serum) and rectal, nasal, and tonsil swabs were collected from each of 10 pigs. Each swab was submerged in a tube of balanced salt solution containing a high concentration of antibiotics. Because of the profuse diarrhea, all pigs were covered with feces, and as a consequence all nasal swabs and at least some tonsil swabs were contaminated with feces. Each of the samples was tested on a variety of cell cultures for virus isolation. Serums also were tested by a nested-set polymerase chain reaction (PCR) for the presence of porcine reproductive and respiratory syndrome virus (PRRSV), and isolates of PRRSV were analyzed by restriction fragment length polymorphism (RFLP). One or more viruses was identified from 1 or more samples from each of the 10 pigs; PRRSV from 10 pigs, porcine respiratory coronavirus from 3 pigs, and porcine enterovirus from 8 pigs. The RFLP patterns indicated that all the isolates of PRRSV were field strains (i.e. not vaccine derived). In addition, a cytotoxin was identified in the fluid into which swabs had been submerged. The toxin was especially damaging in porcine alveolar macrophages that had been exposed to fluids from rectal swabs. Most or all of the macrophages exposed to the toxin were lysed after overnight incubation. A diagnosis of edema disease was made by personnel of the Iowa State University Diagnostic Laboratory on the basis of bacteriologic and histologic examination of additional samples and tissues from the same epidemic. This suggested that the toxin we had identified was a Shiga toxin (Stx), and that porcine alveolar macrophages might be especially susceptible. This hypothesis was confirmed by the following observations. 1) The toxic activity of our field samples and of known Stx preparations was neutralized in both Vero cells (cells commonly used to detect and titrate Stx) and in porcine alveolar macrophages by a bovine polyclonal antiserum against Stx 1, Stx 2, and Stx 2e. 2) Porcine alveolar macrophages were generally more sensitive than Vero cells for detecting Stx in field samples as well as in in vitro preparations. For example the titer of Stx 2 (prepared in vitro) was 10,485,760 in porcine alveolar macrophages and 1,310,720 in Vero cells. In addition to sensitivity, porcine alveolar macrophages were found to have other attributes relative to their use for detecting the presence and titer of Stx. These include the following. 1) The test can be read after overnight incubation (little or no change occurs thereafter) rather than after 48 to 72 hours of incubation as routinely used for Vero-cell assays. 2) Unlike continuous cell lines such as Vero cells, porcine alveolar macrophages need not be continuously propagated to ensure almost instant availability. They can simply be removed from a low-temperature repository, thawed, seeded, and exposed to sample soon thereafter. For the reasons presented above we believe that porcine alveolar macrophages may be diagnostically useful for the identification of Stx.

1Virus and Prion Diseases of Livestock Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, Ames, IA 2Veterinary Medical Research Institute, Iowa State University, Ames, IA Characterization of the Interaction of Escherichia coli Heat-stable Enterotoxin (STa) with its Putative Receptor on the Intestinal Tract of Newborn Calves*

1 2 2 2 A. M. Al-Majali , J. P. Robinson , E. K. Asem , M. J. Freeman , C. H. Lamar2, and A. M. Saeed2

Enterotoxigenic Escherichia coli (ETEC) induce severe diarrhea in newborn calves through the elaboration of heat-stable enterotoxin (STa). We investigated the distribution of the STa-specific receptors on enterocytes and brush border membrane vesicles (BBMVs) prepared from anterior jejunum, posterior jejunum, ileum and colon of newborn calves. We found that STa-receptor density was higher on enterocytes and BBMVs derived from the ileum than enterocytes and BBMVs prepared from other segments of the calf intestine. Additionally, the affinity of the ileum STa-receptors was higher than the affinity of receptors present on the epithelium of other intestinal segments. This study suggests that, in newborn calves, the ileum is the major part of the intestinal tract that is affected in the course of secretory diarrhea caused by STa-producing ETEC strains.

1Research and Development, Merial Limited, 115 Transtech Drive, Athens, GA 2Department of Veterinary Pathobiology, School of Veterinary Medicine, Purdue University, West Lafayette, IN

* Veterinary Research Communications 28:97-104

Evaluation of the ImmunoCardSTAT Rotavirus (ICS-RV) Assay for On-site Detection of Subgroup A Bovine Rotavirus

C. Han1, V. Ciesicki1, L. Brown1, A. G. Wise1, M. L. Vickers2, C. L. Kanitz 3 , and R. Maes1,4

Rotavirus infections are an important cause of neonatal diarrhea in calves, piglets and foals. The majority of these are caused by subgroup A rotavirus. Current laboratory diagnostic methods to diagnose rotavirus infections include fluorescent antibody staining, electron microscopy, antigen detection ELISA, latex agglutination, electropherotyping and RT-PCR. The objective of this study was to evaluate the ICS- RV assay (Meridian Diagnostics Inc., Cincinnati OH) for its usefulness as an on-site diagnostic test for veterinary specimens. A reference strain of bovine rotavirus with known titer was obtained from NVSL. Serial tenfold dilutions were tested by antigen detection ELISA (Sanofi Diagnostics Pasteur, Redmond WA) and the ICS-RV test. The reference test used was a RT-PCR assay, which amplifies a 294 bp fragment of the VP6 gene of type A rotavirus. The detection limits of the ELISA and ICS-RV assays were comparable, but 10- fold lower than the detection limit of RT-PCR. A collection of bovine fecal specimens was subsequently tested by ELISA, ICS-RV and RT-PCR. Compared to RT-PCR, the sensitivity, specificity, positive predictive value and negative predictive were 95.0, 97.4, 95.0 and 97.4 % for ICS-RV and 92.2,79.5, 73.8 and 94.3 % for the ELISA test. The accuracy and kappa values were 95.9% and .99 for the ICS-RV test and 82.2% and 0.69 for the ELISA test. The results show that the ICS-RV would be an excellent on-site test for the detection of type A bovine rotavirus. We are currently in the process of evaluating its use for the detection of subgroup A rotavirus infections in piglets and foals.

1Animal Health Diagnostic Laboratory, MSU, E. Lansing, MI 2Livestock Disease Diagnostic Center, Univ. of Kentucky, Lexington, KY 3Animal Disease Diagnostic Laboratory, Purdue Univ, W. Lafayette, IN 4Dept. of Microbiology, MSU, E. Lansing, MI Simultaneous Detection of Bovine Coronavirus, Bovine Rotavirus and Cryptosporidium parvum in Fecal Samples by Multiplex RT-PCR

A. G. Wise1, and R. Maes1,2

The infectious agents most commonly involved in the induction of neonatal diarrhea in calves are rotavirus, coronavirus, Cryptosporidium parvum and E. coli K99. The relative incidence of these in a multi-state survey of causes of scours in beef calves was 35% for rotavirus and coronavirus combined, 24% for cryptosporidium, 22% for E.coli. and 19% for others. The percent involvement in a study of causes of scours in dairy calves was 46 % for rotavirus, 15% for coronavirus, 23% for cryptosporidium, 13 % for salmonella and 3% for E.coli. The objective of this study was to develop a multiplex RT- PCR/PCR assay for bovine rotavirus, bovine coronavirus, and Cryptosporidium parvum providing rapid and sensitive laboratory diagnosis of individual or combinations of these agents in a single assay. Both RNA and DNA were simultaneously extracted from clinical specimens (fecal material) using a commercial kit. Primer pairs selected were specific for a 294 bp fragment of the VP6 gene of type A rotavirus, a 406 bp fragment of the nucleocapsid gene of bovine coronavirus, and a 194 bp DNA fragment of Cryptosporidium parvum. The multiplex reaction was carried out using a commercially available one- tube RT-PCR system. The PCR detection limit for each agent was determined by testing serial ten-fold dilutions of reference preparations with the three primer pairs of the multiplex. The results showed that the detection limits were 1 TCID50 for rotavirus, 0.1 TCID50 for coronavirus and 10 oocysts for Cryptosporidium parvum. It is anticipated that this assay will be a valuable tool in the differential diagnosis of neonatal diarrheas of infectious origin in beef and dairy calves.

1Animal Health Diagnostic Laboratory, MSU, E. Lansing, MI 2Dept.of Microbiology, MSU, E. Lansing, MI BVDV Nested RT-PCR Technique to Screen Herds Using Pooled Buffy Coat Samples

L. Braun, D. Peterson, and C.Chase

Bovine viral diarrhea virus (BVDV) is the leading infectious cause of abortions, stillbirths and weak calves in South Dakota. The ability to detect and identify the BVDV persistently infected animal is imperative for good herd management. There are several methods to detect BVDV in infected herds. Diagnosis of BVD infection is performed by virus isolation (VI), reverse transcriptase-polymerase chain reaction (RT-PCR), antigen capture enzyme-linked immunosorbent assay (AC-ELISA). The microtiter test or the AC-ELISA in a multi-well tissue culture plate has been the most popular for herd screens. The BVDV RT-PCR is the most sensitive of the assays. Due to the high cost associated with RT-PCR, it has primarily been used as an individual test and currently has not been used as a herd screening method. Dr. Kenny Brock, has designed a BVDV RT-PCR to test bulk milk samples from dairy herds. In our efforts to make BVDV RT-PCR more economically feasible as a screening assay, we have performed several experiments involving pooled buffy coat (BC) samples. We obtained EDTA-whole blood samples from 54 animals in a BVDV negative herd and from 2 persistently infected (PI) animals (kindly provided by Dr. Dan Grooms, Michigan State University). The whole blood was centrifuged and the buffy coat was harvested. The RNA was extracted from these samples using a method developed by Chomonski. The RT-PCR method was a modification of the method of Sullivan and Akkina that produces a product in the Erns region. This method not only detects BVDV but also determines the genotype, type 1 or type 2, of the virus. The RT reaction and the first step of PCR, the outer PCR reaction, were performed. The outer PCR products were used in a second or nested PCR reaction was performed. The outer or single step PCR products and the nested or two step PCR products were separated by electrophoresis on a gel. Of these 56 animals tested, only the two PI samples were RT-PCR positive. There were PCR products visible in both the single (outer) step and nested PCR reaction. Five pools were created by adding 100µl of the following samples; 1) five negative samples, 2) four negative and 1 positive sample, 3) nine negative and 1 positive sample, 4) 19 negative and 1 positive sample, 5) 49 negative and 1 positive sample. An aliquot of 300µl was removed from each of the pools and was processed for RNA extraction and BVD RT-PCR as above. The outer reaction did not produce any bands, however the more sensitive nested reaction produced a positive band in the pooled samples in each of the 3 separate runs. The result of these experiments demonstrated the ability to detect one positive sample from a pool of 50 samples. This appears to be an excellent method for herd screening. On initial test of a herd with an unknown BVDV PI status, we feel the pool size should not exceed 20 samples. Any pool that is positive must be retested. There could be several approaches but the two most likely would be 1) retesting the original pool with RT-PCR using smaller pools i.e. 5 samples/pool and testing all positive pool samples with microtiter test or 2) testing all of original pool with microtiter test or AC. In herds that have been previously screened for PI animals and the prevalence of BVDV PI animals is <1%, 50 sample/pools are a reasonable approach for an annual BVDV PI monitoring program.

South Dakota State University, Brookings, SD 57007 Bovine Viral Diarrhea Virus Infections in Calves from Auction Markets and a Ranch

R. W. Fulton1, J. T. Saliki1,2, A. W. Confer1, L. J. Burge1, C. W. Purdy3, R. E. Briggs4, G. C. Duff5, and R. W. Loan6

The objective of the study was to determine the prevalence of bovine viral diarrhea virus (BVDV) persistently infected (PI) calves and acutely infected calves. The calves were purchased from local auctions in east Tennessee (121) and transported to an experimental feedlot in Clayton, NM where they were mixed with calves from a New Mexico ranch (84). All calves were vaccinated at day 0 with a monovalent vaccine containing modified live virus (MLV) infectious bovine rhinotracheitis virus (IBRV) vaccine. The New Mexico ranch calves had previously received one dose of parenteral vaccine containing IBRV, bovine viral diarrhea virus (BVDV), parainfluenza-3 virus (PI-3V), and bovine respiratory syncytial virus (BRSV). The calves were divided into 10 pens, 20-22 calves per pen: 4 pens Tennessee only; 4 pens mixed with 10 calves each from Tennessee and New Mexico; and 2 pens New Mexico only. The calves were processed on September 30/October 1 and held until 2 November. Sera, EDTA blood samples for peripheral blood leukocytes (PBL), and nasal swabs (NS) were collected weekly from day 0 through day 35, and when the calves were treated. The PBL and NS swabs were tested for viruses by cell culture inoculation and the serums for neutralizing antibodies to IBRV, BVDV types 1 and 2, PI-3V, and BRSV. Lung samples from calves dying in the study were examined microscopically for lesions and viruses by cell culture inoculation. During the study, 152/205 (67.6%) calves were treated for illness associated with acute respiratory disease, and 4/205 (1.8%) calves died. There were differences in the morbidity between the Tennessee, 99/121 (81.8%), calves and the New Mexico, 53/84 (63.1%) calves. One pen of New Mexico only calves had less morbidity than the other 9 pens. For sera from 201 surviving calves, 78 (38.8%) seroconverted to BVDV. With only one exception, the type 1 titers in seroconverting calves were greater. There were 60/117 (51.3%) Tennessee and 18/84 (21.4%) New Mexico calves seroconverting to BVDV1. Seroconversions occurred to both BRSV and PI-3V. One calf was BVDV PI, with repeated positive samples from NS and PBL collections while remaining seronegative to BVDV1 and 2. At least 8 animals were BVDV positive in the NS and/or PBL. BVDV positive cattle were primarily detected in the treated group. Five calves were IBRV positive in the NS within 2 weeks of MLV vaccination. Additional viral isolates, from the NS or PBL, included PI-3V and a bovine adenovirus. BVDV was isolated from one of the 4 lung samples from calves dying in the study. The BVDV isolates are being typed by a nested PCR assay. Pasteurella spp. were isolated also from diseased animals and lungs. The study results underscore the involvement of several viruses plus Pasteurella spp. in acute respiratory diseases of cattle. Also, viruses such as IBRV may be found in NS, even in vaccinated cattle. The study also suggests that BVDV vaccination should be considered in infectious disease control programs under these management conditions.

1Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 2Oklahoma Animal Disease Diagnostic Laboratory, Stillwater, OK 3USDA ARS, Conservation and Production Research Laboratory, Bushland, TX 4USDA ARS, NADC, Ames, IA 5Clayton Livestock Research Center, New Mexico State University, Clayton, NM 6Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX Isolation of Bovine Adenovirus Type 7 from Calves with Respiratory Disease

J. T. Saliki1,2, S. L. Caseltine1, R. W. Fulton2, and H. D. Lemkuhl3

Ten serotypes of bovine adenovirus (BAV), designated BAV-1 through BAV-10, are recognized worldwide. Of these, BAV-1, -2, -3, -4, -7 and –10 have been isolated from cattle in the United States. Isolates have been obtained from apparently healthy animals as well from clinically ill animals, most often associated with pneumonia and enteritis. This report concerns the isolation of BAV-7 from clinically ill animals from four states (NM, NY, OK and TX). The BAV-7 isolates in this report were obtained when peripheral blood leukocytes (PBLs) were inoculated onto bovine turbinate (BT) cells for bovine viral diarrhea virus (BVDV) isolation. The same samples did not yield any BAV growth in Madin Darby Bovine Kidney (MDBK) cells. In BT cells, the virus caused extensive cytopathic effects characterized by cell rounding, increased refractiveness, clumping and rapid destruction of the cell monolayer. Virus identification was done by electron microscopy and serotyping with the serum neutralization test using a panel of control BAV antisera representing the 10 recognized serotypes. The case histories for these isolations were as follows: Case 1: A group of 121 recently weaned calves were purchased from local auctions in TN and transported to NM. About 3-5 weeks after arrival, 99 calves were observed with acute respiratory disease. BAV-7 was isolated from 8 of these. Case 2: Two- to four-month old dairy calves from NY were presented with acute respiratory disease, diarrhea and mortality. PBLs from 13 calves were tested and resulted in BAV-7 isolation from 5 calves, BVDV isolation from 5 calves and a mixed BAV-7/BVDV infection in 1 calf. Cases 3 and 4: Two weaned beef calves from two herds in Oklahoma and Texas were presented with histories of acute respiratory disease with high morbidity (about 20%) and case-fatality (>30%). PBLs cultured for BVDV isolation yielded BAV-7 in BT cell cultures but were negative for BVD virus. The common clinical sign in all these cases was respiratory disease. BAV-7 has been associated with both respiratory disease and enteric diseases in calves and experimental inoculation has reproduced mild respiratory disease. However, the virus has also been isolated from apparently healthy calves. Therefore, it is difficult to establish a cause-effect relationship between the virus and the syndromes observed in this report. The possible role of these isolates of BAV-7 in cattle disease, especially respiratory disease, will be investigated in a future study.

1Oklahoma Animal Disease Diagnostic Laboratory, Oklahoma State University, Stillwater, OK 2Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 3NADC, ARS, USDA, P.O. Box 70, Ames, IA

A Survey of Antimicrobial Susceptibility Testing Practices of Veterinary Diagnostic Laboratories in the U.S.A.

M. B. Brooks* P. S. Morley, D. A. Dargatz, and D. R. Hyatt

There is increasing public concern regarding the frequency and distribution of antimicrobial resistance, particularly as it affects public health. Exposure of bacteria to antimicrobial drugs increases the likelihood of developing resistance, and imprudent use of antimicrobial drugs in humans or animals may affect the rate at which resistant organisms develop. These resistant organisms may be more difficult to combat with available drugs, which in turn can detrimentally impact public health and animal well being. The extent to which resistance among bacteria in animals affects the health of humans or animals is unknown. Scientific studies are urgently needed on this issue to determine the significance of the problem, so that regulatory policies regarding prudent antimicrobial use in animals can be developed based upon factual risk information rather than anecdote and speculation. One way in which information could be generated rapidly and efficiently on this issue would be to evaluate data that have already been generated in the regional veterinary diagnostic laboratories that are located throughout the country. These laboratories are believed to evaluate the vast majority of biologic specimens submitted from animal agriculture in the U.S. for bacterial isolation. Unfortunately, there is no existing monitoring system in place to gather the data produced in these laboratories. Further, there may be considerable variation in types of data collected and how it is archived. The goal of this study is to describe the laboratory practices used at veterinary diagnostic laboratories across the country, and to evaluate the ability to collate this information for the purpose of monitoring antimicrobial resistance in animals. To meet these objectives a survey is currently being conducted through joint sponsorship of USDA:APHIS:VS:Centers for Epidemiology and Animal Health and the Antimicrobial Resistance Research Group from the College of Veterinary Medicine and Biomedical Sciences at Colorado State University. The survey was sent to diagnostic laboratories that are accredited by the American Association of Veterinary Laboratory Diagnosticians (AAVLD). Information is gathered about methods used to determine antimicrobial susceptibility (e.g., broth dilution or disc diffusion) and how the results are reported and archived (e.g., SIR interpretation, zone sizes, MIC values). Data regarding the demographics of the animal populations from which samples are collected, and what types of services are routinely provided are also collected. Data are also collected regarding protocols that are used in evaluating antimicrobial susceptibility. For instance, does the laboratory use standardized protocols such as National Committee on Clinical Laboratory Standards (NCCLS) guidelines in measuring susceptibility? Questions are asked about whether methods of testing are altered in some circumstances or for certain organisms, such as nosocomial organisms (e.g. Salmonella, Staphylococcus). These data will allow an assessment of whether future studies can compile data from these sources efficiently and how meaningful the data will be.

Department of Environmental Health, College of Veterinary Medicine and Biomedical Sciences, Fort Collins, CO *To be considered for graduate student award.

A Simulation Model to Assess the Effect of Age at Vaccination on Transmission of Bovine Viral Diarrhea Virus in Dairy Heifers

C. A. Muñoz-Zanzi*1, C. Thurmond1, and S. K. Hietala2

Bovine viral diarrhea virus (BVDV) is widely distributed and represents one of the cattle diseases with major economic impact for the industry. Findings on the potential long term effects of BVDV post- natal infection (PNI), and observation of a wide antigenic variability of the virus, including emerging more virulent strains, have prompted questions about the benefit of current vaccination programs in preventing BVDV transmission. The objective of the study was to assess the effect of different vaccination programs on the incidence of PNI before first breeding. A stochastic, modified Reed-Frost model was developed to simulate incidence of PNI in heifers from 3 to 10 months of age. The model included decay of colostral antibodies, transmission of BVDV from persistently infected (PI) and PNI heifers, varying duration of shedding after PNI, vaccination at various ages, and varying duration of immunity after vaccination or infection. Graphical and statistical evaluation of the model with observed data demonstrated a good fit (p > 0.5). Results showed effects of PNI and of PI on BVDV transmission, and verified the importance of protection provided by colostral antibodies. Assuming a PI prevalence of 1% and average shedding after PNI of 4 days, vaccination at 130-150 days predicted the greatest reduction in BVDV PNI cases, coinciding with the age at which a critical number of heifers was expected to lose colostral protection. This simulation model provides a tool to quantitatively assess the benefit of different BVDV control programs that could be implemented in the field.

1Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 2California Animal Health and Food Safety Laboratory System, Davis, CA *To be considered for graduate student award. Prevalence of Johne’s Disease in a Subpopulation of Alabama Beef Cattle

B. B. Hill*1, M. West2, and K. V. Brock1

Johne’s disease is a transmissible, insidious, chronic granulomatous enteritis caused by the host immune response to an infection with the bacterium Mycobacterium avium ssp. paratuberculosis. The objective of this study was to estimate the overall prevalence of animals that were infected with M. avium ssp. paratuberculosis in a subpopulation of Alabama beef cattle. This was determined using an IDEXX ELISA for the detection of M. avium ssp. paratuberculosis-specific antibodies in serum. Serum was collected from 79 herds that were participating in the Alabama Brucellosis Certification program. A total of 2,073 beef cattle were tested by randomly selecting 30 animals per herd in herds greater than 30 and selecting all animals in herds 30 and less. It has been estimated that the IDEXX ELISA test has a 60% sensitivity and a 97% specificity. Of the 79 herds tested, 29 herds were seronegative, 24 herds had 1 to 2 positive animals, and 26 herds had 3 or more seropositive animals. The average number of infected animals per positive herd was 3.3. In addition, a calculated minimum of 53.5% of the herds were identified as Johne’s positive herds with a 95 % confidence level. Of the total number of animals tested 8.0% (166/2073) were positive by the ELISA for antibody. After adjustments for test sensitivity and specificity and the proportion of animals sampled per herd, the true prevalence was calculated to be 8.75%. These data suggest that approximately 50 % of the herds are infected with M. avium ssp. paratuberculosis and the overall prevalence of infection in Alabama beef cattle is approximately 8 % which correlates with other previously published regional estimates.

1Department of Pathobiology, Auburn University College of Veterinary Medicine, Auburn, AL 2Department of Discrete and Statistical Sciences, Auburn University, Auburn, AL *To be considered for graduate student award.

Evaluation of PRRS Diagnostics in Detecting Persistently Infected PRRSV Carrier

D. C. Horter*, R. Pogranichnyy, C.-C. Chang, K.-J. Yoon, and J. J. Zimmerman

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) continues to plague the swine industry worldwide with reproductive and respiratory problems. The fact that PRRSV leads to clinically normal, persistently infected animals greatly complicates PRRS eradication and control. Persistently infected animals maintain the virus in the herd by transmitting the infection to in-herd or purchased susceptible animals. Therefore, identifying persistent carriers is essential to the control of PRRS. The objective of this study was to evaluate the diagnostic sensitivity and specificity of PRRSV diagnostic tests (PCR, virus isolation, and ELISA) in persistently infected animals in an experimental population. The experiment was designed as a longitudinal study in which samples were collected over time. 180 three-week-old commercial feeder pigs free of PRRSV were obtained and randomized into two groups: PRRSV inoculated (n = 90) and uninoculated control (n = 90). Inoculated animals received 2 ml of North American prototype ATCC-VR2332 PRRSV at a concentration of 103 fluorescent foci units per ml intranasally. Ante-mortem diagnostic samples taken include serum (sampled bi-weekly), peripheral blood leukocytes (63, 77, 91, 98, 105 days PI), and oropharyngeal scrapings (63, 77, 91, 98, 105 days PI). Twelve inoculated and 12 uninoculated control animals were euthanized on day 63, 77, 91, 98, and 105 days PI. Post-mortem samples collected included tonsil, lung, lung lavage, and tracheobronchial lymph nodes. All samples collected were aliquoted, randomized, and stored at –80oC. Infectious carrier status was established using virus isolation and swine bioassay on oropharyngeal scrapings and tonsil homogenates. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on serum, oropharyngeal scrapings, and tonsil homogenates. Serum samples were assayed for the presence of PRRSV specific antibody using a commercial ELISA. The diagnostic sensitivity and specificity of RT-PCR, VI, and ELISA were calculated for each sampling day PI. Uninoculated control animals remained PRRSV negative throughout the study. 100% of inoculated animals harbored infectious PRRSV at day 63 PI. The proportion of carriers in the inoculated group decreased slightly over time, but infectious virus was detected in approximately 90% of animals on day 105 PI. Based on samples collected between day 63 and 105 PI, the diagnostic specificity of PRRSV RT-PCR on oropharyngeal scrapings and tonsil homogenate was 100%. The mean diagnostic sensitivity of RT-PCR on oropharyngeal scraping samples was 81% while the mean diagnostic sensitivity of RT-PCR on tonsil homogenate samples was 66%. Virus isolation on oropharyngeal scraping yielded a mean diagnostic sensitivity of 47% while the mean diagnostic sensitivity of virus isolation on tonsil homogenate was 27%. Mean diagnostic sensitivity and specificity for ELISA was 98% and 98%, respectively. Over the period of time post inoculation in which animals were observed, ELISA appeared to be the most sensitive and specific assay. PCR was useful for detecting viral RNA but tissue sample selection was an important consideration. Additional work is needed to evaluate assay performance beyond 105 days PI.

Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA * To be considered for graduate student award. The Use of One Tube Nested (OTN) PCR for the Detection of Mycobacterium bovis in Bovine Milk

M. C. Antognoli*1, J. Triantis1, J. Hernandez2, and M. D. Salman1

The polymerase chain reaction (PCR) has been used to detect Mycobacterium species in a variety of clinical samples. PCR can be utilized to detect Mycobacterium bovis in unpasteurized milk, which is an important source of bovine tuberculosis (TB) infection for both animals and humans, and thus prevent the spread of the agent to other individuals. The sensitivity of PCR when used directly on clinical samples needs to be improved. The nested PCR (two sets of primers) increases the sensitivity and the specificity of the PCR system due to the presence of two amplification cycles. The development of a closed lid-one tube nested (OTN) PCR helps reduce cross contamination between samples, which is the main problem encountered when using the nested method. The sensitivity of PCR can also be improved by increasing the efficacy of the DNA extraction and by the removal of PCR inhibitors. Several procedures, ranging from different centrifugation protocols to complicated DNA extraction techniques, have been reported to improve the detection by PCR. This study was designed to evaluate the efficacy of 5 sample treatments combined with OTN PCR in milk samples experimentally inoculated with a phenol-killed M. bovis strain. The treatments evaluated in this study were the following: (1) centrifugation (13,000 x g for 30 minutes), (2) Chelex-Proteinase K (CH-PK), (3) CB18-Chelex-Proteinase K (CB18-CH-PK), (4) Genereleaser-Proteinase K (GNR-PK) and (5) immunomagnetic separation (IMS). Genereleaser and Chelex are resins, which act by sequestering PCR inhibitors and contribute to the purification of the target DNA in the sample. CB18 is a zwitterionic detergent with surface-active properties that is actively sequestered by the mycobacterial cell wall. Because of these characteristics, CB18 causes a reduction in mycobacterial buoyancy and surface tension, which helps to concentrate mycobacteria in a pellet for DNA extraction. The primers utilized in OTN PCR target the 38 kilodalton protein antigen b (Pab), which is highly specific for M. tuberculosis and M. bovis. Milk samples were experimentally inoculated with serial 1:2.5 fold dilutions of M. bovis ranging from 5000 to 20.1 cells/ml of milk. The lowest detection limit for OTN PCR combined with CB18-CH- PK and IMS was approximately 50 cells per ml of milk. These treatments are currently being evaluated in field milk samples from a naturally TB-infected herd. Preliminary results on samples from 40 tuberculin positive and 40 tuberculin negative animals indicate that 2.5% of the cows in each group are shedding either M. bovis or M. tuberculosis in their milk. Positive PCR results will be confirmed by southern blot and/or sequencing. Since M. tuberculosis has been rarely found in cattle it can be assumed that a positive PCR result indicates the presence of M. bovis in milk. In terms of control of bovine TB, differentiation between these species is not an important issue since they are both pathogenic. While a positive tuberculin test indicates sensitization by exposure to either pathogenic or environmental Mycobacteria, a positive PCR test would provide information about the presence of M. bovis and /or M. tuberculosis in milk. Additionally, a positive PCR result from a tuberculin negative cow would suggest the presence of a false negative tuberculin test (anergy). The successful optimization of OTN PCR combined with either IMS or CB18-CH-PK in field milk samples could provide a rapid and sensitive test for milk classification. The identification of anergic animals shedding M. bovis in milk would also accelerate the process of controlling the disease in TB- infected herds.

1Center of Veterinary Epidemiology and Animal Disease Surveillance Systems, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 2Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL *To be considered for graduate student award. Epidemiologic Study of Neonatal Equine Clostridia Enterocolitis

D. Hyatt, K. Tillotson, J. L. Traub-Dargatz, C. E. Dickinson, R. Ellis, P. Morley, M. Salman, G. Thompson, D. Bolte, and R. Magnuson

Clostridia-associated enterocolitis has been increasingly recognized in neonatal foals and young adult horses with colitis. Clostridium perfringens is most commonly implicated as the cause of hemorrhagic enterocolitis in neonatal foals. This disease is associated with a high mortality rate resulting in considerable economic losses. It has been proposed that the disease is multifactorial. However, there is minimal information available concerning the epidemiologic characteristics of this disease. The purpose of this study is to estimate the prevalence of C. perfringens fecal shedding in the general brood mare and foal population as well as in affected foals and their dams using culture methods to optimize recovery of the organism. The association between the presence of the organism, its toxins, and/or spores and hemorrhagic enterocolitis in neonatal foals will be evaluated as well as risk factors associated with development of the disease. To accomplish this goal, a field-based study of brood mares and neonatal foals was implemented in the 2000 foaling season. Feces were collected from brood mares once during the 3rd trimester, at the time of foaling (first feces passed after foaling), and one month post-foaling. Feces were collected from all foals born on participating premises during the first day of life (after meconium was passed), when they were three days old, and when they were one-month old. Two composite swabs, one from the surface of the mare’s teats and one from the hair-coat of the mare’s muzzle and medial surface of the legs was collected within 24 hours after foaling. An environmental sample taken from the surface on which the foal was born was also collected. A panel of tests was performed on all fecal samples including a Gram stain, spore stain, aerobic and anaerobic culture. Fecal and environmental samples were cultured to optimize recovery of clostridial organisms whether it was in its vegetative or spore form. Clostridium perfringens from fecal samples were semi-quantitated by direct plating as well as enriched. Environmental samples were enriched and heat-shocked. All C. perfringens isolates were genotyped using PCR primers. Preliminary fecal culture results from the 115 mare / foal pairs enrolled as of June 15, 2000 indicated that C. perfringens is commonly recovered from the feces of healthy three-day-old foals. Compared to other sample types in the study (mare 3rd trimester, mare at foaling, mare at 30 days post-foaling, foal at 8-12 hours of age, and foal at 30 days of age), feces from foals at 3 days of age were the most likely to contain C. perfringens. The predominant type of C. perfringens shed in the feces of both brood mares and foals was type A. In summary, based on preliminary results, the gastrointestinal tract of foals less than 10 days of age appears to be more highly permissive for C. perfringens type A compared to brood mares.

Veterinary Diagnostic Laboratories, Colorado State University, Fort Collins, CO

Molecular Epidemiology of Year 2000 Pseudorabies Outbreaks in Illinois and Tennessee

E. C. Hahn, B. J. Paszkiet, R. M. Weigel, and G. Scherba

In late winter and early spring of 2000, new outbreaks of pseudorabies occurred in Iowa, Illinois and Tennessee. At the time of these outbreaks, Illinois and Tennessee had been free of the virus. The virus spread to several farms before it was contained in Illinois. A single farm was infected in Tennessee. Possible sources of infection were investigated, because knowledge of the source would enable proper modifications of procedures to prevent repeated infections. Two sources of infection were considered likely: reemerging infection from a pocket of local residual infection or transmission by cross- contaminating trucking. To help distinguish between these two possibilities, virus isolates were obtained from all three states. Each isolate was analyzed by direct sequencing of the PCR-amplified gene for the immunodominant viral glycoprotein, gC, and by restriction endonuclease analysis of the complete genome. Preliminary cleavage patterns of the IL and IA isolates with KpnI and BamHI showed that 4/5 IL isolates were identical and 2/3 IA isolates were identical to the 4 IL viruses. Further endonuclease analysis was performed using 4 different nucleases (Alw44I, BamHI, SalI, and XhoI), followed by molar ratio labeling of the digestion fragments. Data were analyzed with a computer algorithm. An RNase cleavage assay was used to detect single base pair changes among the gC genes of different isolates. Temporal and animal trafficking patterns suggested a trucking connection among the three outbreaks. Results of gene sequencing, RNase cleavage assay and restriction endonuclease analysis confirmed that the outbreaks in the three states were genetically related. There was less similarity with virus isolates that had been previously circulating within the county. Minor differences suggested that some genetic changes had occurred as the virus moved from farm to farm. The molecular techniques complemented one another, but were not equivalent in detecting genetic relationships. The results indicate the utility of molecular epidemiology in tracking viruses and the importance of sanitizing trucks that contact infected animals.

Department of Veterinary Pathobiology, University of Illinois at Urbana-Champaign, IL

The Future of Diagnostic Laboratories in Surveillance: More than Just Sentinel Chickens

AAVLD Epidemiology Committee1,2,3,4

Surveillance is a component of most diagnostic laboratory mission statements, however the question of who benefits and who pays for surveillance programs remains controversial or unanswered. Surveillance is a strategically planned early warning system involving systematic collection, collation, analysis and interpretation of information about infection and disease, that provides immediate dissemination of the information to those responsible for prevention and control. Surveillance programs reliant on reports of laboratory data, where no action is taken to initiate collection of the samples are identified as “passive monitoring,” and the diagnostic laboratories that provide the information are often referred to as the “sentinel chickens” of the system. The term, though meant to emphasize the limitations of information derived from passive monitoring, tends to undervalue the considerable contributions of such programs. A review of the JVDI, AAVLD abstracts, and the AAVLD email list-serve show diagnostic laboratory surveillance to be an effective first step in a process to identify the animal disease problems that merit more detailed investigation. Diagnostic laboratories, as part of their routine work, have excelled in identifying infectious agents and toxicoses that are new, or of very low prevalence, but that have significant economic impacts. They have additionally provided an early warning system of public health threats and food safety concerns, by recognizing and reporting potential zoonotic agents in the animal population. The agents identified often have direct food safety concerns, or may serve as indicators of potential exposure of humans to pathogens, that may or may not cause significant clinical disease in the animal population. Similar early warning, based on recognition of changing patterns of disease, such as seen with antigenic drift of equine influenza virus or recognition of the pathogenic BVDV type II virus in cattle, has been credited with limiting the severity or extent of disease spread in animal populations. It is apparent that veterinary diagnostic laboratories have the key components of an effective surveillance system, including diagnostic curiosity, technical training and expertise, and the skills to identify new, emerging or re-emerging diseases. The current limitations, and source of considerable criticism of the existing laboratory-based surveillance programs, are the lack of a strategy or standard for use of the “passively” derived data. Existing laboratory-based surveillance could be significantly improved by addition of epidemiological techniques to identify and minimize sampling and misclassification bias, as well as to further develop hazard identification, risk assessment, and effective communication. Epidemiological approaches would strengthen and formalize the current surveillance provided by diagnostic laboratory data, and highlight the value of public investment in these programs.

1B. Akey, VA Department of Agriculture, Richmond, VA 2J. Case and S. Hietala, California Animal Health and Food Safety Lab, UC Davis, Davis, CA 3F. Elvinger, VMRCVM, Virginia Tech, Blacksburg, VA 4C. Munoz-Zanzi and M. Thurmond, Dept. Medicine and Epidemiology, UC Davis, Davis, CA

Cloning of Internalin A and Listeriolysin as Antigens in Listeria Serology

P. Boerlin1, F. Boerlin-Petzold2, and T. Jemmi2

Complex antigens used in the past for Listeria serology showed many cross-reactions with antibodies directed against other microorganisms and a poor sensitivity. Listeriolysin (LLO), the hemolysin of Listeria monocytogenes has been proposed as a more suitable antigen for serology of human and animal listeriosis. Internalin A (INL), another virulence factor in L. monocytogenes, also represents a potential antigen for serological tests. Hexahistidine-tagged LLO and INL was cloned in an E. coli expression system for large scale production and purification. Preliminary tests using immunoblots with bovine sera showed promising results. The aim of the present work was to develop a quantitative ELISA system for seroepidemiological studies with bovine sera. An antibody catching ELISA was developed using LLO and INL as antigens and monoclonal anti- bovine IgG labeled with alkaline phosphatase. Cut-off values were determined using ROC-curves based on sera from bacteriologically proven L. monocytogenes infections (encephalitis, abortion, mastitis) and a normal dairy cattle population. Reproducibility testing was used to define a “doubtful range”. The sensitivity for clearly positive results was 91% with INL and 46% with LLO respectively, while the specificity for clearly negative results was 78% (INL) and 71% (LLO). Thus, INL seems to perform better than LLO as an antigen for listeriosis serology, both in terms of sensitivity and specificity. Testing of 1657 sera from 113 randomly selected dairy herds in Switzerland showed that 4% of the animals were simultaneously positive for both LLO and INL. Fourteen percent of the animals were clearly positive against either LLO (11%) or INL (3%) alone. When testing sera from 31 animals with clinical suspicion of cerebral listeriosis, only 3 sera were positive for both antigens, 5 for LLO alone, and 1 for INL alone. Follow up of a successfuly treated case of cerebral listeriosis showed that antibodies directed toward LLO and INL may be only transiently elevated and rapidly decrease again to low levels. In contrast, antibody levels following a listeria abortion remained elevated for more than one year. In conclusion, INL may perform better than LLO in serological tests for the detection of listeria infections. Serological tests based on the detection of IgG antibodies directed toward these antigens may be of interest for the detection of previous listeria infections, including abortion and mastitis, but probably not for the diagnosis of acute cerebral listeriosis in cattle.

1 Institute of Veterinary Bacteriology, University of Bern, CH – 3012 Bern, Switzerland 2 Federal Veterinary Office, CH – 3003 Bern, Switzerland

Outbreak of Multidrug Resistant Salmonella typhimurium (DT104) in Cats in an Animal Shelter with Spread to Humans

R. K. Frank1, J. B. Bender2, K. Culbertson3, and K. Smith4

Salmonellosis was diagnosed at the Minnesota Veterinary Diagnostic Laboratory (MVDL) in 9 kittens from a humane society during September and October 1999. Humane society personnel submitted 8 of the kittens and a private veterinary practitioner submitted one kitten. The kittens were 6-to-14 weeks old (median = 8 weeks), had been housed at the humane society, and originated from multiple sources. Six kittens were adopted out, and 5 were returned to the humane society because of illness. Clinical signs included emaciation (6 cats), failure to thrive (4 cats), anorexia (3 cats), upper respiratory disease (2 cats) and depression (2 cats). Necropsy findings were enlarged and reddened mesenteric lymph nodes (6 cats), soft red intestinal contents (3 cats), subcutaneous edema (1 cat), reddened wet lungs (1 cat) and splenic infarct (1 cat). One kitten had no gross lesions. Microscopic findings included enteritis (3 cats), lymphadenitis (2 cats), and splenic infarct/necrosis (2 cats). Four kittens had concurrent infections: enteric coronavirus (3 cats), parvovirus (1 cat), E. coli (suppurative pneumonia; 1 cat), tapeworms (1 cat), and Bordetella bronchiseptica (from trachea; 1 cat). Salmonella typhimurium was isolated from intestine (9 of 9 cats), liver (6 of 6 cats), lung (5 of 7 cats), mesenteric lymph node (5 of 5 cats), spleen (3 of 4 cats) and kidney (2 of 2 cats). The National Veterinary Services Laboratory at Ames, Iowa confirmed the Salmonella isolates to be S. typhimurium. As part of a routine animal surveillance system for S. typhimurium, isolates were forwarded to the Minnesota Department of Health (MDH) for molecular subtyping and susceptibility testing. Isolates from kittens were indistinguishable by pulsed-field gel electrophoresis (PFGE; subtype TM254) and were resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (R-type ACSSuT). Phage typing performed at the Centers for Disease Control and Prevention identified phage type DT104. Immediately following characterization of the first 5 kitten Salmonella isolates, two human S. typhimurium isolates with the same PFGE pattern (TM254) were identified through routine typing at the MDH Public Health Laboratory. One of these human cases was from a household having recently adopted a kitten from the humane society. A subsequent investigation by the MDH linked 4 additional human cases of salmonellosis. In total, three children and 1 adult were from households who adopted cats from the same humane society. Another two children in a daycare home were infected indirectly from an infected child who previously had adopted a kitten. Following the identification of human illness linked to recent adoptions of kittens, MDH personnel obtained environmental and stool samples from housed cats at the humane society; no Salmonella sp. was isolated from any of the samples. In conclusion, multidrug resistant Salmonella typhimurium (DT104) infection was associated with severe illness in 9 cats from a regional humane society and 6 cases of human salmonellosis were linked to adoptions of cats from this source.

