VETERINARY DIAGNOSTIC LABORATORIES

Diagnostic news and trends from the Colorado State University Veterinary Diagnostic Laboratories Volume 15, Number 1 Spring/Summer 2010 SPECIAL ISSUE INSIDE: Advances in Molecular Diagnostics FOCUS ON PCR PCR detection of infectious diseases iagnosticians use two primary methods to detect Michael R. Lappin, DVM, PhD, DACVIM, Dinfectious diseases: detect the organism or detect CSU VDL Endocrinology and Special Serology Lab Supervisor antibodies against the organism. Most frequently, they detect infectious agents in biological specimens by disease the lower the predictive value of a positive. Dis- culture, cytology, fecal examination, histopathology, ease prevalence has little effect on negative predictive immunological techniques and nucleic acid amplifi ca- values. tion techniques. Depending on the infectious agent in question, PCR The polymerase chain reaction (PCR) assay ampli- and RT-PCR assays can be more sensitive than other CLINICAL TIPS fi es DNA. When a reverse transcriptase step is incorpo- available assays. In addition, PCR assay results can Sensitivity, specifi city, positive rated, it can convert RNA to DNA and then amplify it often be returned within 24 hours of sample submis- predictive value and negative (RT-PCR). These are the most commonly used nucleic sion, which is generally quicker than culture. However, predictive value vary with each acid amplifi cation techniques. Detection of the organ- because special equipment is required, the assays must PCR assay. Many of the ism gives the most information supporting a clinical be shipped to a diagnostic lab. If the organism in ques- infectious disease agents diagnosis of an infectious disease, but these assays tion is diffi cult to culture, can’t be cultured or takes encountered in practice colonize aren’t available nor optimal for all agents. Thus, anti- a long time to culture, such as some normal animals as well as body detection is still commonly used to diagnose viruses, Leptospira, Ehrlichia or myco- induce disease in some infectious diseases. In some situations, the combina- bacteria, PCR assays are of particular individuals. Thus, the PPV of tion of organism detection and antibody detection benefi t. In contrast, some PCR assays many PCR assays can be low. assays is indicated. Bartonella spp. infection in cats are less sensitive than other currently Veterinarians generally need to and Ehrlichia spp. infections in dogs, for example. For available organism demonstration tech- use a combination of fi ndings these organisms, PCR-positive results can occur prior niques. For example, Giardia spp. IFA to help diagnose infectious to seroconversion, so the PCR can be benefi cial to or antigen tests are both more sensitive disease: prove current infection in the acute cases. In contrast, than Giardia spp. PCR assays, probably ■ Appropriate signalment and the PCR assays can become negative later in the course because of PCR inhibitors in feces. history for the infectious agent of disease as the immune responses develop; however, Specifi city of PCR assays can be high, suspected serum antibodies are generally detectable at that time. depending on the primers used in the ■ Clinical signs referable to the This situation may occur in many other infectious die- reaction. For example, primers can be agent seases, as well. designed to detect one pathogen genus ■ Detection of the agent but not others. Primers can also be (cytology, culture, antigen MANAGING FOR PREDICTABILITY designed to identify only one species. For assay, PCR assay) or When evaluating infectious disease diagnostic test example, a PCR assay has been developed antibodies against the agent results, the analytical sensitivity defi nes the minimum to detect all Trichomonas spp. or just one ■ Exclusion of other causes of detectable amount of the substance in question that species, such as T. foetus. the clinical syndrome can accurately be measured; the analytical specifi city ■ Response to an appropriate defi nes whether the substance detected cross-reacts. QUALITY CONTROL IS CRITICAL treatment The diagnostic sensitivity is the proportion of positive PCR assays are prone to false-positive When these criteria are met, test results from known infected animals; the diagnos- results if sample contamination occurs the suspected infectious agent tic specifi city is the proportion of negative test results during collection, transportation or test- might have been the cause of the from known uninfected animals. The predictive value ing. Some PCR assay results may also be clinical disease. However, it is of a positive test, or PPV, is the probability that a affected by administration of anti-micro- always possible that the disease test positive animal is diseased; the predictive value of bial drugs prior to sample collection. For process resolved in spite of the a negative test, or NPV, is the probability that a neg- example, haemoplasma or Bartonella spp. therapy prescribed. ative animal is normal. The lower the prevalence of Continues on page 2 2 Volume 15, Number 1

PCR DETECTION OF INFECTIOUS DISEASES Continued from page 1 PCR results can be transiently negative during anti- determine the amount of microbial DNA in a sample. biotic treatment even though infection persists. When used quantitatively, this technique can be used to Acute infections generally have higher DNA copies monitor response to drug treatment.5 It is possible the in samples than chronic infections, because in DNA or RNA load in a sample will correlate to the pres- chronic infections the immune response has ence of disease for some agents. attenuated the organism. Thus, the optimal PCR assay sample is usually one collected A VALUABLE TOOL...WHEN APPLIED RIGHT during the acute phase prior to antimicrobial Depending on the lab, PCR assays can be offered as indi- treatment. vidual tests or in panels. CSU VDL generally offers indi- While many commercial laboratories offer vidual assays that nucleic acid amplifi cation assays, they may can be coupled as ‘ At CSU VDL, an not be standardized. In addition, some indicated by the appropriate faculty labs offer little external quality control. For individual case. For example, samples from cats with and with- example, it has been specialist in bacteriology, out FIV infection were sent to four different documented that virology or parasitology 1 laboratories offering FIV PCR assay. While haemoplasmas, but oversees the performance the lab with the best performance was right not Bartonella spp. on 90 percent of the samples, two fell below or Rickettsia spp., of each assay and is 60 percent. For this reason, CSU VDL main- are associated with available for consultation.’ tains stringent quality control and standard- hemolytic anemia ized protocols for all PCR assays. in cats. Thus, for cats with hemolytic anemia, only hemo- While PCR assays are very sensitive, the PPV plasma PCR is needed. In contrast, all three genera are of many assays can be very low. For example, associated with fever of unknown origin, and the assays because the technique detects DNA or RNA of can be selected alone or in appropriate combinations. both live and dead organisms, positive test results It’s important practitioners carefully assess the pre- may be achieved even if the infection has been dictive values of available PCR and the expertise and controlled. When the organism being tested for reliability of the lab. New PCR assays are being devel- commonly infects the background population oped almost daily. At CSU VDL, each PCR assay is opti- of healthy pets, interpretation of results for a mized prior to being offered commercially, to ensure single animal can be diffi cult. For example, quality control is stringently maintained. In addition, an healthy cats commonly carry FHV-1, and appropriate specialist in bacteriology, virology or para- modifi ed live vaccine strains colonize cats. sitology oversees the performance of each assay and is Thus, although PCR is a sensitive way available for consultation. ▲ to document FHV-1 infection, its FHV-1 2-4 PPV is actually very low. In one study REFERENCES of cats with and without conjunctivitis,2 1 Crawford PC, Slater MR, Levy JK. Accuracy of polymerase chain more FHV-1 positive tests were detected reaction assays for diagnosis of feline immunodefi ciency virus infection in cats. J Am Vet Med Assoc. 2005 May 1;226(9):1503-7. in the healthy control group than the 2 Burgesser KM, et al. Comparison of PCR, virus isolation, and group with conjunctivitis. The same prob- indirect fl uorescent antibody staining in the detection of naturally lems exist with feline calicivirus. The occurring feline herpesvirus infections. J Vet Diagn Invest. 1999 available RT-PCR assays can’t distinguish Mar;11(2):122-6. between regular calicivirus, vaccine strains 3 Low HC, et al. Prevalence of feline herpesvirus 1, Chlamydophila felis, and Mycoplasma spp DNA in conjunctival cells collected of calicivirus and virulent systemic cali- from cats with and without conjunctivitis. Am J Vet Res. 2007 civirus. This situation may occur with Jun;68(6):643-8. herpesvirus and other viral diseases in 4 Maggs DJ, Clarke HE. Relative sensitivity of polymerase chain other species, such as cattle and dogs. reaction assays used for detection of feline herpesvirus type 1 DNA in clinical samples and commercial vaccines. Am J Vet Res. 2005 Consider these realities when inter- Sep;66(9):1550-5. preting test results. 5 Tasker S, et al. Effect of chronic feline immunodefi ciency infection, Real-time PCR, or fl uorogenic and effi cacy of marbofl oxacin treatment, on ‘Candidatus Mycoplasma haemominutum’ infection. Microbes Infect. 2006 Mar;8(3):653-61. PCR, can be more sensitive than con- 6 Clark M, et al. A commercially available Giardia spp. antigen assay ventional PCR for some organisms. detects the assemblages isolated from dogs. Proceedings of the In addition, this assay can be used to ACVIM Forum, 2008. Spring/Summer 2010 3 LABLINES

Innovative PCR Applications Help target IMA case management

easuring antibodies against ■ Dogs with up to 10 percent Michael R. Lappin, DVM, PhD, DACVIM, Merythrocytes has classically IgM or IgG binding can have CSU VDL Endocrinology and Special SAMPLE relied upon spherocyte estimation, either IMA or infectious hemo- Serology Lab Supervisor presence of agglutination in saline or lytic anemia. ■ 1 ml of EDTA blood direct Coomb’s test. However, eryth- ■ Dogs with greater than 10 percent cifi c DNA, offers clinics high positive for fl ow alone rocyte antibodies can also develop binding are likely to have IMA. and negative predictive values. The ■ 2 ml if combining during infection by blood borne Flow cytometry used on acutely protocol can be used to help deter- with the PCR assays agents, notably Babesia spp., Barton- anemic dogs sampled before treat- mine whether to emphasize immune ella spp., Ehrlichia spp., Mycoplasma ment, combined with appropriate suppression or anti-microbial ther- Shipping haemocanis (previously Haemobar- PCR assays to amplify species- spe- apy in case management. ▲ Deliver on a cold pack to tonella canis) and M. hematopar- arrive within 48 hours of vum. Because those classic tests are collection and before noon only semi-quantitative, they haven’t PCR ASSAY SELECTION RECOMMENDATIONS Fridays. Samples that arrive proved accurate in distinguishing after noon Friday will not be Babesia spp. Neither B. canis nor B. gib- Ehrlichia group. Ehrlichia spp., primary immune-mediated anemia assayed by fl ow cytometry, but soni infections are common in Colorado Anaplasma spp., and Neorickettsia are (IMA) from hemolytic anemia the PCR assays will still be dogs. However, PCR for these agents are uncommon causes of hemolytic anemia. induced by infectious agents. completed. indicated if the dog with hemolytic anemia However, if a dog is seropositive or has Flow cytometry can be used to is a greyhound (B. canis), a pitbull or recent known exposure to Rhipicephalus quantitatively determine the percent- Schedule pitbull cross (B. gibsoni), has been bitten ticks, the assay may be indicated. These age of erythrocytes that have IgM or Monday through Friday with by a pitbull terrier (B. gibsoni), or has a agents are more commonly associated results returned the next day recent history of Rhipicephalus ticks (B. with thrombocytopenia. However, in acute canis). cases with fever, epistaxis, polyarthritis Prices CANINE Bartonella spp. Recently, B. henselae and or thrombocytopenia, Ehrlichia group PCR ■ Flow cytometry alone $50 B. vinsonii have been detected on dogs in assay is indicated, particularly if antibody IgG antibodies bound to the surface. Flow cytometry... Colorado and Wyoming. These agents are tests are negative. Experiments performed over the last ■ Plus one PCR assay $75 uncommon causes of hemolytic anemia Hemoplasmas. All dogs suspected to several years at Colorado State Uni- ■ Plus two PCR assays $100 but have been associated with cases have immune or infectious hemolytic versity have determined: ■ Plus three PCR assays $125 ■ presumed to have primary immune medi- anemia should be screened for The assay is accurate for up to ■ Plus four PCR assays $150 72 hours after the sample is col- ated disease that then fail to respond to Mycoplasma haemocanis and M. hae- lected. Shipping is no longer a immune suppression. All dogs with sus- matoparvum DNA by PCR assay. These problem. pected infectious endocarditis should be agents are commonly negative by cytol- ■ Dogs with 0 percent IgM and IgG screened with this PCR. ogy, and there is no antibody assay. binding are unlikely to have IMA. 4 Volume 15, Number 1