1Dept. of Veterinary Diagnostic Medicine, College of Veterinary Medicine, University of MN, St. Paul, MN 2Dept. of Clinical and Population Sciences, College of Veterinary Medicine, University of MN, St. Paul, MN 3Animal Humane Society, Golden Valley, MN 4Minnesota Department of Health, Minneapolis, MN Molecular Epidemiology of Mycobacterium avium subsp. paratuberculosis

S. R. Pillai1, J. D. Gummo1, E. C. Hue, Jr.2, D. Tiwari 2, J. R. Stabel3 , R. H. Whitlock4, and B. M. Jayarao1

A commercially available kit consisting of twenty 10-mer random primers was evaluated to allow selection of a suitable primer that would permit identification and subtyping of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) and Mycobacterium avium subsp. avium (M. avium) by Randomly Amplified Polymorphic DNA (RAPD). A primer OPE-20 (5'-AAC-GGT-GAC-C-3') was identified to be the most suitable primer when tested with 4 ATCC reference strains of M. paratuberculosis and 8 well characterized field strains each of M. paratuberculosis and M. avium. Primer OPE-20 was further tested for its ability to identify and subtype 200 field isolates of M. paratuberculosis . The fingerprint patterns of M. paratuberculosis (n=212) consisted of 5 unique common fragments (620- , 450-, 310-, 230-, 180-bp) and 9 variable fragments resulting in 6 distinct genotypes (Table1).

Table 1. DNA fingerprint profiles of M. paratuberculosis and M. avium using primer OPE-20 Organism Amplified DNA fragments (bp) Primary Variable Genotype Frequency (%) M. paratuberculosis 620,450,310,230, 1300, 1000, 850, 580 mp1 7.5 (n = 212) 180 1300, 1000, 850, 420 mp2 15.5 1300, 1100, 1000, 850, 730 mp3 34.5 1000, 850 mp4 23.5 850, 210 mp5 11.5 350 mp6 7.5 M. avium 620 650, 380, 310 ma1 12.5 (n=8) 1120, 1000, 850, 750, 650, 380 ma2 37.5 1300, 1120, 1000, 980, 910, 850, ma3 12.5 650, 480, 380, 240, 220 1300, 1120, 1000, 850, 750, 650, ma4 12.5 480, 380, 240, 220 1120, 910, 800, 750, 460, 380 ma5 12.5 1120, 460, 380 ma6 12.5

The DNA fingerprints of M. avium (n=8) consisted of a single common fragment of 620-bp, and 15 variable fragments resulting in 6 different genotypes. Cattle, human and goat isolates of M. paratuberculosis were genetically similar, but a sheep isolate had a different RAPD profile as compared to RAPD profiles from other species. RAPD was observed to be a rapid, reproducible and reliable technique for identification and subtyping of Mycobacterium avium subsp. paratuberculosis.

1 Pennsylvania State University, University Park, PA 2 Pennsylvania Veterinary Diagnostic Laboratory, Department of Agriculture, Harrisburg, PA 3 National Animal Disease Center, USDA, Ames, IA 4 University of Pennsylvania, School of Veterinary Medicine, Kennett Square, PA IS900-PCR Assay for Mycobacterium avium subsp. paratuberculosis from Quarter Milk and Bulk Tank Milk Samples Allows Detection of Herds with Johne’s Disease

B. M. Jayarao1, S. R. Pillai1, D. R. Griswold1, D. R. Wolfgang 1, L. J. Hutchinson1, C. M. Burns1, and C. A. Rossiter2

A total of 180 cows from 4 herds with known history of Johne’s disease were examined for M. paratuberculosis. Pooled quarter milk (50 ml), serum (1 ml) and fecal samples (10 g) were collected for analysis. Bulk tank samples (200 ml) were also collected and split into 4 samples (50 ml). Quarter milk and bulk tank milk samples were tested by IS 900-PCR assay and cultured also for M. paratuberculosis on Herrold’s egg yolk medium slants supplemented with mycobactin. Serum samples were evaluated for M. paratuberculosis by a Kinetics based ELISA (KELA). Fecal samples were cultured for M. paratuberculosis on Herrold’s egg yolk medium slants according to the Cornell method. The IS900-PCR assay was optimized to detect M. paratuberculosis directly from milk samples. The results of the study are summarized in table 1and 2.

Table 1. Detection of M. paratuberculosis by fecal culture, KELA, IS900 PCR and milk culture. Herd Samples A (n=91) B ( n=38) C ( n=29) D ( n=32) Feces 19(21%) 6(16%) 1 (3%) 1 (3%) KELA 15(17%) 5(13%) 6 (20%) 7(22%) Pooled Quarter Milk (IS900 -PCR assay) 28 (31%) 16(42%) 11(38%) 13 (40%) Pooled Quarter milk (Culture) 9 (10%) 0 0 0

Table 2. Number of samples positive for M. paratuberculosis by IS900-PCR assay and culture Herd Bulk Tank Milk A B C D IS900-PCR assay 4/4 3/4 1/4 2/4 Culture 1/4 0/4 0/4 0/4

It was observed that 68 of 180 (36%) pooled quarter milk samples were positive for M. paratuberculosis by the IS900-PCR assay, while fecal culture, KELA and milk culture detected M. paratuberculosis in 27/180(14%), 33/180(17%) and 9/180(5%) samples, respectively. Fifteen cows (8%) were positive by both ELISA and fecal culture, and 16 (8%) were positive by both IS 900-PCR from milk and fecal culture. IS 900-PCR assay allowed detection of M. paratuberculosis from 10/16 (63%) samples of bulk tank milk, while milk culture detected M. paratuberculosis in 1 of 16 (6%) samples. Preliminary results suggest that IS 900-PCR assay can be applied to screen herds using bulk tank milk samples and quarter milk samples to detect cows with Johne’s disease. The IS900-PCR assay needs to be further evaluated on both Johne’s positive and negative herds.

1 The Pennsylvania State University, University Park, PA 2Cornell University, Ithaca, NY

Geographically Targeted Survey of Cattle in Northeast Colorado for Evidence of Chronic Wasting Disease

D. H. Gould1, J. L. Voss2, K. I. O'Rourke3, M. W. Miller4, B. A. Cummings1, and A. A. Frank1

Chronic Wasting Disease (CWD) is a transmissible spongiform encephalopathy (TSE) that affects free-roaming deer in areas of northeast Colorado and southeast Wyoming. In the fall of 1998, a survey of adult cattle, geographically targeted to CWD endemic areas was initiated to evaluate the possibility of CWD being transmitted from deer to cattle. Livestock producers representing 22 ranches where cattle co- mingled with free-roaming deer cooperated in this survey. For cattle to qualify for inclusion in the project, they had to be at least four years old and have spent a minimum of four years in the herd. The surveyed cattle were older cows that were being eliminated from the herd due to age-related problems or not being pregnant. Prior to shipment for slaughter, individual animals were identified by unique ear tags. Submission information on each animal included age, years in herd, and grazing locations. After humane slaughter, the intact heads, including the attached ears and ear tags, were delivered to the Colorado State University Diagnostic Laboratory. The brains were removed and portions were preserved in buffered neutral formalin. After fixation, a wide variety of neuroanatomic sites were embedded in paraffin, sectioned at 5 micrometers, routinely stained with hematoxylin and eosin, and examined microscopically for the tissue alterations indicative of a TSE. The neuroanatomic sites examined included those sites commonly affected in the TSEs as well as uncommonly affected areas. Immunostaining with anti-PrP antibody was applied to sections of the commonly affected neuroanatomic sites in the medulla oblongata. Ultimately, 262 brains could be evaluated for the histological alterations indicative of a TSE. Analysis of all 262 brains failed to reveal any indications of CWD or any other TSE. Incidental findings in some brains included mild non-suppurative meningoencephalitis, neuronal lipofuscin accumulation, and occasional neuronal perikaryonic vacuoles in the red nucleus. Prion deposition was not evident immunohistochemically using a method with formic acid and proteinase K treatment prior to application of monoclonal antibody to the bovine prion protein F99/97.6.1. Thus, evidence of transmission of CWD from deer to cattle under free-roaming conditions could not be demonstrated in this group of cattle known to have lived in CWD endemic areas of northeast Colorado. Acknowledgements: This study would not have been possible without the cooperation of the many ranchers in the project area. Funding was provided by the Colorado Beef Board, Colorado Cattlemen’s Association, and CSU Anna Lee White Research Foundation.

1, 2Departments of Pathology and Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 3US Department of Agriculture, ARS, Animal Disease Research Unit, 3003 ADBF, PO Box 646630, Pullman, WA 4Colorado Division of Wildlife, Fort Collins, CO

Future Directions in Probabilistic Diagnostic Assessment as Illustrated by Use of Bayes Theorem in Diagnosing Bovine Viral Diarrhea Virus (BVDV) and Neospora caninum Infections

M. C. Thurmond1, C. A. Muñoz-Zanzi1, S. K. Hietala2, and W. O. Johnson3

Increasingly, clients and practitioners are requesting that diagnostic results be given as the probability that an animal has or does not have a specified disease or infection. Many of these requests refer to results of serology using a cutoff value designating animals as either 'seropositive' or 'seronegative'. Error and bias associated with selection of cutoff values, dichotomization of a continuous measurement scale, and failure to consider infection prevalence will contribute to problems of diagnostic interpretation and preclude assessment of infection probability. We present an approach that avoids use of a single cutoff value, thereby taking advantage of all information inherent in the continuum of serologic responses, and eliminates the need for sensitivity and specificity to define the test. The approach derives probabilities of infection, given a serologic result and herd prevalence. Probability functions of ELISA and SN values were developed for cattle determined by test criteria to be infected or not with N. caninum or with and without BVDV PI. The functions and herd prevalence were applied in Bayes theorem to derive the probability of infection for an animal with a given serologic result. As an illustration, for a BVDV PI prevalence of 0.5%, the probabilities of PI for cows with SN titers of 1:4 and 1:128 were 0.26 and 0.004, and for cows in herds with N. caninum prevalences of 10% and 60% and with an ELISA s/p of 0.40, the probabilities of N. caninum infection were 0.08 and 0.55. Probabilistic assessment can be applied generally to diagnostic medicine in the context of risk analysis and biosecurity.

1Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 2California Animal Health and Food Safety Laboratory, Davis, CA 3Division of Statistics, University of California, Davis, CA

Plenary Scientific Session B Monday, October 23, 2000 8:00 AM – 12:00 NOON Birmingham Ballroom – XI/XII

Expectations of AAVLD Labs in the Next Century “A special-focus Plenary Session”

INTRODUCTION: As we enter the next century, it seems to be an opportune time to pause and reflect on the changes that are happening around us and attempt to predict how they will specifically impact veterinary diagnostic medicine. Change is nothing new, but the rate of change seems to be ever increasing. There are numerous factors that have great potential to impact the “norm” in veterinary diagnostic medicine today. Some of the major factors include: • Changes in food animal production trends and in production systems • Changing demand for foods of animal-origin and global trade issues • International standardization of diagnostic testing • Enhanced public concern over food-safety, zoonotic diseases, and animal welfare • Changes in biomedical and diagnostic technology • Higher expectations in the areas of quality assurance and quality control • Changes in information exchange systems • Changes in the practice of veterinary medicine in North America

In the first half of this session, we have invited a panel of experts to update us on some of these timely issues. The second part of the session features leading practitioners from the major practitioner associations, sharing their viewpoints on future expectations of AAVLD service labs. The latter is our opportunity to hear directly from the customers we serve everyday. We hope this session helps prepare you and your lab for future expectations.

David H. Zeman Program Chair 2000

======Generous financial sponsorship of this special-focus plenary session has been provided by the following animal health companies:

• Pharmacia & Upjohn Co.

• Pfizer Animal Health

• Boehringer Ingelheim Vetmedica

• IDEXX Laboratories, Inc.

• Grand Laboratories Future Trends in Food Animal Production in North America

Maynard Hogberg, Ph.D., PAS Department of Animal Science Michigan State University

Food animal production in North America has been undergoing significant changes. Changes that we have seen in the past 10 years have far surpassed the total changes observed over the previous 40 years. These changes have come about through the impact of technology (how things are done), the impact of structure (how things are organized) and the impact of external forces. The rapidity and complexity of change is making it very difficult to accurately project what our animal agriculture industries will look like in 5 years let alone projecting further into the future. However, I will try to look at some of the critical trends and then cast some thoughts as where these trends and directions will take our food animal industries in the years ahead.

Our first trend is to look at the potential for growth in the consumption of animal products. World population is expected to increase to 7.7 billion people by 2020. The majority (95%) of this growth is expected to be in the developing countries where 77% of the current world population resides. In the CAST Report 135, it is projected that meat consumption in the world will more than double by the year 2020 as compared to consumption in 1983. This projected increase in meat consumption will be almost entirely in developing countries. Projected annual growth in meat consumption from 1993-2020 for developed countries is 0.6%/year while growth in developing countries is projected at 2.9%/year. Little growth is projected for the United States in meat consumption. If we are to increase animal agriculture in this region, there will be a need to export products. This will have an impact on how we produce livestock.

Impact of technology: Advances in genetics, nutrition, health and facilities have greatly aided in allowing farm sizes to grow to levels once thought impossible. We have seen a real revolution in mass- producing genetics in poultry and swine. These genetics gains have resulted in improved biological efficiency as well as an improved product for consumers. Like most changes, all problems have not been eliminated and all opportunities lost. The dramatic improvement in the leanness in swine carcasses has also lead to carcasses that have negative effects on consumer acceptance. We now get carcasses that are soft, pale and exudative, have low marbling, poor color and high drip loss which has lowered shelf life and created a product that is dry and less tasteful. Opportunities still exist when quality of final product is enhanced.

We have just started to see the impact of biotechnology on animal agriculture. Sexed semen and sexed embryos are likely to be available which will have a major impact on the dairy and beef industries. Identifying the genetic code of animals will allow us to select animals for disease resistance as well as for improving efficiency of production. Selection through the use of DNA markers will be commonplace in the future. Nutritional programs have been designed to more closely fit the biological needs of the animal with less need for over formulating as a safety factor. Enhanced digestibility of nutrients with products such as phytase and more accurate formulations has not only lowered costs but improved environmental impacts. Environmental nutrition will be a major emphasis in the future.

Improved health standards has lead to an increase in performance and a reduction in the use of antibiotics and vaccines, thus creating an opportunity to improve food safety on the farm. Computer controlled ventilation systems now more accurately provide the proper environment for animals raised in confined systems. This is one area where I would predict that we will see considerable emphasis in the future. We do not know the impact that the odors and gases in confined animal facilities have on the health of the animals and quality of products that they produce. Technologies will continue to push the cost of production lower and the quality higher. Successful farms will adopt new technologies quickly as they become cost-effective.

Impact of Structure: The adoption of biological and informational technologies have greatly improved profits which in turn has changed the face of animal agriculture at a rate that none of us would have predicted 10 years ago. Farms that raise livestock are becoming larger in size but fewer of them exist (Table 1). We have seen one firm gain control of 14% of the nations sow herd and pig supply. This implication goes even further when you consider that this same firm controls the slaughter capacity of 21% of the total hogs produced in the United States. Operations with more than 5000 head of inventory account for only 2% of all operations but 46% of the total inventory. Beef feedlots, broilers, turkey production and dairies are also concentrating in size at a similar rate. Merger mania has also hit the processing industry resulting in fewer but larger processors. In the beef packing industry, 83% of all cattle slaughtered are in the top 5 firms while the top 6 pork processors control 75% of the US market. There is no doubt that the food industry is consolidating. It is thought that as few as 5-6 firms will someday control the world food system from dirt to dinner plate or conception to consumption. It this good for American Agriculture and the livestock industry in this country? The world?

Marketing: What was normally known as an open market for commodity pork or beef is rapidly becoming a thing of the past. Contractual arrangements now include 75% of the pork produced, a trend that is rapidly following the broiler and turkey industries. Marketing programs have moved from being independent to being interdependent with alliances, contracts, consolidations and networks increasing at a rapid pace. Alliances and networks are seen as a means for independent producers to join forces to produce a product of demand and be able to collectively supply a volume of product that will attract an acceptable price. Today it is difficult to get a true price discovery for commodity beef and pork. We are rapidly reaching the point where products will be sold before they are produced. Niche markets are developing and offer an opportunity to firms or farms that can produce a differentiated product of quality that will sell that is difficult to duplicate by those involved in commodity pork, beef, poultry or milk production.

Availability of Labor: The availability of quality labor is becoming a major problem in larger livestock operations. Work conditions and benefits are changing rapidly in order to be competitive for the labor that is available. It is not unusual for farms to provide housing, health benefits, a retirement package, improved working conditions and many other incentives. All of this is driving up the cost of labor. Increased mechanization will happen as soon as technologies are developed to replace labor. It is not known if the shortage of labor at the farm is a result of the low unemployment in our nation and whether this will change when we have a down turn in the economy. If we are unable to find the necessary labor and can not improve the efficiencies sufficient to be competitive, we will be in danger of seeing our livestock production and processing industries move to developing countries where labor is cheaper and more readily available.

Impact of External Forces: During the past 20 years external forces have played an increasingly larger role on the production and processing systems of the livestock industries. Environmental issues are everywhere. Animal rights/welfare have greatly altered Europe’s livestock production systems and could have a major impact on North America in the future. Food safety is quickly becoming the emotional issues around the production and processing systems that exist today. All of these become even more important as the globalization of food systems progresses.

Environmental Issues: Animal agriculture is under tremendous pressure to create an environmentally benign system of production. CAFO’s, AFO’s and water quality are a major thrust of the EPA . Land use, water use, global warming and air quality are emotional issues often aimed at animal agriculture. It is paramount that animal agriculture aggressively addresses these issues in a pro-active manner. We need an environmental policy that promotes good stewardship and the commitment to designing and managing production systems in an environmentally sound manner. This policy must be based on sound science. I believe that we have the know-how to protect and improve water quality with large livestock systems. Less is known how to minimize odors from these large production systems in a cost-effective manner. A critical evaluation is needed for manure storage and handling technologies to make sure that the appropriate technologies are being used for the size and location. For example, lagoons have been a good technology for storage of manure in a cost-effective manner during the latter part of the 20th century. The question needs to be asked, can units become so large that this technology is no longer appropriate? Is there a size where the breaking of the lagoon wall will cause immeasurable damage to the environment? If the answer is yes (and I believe that it is) then the technology is no longer appropriate for that situation.

Animal Rights/Welfare: A real unknown is how our livestock production systems will be shaped in the future by the social concerns of the American public. Significant changes on how animals are produced have been activated in the European community. By and large, legislation in this country has been considerably less. Part of this may be due to the fact that we have seen changes being voluntarily implemented over the past twenty years, thus reducing the need as seen by the consumers to increase the amount of regulations. I feel that we must expand the research base and implementation in such areas of transportation, handling and slaughter. We must also develop and define better scientific measures to assess animal well-being, including stress, pain and behavioral needs based on sound science

Food Safety: We have been blessed with the safest and cheapest food supply in the world. We have far fewer illnesses and deaths due to food safety that in other countries. However, this is not enough and we will be challenging our production, processing and distribution systems to do even better. We have shifted to a more science-based system and implemented Hazard Analysis Critical Control Points program that relies more on microbial testing and verification of safety. This will quickly be pushed back to the production site. Already, many hog packers are requiring that hogs they purchase come from PQA III herds. It is easy to see that in the future, the consumer will know how the animal was produced, what antibiotics and vaccines that were used and the microbial history of the farm and processing plant when buying a steak to serve to their family. Farms and processors will be equipped with rapid detection devices that will allow them to constantly monitor their products. Herd health programs and monitoring systems will be mandated back through the system to insure food safety.