Canine Virology PCR to refi ne differential diagnosis e are often asked by a referring DVM or an Tawfi k Aboellail, BVSc, MVSc, PhD, DACVP, Wowner: “Hi Doc. What killed my puppy (or CSU VDL Pathologist puppies) and what is the best diagnostic protocol?” Among the numerous potential causes of stillbirth neys. Also, necrosis and or post-natal death, including toxic milk syndrome, hemorrhage can occur FOCUS mycoplasmosis, neonatal septicemias and in the liver and lungs. ON PCR canine brucellosis, an investigative necropsy should include investigation for canine MOLECULAR DIAGNOSIS CAN HELP herpes virus. Because the causative CHV virus is quite labile and CHV, also known as “fading puppy syn- is shed only occasionally and in small amounts, rou- drome,” is a viral infection of the adult tine rapid diagnosis of CHV infections by conventional reproductive and respiratory tracts. While viral culture or serology is becoming more diffi cult adult dogs with latent infections may remain and unreliable. Antibody levels are often very low and asymptomatic for a long period of time, they sometimes undetectable. usually pass the virus in their vaginal secre- Polymerase chain reaction offers a highly sensitive, tions or as a droplet during sneezing. Pup- rapid and specifi c method to identify the virus in dead pies contracting the infection during the fi rst puppies or clinical samples from a symptomatic or three weeks of life are the most vulnerable. asymptomatic bitch or stud. Genital, nasopharyngeal, “Turkey-egg” appearance of An entire litter may succumb to the infection within conjunctival or throat swab can be suffi cient for diag- kidneys is due to hemorrhage a 24-hour period. Death may occur abruptly without nosis. Also a small amount of blood, tracheal wash, and and tissue necrosis. Diagno- any premonitory signs. frozen or fresh tissue can prove recent exposure to the sis was confi rmed in less Diagnosis of CHV infection in puppies is usually virus. than 24 hours by PCR. made at necropsy. Characteristic gross lesions are CSU VDL offers a reasonably priced molecular diag- evident in the kidneys as petechial to ecchymotic nosis to help you quickly evaluate whether the rest of hemorrhage and necrosis producing “speckled” kid- the litter is at risk of CHV. ▲

Molecular Parasitology Need the ability to rule something out? e often think of using the PCR technique to Lora R. Ballweber, DVM, MS, Wconfi rm that a particular organism is present. CSU VDL Parasitology Section Head But what about using it to determine that an organism is not? them into areas where they once thrived is one factor Such was the case with a wolf shedding taeniid responsible for the spread of many parasites. Thus, tapeworm eggs. This animal had been rescued it was imperative that the true identity of these tape- from a property in the upper midwest worm eggs be determined. and brought to Colorado. Fecal fl otation Eggs of these species are quite diffi cult to tell indicated the animal was shedding taeniid apart based just on morphology. Therefore, CSU VDL tapeworm eggs. These types of eggs are usu- employed molecular techniques. After extracting the ally thought to be a species of the genus DNA, amplifying it through PCR and then sequenc- Taenia, a common tapeworm of canids throughout ing the resulting amplicons, it could be determined

© Geoffrey Kuchera | Dreamstime.com the United States. However, this particular animal that these eggs were not E. multilocularis, but were A carrier or not? CSU VDL used came from an area in which wolves harbor both Taenia krabbei, a common species of tapeworm found PCR to rule out the presence of Taenia and another tapeworm: Echinococcus mul- in wolves and other wild canids. Transporting this a potentially zoonotic tapeworm tilocularis. While most species of Taenia found in animal from an E. mulilocularis-endemic area into a species in a transplanted wolf, canids are not zoonotic, E. multilocularis is. Plus, this non-endemic area does not appear to have spread this ensuring the animal wasn’t intro- parasite is not known to exist in Colorado. Trans- particular parasite. ▲ ducing the parasite into Colorado. porting animals into new areas or reintroducing Spring/Summer 2010 5 LABLINES

Canine Oncology Innovations New canine cutaneous mast cell tumor profi le helps guide therapy Mast cell growth process n 1999, a group of researchers at Harvard Medical Anne Avery, DVM,PhD, CSU Microbiology, Immunology Normal Mutated ISchool led by a veterinary oncologist discovered an and Pathology Assistant Professor; EJ Ehrhart, DVM, c-kit c-kit important clue to the pathogenesis of mast cell tumors PhD,DACVP, CSU VDL Pathologist; and Debra Kamstock, in dogs.1 Eventually, the discovery of that mutation and PhD,DVM,DACVP, CSU VDL Pathologist its effects on mast cells led to the development of a new drug, Palladia, a tyrosine kinase inhibitor, specifi - Ki67 expression, a marker cally targeted to treating canine mast cell tumors. Test- expressed during all ing now available through the Diagnostic Laboratory, stages of active cell Stem cell as a component of the new Canine Cutaneous Mast division. Plus, it factor binds to c-kit Cell Tumor Profi le, can identify this mutation and help evaluates expres- Mutated c-kit C-kit is guide therapeutic decisions. sion and localiza- is always phosphorylated tion of the c-kit phosphorylated IDENTIFYING GROWTH DRIVERS receptor/protein. Mast cells Mast cell growth is driven in part by the binding Proliferation undergo limited of a growth factor (stem cell factor) to a tyrosine index, both through division kinase receptor (c-kit) expressed on the surface of mitotic index3,4 and mast cells. When the Ki67 expression,5 has Mast cells growth factor binds, the been demonstrated to divide FOCUS continuously ON PCR intracellular portion of inversely correlate with local recur- c-kit becomes phos- rence, incidence of distant metastases and overall phorylated, initiating a signaling cascade that eventu- survival. Additionally, localization of the c-kit ally leads to mast cell division. The Harvard researchers protein — specifi cally, aberrant cytoplasmic expres- 1. London CA, Galli SJ, Yuuki T, et al. sion) — has also been demonstrated to correlate with Spontaneous canine mast cell tumors found about 20 percent of canine mast cell tumors car- express tandem duplications in the ried a mutation in the c-kit gene called an internal local recurrence and overall survival times, where Type proto-oncogene c-kit. Exp Hematol. 1999 tandem duplication, or ITC; part of the gene is dupli- II and III localization (focal and diffuse cytoplasmic, Apr;27(4):689-97. cated. The biological consequence of this duplication respectively) are associated with increased local recur- 2. London CA, Malpas PB, Wood-Follis 6 SL, et al. Multi-center, placebo-controlled, was a change in the protein structure of c-kit, which rence and decreased survival times. double-blind, randomized study of oral causes it to be permanently phosphorylated and consti- toceranib phosphate (SU11654), a tuitively active, even when the growth factor is not pres- PUTTING THE ASSAY TO WORK receptor tyrosine kinase inhibitor, for the treatment of dogs with recurrent (either ent. The unchecked growth we see in mast cell tumors CSU VDL’s DNA assay can be run on air-dried stained local or distant) mast cell tumor following results. or unstained aspirates, as well as on biopsy formalin surgical excision. Clin Cancer Res. 2009 The new tyrosine kinsase inhibitor Palladia inhibits fi xed tissues; however at least 10 percent of the cells Jun 1;15(11):3856-65. c-kit phosphorylation, and therefore effectively inhib- in the aspirate or biopsy must be mast cells in order 3. Romansik EM, Reilly CM, Kass PH, Moore PF, London CA. Mitotic index its mast cell growth. Recent clinical trials have dem- for the mutation to be detected. The proliferative index is predictive for survival for canine onstrated the effectiveness of Palladia as a therapy for and IHC stain for Kit localization are carried out on cutaneous mast cell tumors. Vet Pathol. mast cell tumors that have recurred after excision.2 Pal- biopsy samples. All tests can be run independent of the 2007 May;44(3):335-41. ladia was most effective in patients whose tumors car- MCT profi le, but the combined information obtained 4. Elston L, Sueiro FA, Cavalcanti JN, Metze. The importance of the mitotic index as ried the c-kit mutation, at 60 percent to 70 percent via the complete profi le provides optimal prognostic a prognostic factor for canine cutaneous effective, compared to patients whose tumors did not and therapeutic guidance. mast cell tumors - a validation study. Vet carry the mutation, at only 30 percent to 40 percent For information on this new MCT profi le, includ- Pathol. 2009 Mar;46(2):362-364. ing how to request one and when the test is run, 5. Webster JD, Yuzbasiyan-Gurkan V, Miller response. Detecting the c-kit mutation can help guide RA, et al. Cellular proliferation in canine a decision to use Palladia over other chemotherapeutic visit the website dlab.colostate.edu/webdocs/services/ cutaneous mast cell tumors: associations agents such as vinblastine. MCTProfi leInformation.pdf or call (970) 297-1281. with c-KIT and its role in prognostication. Vet Pathol. 2007 May;44(3):298-308. In addition to the evaluation for a c-kit gene Anne Avery, at [email protected], can answer 6. Webster JD, Yuzbasiyan-Gurkan V, Thamm mutation, the new canine cutaneous mast cell tumor questions on the c-kit mutation assay, and Brad Charles, DH, et al., Evaluation of prognostic (MCT) profi le also assesses the proliferation index of at (970) 297-4087 can provide more information about markers for canine mast cell tumors the tumor via a combination of mitotic index and the mast cell tumor profi le. ▲ treated with vinblastine and prednisone. BMC Vet Res. 2008 Aug 13;4:32. 6 Volume 15, Number 1

Guardians of Public Health Real-time infl uenza PCR testing Contact Kristy Pabilonia s a member of the National Animal Health Labo- Kristy Pabilonia, DVM, DACVM, CSU VDL Avian Diagnostics at (970) 297-4109 if you Aratory Network, CSU VDL is approved to perform and BSL3 Operations Section Head; and Christina have questions about infl uenza real-time reverse transcription PCR. Five assays Weller, CSU VDL Avian Diagnostics and BSL3 Operations are available to NAHLN labs, and CSU VDL performs all Microbiologist infl uenza testing fi ve: infl uenza A viruses (matrix gene), H5 avian infl u- enza viruses, H7 avian infl uenza viruses and the pan- enza virus. We have detected low pathogenic avian infl u- demic H1N1 infl uenza virus (matrix gene enza viruses, primarily in wild bird samples. No highly assay and N1 assay). All positive samples are pathogenic avian infl uenza viruses have been identifi ed. sent to the National Veterinary Services Lab In 2009, with the emergence of pandemic H1N1 in Ames, Iowa, for confi rmatory testing. infl uenza virus, the NAHLN released a modifi ed infl u- In 2004, CSU VDL and the Colorado enza A matrix real-time PCR protocol, as well as a Department of Agriculture started the Col- real-time PCR protocol targeting the 2009 N1 subtype. orado Avian Disease Surveillance Program. While the primary use for these assays is to test swine, Funded by USDA and housed at VDL, we were granted permission to use them to screen the program provides free avian infl uenza companion animals. surveillance testing for commercial fl ocks, To date, we have tested 14 samples from swine, cats backyard fl ocks and live bird markets. The and dogs. All swine and canine samples have tested program also provides free disease inves- negative. CSU VDL diagnosed two cases of H1N1 pan- CSU VDL’s cooperative Avian tigation services for backyard fl ock owners experienc- demic infl uenza virus in domestic cats in November Disease Surveillance Program ing disease and mortality. In addition, the CSU VDL has 2009. These cases were some of the fi rst cases diag- helps monitor the growing also participated in the national USDA-Wildlife Services nosed in companion animals. Both cats presented to urban poultry movement for wild bird avian infl uenza surveillance program. veterinarians with respiratory abnormalities and expe- early warnings of emergent Since 2004, more than 30,000 Colorado wild and rienced a prolonged period of illness. Information on pandemic infl uenza. domestic bird samples have been tested for avian infl u- these two cases is in preparation for publication. ▲