Globalization: As the globalization of the food system continues, this will drive the external force changes as described above. These will become necessary as a condition of trade. With the majority of growth in the consumption of animal products in the developing world, our survival will demand that we change to meet the needs and demands of the consumers worldwide.

Consumer Acceptance of Biotechnology: A real question is whether consumers, home and abroad, will accept food that has been produced with genetic manipulated organisms (GMO’s). This is a highly contested topic in some areas of the world where Europe and Japan have banned the importation of GMO’s. Will food produced using these new technologies be accepted? I believe that they will. A study by Roper Starch Worldwide, Inc. indicated that consumers were willing to accept biotechnology as long as the trade-off was in favor of reducing the chemicals to produce enough food for a growing population. The use of biotechnology to enhance the appearance or to simply increase the supply with no other benefits was less acceptable. This study indicated that we do need to justify the use of biotechnology in a manner that does have positive trade-offs if we are to gain consumer acceptance.

Table 1. Percent of U.S. Farms with Livestock

Species 1950 1964 1974 1992

Hogs 56.0 34.2 20.3 9.9

Dairy 67.8 35.9 17.4 8.1

Beef 75.5 72.3 44.3 41.7

Poultry 78.3 38.3 13.7 4.6

USDA Census on Agriculture

References: 1. Animal Agriculture and the Global Food Supply. Council for Agricultural Science and Technology, Task Force Report No. 135, July, 1999. 2. A2K Ag. Beyond 2000. Exclusive research with analysis by the editors of Beef, Farm industry News. Farm Press publications, Hay and Forage Grower, National Hog Farmer and Soybean Digest. Spring, 2000. 3. FAIR 2002, Animal Products for the next millennium - An Agenda for Research and Education. Animal Agriculture Coalition and Federation of Animal Science Societies. Spring, 2000. 4. Ritchie, H.D. The U.S. Beef Industry: Where Are We Headed? Nineth Alabama Conference for Food Veterinarians. February 4-5, 2000. 5. “1999 GAP Research: Consumer and Farmer Opinions About Food and Agriculture” conducted by Roper Starch World Wide, Inc. on behalf of the Philip Morris family of companies and The American Farm Bureau Federation.

Global Trade Issues and How Science and Politics Shape Animal Health Policies

William D. Hueston, DVM, PhD, DACVPM, Specialty Epidemiology Professor and Associate Dean, University of Maryland Campus Virginia-Maryland Regional College of Veterinary Medicine Avrum Gudelsky Veterinary Center College Park, MD 20742

The completion of the Uruguay Round of the General Agreement on Tariffs and Trade (GATT) heralded a new global trade environment. Decreasing tariffs placed added significance on health requirements as potential non-tariff trade barriers to the movement of animals and animal products. The GATT agreement on the Sanitary and Phytosanitary (SPS) measures was designed to ensure that health requirements are scientifically sound and justifiable. The agreement recognized an international animal health standards organization, the International Office of Epizootics (OIE), and established risk assessment as the method for summarizing the scientific justification for health requirements. Furthermore, the SPS agreement put forth the concepts of equivalence, harmonization, regionalization and transparency.

World trade issues concerning animal health have now reached the headline news. The World Trade Organization is embroiled in dispute resolutions on a myriad of issues from hormones in beef to salmon fillets; the OIE strives to meet the demands of their new status as the international standards agency; and producers and special interest groups alike protest what they perceive to be the inadequacies of the GATT. The clarion call for “science-based” health requirements is trumpeted by all sides of the issues, however, disagreements continue on the interpretation of scientific data, the application of risk analysis techniques, the implications of animal welfare and environmental impacts, and the appropriate and justifiable response of trading nations. The disputes also illuminate the dirth of animal disease surveillance data and the challenge of defining “acceptable risk”.

Selected current issues will be utilized to illustrate the complex and dynamic interactions between science and politics in the shaping of animal health trade policies at the global level. An understanding of the underlying scientific and political issues and an appreciation of the complexity of their interplay will increase the ability of diagnosticians to prepare for and meet the expectations of the next century.

The Role of the Office International des Epizooties in the Standardization of Diagnostic Testing

J. E. Pearson Office International des Epizooties, 12 rue de Prony, 75017 Paris, France

In 1949, the Office International des Epizooties (OIE) established the Standards Commission, the responsibility of which was to harmonize diagnostic methods and biological products. The standardization of diagnostic testing received new emphasis in 1995 when 125 States signed the ‘Final Act embodying the results of the Uruguay Round of the Multilateral Trade Negotiations’ concluded under the auspices of the General Agreement on Tariffs and Trade (GATT). The Round resulted in the formation of the World Trade Organization (WTO) and the implementation of the Agreement on Sanitary and Phytosanitary Measures (SPS Agreement) whereby the OIE is identified as the competent international organization for developing international standards, guidelines and recommendations relating to animal diseases and zoonoses. With the increase in trade in animals and animal products, standard setting by the OIE took on new significance. To meet the WTO requirements, testing that must be done to determine the health status of animals or animal products for international movement should be performed as described by the OIE. Monitoring and surveillance programs, to establish disease prevalence and/or freedom from disease, should also use OIE described standards. The following are the methods used by the OIE to help standardize diagnostic testing:

Manual of Standards for Diagnostic Tests and Vaccines: This Manual was first published by the OIE in 1989. The OIE is now preparing the fourth edition, which will be published in December 2000. The Manual provides standard diagnostic test procedures for the 15 OIE list A diseases, 65 list B diseases, and 13 other diseases. In addition, it contains seven introductory chapters providing general guidelines for diagnostic testing and the control of vaccine production. Each chapter is written and reviewed by a specialist. All chapters are submitted to the 155 OIE Member Countries, and many are submitted to the AVVLD, for review; the final version is approved by the OIE International Committee (IC). The Delegate to the IC is the Chief Animal Health Official from each Member Country.

Prescribed and Alternative Tests: The Standards Commission has developed a list of prescribed tests. An OIE prescribed test should be used if a test is required by the OIE International Animal Health Code to allow animals or animal products for international movement. The procedure that is used to perform the prescribed test is that which is outlined in the Manual. The tests are primarily to detect antibody to confirm that the animal had not been exposed to the agent. Recently, a polymerase chain reaction was approved as a prescribed test for bluetongue. A list of alternative tests is also provided; these tests can be used for testing within a local setting and for international trade by bilateral agreement between the countries involved.

Reference Laboratories: The OIE has established 118 Reference Laboratories for terrestrial animal diseases and 14 laboratories for aquatic animal diseases. These laboratories are located in 28 countries and specialize in the diagnosis of 46 terrestrial and 12 aquatic animal diseases or groups of diseases; 22 of these laboratories are located in the United States of America. The OIE Reference Laboratories provide reference reagents, training, and consultation to the OIE, and perform reference testing, organize scientific meetings, develop new methods, and co-ordinate interlaboratory collaborative studies.

Collaborating Centers: The OIE has nine Centers; five of these support standardization of diagnostic tests. Two of these Collaborating Centers are located in the United States of America. Reference Reagents: The OIE has approved Reference Sera for diagnostic tests for 14 diseases. These are to be used for the international standardization of diagnostic tests. Eleven of these diseases are endemic in the United States of America.

Quality Assurance Standard: The OIE prepared a Standard for use by diagnostic laboratories in developing quality assurance programs.

Guideline for Proficiency Tests: A guideline was developed by the OIE for use by Reference or National Laboratories in preparing and administrating proficiency tests. The National Veterinary Services Laboratories has used this standard for US proficiency tests. Future Expectations of Veterinary Diagnostic Laboratories: Quality Assurance Systems

Dale Polson, Wayne Chittick, & Dianna Jordan Health Management Center Boehringer Ingelheim Vetmedica Ames, Iowa

Introduction:

State and Federal Veterinary Diagnostic Laboratories (VDL’s) have served their food animal industry customers in North America very well for decades. VDL’s have traditionally provided a range of services designed to assist with government disease eradication programs, monitoring for regulated domestic diseases, monitoring for foreign animal diseases (FAD), and providing as wide range of ad hoc diagnostic services designed to determine the infectious, nutritional, and chemical causes of disease/health problems on farms.

As long as governmental disease eradication programs exist, ebb and flow, and come and go – Hog Cholera, Pseudorabies, and whichever disease is next in line – there will always be a need for some level of their related diagnostic testing. As long as animals move from state-to-state, and disease-related health certification laws governing such movement remain in place, there will always be a need for some level of related diagnostic testing. As long as there exist diseases we don’t have but don’t want to get (regardless of whether we’ve had them before of not), there will always be a need for some level of FAD surveillance and its related diagnostic testing. As long as there are production/performance problems that may involve disease agents, there will always be a need for diagnostic investigations and their related diagnostic testing. While there will always be an ongoing level of demand for these diagnostic services, the needs of the industries served by VDL’s are changing.

We have all observed over the past several years, and continue to see, the food animal industries in North America be dramatically transformed before our eyes – becoming more business-oriented, more technically-demanding, and more measurement-savvy. What do these “evolving” food producers and their veterinary advisors need/want from a Diagnostic services provider? What should Diagnostic service providers be doing for these customers now? What should Diagnostic service providers be preparing to do for these customers?

One need/want that has been clearly emerging is the need / desire to use diagnostic information beyond its traditionally qualitative application (positive:negative), and use it in a far more quantitative manner. We’re not simply talking about a herd/site being considered positive or negative (qualitative at the herd level) vs. estimating the within-herd/site prevalence (semi-quantitative at the herd level, but still a qualitative evaluation at the animal level). Rather, we are referring to the need / desire to use diagnostic information (e.g., detectable antibody titers, detectable antigen levels) to:

[1] Evaluate the type, timing, and level of disease exposure and nature of disease circulation within a population of animals and within a production system composed of multiple production stages and many sites; [2] Evaluate compliance to immunization procedures (whether by controlled exposure to endemic live organisms, or by use of commercial vaccine(s)); and [3] Assess the level of protective immunity (either by proxy indirectly and/or directly for the protective levels of humoral and/or cell-mediated immunity).

We would consider this need / desire appropriately referred to as the need for “health management measurement” rather than diagnostics, per se.

Quality control (QC) / quality assurance (QA) procedures and measurement have always been an important and integral part of VDL operations. Most current QC/QA procedures and measurement typically support the accuracy and consistency of qualitative diagnostic results. However, with the increasing demand / desire for health management measurement, QC/QA that supports only qualitative diagnostics are becoming increasingly inadequate.

Quality – What is it? And why do we care?

Some definitions are in order.

These definitions are taken from the teaching materials used at an AOAC International Short Course (September, 1996).

• Quality Control – “Quality Control comprises specific activities whose purpose is to monitor and control discrete laboratory tasks so that they meet predefined criteria.” • Control Point – “A location within a process or procedure at which a control measure can be exercised that will minimize chances for error.” • Critical Control Point (CCP) – “A location within a process or procedure at which the control practice can be measured and adjusted to eliminate, prevent, or minimize variation, and hence optimize analytical performance. • Assessment – “The act or practice of evaluating or appraising.” • Quality Assessment – “Quality Assessment comprises specific audit activities (both internal and external) whose purpose is to review the efficacy of quality control.” • Assurance – “Freedom from doubt or uncertainty…Assurance = ( Control + Assessment )” • Quality Assurance – “Quality Assurance is a SYSTEM of activities which allow an analytical laboratory to demonstrate that it is consistently providing services of definable quality to its clients/customers. Quality Assurance is a MANAGEMENT responsibility which ensures that appropriate quality control and quality assessment procedures are performed and documented in a dependable, timely, and economic manner.”

Quality Control does not equal Quality Assurance

• ISO 9000 Series Standards – “A set of guidelines for developing a documented quality system, encompassing the organizational structure, responsibilities, procedures, and resources necessary to implement quality management.”

Why should we / do we care about all of these aspects of “quality”? Why should we / do we care about developing, implementing, and operating effective quality assurance systems? Because our respective (and collective) futures as diagnostic service and health management measurement service providers depend on it.

As is the case with any service providing business in any industry, our viability as a business (and no matter how we look at it – we are each a business that serves customers) depends very much on the quality we deliver to our customers.

“Quality Assurance means never having to say you’re sorry!” AOAC International

But First, a “Reality Check”:

Before diving into any discussion of quality assurance systems and related topics, it is important that we take a “reality check”. The premise we are operating under is that what customers of veterinary diagnostic service and/or health management service providers increasingly want are:

• high quality (a.k.a., accurate, consistent, trustworthy) results they can be confident in • available in a convenient, understandable (a.k.a., easily accessible and quickly interpretable) form • made available in a timely (a.k.a. immediate, or the next closest thing) manner, • along with knowledgeable technical support that helps them better solve the problem(s) that prompted their “ad hoc” case submissions and/or better understand and manage the health of their farms and/or production systems.

Regarding the quality expectation – There are no “perfect” diagnostic assays. There are no “perfect” diagnostic laboratories. Not now. Not ever. Variation and error are universal and timeless. We must learn to live within these “facts” by minimizing their influence on what we do, and educating those we serve on how to optimize the value of the information we provide given their inherent, but controllable, limitations.

Regarding the form and format of results – Bottom line? Positive/Negative results are no longer good enough. Our collective clients/customers are demanding more from us in the area of quantitative health measurement – and we believe the reality is that we can learn to effectively, accurately, and predictably deliver it.

Regarding the timeliness of results – Our demanding clients/customers will accept immediate, but sooner would be even nicer. And not at the expense of quality.

Regarding supporting the information we provide – Clients/customers not only expect result data/information, they also expect us to know how to help them maximize the extraction of the knowledge these data contain.

Key QC Components for Veterinary Diagnostic Laboratories:

Obviously pursuing high quality is clearly a “good thing”. A key aspect to delivering high quality results is what we have in place for quality control. So what are the essential components of good QC? There are fundamentally two:

• Measurement • Responding to Measurement • Calibration • Maintenance • Records

QC measurement involves establishing a logical and systematic process of acquiring, running, and analyzing the resulting data from assay controls (e.g., positive, negative) and standards (e.g., high positive, low positive, suspect, negative).

Responding to QC measurement involves a process of equipment calibration (routinely evaluating equipment calibration and making adjustments when necessary), routine and regular maintenance of equipment, and maintaining good documentation of calibration and maintenance activities.

Not always considered in the same light as equipment, but no less important, is the “calibration” and “routine maintenance” of the environment within which all diagnostic/health measurement assays are run (temperature, humidity, dust, …), as well as the “calibration” and “routine maintenance” (via technical and procedural education & training) of laboratory personnel.

Elements of Quality Assessment for Veterinary Diagnostic Laboratories:

To regularly evaluate the “efficacy” of a quality control process, a quality assessment process is necessary.

Quality assessment fundamentally involves an auditing process of the site and its procedures, and encompasses the areas of:

• Facilities & Equipment • Environment • Methods & Procedures • As intended or designed • In actual practice (compliance) • Record Keeping • Validation (accuracy & consistency) of results • Training

Together, quality control and quality assessment processes comprise as system of quality assurance.

Quality Assurance (QA) – Read that “Continuous Quality Improvement (CQI)”:

What relevance does continuous quality improvement (CQI) have to do with a discussion on quality assurance for veterinary diagnostic & health measurement service providers? W. Edwards Deming pioneered the development of the philosophy and principles of a CQI system and, during his lifetime, applied this system in a wide range of manufacturing industries throughout the world. So what does that have to do with those of us in the veterinary diagnostic discipline & “business”? Only EVERYTHING!

If you examine the philosophy, principles, and methods which make up a CQI system, you will find that all the elements of “QA” are contained within “CQI”, and then some.

Models which attempt to define and describe a process for CQI are many and varied. However, they all (at least those that would be considered meaningful and appropriate) have several common process phases that are linked together in a continuous cycle:

[1] Measurement, [2] Problem identification and/or quantification of process capability, [3] Identification (diagnosis) of problem assignable cause(s) and/or reasons for an “incapable” process, [4] Resolution of problem assignable cause(s) and/or causes of an “incapable” process (either by intervention, redesign, compliance, or evaluation of benchmarks and a corresponding adjustment of targets & specifications).

In addition to its common elements with a system of QA, a CQI system has also critically important “human” aspects. For further evaluation of the “human” aspects of CQI, we would refer you to the many resources written by Deming, as well as many excellent resources written about the work that Deming pioneered.

Conclusions:

To return to where we began – with the question “What will be the future expectations of veterinary diagnostic laboratories?” – it is clear that is clients/customers are the ones who will be doing the “expecting”, and that what they will expect is for us to help them reach their health management goals – goals they design to support their business goals.

As clients/customers, they don’t exist to serve us and assure our survival. Rather, we exist to (and survive only if we) deliver what they need/want so they can survive and thrive in their respective food-producing industries.

Towards that end, the ongoing delivery of a high level of quality will be the “entry fee” required of everyone wishing to continue to exist in the diagnostic and health measurement business.

References & Worthwhile Reading:

Anonymous; Proceedings of Quality Assurance for Microbiological Laboratories – Short Course; AOAC International, Gaithersburg, Maryland; Orlando, Florida, September 1996

Neave, H.R.: The Deming Dimension; SPC Press; Knoxville, Tennessee; 440pp; 1990

Shewhart, W.A.: Statistical Method from the Viewpoint of Quality Control; Dover Publications, Inc.; New York, New York; 155pp ; 1939

Tribus, M.: The Germ Theory of Management; Exergy, Inc.; Hayward, California; 12pp; 1989

Wheeler D.J., Chambers D.S.: Understanding Statistical Process Control; 2nd Edition; SPC Press; Knoxville, Tennessee; 406pp; 1992

Wheeler D.J.: Understanding Variation – The Key to Managing Chaos; SPC Press; Knoxville, Tennessee; 136pp; 1993

Wheeler D.J.: Advanced Topics in Statistical Process Control; SPC Press; Knoxville, Tennessee; 470pp; 1995

Wheeler D.J., Poling S.R.: Building Continual Improvement – A Guide for Business; SPC Press; Knoxville, Tennessee; 320pp; 1998

Food Safety – Possible New Role for AAVLD Laboratories

Richard E. Breitmeyer, DVM, MPVM Director and State Veterinarian, Animal Health and Food Safety Services California Department of Food and Agriculture Sacramento, California

Production agriculture is facing many new food safety challenges. The expectations of safe food from “farm to table” are placing more responsibility and accountability on farmers and ranchers. The producer is being required by the marketplace as well as government regulators to implement and document new on-farm standards and programs. AAVLD laboratories are well positioned to assist production agriculture in meeting these new challenges.

Livestock and poultry are often the primary reservoirs of foodborne pathogens. However, these pathogens typically cause little or no clinical disease in animals. There is also concern about the use of antibiotics in food animals and that antibiotic resistant organisms can be transferred to people via the food chain. Routine surveillance programs through veterinary laboratories are an excellent means to detect new and emerging pathogens. Though we currently have limited tools to address foodborne pathogens on the farm, as new knowledge becomes available, early detection and intervention strategies will assist to reduce the load of preharvest pathogens.

Epidemiologic investigations of foodborne illness are advancing with investments in national programs such as FoodNet and PulseNet. FoodNet consists of active surveillance for foodborne diseases and related epidemiologic studies designed to help public health officials better understand the epidemiology of foodborne diseases in the United States. PulseNet is a national network of public health laboratories that perform DNA “fingerprinting” on bacteria that may be foodborne. The network permits rapid comparison of these “fingerprint” patterns through an electronic database at Centers for Disease Control and Prevention. As demands increase to trace additional foodborne pathogens back to the farm, animal health officials, in partnership with AAVLD, must develop concurrent technologies, strategies and policies. It will be necessary to correlate human, animal and environmental isolates in investigations and cooperate on methods and approaches to molecular epidemiology. The veterinary profession must address these and other on-farm food safety issues cooperatively with our public health partners to ensure reasonable goals and appropriate actions.