Case Study Infectious hepatitis: Forgotten, not gone young adult, mixed-breed bitch whelped a litter of Jeanette Bishop, MS, CSU VDL Molecular Diagnostics A11 puppies while in a rescue facility. Several became Research Associate, and Hana Van Campen, ill at 5 weeks old. One died and was diagnosed with infec- DVM,PhD,DACVM, CSU VDL Virology Section Head tious canine hepatitis (ICH). Two unrelated 4-month- old puppies were exposed. The virus targets the lymphoid tissues, including the FOCUS Urine samples were sub- tonsils and Peyer’s patches; the liver, causing hepatitis; and ON PCR mitted from nine remaining, the kidneys. Although the immune response clears the 8-week-old puppies, the dam virus in 10 to 14 days, the virus persists in the kidneys of and the two exposed dogs for PCR canine adenovi- survivors and is shed in urine for weeks to months. rus-1 (CAV-1) testing. All nine puppy sample were In addition to dogs, CAV-1 infects foxes, coyotes, positive for CAV-1 by PCR, while the dam and exposed wolves, raccoons, ferrets, skunks and bears. Immuni- puppy samples were negative. zation with CAV-2 containing modifi ed live viral vac- CAV-1 is the etiologic agent for ICH. The clinical cines has been effective in preventing ICH in domestic signs in the acute phase of ICH — fever, anorexia, leth- dogs. While uncommon in vaccinated populations, argy, vomiting and diarrhea — are non-specifi c and may ICH occasionally crops up in unvaccinated dogs in be mistaken for parvovirus-1 or distemper. Frequently, rescue and humane shelters. In suspect cases, whole ICH is suspected only when some dogs develop corneal EDTA blood may be submitted for PCR. Tissues includ- edema or “blue-eye” in the subacute phase, at three to ing liver, spleen, kidney and lymph nodes are suitable four weeks after infection. The disease is more severe in post-mortem samples. Urine samples should be tested younger dogs, and peracute death may occur in puppies. for shedding. The cost of the PCR is $35. ▲ Spring/Summer 2010 7 LABLINES

Lab Update TREKing Johne’s for faster results SU VDL’s Bacteriology Section offers a more Mike Russell, CSU VDL Microbiologist, and Doreene Hyatt, Crapid test for the detection of Mycobacterium PhD, CSU VDL Bacteriology Section Head avium subspecies paratuberculosis. Instead of using the old method of culturing the sample onto solid days. The PCR then will be performed again on day media and checking for 45 to ensure the sample is still negative. ▲ growth over a period of BOVINE 112 days, bacteriology VDL NOW NVSL-APPROVED now performs a liquid FOR JOHNE’S DIRECT PCR DETECTION culture using the TREK para-JEM ESP Culture System ATTENTION: NEW LIMS! II, which checks for bacterial growth over a period of VDL is pleased to inform you it has 45 days. passed the individual 2010 Johne’s Starting July 1 we The TREK system works by continuously monitor- Profi ciency Panel using direct PCR. This switched to a new ing the liquid culture for bacterial growth, notifying update will permit us to conduct offi cial laboratory information the technician of positive growth with a light. Just testing for USDA beginning immediately management system because there is growth does not necessarily mean through the end of 2011. (LIMS), call STARLIMS. the sample contains M. avium paratuberculosis. The You will see new and sample is put through a decontamination process, The real-time PCR detection of M. avium but some organisms, such as other mycobacteria spe- paratuberculosisis directly from a fecal improved formats for cies, are hardy enough to survive the process and will sample will allow clients to receive a your results. Please call grow in the liquid culture, giving a false positive. To result many times on the same day we with questions and ensure no false positives are reported, we perform a receive the sample. comments. Please be real-time PCR detecting M. avium paratuberculosi- We have also passed our profi ciency patient with as we go sis after growth in the liquid culture. If the sample through this transition. is PCR-positive, the client will receive an M. avium test for liquid culture, solid culture and paratuberculosisis-positive report. If the sample is serum ELISA for Johne’s disease and are PCR-negative, the liquid culture will be put back approved by USDA through the end of 2010. onto the TREK system for the remainder of the 45

Real-time PCR testing CSU ROCKY FORD DEMONSTRATES POOLED TRICH SAMPLE RELIABILITY for T. foetus is now avail- From Jan. 1, 2007, to May 5, 2010, the CSU able at the Fort Collins Tritrichomonas foetus Positives VDL at Rocky Ford has tested 27,856 bulls lab. Quicker results with 0.9% by pooled PCR for the presence of T. foetus no change in sample col- (n = 79) and found 288 positive individuals. During the lection or increase in same period, the lab has tested 8,675 by 1.0% price compared to con- individual PCR and found 79 positives. The (n = 288) ventional testing. Email number of positive by pooling is 1 percent, [email protected] or while the number by individual PCR it is 0.9 call (970) 297-1281 percent. Statistical analysis of the test results for details. show no signifi cant statistical difference in Tested by individual PCR (n = 8,675) the number of positives detected by pools Tested versus the number of positives detected by by pooled PCR individual PCR. Pooling can increase the (n = 27,856) cost-effectiveness of Trich testing without compromising diagnostic reliability. 8 Volume 15, Number 1

Reportable Disease Update Colorado horse tests EP-positive SDA has now authorized CSU VDL to perform the sia caballi-negative within 30 days of admission to Arap- UcELISA tests for Theileria equi and Babesia caballi ahoe Park. The piroplasmosis test-positive horse was in for the intra- and interstate movement of equids not dis- a stable with about twenty horses. None of those horses playing clinical signs of piroplasmosis. Tests are are considered positive at this time. Further testing will run each Tuesday and Friday. Submit 1 ml of be done in approximately 30 days from the initial tests. If serum and be sure to include horse’s iden- they are negative, the quarantine will be released. tifi cation. Cost is $16 per sample. Equine piroplasmosis is a reportable disease and As was reported in April, a horse in Col- leads to regulatory consequences. A quarantine and orado tested positive for equine piroplas- subsequent disease-control plan for any test-positive mosis. Although it showed no clinical signs of horses is determined by the State Veterinarian of Col- equine piroplasmosis, the horse tested positive to Theile- orado, USDA-APHIS-VS, along with input from the ria equi on the cELISA test. The serum sample had been owners. Currently, there is no vaccine or approved submitted by a Colorado private practitioner. The posi- treatment for EP in the United States. tive horse has since been euthanized. USDA-APHIS-VS’ Equine Piroplasmosis Info Sheet, Arapahoe Park Racetrack in Aurora instituted EP test- written in English and Spanish, provides further infor- ing requirements for all horses entering the track facil- mation for you and your clients. You can access it ities — the reason the EP-positive sample was on the Web at www.aphis.usda.gov/animal_health/ submitted. Horses must be T. equi- and Babe- animal_diseases/piroplasmosis/index.shtml. ▲ Questions? Call the State Veterinarian’s Offi ce at (303) 239-4161 Bovine Virology Case Study An unusual case of BRSV in a calf sample of lung from a 4-month Lung tissue was also assayed for Christina Gates, CSU VDL Aold dairy calf was submitted BRSV using a reverse transcriptase Microbiologist; Jeanette Bishop, MS, to the diagnostic laboratory for PCR test. It was strongly positive CSU VDL Molecular Diagnostics histopathology and FA for BRSV genomic RNA. Research Associate; Barb Powers, tests. Entire lobules were Due to the histologic fi ndings and FOCUS DVM,PhD,DACVP, CSU VDL Director; ON PCR positively stained for the extensive FA staining in the lung, and Hana Van Campen, bovine respiratory syn- vaccination history of the calf was DVM,PhD,DACVM, CSU VDL Virology cytial virus by FA test. The pattern of obtained. Calves were inoculated by Section Head intranasal route with a modifi ed 1-100 bp ladder live BRSV vaccine labeled for intra- muscular administration. Six calves Calf lung became ill, fi ve responded to treat- ment and one died. 1:10 dilution of calf lung Attenuated live viral IM vaccines may result in disease when admin- Negative extraction control istered by the intranasal route. Unforeseen environmental factors, BRSV positive amplifi cation control such as immune status, and other infectious agents may tip the bal- Negative amplifi cation control ance of health in calves inoculated intranasally with BRSV. Recent BRSV staining is unusual compared reports suggest intranasal vaccina- 1 Ellis JA, Gow SP, Goji N. Response tion for BRSV in young calves may to experimentally induced infection with to that observed in bovine pneu- bovine respiratory syncytial virus following monias. Histologically, we observed not fully protect against subsequent intranasal vaccination of seropositive and viral challenge due to the presence seronegative calves. J Am Vet Med Assoc. a severe interstitial pneumonia with 1 ▲ 2010 May 1;236(9):991-9. multinucleated syncycitial cells. of maternal antibodies. Spring/Summer 2010 9 LABLINES

Gaurdians of Public Health Meeting the challenge of Q fever fever is a zoonotic disease that results from infec- Kristy Pabilonia, DVM, DACVM, CSU VDL Avian Diagnostics Qtion with the organism Coxiella burnetii. This and BSL3 Section Head; and Christina Weller, CSU VDL obligate intracellular Gram-negative bacterium is dis- Microbiologist tributed worldwide. Cattle, sheep and goats are the pri- mary reservoir of the organism. The organism is shed testing should be interpreted with care. Current serol- in milk, urine, feces, amniotic fl uid, birth fl uids and ogy techniques lack desired sensitivity and specifi city, placenta of infected animals. The organism can sur- and seropositivity can be diffi cult to interpret as it is vive for prolonged periods in the environment and diffi cult to differentiate between acute infections and exposure to only a few organisms can result in infec- past exposure. tion. Animal infections are often asymptomatic. The primary clinical manifestations observed in animal CSU VDL MONITORING UPDATE populations are aborted fetuses and birth of weak or Since we began offering a real-time PCR assay for C. bur- nonviable neonates. netii, the number of submissions has grown from 21 in 2007 to more than Q Fever Positives ZOONOTIC POTENTIAL 300 in 2009. Submissions Most human cases results from contact with cattle, have been received from 8.7% sheep and goats, especially during parturition. Human bovine, camelid, canine, cases can also potentially result from consumption caprine and ovine species. of unpasteurized milk. Infection is acquired through We receive samples for diag- inhalation or ingestion of the organism. Companion nostic testing for animals 2.6% animals are also susceptible and have been documented with clinical manifestations as the source of human infections. Q fever can result of Q fever and from animals 0% in acute and chronic cases in humans. In acute cases, on premises with Q-fever 2007 2008 2009 symptoms include fever, vomiting, diarrhea, atypical positive owners. In addition, we receive submissions for Contact Kristy Pabilonia pneumonia, headache, hepatitis, skin rash, myalgia and screening research animals and for export testing. at (970) 297-4109 if you general malaise. In chronic cases, the disease persists As the overall number of submissions has grown, have questions about for six months or longer and can result in severe dis- the prevalence of positive samples has decreased. In Q fever testing ease and endocarditis. Infection during pregnancy may 2007, 8.7 percent of the total samples tested positive; result in abortion, fetal death, premature delivery and however, this amounted to only two samples out of a death. Q fever is a reportable disease, but because total of 21 tested. In 2008, 2.6 percent of samples tested approximately 60 percent of human cases are asymp- positive, with no confi rmed positives in 2009. tomatic, it is often under-reported. We have recently started offer- In 2009, the Netherlands diagnosed more than 2,300 ing the CDC Laboratory Response FOCUS human cases and a large scale effort was instituted to Network’s real-time PCR assay for ON PCR control prevalence of the organism in small ruminants, Q Fever. This assay incorporates with the hope that this would decrease incidence of three primer/probe sets, each of which targets a different human infection. Control measures include culling of sequence of the C. burnetii genome. This PCR will allow tens of thousands of pregnant goats on affected farms us to provide a higher level of diagnostic sensitivity. and mass vaccination. The impact of these actions con- However, the increase in the number of target sequences tinues to be evaluated by the scientifi c community and from one to three means that there is an increased test it is hoped will provide insight on the best way to cost. The cost of this test is $60 per sample. C. burnetii handle this type of situation. organisms can be found in the lungs, spleen and liver, as well as milk and blood, so these samples are best for test- DIAGNOSTIC CHALLENGES ing in non-abortion cases. In abortion cases, placenta Because infected animals do not always shed organ- is the best sample for diagnostic testing. Tissues, swabs isms, diagnostic testing for Q fever can be diffi cult. and milk samples from multiple animals may be pooled A negative PCR result does not guarantee that the for testing, but this may decrease overall sensitivity. In animal or population is not infected. Repeated sam- order to minimize exposure of our laboratory personnel pling and testing should be used before an animal or to this agent, we perform this test in our new biosafety farm is determined to be Q-fever negative. Antibody level 3 laboratory. ▲ 10 Volume 15, Number 1