Preventing chemical contamination of meat, poultry and milk products is the responsibility of producers. In most cases, further processing will not eliminate chemical contamination of food products. Prevention of antibiotic residues, pesticides and toxins are priority issues. Producers must recognize their responsibility to safeguard food products. Immediate investigation and early diagnosis of toxicological events is mandatory to prevent contaminated products from reaching the marketplace. For example, milk from one dairy may be commingled in silos now larger than 200,000 gallons and fluid milk is on store shelves in less than 48 hours of leaving the dairy. Contaminated food endangers public health, can devastate a company economically and has far reaching negative consequences to an entire industry. Veterinary diagnostic laboratories play essential roles in addressing toxicological events, including surveillance and early detection, recommendations for prevention and treatment, laboratory methods development, and assessment of risks of food contamination.

Quality assurance programs for livestock and poultry are being developed to include food safety issues. Locally developed programs can provide the infrastructure for cooperation, within a state or region, among industry, government and academia. A state or regional veterinary diagnostic laboratory is a critical element of the quality assurance team. In order for producers to support a quality assurance program, there must be a perceived benefit for participation. An economic benefit is the most compelling incentive for participation that may include direct price incentives, increased market share or simply continued access to the marketplace. Enhanced status with regulatory agencies may be another incentive. Voluntary compliance may reduce the need for additional regulations or may demonstrate to regulatory officials that producers participating in the program are in compliance of certain described standards.

The participants of a quality assurance program may chose to include a certification component, often by a third party, to assure the marketplace or a government agency that standards have been met. Certification may also require sampling and laboratory analysis to verify the program. For example, in the California Egg Quality Assurance Plan, environmental sampling for Salmonella enteritidis is required and reviewed at time of third party certification by a Veterinary Medical Officer. Our veterinary diagnostic laboratories need to provide support for these certification programs.

Any laboratory protocol used to verify a food safety program must be standardized, and the laboratory performing the analyses certified. Nationally, we would look to AAVLD to provide guidance and support for developing the standards and certification protocols, in cooperation with the USDA National Veterinary Services Laboratory.

While the AAVLD leadership is needed to assist with food safety issues at the national level, many opportunities to support food safety activities will occur locally with individual laboratories. Directors and staff from each laboratory should be encouraged to contact their State Veterinarian, public health agencies, livestock and poultry commodity organizations, food safety researchers and others that are working on food safety issues with production agriculture. Consumer demands and regulatory pressure to address food safety concerns will continue to place more demands on production agriculture in line with policies to address food safety from “farm to table”. There are many exciting opportunities for AAVLD and its member laboratories.

(Note - California has enjoyed an excellent partnership, both financially and administratively, between the California Department of Food and Agriculture and the California Animal Health and Food Safety Laboratory (previously named the California Veterinary Diagnostic Laboratory System) for many years. Many of the comments and recommendations described here are based on our joint experiences gained in California and the opportunities we see in the future.) Profiles of Veterinary Practitioners in the Next Century

Lonnie J. King, DVM, MS Dean, College of Veterinary Medicine Michigan State University East Lansing, Michigan

Animal Health Diagnostic Laboratories (AHDL) are primarily in the service business. As such, the labs need to better understand the expectations and needs of new practitioners who will become their key users in the future. Without question, both the demands for services and the practitioners themselves will differ substantially from yesterday and today.

Obvious changes such as the percentage of women in veterinary medicine, the increase in companion animal interests, and the increase of new practitioners from urban/suburban settings have been occurring for awhile, however, the acceleration of these changes is particularly noteworthy. Yet, other changes will more profoundly affect the future of AHDL.

The level of sophistication and specialization will continue in veterinary practice. For example, over the last three years, approximately 20 percent of the new veterinary graduates from Michigan State University's College of Veterinary Medicine have opted for internships, residencies and/or graduate education. Specialization is driven by continuous breakthroughs in our knowledge and equipment and by clients who themselves are becoming more knowledgeable and sophisticated, as well as demanding and willing to spend more money.

Veterinary practitioners, more and more, understand the human-animal bond and its significance to their practices. There is now empirical evidence that supports the benefit of the bond on human health and wellbeing. New practitioners better appreciate this association and better understand that clients want options other than euthanasia for difficult cases. Oncology services are growing rapidly in veterinary medicine, as clients demand an extended and better quality of life for their pets.

There is also evidence that practitioners of the future may be involved with larger practices. The healthcare delivery system will need to shift from one and two person practices for a variety of reasons. These large practices will better market their services, become more specialized and may use associated services such as AHDL more readily. Practices will also look for savings based on economics of scale and will partner with other practices and/or demand contracts with AHDL for lower fees based on greater volume. For example, genetic testing and treatments are clearly on the horizon and will follow the lead of human medicine.

Diagnostic laboratories will also need to be more supportive and service-oriented with regard to an increase in more exotic pets. Pet birds, "pocket-pets" such as ferrets, reptiles and other exotics are more prominent today; yet, the profession has not adequately kept up with their special needs and medical care. AHDL will also need to adjust and prepare to serve this segment.

Practitioners in the next century will continue to work in an era of emerging diseases. Zoonotic disease and emerging infectious diseases will continue to be critical issues in veterinary medicine. AHDL's must be able to render diagnostic services in this area. There are both public health and legal implications involved in the rapid and accurate diagnosis of these diseases and a need to work closely with practitioners as partners in protecting the public's health.

New practitioners engaged in food animal practice will also change dramatically. They clearly will be viewed as a "business" partner and consultant to owners and producers of larger production operations. Although the projected growth in our food animal populations will not be robust, there will be more testing that will occur within these populations. Trade requirements, food safety, production optimization, environmental quality, and new demands by consumers and retailers for more marketable products are driving forces pushing the trend for more diagnostic testing. At Michigan State, we have just established five new internship programs in partnership with several large dairies in the State. The 4-year DVM program may very well need additional education and learning as an adjunct to our traditional educational component to truly prepare veterinarians for the rigors and demands of specialized food animal practice.

Technology will continue to alter our lives and our careers. In addition to more rapid, improved, and broader diagnostic services, AHDL must be able to communicate with next century's practitioners much differently. Our DVM students are more technologically competent than our faculty and staff. AHDL will have to exchange data and information with practitioners rapidly, conveniently, and effectively. Electronic consultation and easily accessible information are already expected. AHDL will need to be flexible, fast and must keep up with technological advances, both in equipment and in telecommunications.

With technological advancements, especially with regard to new databases and better standardization of tests, it will be easier to evaluate large volumes of data. Hopefully, AHDL and practitioners will collaborate more effectively and engage in clinical research and research and development activities. Our profession will only advance based on our research findings. Outcome assessments, disease prevention strategies, and improved decision-making on healthcare delivery options are all areas that can benefit from large prospective and retrospective studies. New graduates will be more open to participating in clinical trials and research.

Finally, AHDL will be under increasing pressures from competition derived from rapid, easily used, in- hospital test systems. The labs will also need to adopt better business practices. It will become more difficult to receive subsidies from government sources and cost-recovery and profit margins will become even more critical. It's not inconceivable that more consolidation of laboratories may occur and that private ventures will continue to both compete and cooperate with the AHDL of the future. New practitioners have grown up in this environment. Fees, services and quality will become more important than loyalty. Successful AHDL will be characterized as fast, focused, friendly, and flexible and will have different relationships with practitioners – intellectually, technologically and with regard to their service- orientation. What Future Swine Practitioners Need from Diagnostic Labs

Rick Tubbs, DVM, MS, MBA Green River Swine Consultation Bowling Green, Kentucky

Swine practitioners today and in the future need diagnostic results that are timely, accurate and inexpensive. Ideally, tests could be conducted at animal-side, would be highly specific and sensitive and would cost pennies to run. Swine production and breeding stock companies are large, have locations in more than one state or country and move animals and ship semen frequently. Shipments are often based on the results of diagnostic testing. Long lag times and inaccurate test results create havoc with sales and shipments. Currently, timeliness and accuracy outweigh cost in many situations but as the industry continues to mature and margins remain tight, costs will become a bigger issue. Many production companies have established in-house laboratories for routine testing and will expand in-house capabilities in order to have more control over turn-around times and costs. Diagnostic laboratories will find as have breeding stock companies, that their former largest customers are now their biggest competitors, as large production companies begin conducting more diagnostic testing internally. The scope of testing for specific projects will expand. The cost of being wrong will increase. Tables 1 and 2 show the amount of testing and the results for the initial stocking of a new genetic nucleus herd of 2700 sows established in 1999 and 2000. The aim of this project was to maintain freedom from APP, mange, PRV, Brucellosis, TGE, Leptospirosis, AR (toxigenic PmD) and swine dysentery and to eliminate via a MEW program PRCV, PRRS and Mycoplasma hyopneumoniae. Table 3 shows the results of PRRS testing for one specific group in this project. Table 4 shows the cost of closing a boar stud for 3 days due to false positive PRRS PCR tests.

Table 1. Diagnostic testing performed 1999-2000 for initial stocking of a 2700-sow genetic nucleus farm.

PRRS PCR 8500 (13,000 animals represented) PRRS ELISA 18000 APP types 1, 5 and 7 4000 TGE SN (PRCV) 1500 Pm T ELISA 650 M hyo DAKO 2100 PRV 14000 Brucella 14000 SIV (H1N1, H3N2) 500 Necropsies 400

Total 63,500

Table 2. Results of diagnostic testing described in Table 1.

All test results negative except PRRS • 9500 pigs produced • 31 nursery batches over 44 weeks • 7 grower batches • Number of rejected batches – 8 3 in the nursery (PRRS positive) 3 in the grower (PRRS positive) 2 in the grower (diagnostic concerns) • 3415 pigs moved to the GN farm

Table 3. Positive test results from the last 2 rejected batches.

No. Tested ELISA lab A ELISA lab B PRRS ELISA lab C IFA FFN 711 61/711 47/56 16/61 0/61 8/61

Table 4. Cost to close a boar stud for 3 days due to false positive PRRS PCR results.

Number of boars in stud = 490 Doses of semen sold per week = 12,000 Cost of semen at commercial level = $7.00 per dose Closure for 3 days = 7,000 doses x $7.00/dose = $49,000

In conclusion, swine practitioners need timely, accurate and inexpensive laboratory tests. Results posted to a web page or provided via e-mail as soon as tests are completed will be necessary. Lab support for development of in-house diagnostics will be necessary. Tests that detect specific agents or antigens as opposed to traditional antibody tests are needed. Continued development and application of biotechnology will be critical to providing timely, accurate and inexpensive tests.

What Future Bovine Practitioners Need from Diagnostic Laboratories

Robert (Bob) A. Smith, DVM, MS Diplomate, ABVP (Food Animal) Stillwater, Oklahoma

Many innovative diagnostic tests for various diseases or maladies have been introduced to the veterinary medical community during the recent past. As a result, bovine practitioners have enjoyed a wider range of diagnostic services and have received results on a more timely basis, while the information is still useful. As we enter the new millennium, the potential for diagnostics seem almost unlimited. From the bovine practitioner perspective, we have many expectations for diagnostic laboratories in the future.

Maintain the "Gold Standard": More and more diagnostic kits are becoming available to practitioners. Cow-side and chute-side diagnostics are attractive to the practitioner as test results are available immediately. These commercially available test systems will directly complete with diagnostic laboratories. Near term, much of the technology utilized for complicated diagnostic tests will be cost prohibitive for use in veterinary clinics, but scientific ingenuity will continue to create more and more testing systems affordable for the practicing veterinarian.

Diagnostic test kits intended for use in veterinary clinics often lack the sensitivity and specificity expected by the medical community. As a result, many are useful for screening or for development of a tentative diagnosis, but the diagnostic laboratory will remain as the "Gold Standard". Diagnostic laboratories accredited by the American Association of Veterinary Laboratory Diagnosticians provide test results that will withstand scrutiny by the scientific, regulatory and legal communities. It will be important to maintain funding levels to support adequate professional staffing and upgrading of equipment so that the diagnostic laboratory can continue to offer "Gold Standard" services.

Expand the Scope of Involvement: Traditionally, diagnostic laboratories have provided services focusing primarily on diagnosing diseases, largely those caused by infectious agents or toxins. The ever increasing concerns over water quality, environmental management and food safety offer many opportunities to diagnostic laboratories. Most laboratories already own expensive, modern equipment and employ professionals that enable the laboratory to expand their role in all of these areas.

One possibility would be to offer services to assist livestock operators monitor the effectiveness of their required Pollution Prevention Plan. These livestock owners are our clients, and we are being asked to provide input into their water quality and environmental management programs. Hazard Analysis Critical Control Points (HACCP) is already required in packing plants and food processing facilities in the U.S. and there are strong indications that similar process control systems will be commonplace on beef operations and dairies in the future. The diagnostic laboratory can provide third-party verification of the effectiveness of these food safety programs.

Become more Production Animal Oriented: Traditionally, diagnostic laboratories have focused on diagnosing causes of illness or death in individual animals. Today and in the future, more and more beef and dairy operations are increasing in size. Profit margins have narrowed substantially, necessitating more prudent business management. Increasing numbers of bovine practitioners are practicing production medicine.

While individual cow problems contribute to herd production problems, the diagnostic laboratory must be poised to help diagnose and solve production problems on a herd basis. Adding an epidemiology section to the diagnostic laboratory can help the production medicine veterinarian in the field solve herd production problems, track disease trends, make herd disease management decisions and better understand the cost-return relationships of management recommendations.

Be Innovative in Both Service and Communication: Historically, samples have been shipped by the postal service, package carrier or personally delivered by the veterinarian or producer. In this rapidly developing computer age, look for technology whereby the sample may be "submitted" locally over the Internet, using satellites or other modern means to the diagnostic laboratory. A current crude example is the transfer of electrocardiogram tracings from a rural hospital to a referral hospital miles away with a board certified cardiologist on staff to interpret it. In the future, perhaps "modules" can be leased to veterinary clinics whereby certain samples can be placed in these modules, and then data is transferred to a diagnostic laboratory for further analysis.

Develop Specialties in the Diagnostic Laboratory: While there are several routine tests each diagnostic laboratory should be able to perform, some tests utilize equipment that requires a huge capital investment, yet the demand is insufficient to generate income to offset this cost. Because package delivery services can move samples anywhere in the nation within 24 hours, many diagnostic laboratories can better utilize funds and professional staff by referring some tests to other laboratories. In essence, laboratories should avoid duplicating expensive, but seldom used services and invest the savings in areas where they excel.

Summary: The future will hold many challenges for the bovine practitioner and the beef and dairy clients they serve. Opportunities abound in food safety, control or elimination of zoonotic diseases, environmental management, and competing in the world trade arena. Terms such as HACCP, source verification, process verification, and total quality management are becoming commonplace in the beef and dairy industry. Expectations by consumers and regulatory officials will continue to increase.

Beef and dairy practitioners will need specialized partners, such as diagnostic laboratories, to meet these demands. Diagnostic laboratories must be poised to have the professional staff and equipment to be a part of these programs. Future Needs of Equine Practitioners for Veterinary Diagnostic Laboratory Services

Nat T. Messer IV, DVM Diplomate, ABVP (Equine Practice) Columbia, Missouri

Equine practitioners have some unique needs relative to diagnostic laboratory services simply because of the nature of equine practice itself. For the most part, equine practitioners examine and treat individual animals on a case-by-case basis, similar to small animal practitioners. However, equine practitioner’s patients are frequently members of a herd (eg: breeding farms, ranches, community pastures) or are housed in fairly close quarters with large numbers of other horses, some of which make up a relatively transient population of horses (eg: boarding stables, horse shows, racetracks).

These conditions allow for the transmission of infectious and contagious disease to occur with regularity in populations of horses. Thus the need for services related to rapid diagnosis of the common infectious diseases of horses is of paramount importance to equine practitioners. For example, such respiratory tract diseases as equine influenza, equine rhinopneumonitis, equine viral arteritis, Streptococcus equi var. equi (“strangles”), and Rhodococcus equi or such enteric diseases as rotavirus, Salmonella, Clostridium difficile (type A and type B toxin analysis), Clostridium perfringens (enterotoxin analysis), Erlichia risticii all require either prompt institution of therapy or methods to prevent spread of the disease to others once a specific diagnosis is made. Thus, validated, sensitive and specific tests for these diseases with a rapid turnaround time (<48 hours) are much needed and highly desirable. In many cases, tests have been developed, but they not readily available throughout the US, eg: PCR for Salmonella, PCR for Rhodococcus equi. In other instances, tests still need to be developed.

Based on an informal survey of equine practitioners via two equine oriented list serves (Equine Clinicians Network, AAEP), equine practitioners frequently requested more access to hormone analysis including inhibin, estrone sulfate, progesterone, LH, TSH, T3, T4, insulin, cortisol, and PTH. They also requested drug screening/testing panels for detection of medications in performance animals as well as therapeutic monitoring for serum levels of antimicrobial drugs. Nearly all equine practitioners requested a more reliable test for equine protozoal myelitis.

One veterinarian had a very interesting request that may or may not fall under the purview of a diagnostic laboratory. That request involved a “diagnostic support service” for such things as radiographs, ultrasound images, biochemical profiles, electrodiagnostics, etc. where the practitioner would submit a case profile (history, physical examination, diagnostic tests) to the diagnostic laboratory and obtain diagnostic support in interpretation of the specialized diagnostic tests as they relate to that case. Academic clinicians and private practitioners in referral hospitals frequently perform this function for colleagues, but this individual envisioned the diagnostic laboratory having a “diagnostic support team” that would perform this function upon request.

Expectations of AAVLD Labs in the Next Century: The Small Animal Practitioner’s Perspective

Paul Glouton, DVM Lilburn, Georgia

Small animal diagnostic laboratory needs are directly linked to what our clients will expect from us in the future. What will the client of tomorrow expect from their veterinary health care facility? What will be the barriers in permitting small animal practices from delivering the type of services that clients would like to have available? How can laboratory diagnosticians help us to overcome the barriers and enable us to provide the types of services clients would like to utilize? What are the services small animal practitioners will need from laboratory diagnosticians in the future?

If we can answer these questions and deliver these services in a cost effective manner, we can meet our clients’ needs, enhance our patients’ well being, and improve our staffs’ feelings of accomplishment along with improving our financial health and that of our staff and suppliers.

What will the client of tomorrow expect and want from a modern veterinary facility? Several recent surveys have shown that at least 70% of pet owners consider their pets as part of the family. Surveys also have shown that clients are willing to spend money on their pets with 50% of them willing to spend up to $1,000 on a major illness or injury. Clients are watching cable television shows that depict very sophisticated veterinary care. They are bombarded with programs that not only educate them regarding the availability of a variety of pet related medications, but more importantly reminds them of the bond they have with their pet and the importance their pet plays in their daily life.

Clients who consider their pet as part of their family now and more so in the future will expect their animal health care facility to provide cutting edge preventative health care, non-invasive diagnostics to diagnose maladies early in their course, and be capable of providing the most up to date care available anywhere in the country. They will not be content with “shotgun medicine” but rather will insist on a specific diagnosis and appropriate therapy for the disease. Even if they are located far from a major animal health care center, they will still expect the most up to date diagnostics and treatment modalities to be offered for their pet.

The myriad forms of communication and especially the Internet has dramatically altered clients’ perception of what they consider appropriate medical care for their four-footed family members. Tomorrow’s client will certainly expect a much higher level of sophistication than yesterday’s client.

Tomorrow’s client will also expect more convenience. They will expect a rapid accurate diagnosis of illnesses. They will want access to 24/7 care. They will not be content to wait several days for answers nor will they be happy if they have to make multiple trips to the hospital to solve their pet’s minor illnesses.

The client of today and more so the client of the future will demand more non-invasive diagnostic procedures for their pet. Endoscopies, bronchscopy, laproscopy, radiography, ultrasound and ultrasound guided biopsies; along with echocardiography and increased use of cytology and biopsy will be the accepted standard of diagnostics.

Clients will expect the same level of intensive and critical care management of complex maladies as they expect from their own physicians. Chemotherapy for oncology cases and immunology disorders will become a commonplace procedure in large multi doctor practice settings. Sophisticated diagnosis, monitoring, and treatment will be the standard of care for many disorders that today we think of as non- treatable.

More primary care veterinarians will be practicing in large multi doctor facilities. Emergency centers, critical care centers, and boarded specialists will play a much larger role in the delivery of everyday animal health care. Our knowledge and abilities will dramatically improve both the quality and the length of our patients’ lives.

What are the barriers veterinary practitioner managers and practitioners face in delivering an increasingly sophisticated level of care in a traditional practice setting? There are many as you might imagine but they are not insurmountable. The advances in medical discoveries and technology in the recent past and those certainly to come in the future may make the impossible of today the standard of care for tomorrow.

A few of the barriers faced by small animal health care providers in delivering the type of sophisticated animal health care envisioned will include the following:

• Trained support staff needed for sophisticated medical care • Accurate laboratory capabilities with rapid turn around times • Access to specialists for timely second opinions or consultations • Lack of veterinarians with hands on experience in sophisticated diagnostic modalities • Initial capital expense and maintenance expense of non invasive diagnostic equipment • Consolidating a fragmented animal health care delivery system • Veterinarians reluctance to charge for sophisticated medical care • Financing options for clients unable to pay large bills in lump sum

How can the American Association of Veterinary Laboratory Diagnosticians, its members and veterinary practitioners work together to overcome these barriers and help us to provide the services of tomorrow’s client? Many of the barriers may seem outside the scope of a veterinary laboratory diagnostician’s area of expertise but if one thinks out of the box, a solution may be attainable.

Lack of trained support staff is the major barrier in improving the practitioner’s level of sophistication for delivering animal health care. This directly impacts our needs from diagnostic laboratories. By providing training aids regarding diagnostic protocols and procedures, along with on site, Internet, and remote training sessions, laboratory diagnosticians can play an important part in alleviating a major bottleneck in improving service to our patients. The majority of this training should be geared towards the veterinary assistant staff members.

Practitioners face the decision on how much laboratory testing needs to be in house and what can be outsourced to an outside laboratory. Turn around time frequently dictates that in house testing is chosen over an outside source. Accuracy is of paramount importance. With a critical shortage of trained veterinary technicians, it would seem prudent to utilize the services of an outside lab with a board certified veterinary pathologist as the medical director. However, in many locations, turn around time for samples for an outside lab dictates that practitioners provide in house CBC, urinalysis, biochemical profiling, along with Elisa snap testing.

Outside labs must work to reduce turn around time to increase their attractiveness to practitioners. Enabling remote access to patient test results via the Internet or computer improves accessibility and efficiency. Rapid access to clinical pathologists for consultations regarding lab results will become increasing important as we become more sophisticated in the future. Laboratories and pathologists who are adept at interpreting micro samples will become even more important as more practitioners move towards non-invasive diagnostic techniques and are more focused on a definitive diagnosis for their patients.

Providing information and recommendations on in house laboratory equipment could be a tremendous service to the practitioner. The practitioner does not possess the expertise to know what criteria are important in selecting equipment other than how fast and what tests are available. Assisting the practitioner in facilitating how they can best provide appropriate laboratory testing capabilities utilizing both in house testing and an outside lab could be a tremendous service laboratory diagnosticians could provide.

Using the Internet for transmission of images has already become commonplace. Bringing specialists into the practice via remote image technology can be a way for the everyday practice to step up their level of care relatively easily and inexpensively. By embracing the technology, laboratory diagnosticians and their business organizations can play a major role in veterinarians providing more of a team health care approach utilizing the most up to date diagnostics and treatment modalities, while also keeping the small animal practitioner in the smaller practices current with newer techniques. Emailing interesting cytology images to referring practitioners and accepting email images of lesions could be both useful and educational along with increasing the yield of submitted samples.

Using electronic transmission for submitting sample data, images and patient information would increase efficiency and data recoding at both the practice and the lab sites. Reporting lab results in an electronic format that could be recorded in the electronic patient record could also increase efficiency for tomorrow’s paperless practitioner. This will be especially applicable for practice entities owning multiple sites and those that need to access patient information at remote sites.

Both the equipment and the service must be cost effective to be utilized. Every practice may not need remote capabilities or the very sophisticated expensive equipment capabilities. Courier services of existing labs could pick up samples to be taken to remote lab sites for preparation and image transmission. Specialists visiting remote sites on a periodic basis to provide diagnostic assistance, such as many areas currently provide for ultrasound and echocardiograph capabilities could be a very useful service.

Training at less than a board certified level for veterinarians on sophisticated diagnostic modalities could be useful and improve the level care for future practitioners. Providing brief sabbatical experiences would not only raise the level of veterinary care for the future but also raise the self-esteem of the veterinarians participating in submittals.

Teaching veterinarians to charge for the increasing level of sophisticated care remains a major stumbling block towards a successful future for the veterinary medical profession. Increased use of programs such as AAHA’s Art of Practice need to be expanded to teach veterinarians self worth and the value of their services. Laboratory diagnosticians are a very small part of this equation. However, to reach the future that our pet owners envision, we should all encourage charging a fee that includes a profit for the practitioner, the lab, and the manufacturer. Without profit there is no reason for any business to invest in new procedures. An efficiently run veterinary practice has a twelve per cent profit. Very few veterinarians providing quality veterinary care are making large profit percentages. It is one of the major problems facing our profession. Profit is not a dirty word!!

Finding innovative methods for clients to pay for sophisticated services will continue to be a problem for tomorrow’s veterinarian. Clients keep telling us in surveys that they are willing to pay more for veterinary services than we are charging. However, they do need payment plans. Third party financing along with insurance will become a much larger part of the veterinary business for tomorrow’s veterinarian. Laboratory diagnosticians probably play no role in this arena but it is important they understand it is a critical aspect of the business chain if they are to be successful in collecting their fees from veterinarians.

Specific tests that small animal practitioners would like to see for the future implementation would include the following: • More and better rapid immunological based tests for specific canine and feline diseases • Improved serum skin testing capabilities • Accurate electrolytes dry chemistry testing capabilities • Improved methods for detecting metastasis cancer

By looking into the future to envision what the end client will want and realizing the barriers faced by practitioners, the American Association of Laboratory Diagnosticians and its members can provide the diagnostic services to attain the goal of improved care of our four-footed friends.

Maximizing Our Service to the Public by Thinking Out of Our Traditional Boxes

David H. Zeman, DVM, PhD President-Elect, AAVLD Head/Director, Veterinary Science Dept./Animal Disease Research and Diagnostic Laboratory South Dakota State University, Brookings, SD 57007

There are currently 36 AAVLD accredited veterinary diagnostic laboratories in the United States and two in Canada. These public laboratories comprise an extensive animal health support system that has proven itself essential to animal owners and allied industries. AAVLD laboratories work closely with referring veterinary practitioners, state and federal regulatory officials, and animal owners. Together these groups have been successful in maintaining large populations of relatively healthy and productive animals, while providing a safe economical and abundant supply of foods of animal-origin.

However, dramatic changes have been occurring in recent years. These changes have already been impacting veterinary diagnostic labs and will continue to alter what we might currently consider standard operating procedures in our labs. Major change-factors include: • Changes in food animal production trends and in production systems • Increasing world-wide demand for foods of animal-origin • Global trade issues • International standardization of diagnostic testing • Enhanced public concern over food-safety and zoonotic diseases, driven by a seeming desire of the public to be 100% free of risk • Enhanced concern over animal welfare; including scrutiny of the traditional way we raise food animals • Elevation of the status of our domestic pets and higher expectations regarding their care • Increased sophistication of biomedical and diagnostic technology; the molecular era • Higher expectations in the areas of quality assurance and quality control • Improved information exchange systems; • Elevated public expectations that everything can and should be done extremely fast • Downsizing of federal and state government; trends toward privatization where practical • Diversification of economies in many states that were previously agriculturally dominated • Changes in the practice of veterinary medicine in North America

As we enter the next century, it seems to be an opportune time for AAVLD laboratories to pause, reflect, and listen to our customers. As professionals in public service laboratories, we have dedicated our talents and careers to serving the best interests of the public. Are we on track? Are we adjusting and fulfilling our leadership responsibilities relative to veterinary diagnostic medicine and animal health? The purpose of this special-focus plenary session is to aid us in this evaluation. As I attempt to digest what our future- session speakers, have shared, I will first make some general statements regarding past and current activities of AAVLD veterinary diagnostic labs in the United States, and then speculate on our future opportunities.

Our Roots: Since most public veterinary diagnostic labs are part of individual state government systems, it is not surprising their origins and lines of accountability are highly variable. Many labs can trace their diagnostic service roots back to the 1800s. For example, the South Dakota lab in Brookings has been providing diagnostic services since 1887, shortly after the origins of the state’s first land-grant university (currently South Dakota State University). One early common thread was that veterinary diagnostic labs were born out of the necessity to identify, monitor, control and hopefully eliminate major diseases of domestic animals, especially livestock. As our nation’s human population grew during the 20th century, ensuring a safe and stable food supply (including our food-animal supply) was considered necessary by state governments and they funded our activities. State government support was essential since the work required a local laboratory; i.e. efficient overnight shipping was not yet available. Close working relationships with state agencies such as the livestock sanitary boards were typical. In fact the initiation of many state veterinary diagnostic labs was at the request of state veterinary regulatory officials (State Veterinarians) to aid them with their mandated tasks of monitoring and controlling diseases of animals within their states. Federal agencies were charged with the same for the nation and tackled large problems such as Brucellosis, Tuberculosis and Contagious Bovine Pleuropneumonia.

Laboratory administration varied from state to state. Some labs arose as part of the university system, typically at the state agricultural college, and thus reported to the Dean of the College of Agriculture. While other labs became affiliated with state agencies and thus reported to the Secretary of Agriculture or the State Veterinarian. The mission of veterinary diagnostic laboratories also varied somewhat in these different administrative settings. Some department of agriculture labs were more restricted in the scope of their mission; for example non-livestock cases may have been discouraged or were charged a higher fee. In general, taking care of our food supply was considered essential but taking care of pets and other animals may have been viewed as more of a luxury. Again, such rules and roles varied from state to state and even today change from time to time with the political winds.

University labs were typically located at land-grant universities, within Colleges of Agriculture. Most were affiliated with a veterinary science department or a school of veterinary medicine. University labs often had fewer restrictions relative to the types of animals they serviced, taking a more holistic approach to veterinary diagnostic medicine. As veterinary teaching-hospitals grew in popularity, a broader variety of animals were submitted for necropsy and diagnostic services. This resulted in the rapid accumulation of disease information in species other than livestock.

Regardless of institutional affiliation, all veterinary diagnostic labs were directed to make rapid and precise diagnoses, as well as discover new disease syndromes. In addition, university veterinary diagnostic labs became involved with research projects in the traditional areas of pathology, microbiology and toxicology. Research was also directed at improving test methods, reagents or vaccines. University laboratories also assumed and carried out the essential function of training future veterinary diagnosticians in the traditional specialties.

After World War II, the country grew rapidly and by the 60’s the need to modernize and/or expand veterinary diagnostic laboratories became necessary in order to handle the growing demand for diagnostic services. A definite trend by veterinary practitioners and animal owners was the desire to discover precisely what disease they were dealing with, rather than making assumptions based on clinical signs. Many states improved their physical facilities during the decades of the 60s, 70s and 80s. The caseload of most labs increased steadily during the same period, with the usual fluctuations associated with livestock market prices. During the 90’s caseloads have remained steady or grown slightly, with some states experiencing major shifts reflected by the changing livestock industry, especially the swine industry.

Our Present Status

Currently there are 38 AAVLD accredited veterinary diagnostic laboratories in the US and Canada: • 16 are affiliated with universities, and located at or near colleges/schools of veterinary medicine • 10 are affiliated with universities, but not located at colleges/schools of veterinary medicine • 12 are affiliated with state government (such as state departments of agriculture)

The AAVLD is composed of approximately 1200 members representing numerous specialties, such as pathology, clinical pathology, bacteriology, virology, toxicology, and epidemiology. In addition numerous support personnel bring expertise in areas such as administration, clerical, accounting, computer support, animal care, and maintenance.

University labs are heavily involved in research and professional training, as mentioned above. University employees frequently carry joint responsibilities in their individual positions (such as diagnostic service, research, teaching, extension, etc). As a diagnostic system, all species of common animals are being served. There is still a heavy emphasis on food animals. There is also still a heavy emphasis on postmortem testing (reactive diagnostics) versus antemortem testing (proactive preventative diagnostics). However, these trends certainly began to change in the 90s, especially in certain species. The strong antemortem trend seems to be driven by the fact that more animals are at risk in larger units and thus the cost of introducing disease into “clean groups” of animals is higher. Current disease management practices in large production units seem to drive a hunger for more diagnostic data (both postmortem and antemortem). Cutting-edge practitioners are applying epidemiological principles to their monitoring strategies in an attempt to contain diagnostic costs and improve test interpretation.

Advances in biotechnology gained during the previous 20 years are being applied in diagnostic labs. Many advances have resulted in better tests with improved sensitivity and specificity. Yet, even these advanced tests have practical limitations. Picking the correct diagnostic strategy, test cost and test-speed are important factors in diagnostic decision making. The correct level of sensitivity is now a consideration for ultrasensitive tests. Using the correct test to match the desired question is increasingly important since test options continue to increase.

Applying new technologies to the routine business of veterinary diagnostic medicine has required expanding, upgrading, or constructing new facilities. New technology often comes with the need for more bench space. In addition, whenever remodeling or new construction occurs, labs must consider improvements for employee safety and environmental impact. Such necessary improvements greatly add to the construction costs of new biomedical laboratories of all types.

Besides applying new technology to what we have traditionally done, broader roles are being defined. In recent years, more labs have moved into or expanded their activities in the areas of companion and exotic animals, wildlife health, genetic testing, public health (zoonoses) and food safety. Some labs have even changed their names to better reflect these expanded roles (for example the “California Animal Health and Food Safety Laboratory”). All seem to be legitimate components of veterinary diagnostic medicine.

Quality assurance and quality control issues have been increasingly emphasized over the last 10 years. There is a growing expectation from the public that whoever serves them does it perfectly, with little sympathy for errors. Many labs have made significant strides towards formalizing their QA/QC procedures, but much more needs to be done across the system. The OIE has been given the charge to standardize procedures among nations and will thus have significant influence in labs that conduct required exportation tests.

Our Future Opportunities

Is the glass half-full or is it half-empty? Looking towards the future can be unsettling or invigorating, depending on one’s perspective. Regardless of personal perspectives, most will agree that it is better to be prepared than to be surprised by the future. It is better to mold, rather than be molded by chance. Looking back to our roots showed us that society previously valued the gathering of information that would support animal health in species considered essential to them (food animals at least). Will society in the future fund a veterinary diagnostic lab system to support the health of animals that they consider essential or at least important to them? I think the answer is YES and every time we hear of another public diagnostic lab being newly constructed, renovated or expanded, we have received affirmation that society has again agreed.

An accurate forecast of the future for AAVLD labs depends on the answer to two major questions: Is our service considered valuable to society? Is the best interest of society served by funding non-biased public laboratories? Each of us can answer these questions based on our own experiences with the following: • Does society value a stable, abundant and economical food supply? • Does society value safe food? • Does society value companion animals? • Does society value wildlife? • Does society value public health, as it relates to interactions with animals?

I believe the answers to all of the above questions are definitely YES, and that the scope of the value is rapidly growing in all categories. As I read the news over the past several years, I sense: • Society expects a stable economical food supply, even as the world rapidly grows • Society expects an absolutely safe food supply • Society expects a high quality support system for their companion animals (this involves the DVM practitioner and what they need to do their job = Dx labs). • Society considers wildlife a public treasure, and they expect support for wildlife health issues • Society expects support systems to deal with public health issues that arise from their interactions with animals (zoonotic diseases)

Based on these values and expectations, I believe there are numerous new opportunities for AAVLD veterinary diagnostic labs to maximize their services to the public. I believe that as long as the economy allows, society will be willing to fund our activities in the traditional areas we have always served, as well as in areas I call new opportunities. I am also convinced that public laboratories serve the public’s best interest. AAVLD labs have already accumulated the professional expertise and the physical infrastructure, and have the operational mechanisms in place to serve the public efficiently and economically. Expanding into new areas of opportunities merely increases our value to society, for a relatively small amount of additional overhead.

Here are the best opportunities for expanded diagnostic service from my perspective:

1. Expanded Food Animal Test Services: The demand for more antemortem testing services continues to grow. New technologies have created new opportunities for diagnosticians to assist food animal practitioners with proactive monitor and surveillance programs. Such testing provides critical base-line surveillance data for regulatory officials as well; an ever-increasing issue relative to global trade and emerging diseases. Cooperative alliances with state and federal agencies are as essential as ever. New test technology includes genetic testing to identify traits related to specific problem diseases. In addition, the quality and thus the value of traditional postmortem test services will benefit from new testing technologies.

2. Food Safety: Veterinarians have been involved with food safety programs involving foods of animal-origin from the beginning of modern veterinary medicine. Therefore, it seems quite logical that DVMs and veterinary diagnostic labs are key to future food safety solutions. This will not only involve slaughterhouse issues, but monitoring and surveillance of live animals and their environment. Many labs are already heavily involved with food safety testing. The expertise, equipment and facilities are already in place within veterinary diagnostic labs. It seems that establishing a separate system of lab facilities across the nation just to handle food safety would be redundant. In my opinion, AAVLD labs should step-up and maximize our service to the public by providing these services.

3. Companion Animal, Equine, Aquaculture, and Wildlife Health: Agricultural support has been key to political support and thus key to strong financial support for many AAVLD labs over the years. As many state economies have become more diversified and as the number of people running agriculture have dramatically declined, we essentially have significantly fewer voices to politically support and appreciate our efforts. For most labs, it is essential to maintain our important services to and liaisons with agriculture. However in these changing times, it is also necessary for us to gather all friends of animal health including food animal producers; companion animal owners; equine owners; aquaculturalists; wildlife managers; public health allies; and food safety advocates. We must leverage the combined political influence of these allies to maintain the funding necessary to do our traditional jobs and to be able to expand our efforts towards new opportunities for service. I am convinced we are well qualified to serve in these additional areas if adequately supported. Not only does it make sense professionally, but it also makes sense economically to fully utilize our expensive facilities for the maximum public good. This transition may require hiring some professional staff with specific species expertise.

4. Public Health – Zoonoses: AAVLD laboratories are professionally staffed and equipped to handle all animal health issues, including testing for zoonotic diseases such as rabies. Zoonotic disease is a legitimate component of diagnostic veterinary medicine. AAVLD labs must be ready, willing and prepared to step-up and offer their expertise to their states regarding these sometimes “hot issues of zoonoses”. Being on the cutting edge of our expertise should prepare us well to serve the public, whether it be rabies, cryptosporidiosis, or West Nile Virus. New diagnostic labs should be built with this potential in mind. Our willingness to come forward and be involved will only increase our value to the public.

Forecast

Future forecasting is risky business. Perspectives can widely vary among individuals while looking at the same data. Some may be overwhelmed by changes and see a dark future no matter what. Some may be having so much fun with all of our high-tech gadgets, that they blindly think the future is bright no matter what. I close with these key thought about the future. • The future can be molded. Frequently, the future is what we make it. • We must continue to provide the high quality trusted traditional services the public has come to rely upon during the past century. • In addition, we must be willing to apply our expertise and our facilities to new areas of service. • I believe that AAVLD laboratories are poised for a bright future, if we are willing to maximize our service to the public by thinking out of our traditional boxes!

Coronavirus Associated Epizootic Catarrhal Enteritis (ECE) of Ferrets

M. Kiupel*1, B. H. Williams2, J. T. Raymond1, C. K. Grant3, and K. H. West4

Since 1993, epizootics of a green mucous diarrhea have caused significant morbidity and variable mortality in ferret breeding colonies and rescue operations throughout the U.S. and Canada. Necropsy material from naturally infected ferrets from a wide range of sources was examined in order to identify an etiologic agent as well as to elucidate pathologic lesions associated with this disease. Characteristic microscopic lesions consistent with coronavirus infection in other species were consistently present in affected ferrets and included vacuolar degeneration and necrosis of villar enterocytes, and goblet cell hyperplasia in acute cases. Villous atrophy, fusion, and blunting occur. Varying degrees of lymphocytic enteritis are seen in subacute and chronic infections. Coronavirus particles were identified by transmission electron microscopy in both feces and jejunal enterocytes from affected animals. Immunohistochemical staining of jejunal sections from affected ferrets were positive for coronaviral antigen. Antigen staining was absent in clinically normal ferrets and in ferrets with other gastrointestinal syndromes. The pathogenesis and clinical progression of this condition in ferrets mirrors coronaviral enteritides in other species. The findings presented here strongly support a coronavirus as the etiologic agent of ECE.