CSU VDL In Press A roundup of VDL faculty research Spraker TR, et al. Detection of the Abnormal excision may provide a good outcome: All three pigs that Isoform of the Prion Protein Associated were treated surgically survived at least nine months with Chronic Wasting Disease in the Optic Path- after surgery with good quality of life. ways of the Brain and Retina of Rocky Mountain Elk. Vet Pathol. 2010 May;47(3):536-46. Ballweber LR. Taenia crassiceps subcutaneous CSU VDL Pathologist Terry Spraker examined the eyes cysticercosis in an adult dog. Vet Rec. 2009 Dec and nuclei of the visual pathways in the brains of 30 5;165(23):693-4. Rocky Mountain elk (Cervus elaphus nelsoni) for the Lora Ballweber, CSU VDL Parasitology Section head, presence of the abnormal isoform of the prion protein details the case of an eight-year-old adult blue heeler associated with chronic wasting disease, PrP.CWD The cross presented to the referring emergency clinic with an research team found the prion in the retina of 89 per- abscess on the left hindlimb. A large, fl uctuant swelling cent of the elk showing an obex score higher than 7 was present on the medial left thigh; cytology revealed (ranging from a disease-free 0 to a terminal 10), com- numerous degenerative neutrophils and macrophages pared to no detection in disease-free elk or those with an obex below 6. The elk with an obex score of less than 6 had no evidence of PrPCWD immunoreactivity in the optic nerve or retina but did have PrPCWD immu- noreactivity in neural regions of the visual pathway in the brain. The data demonstrate detectable PrPCWD immunoreactivity fi rst accumulates in the visual path- ways of the brain, before the retina. Ballweber documents the budding cysticerci of Taenia crassi- ceps from a swelling on the leg of a dog (left) and a squash McCoy AM, Hackett ES, Callan RJ, Powers BE. mount of an inverted scolex showing typical rostellar hooks. Alimentary-associated carcinomas in fi ve Viet- namese potbellied pigs. J Am Vet Med Assoc. with no other abnormalities. Ovoid parasites containing 2009 Dec 1;235(11):1336-41. clear watery fl uid with a single invaginated scolex identi- VDL Director Barb Powers assisted a CSU Department fi ed upon diagnostics were identifi ed as T. crassiceps cys- of Clinical Sciences team in documenting the cases of tice. T. crassiceps cysticercosis in dogs as been reported fi ve geriatric Vietnamese potbellied pigs presenting to the only six times previously in Europe and only once in CSU Veterinary Hospital with complaints of abdominal the United States. T. crassiceps not only poses a fatal distress that had not responded to medical treatment, threat for dogs and cats, it is a clear zoonotic risk as well. in the case of four, and a draining tract of Ballweber reminds veterinarians they should remember the cranial abdomen of unknown duration, in dogs are defi nitive hosts for multiple species of Taenia, the fi fth. Although neoplasia of gastrointes- including crassiceps. Comprehensive pet parasite control tinal or hepatobiliary origin in pigs is infre- should continue to be a priority. quently reported — and never yet reported in Vietnamese potbellied pigs — the study Ballweber LR, et al. Giardiasis in dogs and cats: team tracked the cases of the fi ve pigs with update on epidemiology and public health primary intestinal tumors (in three), gastric signifi cance. Trends in Parasitology 2010 carcinoma (in one), and undifferentiated Apr;26(4):180-9. carcinoma of billiary origin (in one). Ballweber updates the status of molecular identifi cation Neoplasia is generally considered rare of the seven genetic assemblages of Giardia duodenalis, in pigs, but it shouldn’t be overlooked named A to G. Humans are infected with assemblages in the differential diagnosis of generalized A and B, dogs primarily with C and D, and cats with abdominal distress in middle-aged and older F. Recently, small numbers of dogs and cats have been potbellied pigs. As was evident in the report, reported to also carry assemblages A-I or B. Although tumors can potentially arise from multiple the role of dogs and cats as a source of human giardia- components of the gastrointestinal and hepato- sis remains unresolved, the potential role of pets can’t be biliary tracts. If neoplasia is suspected, tumor conclusively excluded, she cautions. ▲ Spring/Summer 2010 11 LABLINES

NEW DIAGNOSTIC SCREENS CSU VDL has introduced new diagnostic on the species and type of panel, but all respiratory, diarrhea and abortion are offered at a discount from the cost of screens. These species-specifi c screens running all the included tests individually. combine tests for the most commonly Look for these new tests on Page 2 of the diagnosed viral, bacterial, parasitic and General Submission Form. toxicological agents of respiratory, Detailed descriptions of these panels can diarrhea and abortion cases. In most be viewed at dlab.colostate.edu. Scroll cases, histology is also included. down to the In the News section and click The costs of these panels vary depending on Panels and Screens.