1Animal Disease Diagnostic Laboratory, Purdue University, West Lafayette, IN 2Department of Veterinary Pathology, Armed Forces Institute of Pathology, Washington, DC 3Custom Monoclonals International, 813 Harbor Blvd, Suite #284, W. Sacramento, CA 4Prairie Diagnostic Services, Saskatoon, Saskatchewan, Canada *To be considered for graduate student award. Effect of Transport Enrichment Media, Transport Time, and Growth Media on the Detection of Campylobacter fetus subsp. venerealis

H. J. Monke*1, B. C. Love2, B. A. Byrum2, T. E. Wittum1, and D. R. Monke3

Bovine genital campylobacteriosis is a contagious venereal disease of cattle caused by Campylobacter fetus subsp. venerealis. Semen collected from a bull infected with C. fetus subsp. venerealis can be contaminated and the bacteria transmitted to thousands of cows by artificial insemination. Reliable diagnostic procedures are required to accurately test semen donor bulls and to prevent seminal transmission of disease. The purpose of this study was to determine the combination of media and growth conditions, including transport enrichment media (TEM), transport time, TEM incubation time, and culture media, that best supports C. fetus subsp. venerealis while inhibiting contaminants. TEMs evaluated include Weybridge, Cary-Blair, and 0.85% saline solution. Each TEM was inoculated with preputial smegma spiked with C. fetus subsp. venerealis and transported for 4 or 24 hours before being inoculated onto growth media with and without overnight incubation at 37 C. Campylobacter fetus subsp. venerealis and contamination were evaluated on a scale of 0-4. Median scores of C. fetus subsp. venerealis and microbial contamination were compared within TEM, transport time, overnight incubation, and growth media groups using the Mann-Whitney U test and Kruskal-Wallis test. The proportion of samples with any growth or contamination within each group was compared using the Chi Square test. The results suggest that recovery of C. fetus subsp. venerealis was significantly influenced by three of the four criteria evaluated. Weybridge TEM more effectively maintained the organism than either Cary-Blair or 0.85% saline solution (P<0.0001). Transport time of 4 hours rather than 24 hours was superior (P<0.0001). Benefits were associated with avoiding overnight TEM incubation at 37 C (P=0.0002). Significant differences were not identified for growth media; however, Skirrow's Campylobacter agar yielded slightly better growth than either blood agar or Greenbriar Plus agar. Contaminant growth was also significantly influenced by three of the four variables. Differences associated with TEM indicated Weybridge medium inhibited contaminant growth more effectively than either Cary-Blair or 0.85% saline solution (P<0.0001). Transport times of 4 and 24 hours did not significantly influence contaminant growth. Elimination of overnight incubation of transport medium reduced contamination (P=0.0032). Skirrow’s agar was superior to blood agar and Greenbriar plus agar (P<0.0001) for suppression of contamination on solid medium. These results suggest that the detection of Campylobacter fetus subsp. venerealis is enhanced when preputial smegma samples arrive at the diagnostic laboratory within 4 hours after inoculation into Weybridge TEM, and are transferred to Skirrow’s agar the same day they arrive in the laboratory. Adherence to these guidelines will facilitate accurate diagnosis of bovine genital campylobacteriosis in bulls, thereby reducing the potential for seminal transmission of C. fetus subsp. venerealis and subsequent occurrence of infertility, early embryonic death, and abortion.

1Department of Veterinary Preventive Medicine, Ohio State University, Columbus, OH 2Animal Disease Diagnostic Laboratory, Ohio Department of Agriculture, Reynoldsburg, OH 3Select Sires, Inc., Plain City, OH *To be considered for graduate student award.

Sensitivity and Specificity of Pooled-sample Testing for Diagnosis of Low Prevalence Diseases and Effect on Cost and Predictive Values

C. A. Muñoz-Zanzi*1, M. C. Thurmond1, S. K. Hietala2, and W. O. Johnson3

Testing of pooled samples has been proposed as a lower cost alternative for diagnosis of low prevalence diseases with direct application to disease control programs. The present study extended our previous work, in which we described strategies for pooled-sample testing (PST),# to evaluate the effect of prevalence, pool size, antigen detection limits, and laboratory error on pooled sensitivity (Se) and specificity (Sp), and subsequently, on the least cost and predictive values of the PST procedure. Probability theory and Monte Carlo simulations provided estimates of mean least cost per sample, while maximizing overall positive and negative predictive values as follows, , where k = pool size, π = prevalence, c = test cost, η = pooled Se, and θ = pooled Sp. Increasing pool size affected Se and Sp through dilution and increased probability of contamination. Assays with lower pooled Sp had substantially higher costs due to false positive pools. Assays with lower pooled Se had slightly lower costs, but with higher probability of not detecting all infected samples. At a prevalence of 0.5% and 15 samples/pool, cost per animal increased from $2.78 to $6.49 when Sp decreased from 100% to 80% and decreased to $2.47 when pooled Se decreased from 100% to 80%. The simulations provided a quantitative assessment of how using assays of various Se and Sp can affect the cost-benefit of PST allowing application to a broad range of low prevalence diseases and assays available.

1Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 2California Animal Health and Food Safety Laboratory System, Davis, CA 3Division of Statistics, University of California, Davis, CA #Muñoz-Zanzi CA, Johnson WO, Thurmond MC, Hietala SK. Pooled-sample testing as a herd-screening tool for detection of bovine viral diarrhea virus persistently infected cattle. J Vet Diagn Invest 2000, 12:195-203 *To be considered for graduate student award. Oral and Intravenous Inoculation of Greyhound Dogs with Escherichia coli O157:H7 Did Not Result in Cutaneous Renal Glomerular Vasculopathy

M. L. Renninger1, 2*, M. R. White1, 2, A. M. Saeed2, C. C. Wu1, 2, J. A. Christian2, and S. R. Albregts1

Cutaneous renal glomerular vasculopathy (CRGV) is a disorder of unknown etiology that occurs in Greyhound dogs. CRGV is characterized by microangiopathic hemolytic anemia, thrombocytopenia, acute renal failure, and cutaneous ulceration. Hemolytic Uremic Syndrome (HUS) is a systemic disease in humans and other mammals that is also characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Escherichia coli O157:H7 is one cause of HUS. The effects of oral and intravenous inoculation of Escherichia coli O157:H7 in greyhound dogs and the effects of oral inoculation of E.coli O157:H7 with concurrent corticosteroid immunosuppression were examined. Eight adult male Greyhound dogs were inoculated orally with Escherichia coli O157:H7 at a dose of 1 x 1010 colony forming units (CFU). Following inoculation, the dogs were monitored daily for three days for changes in packed cell volume (PCV), platelet count, blood urea nitrogen (BUN), creatinine, urine specific gravity, temperature, pulse, respiration, and fecal output. On the second day post- inoculation (DPI), changes in complete blood cell count (CBC), chemistry panel, urinalysis, one-stage prothrombin time (PT), and activated partial thromboplastin time (PTT) were assessed. At 3 DPI, blood, fecal, and urine samples were collected for bacterial culture. No clinical disease developed and no consequential changes in measured parameters occurred after oral inoculation at a dose of 1 x 1010 colony forming units (CFU). Escherichia coli O157:H7 was cultured from the feces of 5 of the 8 dogs at 3 DPI. A second oral dose of 2 x 1010 CFU of Escherichia coli O157:H7 was given at 7 DPI. The same parameters were monitored following this dose. No clinical disease developed and no consequential changes in measured parameters occurred. Escherichia coli O157:H7 was cultured from the feces of 6 of the 8 dogs at 3 DPI and all dogs at 17 DPI after the second inoculation. At 6 DPI after the second inoculation, immunosuppressive doses (2.2mg/kg) of prednisone were given to each dog orally for 15 days. Changes in the CBC of each dog indicated a corticosteroid response characterized by neutrophilia, lymphopenia, and eosinopenia. The dogs were then given an oral dose of 5 x 1010 CFU of Escherichia coli O157:H7. The same hematologic and physiological parameters were measured. No clinical disease developed, and no consequential changes in measured parameters occurred. Fourteen days after the third oral dose, the dogs were given 1 x 108 CFU Escherichia coli O157:H7 intravenously. Following inoculation, the dogs were monitored daily for three days for changes in CBC, chemistry panel, and urinalysis. Changes in PT and PTT were monitored at 2 DPI. At 6, 12, and 18 hours post inoculation (HPI), the dogs were moderately depressed and febrile. Seven of the eight dogs vomited by 6 HPI. At 12 HPI, significant changes in the CBC included lymphopenia in eight dogs, left shift in six dogs, and neutrophilia in two dogs. Consequential changes in platelet count, BUN, creatinine, urine specific gravity, PT, and PTT did not occur. At 24 HPI, the dogs were not febrile or depressed. At 36 HPI, the neutrophilia and left shift were not present. At 3 DPI, blood, fecal, and urine samples were collected for bacterial culture, and Escherichia coli O157:H7 was not isolated. All eight dogs were euthanized and necropsied. Morphologic lesions of Cutaneous Renal Glomerular Vasculopathy were not present. We conclude that healthy adult male Greyhound dogs inoculated orally or intravenously with Escherichia coli O157:H7 do not develop systemic disease or lesions attributable to Cutaneous Renal Glomerular Vasculopathy. In addition, adult male Greyhound dogs immune-suppressed with corticosteroid administration do not develop CRGV after oral inoculation of Escherichia coli O157:H7. Factors other than immune suppression, including genetics, concurrent infections, and health status, may play a role in development of this disease.

1Animal Disease Diagnostic Laboratory, Purdue University, West Lafayette, IN 2Department of Veterinary Pathobiology, Purdue, University, West Lafayette, IN *To be considered for graduate student award.

RT-PCR RFLP Patterns of Various PRRSV Isolates Recovered from Ontario Farms, 1998-2000

H. Alexander, S. Carman, D. Lloyd, G. Maxie, G. Josephson, and H. Cai

From January 1998 to July 2000, porcine reproductive and respiratory syndrome viruses (PRRSV) were detected and typed in 2456 samples from 760 submissions made to the Animal Health Laboratory, Ontario, Canada using RT-PCR and RT-PCR RFLP assays. An RT-PCR assay (PRRSV ORF7-RT-PCR) amplifying a 433bp region of ORF 7 of PRRSV was used to detect PRRSV in clinical samples, including lungs, tonsils, lymph nodes, sera, kidneys and livers. Specimens submitted were from pigs with clinical signs of respiratory disease and histological evidence of interstitial pneumonia, or from aborted fetuses. A total of 516(20.3% samples from 284 cases (37.4%) were PRRSV ORF7-RT-PCR positive. An RT-PCR RFLP method (ORF5-716bp-RT-PCR-RFLP) was used to type the viruses in the positive samples using the primers that amplified a 716bp region of ORF 5. If 716bp-RT-PCR-RFLP failed to type the virus, a back up method (ORF5-933bp-RT-PCR) was used to amplify a 933bp region of ORF5. This method amplified the area from 47bp upstream to 170 bp downstream of the above 716bp fragment. Each RFLP pattern was assigned a code according to Wesley et al.1 or a code (capped with a "*") adopted from Wesley's for ORF5-933bp-RT-PCR-RFLP. By using both typing methods, our laboratory was able to type samples from 254 cases (89.4%) which consisted of 45 different patterns. Of these, 164 cases (66.1%) had 43 different RFLP patterns of field or intermediate strains; 86 (33.8%) had the pattern 252, similar to the vaccine Resp/PRRS or VR 2332 strain of the virus; 4 (1.6%) were similar to the vaccine PrimePac or a field strain which shared pattern 144. In 4 cases (1.6%), 2 different RFLP types were identified from tissues from different pigs that were submitted at the same time. The co-exiting types were 212 and 222, 132 and a mix of 172 and 132, 122* and 124*, 114* and 114* with deletion. Of the 195 farms that submitted PRRSV PCR positive samples, 48 submitted samples on more than one occasion during the specified time frame. Different patterns of PRRSV were recovered from 23 (47.9%) farms whereas in the other 25 (52.1%) farms, the RFLP pattern remained unchanged. Using the ORF5-716bp-RT-PCR-RFLP, 36 different PRRS virus patterns were identified from 236 cases. There were 18 cases, consisting of 9 different patterns, typeable only by ORF5-933bp-RT-PCR- RFLP. This study provides information on the number and characterization of the different strains of PRRSV circulating in the Ontario swine population. Many RFLP types of PRRSV exist on Ontario farms. About half of the farms with multiple submissions had different types of PRRSV during the time period of this study. Further investigation is required to determine if co-infection or mutation was responsible for this phenomenon and determine if further changes of the virus occurred on these farms. The use of more than one typing scheme (i.e. ORF5-716-RT-PCR-RFLP and ORF5-933-RT-PCR-RFLP) appeared to be more helpful in clarifying PRRSV genotypes than the use of one scheme alone.

Animal Health Laboratory, University of Guelph, Box 3612 Guelph, Ontario, Canada Effect of Iron Dextran on Nested RT-PCR Assay for PRRS Virus

G. D. Appleyard, J. McMurchy, and B. Yue

Soluble iron dextran is commonly administered to one to three day old piglets to prophylactically treat iron deficiency anemia. Pigs of this age may need to be tested for the presence of PRRS virus as they are moved from quarantine into a disease-free barn but serum samples collected within days of this administration can contain a significant amount of iron dextran. Testing for the presence of porcine reproductive and respiratory syndrome virus by nested RT-PCR is frequently carried out using serum as a sample source however, standard extraction procedures efficiently co-purify both RNA and iron dextran. PRRS specific nested RT-PCR was conducted using viral RNA spiked with iron dextran at four different concentrations to determine the effect that iron dextran might have on assay sensitivity. Results suggest that iron dextran, at concentrations comparable to levels observed in young piglets, increased test sensitivity by as much as 5 fold. Serum samples collected following administration of iron can therefore still be used as a sample source since the presence of iron dextran does not appear to inhibit either reverse transcriptase or Taq polymerase. One hypothesis for the increased sensitivity is that the dextran is acting as a carrier aiding precipitation of RNA as has previously been described.

University of Saskatchewan, Dept. Veterinary Pathology, Saskatoon, Saskatchewan, Canada Design and Evaluation of a Diagnostic PCR Test to Detect Clostridium piliforme

D. C. Bolin, J. M. Donahue, M. Hiles, and S.F. Sells

A diagnostic PCR test targeting the 16S rDNA of Clostridium piliforme was designed and evaluated. The oligos were designed to recognize all published C. piliforme sequences and exclude cross reactivity with other DNA sequences including the closest phylogenetically related species, Clostridium colinum. Paraffin embedded tissues from 62 diagnostic cases submitted to the University of Kentucky Department of Veterinary Science Livestock Disease Diagnostic Center since 1995 were used to evaluate the test. Clear amplification of a single 158 bp amplicon was observed from all 30 cases previously diagnosed as Tyzzer’s disease in a serval kitten and 29 equine cases. No amplification was observed using tissues from 32 age-matched (less than one year old) equine cases known to have expired from other causes (bronchopneumonia, fracture, etc). This test should be beneficial as a rapid diagnostic tool and presumably will prove useful to researchers studying non-lethal C. piliforme infections in horses.

University of Kentucky, College of Agriculture, Veterinary Science Department, Livestock Disease Diagnostic Center, Lexington, KY Presence of Salmonella in Pig Ear Dog Treats Obtained from Retail Stores

L. L. English, S. L. Ayers, S. Zhao, S. L. Friedman, D. G. White, D. D. Wagner, S. D. McDermott, and M. J. Myers

Between March and August in the year 1999, dried pig ear dog treats were implicated in outbreaks of human salmonellosis in Canada. In one outbreak of Salmonella Infantis in Calgary, 8 of 12 cases were pet owners who had fed pig ear treats to their dogs. Of 60 total S. Infantis cases in Canada, 25 were linked to exposure to pig ear dog treats. Canadian health agencies subsequently isolated several other Salmonella serotypes from retail samples of dried pig ears, from fresh pig ears, and from samples collected throughout the manufacturing process. Farm Meats Canada Ltd., the firm responsible for producing the dog treats implicated in the Calgary outbreak, distributed this product throughout Canada and to the United States. The purpose of this study was to determine whether dried pig ear dog treats offered for sale in U.S. stores also were contaminated with Salmonella. Retail samples were collected from 8 different stores in the Baltimore-Washington metropolitan area. These consisted of smoked, “naturally roasted”, or non-specified whole pig ears or pig ear pieces packaged in boxes, mesh bags, plastic bags, or bulk bins. Samples were screened for Salmonella using a commercial EIA kit (Tecra®), as well as conventional culture methods. Presumptive Salmonella isolates were identified using API 20E® test strips, followed by serotyping using Difco antisera. In addition, confirmed Salmonella isolates were tested for susceptibility to a number of antimicrobials of human and veterinary importance using the Sensititre system (Trek Diagnostics). Salmonella isolates were further analyzed by pulsed-field gel electrophoresis and screened for the presence of class 1 integrons via PCR. A total of 10 samples were collected from the 8 stores; 4 of the 10 samples were positive for Salmonella. The serotypes that were isolated include Salmonella London, Salmonella Muenchen, Salmonella Manhattan, Salmonella Rissen, Salmonella Typhimurium, and Salmonella Typhimurium var. Copenhagen. All of the serotypes had distinct PFGE patterns. No differences were seen for PFGE patterns within a serotype. The Salmonella isolates displayed resistance to several antimicrobials; one isolate had a susceptibility profile, PFGE pattern, and phage type consistent with S. Typhimurium DT104. The S. Typhimurium DT104 isolate contained two chromosomal integrons of 1,000 and 1,200 base pairs, encoding resistance to streptomycin (aadA2 aminoglycoside 3" adenyltransferase) and ampicillin (bla PSE-1 β-lactamase), respectively. None of the other Salmonella isolates assayed possessed integrons. This study indicates that pig ear dog treats may contain Salmonella strains that have the potential to cause salmonellosis in humans and animals and that some of the isolates may be resistant to multiple antimicrobials.

Division of Animal and Food Microbiology, Office of Research, Center for Veterinary Medicine, U.S. Food and Drug Administration, Laurel, MD Brain Cholinesterase Activities in Exotic/Zoo Species

R. J. Everson1, J. A. Raymond2, R. R. Bedel1, and S. B. Hooser1

Brain activity of acetylcholinesterase can often be diagnostic for toxicoses in animals exposed to organophosphorus or carbamate insecticides when evaluated in combination with appropriate adverse clinical signs (e.g., salivation, lacrimation, urination, defecation, dyspnea or found dead). When brain cholinesterase activities are calculated using the modified Ellman method, it is necessary to express the sample cholinesterase activity as a percentage of the accepted normal activity. Since brain cholinesterase activity can be highly variable between different species, normal values are needed for each species. Following a review of the literature, we found a dearth of publications evaluating normal brain cholinesterase activities in exotic/zoo animals. Therefore, sections of brains from a: P.D. deer, muntjac (2), gazelle (2), blesbok, oryx, duck, spoonbill, eagle, swan, screech owl, wood duck, parrot and a golden eye duck were obtained from the Smithsonian National Zoological Park. Since these samples had been obtained at necropsy, frozen and archived, but had not been taken specifically for the purpose of running brain cholinesterase activities, the brain sections were not uniformly cut from the same areas in each animal. Samples of brain from each species (n = 1 unless otherwise specified) were thawed and minced using a scalpel (most sections were too small to use a homogenizer). In avian species, 0.1 mg of minced brain, and in mammals, 0.2 mg of minced brain were suspended and vortexed in 1% Triton X-100 buffer, pH = 8.0. Cholinesterase activity was measured using a modified Ellman method with spectrophotometric detection at a wavelength of 412 nm. All samples were run in duplicate and the activities averaged for each individual.

Brain Cholinesterase activities were:

Species Brain Cholinesterase Species Brain Cholinesterase Activity Activity µmol/g/min µmol/g/min Bald Eagle 13 Blesbok 0.70 Duck (nos) 16 Gazelle 1 3.0 Golden Eye Duck 16 Gazelle 2 1.4 Parrot 17 Muntjac 1 3.7 Screech Owl 10 Muntjac 2 1.6 Spoonbill 18 P.D. deer 1 1.2 Swan 17 P.D. deer 2 0.82 Wood Duck 17 Oryx 0.71 nos = not otherwise specified

Conclusion: Brain cholinesterase activities vary between different species. To determine whether the activity has been suppressed in an animal exposed to organophosphorus/carbamate insecticides, the normal activity for that species is needed. Therefore, we have begun to develop a database of normal brain cholinesterase activities for exotic/zoo animals.

1Animal Disease Diagnostic Laboratory, Purdue University, West Lafayette, IN 2Smithsonian National Zoological Park, Washington, DC Evaluation of the SvanovirTM ELISA for Bovine Leukosis, Bovine Viral Diarrhea and M. paratuberculosis

G. Keefe1, J. VanLeeuwen1, and S. Hotham2

Three novel serologic tests (SVANOVA Biotech, Uppsala, Sweden) were evaluated: an indirect ELISA for Bovine Leukosis Virus (BLV), an indirect ELISA for Bovine Viral Diarrhea (BVD), and an indirect ELISA for M. paratuberculosis (Johne’s). Each test was compared to industry standards in North America: IDEXX ELISA for BLV and Johne’s (IDEXX, Westbrook, Maine USA) and serum neutralization for BVD. Where appropriate, sensitivity and specificity values or Kappa scores were calculated. Serum samples were collected as part of a seroprevalence survey for production limiting diseases in Eastern Canada. Thirty lactating cows in each of 30 herds in Nova Scotia, New Brunswick and Prince Edward Island were sampled. Both herd and cow selection was by formal random sampling. From this serum bank, 528 cows were selected for the comparative BLV testing, 498 cows for M. paratuberculosis, and 218 unvaccinated animals, greater than 6 m of age, for BVD testing.

Table 1 is a contingency table of the Table 1. SVANOVA and IDEXX ELISA for BLV BLV results. Serologic diagnosis of infection IDEXX IDEXX Total with BLV is very accurate, therefore, +ve -ve sensitivity and specificity could be calculated. SVANOVA 225 0 225 There were no false positives or false negatives, +ve therefore, the test scored 100% on both these SVANOVA 0 303 303 parameters.

Table 2 contains the BVD results. SN Table 2. SVANOVA ELISA and SN for BVD is considered the gold standard for BVD. SN $ SN # Total When compared to a SN dilution $1:256, 1:256 1:256 the sensitivity and specificity of the ELISA SVANOVA 33 7 40 was 94% and 96%, respectively. +ve SVANOVA 2 176 178 Table 3 contains the Johne’s results. The reported sensitivity and specificity of the IDEXX method is 47.3% and 99%, Table 3. SVANOVA and IDEXX ELISA for Johne’s respectively. There was a relatively low agreement, beyond what is expected due to IDEXX IDEXX Total +ve -ve chance alone, between tests (Kappa 0.27). SVANOVA 22 83 105 It is interesting to note, however, that the +ve SVANOVA test identified many more animals SVANOVA 5 388 393 as positive. This may be an improvement on the high false negative rate of the IDEXX test. The SVANOVA ELISA for BLV and BVD appear to be equivalent to the current industry standards. The SVANOVA ELISA for M. paratuberculosis shows some promise of being an improvement on the current testing methods utilized in North America. Additional evaluation of this test is necessary to truly discern its capabilities.

1Department of Health Management, Atlantic Veterinary College, Charlottetown, PEI, CANADA 2Diagnostic Chemicals Limited, Charlottetown, PEI, CANADA

Evaluation of IS 900-PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis Directly From Raw Milk

S. R. Pillai, and B. M. Jayarao

Johne's disease which is caused by Mycobacterium avium subsp. paratuberculosis results in losses exceeding $1.5 billion/year. Current detection methods for Johne’s disease rely on culture and ELISA- based assays. However, both of these methods have poor sensitivity. DNA probes based on the insertion sequence 900 (IS 900) of M. paratuberculosis offer an alternative diagnostic test that is rapid and specific. The objective of this study was to standardize the IS 900-PCR assay for detection of M. paratuberculosis directly from raw milk. Raw milk and Middlebrook’s 7H9 (M7H9) broth samples were inoculated with 1 ml of a suspension containing 108–0 CFU/ml (Table 1) of each of 4 American Type Culture Collection (ATCC) strains of M. paratuberculosis (ATCC19698, ATCC 43544, ATCC 43545, ATCC 43015). Milk and M7H9 samples were centrifuged at 1950 x g for 30 min, and pellets obtained were divided equally. One half of the pellet was used for IS 900-PCR. The other half was decontaminated in HPC (0.75%) and cultured (150 µl) on 2 slants each of Herrold’s egg yolk medium (HEYM). All recovery and detection experiments were repeated 6 times.

Table 1. Comparison of culture and IS 900-PCR for detection of M. paratuberculosis. Milk M7H9 (n=6 replicates, 2 HEYM slants per replicate) (n=6 replicates, 2 HEYM slants per replicate)* CFU/ml +ve by Counts Culture CFU/ml +ve by Counts Culture IS 900 (Avg CFU/ml) +ve IS 900 (Avg CFU/ml) +ve PCR PCR 108 24/24 TNTC# 48/48 108 24/24 TNTC 48/48 106 24/24 TNTC 48/48 106 24/24 TNTC 48/48 104 24/24 TNTC 48/48 104 24/24 TNTC 48/48 102 24/24 53 40/48 102 24/24 75 44/48 10 12/24 2 10/48 10 24/24 7 20/48 1 0/24 - 0/48 1 0/24 - 0/48 0 0/24 - 0/48 0 0/24 - 0/48 * 6 replicates each of 4 ATCC reference strains of M. paratuberculosis: ATCC19698-Bovine, ATCC 43544-Ben, ATCC 43545-Dominic, ATCC 43015-Linda. # Too numerous to count

Under experimental conditions, IS 900-PCR assay could detect counts as low as 10 CFU/ml - 100 CFU/ml of M. paratuberculosis (Table 1). Further, IS 900-PCR detected M. paratuberculosis in 5/8 farm bulk tank milk samples from herds with known clinical history of Johne’s disease. Two out of the 5 samples positive by IS 900-PCR were also positive by culture for M. paratuberculosis. The results of this experimental study are currently being validated by testing bulk tank samples from several herds in Pennsylvania.

Pennsylvania State University, University Park, PA

Comparison of Nested PCR, Pepsin/Trypsin Digest and Histology for the Diagnosis of Whirling Disease in Salmonid Fish

T. Qureshi1, M. R. White1,2, and C. Santrich1,2

Whirling Disease (WD), caused by Myxobolus cerebralis, is an important disease of salmonid fish throughout the world. This disease is now well established in many areas of the United States and has been implicated in the decline of some salmonid populations. The clinical signs of this disease include skeletal deformity, blackening of the tail, and erratic swimming patterns (i.e., whirling). Several species of myxozoans infect fish, but only M. cerebralis has affinity for cartilaginous tissue of the cranium resulting in WD. Diagnosis of WD is based upon observing spores in the cytological preparations obtained from the sediment following pepsin/trypsin (P/T) digest, and/or on histological examination of cartilaginous head tissue. Different DNA testing methods have been developed, including nested PCR, single round PCR, and in situ hybridization. These methods are highly sensitive and specific, but are not yet recognized by the American Fisheries Society, Fish Health Section, as acceptable tests. The objectives of this study were to survey fish from state hatcheries in Indiana and private hatcheries in Michigan for WD using nested PCR, P/T digest, and histopathology. One group of forty and five groups of sixty fish heads, for a total of 340 samples, were submitted from hatcheries in Indiana and Michigan. These samples were examined for myxozoan spores using histopathology, P/T digest and PCR tests. The heads were hemisectioned, and one-half was fixed in 10% neutral-buffered formalin. Sections were cut and stained with hematoxylin and eosin stain for histological examination. The other half was processed for P/T digest and PCR. With immature fish, the entire half of the head was processed for P/T digest, and with mature adult fish, a biopsy punch sample was collected from the top of the eye to include cranium cartilage. All the flesh was removed, and the cartilage and bone were subjected to the P/T digest. The sediment of the digest was placed on slides as wet mounts and examined for the presence of myxozoan spores. If spores were observed, then the sediment was prepared for the nested PCR analysis according to published protocols. Histological examinations did not reveal M. cerebralis positive in any of the 340 samples. Serial step sections, taken at five micron intervals, were evaluated for those samples that had positive spore identification using P/T digest. However, histologic examination of these sections failed to reveal any myxozoan parasites. Myxozoan spores were observed in 28.8% (98 of 340) of samples on examination of the sediment after P/T digest. Spores morphologically similar to M. cerebralis were observed in six P/T digest samples, with a prevalence of 1.7 %. Nested PCR was performed on all samples in which myxozoan spores were observed in the P/T digest and indicated M. cerebralis infections in only 3 samples (prevalence of 0.9%). P/T digest correctly diagnosed M. cerebralis spores in only one sample, a mixed infection in another sample, while M. cerebralis spores were not observed in the third PCR positive sample. All the three positive samples came from the same hatchery. Diagnosis of M. cerebralis in this study indicated a low prevalence of the disease. Histopathology was a very insensitive indicator of WD. The PCR was highly specific and was able to differentiate M. cerebralis from similar spores of other species. Other studies with Whirling Disease have also shown that the PCR was more sensitive and specific than either the P/T digest or histopathology, and was also able to diagnose infections earlier than the other tests.

1Department of Veterinary Pathobiology, School of Veterinary Medicine, Purdue University, West Lafayette, IN 2Animal Disease Diagnostic Laboratory, Purdue University, West Lafayette, IN

Three Soft Tissue Sarcomas in Two African Hedgehogs (Atelerix albiventris)

J. A. Ramos-Vara

Reported neoplasms in hedgehogs include cutaneous squamous cell carcinoma, pituitary adenoma, mast cell tumor, myelogenous leukemia, bone-associated sarcomas, intestinal lymphosarcoma, intestinal plasmacytoma, transitional cell carcinoma, hepatic adenoma, and oral carcinoma. Connective tissue sarcomas of soft tissues have not been reported in this species. The purpose of this report is to describe 3 soft tissue sarcomas in 2 hedgehogs. Case 1. A 4.5-year-old hedgehog developed a fast growing subcutaneous mass on the medial aspect of its right front leg. Histologically, this mass was ulcerated and consisted of short intersecting fascicles of spindle cells with eosinophilic cytoplasm and oval nucleus with reticulated chromatin and small or inapparent nucleolus. Clefts and occasional perivascular whorls were observed. The tumor stroma was scant to moderate. It had few mitoses and extensive necrosis. Numerous neoplastic cells were positive for vimentin, CD10 antigen, and neuron specific enolase (NSE); fewer cells were positive for chromogranin A and smooth muscle actin (less than 10%). Other markers (S100 protein, GFAP, factor VIII-rAg, somatostatin, and desmin) were negative. Ultrastructurally, neoplastic cells had abundant cytoplasm with irregular cytoplasmic processes and numerous RER profiles. There were also free ribosomes and large mitochondria with prominent cristae. Extracellular matrix was abundant. Case 2. A 3-year-old female hedgehog developed a subcutaneous mass on its lumbar area and a vaginal mass. The subcutaneous mass consisted of intersecting bundles and whorls of spindle cells with eosinophilic cytoplasm, oval nucleus with reticulated chromatin and small or inapparent nucleolus. Stroma was scant and multinucleated giant cells were rare. The mitotic index was intermediate and necrotic areas were uncommon. The vaginal mass consisted of an infiltrative growth of loose bundles of spindle, polygonal and stellate cells with basophilic and finely vacuolated cytoplasm and large oval nucleus with reticulated chromatin and single nucleolus. Binucleate and karyomegalic cells were common. This tumor had low mitotic index and extensive areas of necrosis and ulceration. Both tumors were positive for vimentin and negative for muscle actin, smooth muscle actin, S100 protein, synaptophysin, chromogranin A, and desmin. The vaginal tumor, but not the cutaneous mass, was also focally positive for NSE. Microscopically, none of these tumors had diagnostic features to allow a more precise classification, although case 1 had features suggestive of neurofibrosarcoma. Immunohistochemistry did not contribute substantially to classify these tumors.

Veterinary Medical Diagnostic Laboratory, University of Missouri, PO Box 6023, Columbia, MO Glucagonoma and Degenerative Joint Disease in a Jaguar (Panthera onca)

J. A. Ramos-Vara1 , M. A. Miller1 , and D. Preziosi2

Endocrine neoplasms, which are infrequently reported in large felidae, include thyroid carcinoma, adrenocortical carcinoma, pheochromocytoma, and neuroendocrine tumor. This report describes a case of glucagon-producing islet cell tumor in a jaguar (Panthera onca) with degenerative joint disease. To the best of our knowledge, neither of these conditions have been reported previously in this species. A 15.5-year-old, obese, intact male, melanistic (all black) jaguar (Panthera onca) presented with a 1- year history of progressive forelimb lameness, during which it developed a pronated stance and eventually was unable to rise. It was euthanized because of progressive lameness. Both elbow joints had irregular bony proliferation of the joint margins as well as articular erosions and a joint mouse. The jaguar had two, 3 cm in diameter pancreatic nodules that were islet cell tumors immunohistochemically positive for glucagon, neuron specific enolase, chromogranin A and synaptophysin. Multiple synovial membranes had villous hyperplasia and were infiltrated with lymphocytes and plasma cells. Focal chronic pancreatitis was also present. Lameness was most likely the result of degenerative joint disease due to obesity. The jaguar did not exhibit clinical signs consistent with hyperglucagonemia.

1Veterinary Medical Diagnostic Laboratory, University of Missouri, PO Box 6023, Columbia, MO 2Department of Veterinary Medicine and Surgery, University of Missouri, Columbia, MO

Development of a PCR-based Test for Detection of Leptospira in Bovine Semen

E. E. Tanaka, S. E. Druhan, and E. J. Golsteyn-Thomas

Pathogenic strains of Leptospira are the etiologic agents of leptospirosis, a zoonotic disease associated with abortion, stillbirths, infertility and decreased milk production. Transmission usually results from direct or indirect contact with urine, kidneys or conception products of infected animals. Additionally, venereal transmission has been reported with recovery of leptospires from bovine genital tracts and from semen of naturally and experimentally infected bulls. In Canada, bulls must be “seronegative” for specific serovars of Leptospira in order for their semen to qualify for export. The universally accepted serological test, the microscopic agglutination test (MAT), is useful as a screening test but cannot differentiate between previously infected animals and vaccinated animals nor can it reliably determine the shedding status of an animal. There have been several examples in the literature of seronegative animals which shed leptospires in their urine. For this reason, the direct detection of leptospires in semen may be the most practical way to ensure that semen used for artificial insemination (AI) purposes does not contain these leptospires. This study employs an optimized PCR-based assay to detect pathogenic leptospires in bovine semen. Clinical samples invariably contain compounds that are inhibitory to the PCR process. It is believed that this protocol overcomes potential sources of inhibition while maintaining test specificity and sensitivity. In brief, leptospiral DNA is extracted from semen samples using a QIAamp kit (QIAGEN Inc., Mississauga, Ontario) and analyzed using a PCR assay that was developed to specifically detect pathogenic Leptospira spp. This PCR assay can differentiate between pathogenic species of Leptospira when used in conjunction with restriction endonuclease analysis of the PCR product. When combined with the QIAGEN extraction procedure, this assay has the capability of detecting as few as 103 leptospires in raw (neat) semen and 102 leptospires in extended (processed) semen when starting with 50 µl and 500 µl, respectively. The ability to test raw and extended semen could allow managers of AI studs to test vaccinated bulls and animals with persistent antibody titers for entry into their units. Thus, superior sires, previously excluded from AI studs by traditional serological testing, may be included in the gene pool. This test could also aid in the diagnosis of acute and chronic (carrier state) infections thereby offering cattle producers a useful tool for better herd health management. .

Canadian Food Inspection Agency, Animal Diseases Research Institute, Lethbridge, Alberta, Canada Melanocytic Schwannoma in a Brown Bullhead Catfish (Ictalurus nebulosus)

M. R. White, and K. Sakamoto

One brown bullhead catfish, (Ictalurus nebulosus), collected as part of a wild fish survey by the Indiana Department of Natural Resources, was submitted to the Animal Disease Diagnostic Laboratory, Purdue University. This catfish was dead upon arrival. The most striking gross lesion was the presence of a large mass (3.3 x 4.0 cm) on the dorsal midline, just caudal to the dorsal fin. This mass was spherical, and diffusely black on both the surface and on section. The mass was covered with skin, and attached to the underlying epidermis by a broad stalk. Other gross lesions included: discoloration of the skin characterized by patches of hyperpigmentation and hypopigmentation, an irregular concentric thickening of the left barbel and an irregular thickening of the distal portion of the left pectoral fin. Histologic evaluation of the mass revealed two distinct neoplastic cell populations: a pigmented (melanocytic) cell population and a Schwann cell population. Ultrastructural findings confirmed two distinct cell populations. To our knowledge, melanocytic schwannomas have never been previously reported in fish. Schwannomas have been reported in a few fish species. Melanocytic schwannomas are described in terrestrial mammals, and occur commonly in humans.

Department of Veterinary Pathobiology, School of Veterinary Medicine, and Animal Disease Diagnostic Laboratory, Purdue University, West Lafayette, IN Heavy Metal, Organochlorine and Polyaromatic Hydrocarbon Burdens in Steelhead Trout from Lake Michigan Compared to Rainbow Trout from the Pristine Waters of Northeastern Indiana

C. R. Wilson1, M. R. White1,2, R. J. Everson1, J. O. Hall3, M. A. Greeley1, R. R. Bedel1 and S. B. Hooser1,2

In order to have a better understanding of potential environmental contaminants of fish and obtain baseline data for some species of fish in our region, we obtained liver samples from adult Lake Michigan Steelhead trout (Oncorhynchus mykiss, Bodine State Fish Hatchery, taken January and March 1998, n=12) and Rainbow trout (Oncorhynchus mykiss, Curtis Creek Trout Rearing Station in a relatively pristine area in northeastern Indiana, taken December 1997, n=11). These were analyzed for chlorinated hydrocarbons (by gas chromatography-mass spectroscopy (GC-MS)), polyaromatic hydrocarbons (fluorometrically in bile and by GC-MS in liver), and heavy metals by ICP-MS. Bile samples were fluorometrically analyzed for polyaromatic hydrocarbons. It was found that DDE, a metabolite of DDT, was not detectable in the livers of any trout from Curtis Creek. However, DDE was detected in the livers of trout from Bodine at 1.2 ± 0.2 ppm. In addition, PCBs were not detected in the livers of any trout from Curtis Creek. PCBs were only detected in trace amounts in the liver of one trout from Bodine. Although low in most heavy metals and organochlorine compounds, bile from Curtis Creek trout contained the polyaromatic hydrocarbon benzo(a)pyrene (B(a)P) at 0.6 ± 0.34 µg/mg of protein while B(a)P was not detected in the bile of any Bodine trout. In addition, naphthalene was detected in the bile of Curtis Creek trout at 52.1 ± 28.2 µg/mg protein. This was significantly higher (p ≤ 0.02) than the naphthalene in the bile of Bodine trout which was 1.2 ± 0.2 µg/mg protein. Finally, substituted naphthalenes (also polyaromatic hydrocarbons) were detected in the livers of all trout from Bodine, but only two trout from Curtis Creek. Following ICP-MS analysis, the following metal concentrations were statistically the same in the livers of both the Curtis Creek and Bodine trout: beryllium (Be), cobalt (Co), potassium (K), lithium (Li), lead (Pb), antimony (Sb), strontium (Sr) and vanadium (V). However, the following metal concentrations were statistically higher in the livers of the Curtis Creek trout than in the Bodine trout: aluminum (Al) and zinc (Zn). Finally, the following metal concentrations were statistically higher in the livers of the Bodine trout than in the Curtis Creek trout: silver (Ag), arsenic (As), boron (B), barium (Ba), calcium (Ca), cadmium (Cd), chromium (Cr), copper (Cu), iron (Fe), mercury (Hg), magnesium (Mg), manganese (Mn), molybdenum (Mo), sodium (Na), nickel (Ni), phosphorus (P), selenium (Se), silicon (Si), tin (Sn) and thallium (Tl). Numerous differences in hepatic DDE and heavy metal concentrations were detected between the two groups. In general, trout from Bodine had higher liver concentrations of heavy metals and DDE while trout from Curtis Creek had higher concentrations of polyaromatic hydrocarbons in their bile. While the physiological significance of these differences is not understood at this time, continued monitoring of trout for environmental contaminants can only lead to a deeper understanding of the role of these contaminants in the health of salmonids in Indiana.

1Animal Disease Diagnostic Laboratory, Purdue University, West Lafayette, IN 2Department of Veterinary Pathobiology, Purdue University, West Lafayette, IN 3Utah Veterinary Diagnostic Laboratory, Utah State University, Logan, UT