Veterinary Community Outreach

WIN A $50 CREDIT External advisory committee ON LAB WORK ur External Advisory Committee members vol- future directions. We are grateful for their time and Take a short fi ve-minute survey to unteer their time to meet with us annually and advice, and hope they feel they are an integral part O tell us how to improve LabLines assess our progress, as well as provide input to our of the laboratory. and we’ll enter you in a drawing ■ ■ ■ Dr. Joan Bowen, Small Ruminant Mr. Ed Hansen, Beef Cattle Dr. Del Miles, Beef Cattle to win a $50 credit on CSU VDL Wellington Livermore Greeley services. ■ Mr. Norm Brown, Equine ■ Dr. Dean Hendrickson, Director ■ Dr. Mike Miller, Wildlife Wellington CSU Veterinary Teaching Hospital Colorado Dept To begin the survey, use your ■ Dr. Keith Roehr, State Vet Fort Collins of Wildlife Internet browser to go to: ■ CO Dept of Agric, Denver Dr. Ron Kollers, Small Animal Fort Collins FoodChainCommunications.com/ ■ ■ Mr. Terry Fankhauser, Executive Fort Collins Dr. Roger Perkins Lablines Director, CCA ■ Dr. Elisabeth Lawaczeck USDA/AVIC/APHIS Arvada Colo. Dept of Public Health Lakewood Some restrictions apply. See website for details. ■ Dr. Mike Gotchey, Equine and Environment ■ Mr. Kenny Rogers, CCA Steamboat Springs Denver Yuma ■ Dr. Bob Davies, Wildlife ■ Dr. Larry Mackey, Large Animal ■ Dr. Steve Wheeler, Small Animal Colorado Dept of Wildlife Greeley Englewood Denver ■ Dr. Leesa McCue, Mixed Animal ■ Dr. Deb Young, CSU Extension ■ Dr. Marv Hamann, Mixed Practice Limon Fort Collins Pueblo West

We would like to extend our sincere gratitude to our initial on this mission to establish and grow the Endowment Fund. donors to the new CSU VDL Endowment. Thank you for Contribute by visiting us online at www.dlab.colostate.edu and supporting the VDL and our mission. We invite you to join us clicking “Support the DLAB online.”

2008 NOVEMBER 2009 ■ Amanda Hosny, CVT, in honor of JOE MCDOWELL ■ Barbara E. Powers, DVM ■ Barbara E. Powers, DVM Dr. Jenna Burton Craig, Colo., native and University APRIL 2009 ■ Fisher’s Peak Veterinary Clinic on ■ Ferris Veterinary Services on of Northern Colorado grad Joe ■ Hana Van Campen behalf of William C. Aaroe, DVM behalf of Martha A Ferris, DVM McDowell joined the VDL in MAY 2009 DECEMBER 2009 MARCH 2010 February. Joe will help VDL ■ James A. Kennedy on behalf ■ Foothills Animal Hospital on ■ Colorado Cattlemen’s Association maintain its commitment to quality of Ms. Kathy Gilbert behalf of Roger A. Liehr, DVM assurance and customer service AUGUST 2009 ■ Constance M. Hvass, PhD, in through various lab support ■ Kristy L. Pabilonia, DVM honor of Dr. Linda Sue Wiest functions. Welcome, Joe. REGULAR COLUMNS INSIDEINSIDE THISTHIS ISSUEISSUE

■ DIAGNOSTIC UPDATE p. 5 FOCUS ON PCR ...... p. 1 EP TESTING UPDATE . . . . p. 8 Canine mast cell profi le. How PCR improves the Equine piroplasmosis appears detection and management in Colorado. What you need ■ PUBLIC HEALTH p. 6 of infectious diseases. to know about testing. Real-time infl uenza PCR. ■ DIAGNOSTIC UPDATE p. 7 IMA MANAGEMENT . . . . . p. 3 AVIAN FLU ...... p. 6 Johne’s testing additions. How CSU VDL’s PCR can help Employing PCR to monitor clinics better manage for avian infl uenza. ■ VDL IN PRESS p. 10 immune-mediated anemia. The latest research roundup Q FEVER REVIEW ...... p. 9 from the VDL faculty. WHAT KILLED MY PUP? . . p. 4 VDL’s experts detail what we PCR improves differential know about this

■ MEET THE LAB p. 11 diagnosis of neonatal deaths. zoonotic pathogen.

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Updatep from the Director olecular Diagnostics, specifi cally In other news, our 2009 Annual Report MPolymerase Chain Reaction (PCR), is available on our website or as hard copy is certainly not new to our laboratory or upon request. In January, we met with laboratories across the country. However, in our External Advisory Committee, which the recent years, requests for this technol- as always provided us with guidance on ogy have dramatically increased. In general, moving the laboratory forward. Our labo- PCR technology is accepted to be a very sen- ratory, as well as other diagnostic labora- sitive and specifi c testing procedure. It has tories, veterinary clinics and people across replaced many of the older diagnostic tech- the country have struggled in these eco- niques, which were either less sensitive or nomic times. But recently, we have seen took a long time to complete. Does a posi- BARBARA POWERS, an increase in laboratory use, signaling the tive PCR test always conclusively show the DVM, PHD, DACVP economy is truly on its way to recovery. DIRECTOR animal has disease? Likewise, does a nega- We hope you all have an enjoyable tive PCR test always indicate the animal does not have summer and enjoy the disease? This issue of LabLines explores these ques- warm weather which LAB tions and demonstrate the proper use of PCR tech- has fi nally just arrived NEWS nology to either make or confi rm a diagnosis of a sus- to Colorado after a very FOLLOW OUR pected disease. The lead article by Dr. Mike Lappin also long, cold winter. We look forward to seeing you this ANNUAL PROGRESS details some of the issues regarding interpretation of fall at the Colorado Veterinary Medical Association Check out our 2009 PCR diagnostics. As always, a practitioner and diagnos- meeting in Loveland and the Annual American Asso- tician, working as a team, is critical in interpreting the ciation of Veterinary Laboratory Diagnosticians meet- annual report, at fi nal result of this — and any — test. That’s why CSU ing in Minnesota. www.dlab.colostate.edu/ VDL always includes close oversight and consultation webdocs/news/ with an appropriate faculty specialist in bacteriology, virology or parasitology for each assay