Washington Animal Disease Diagnostic Laboratory Research Articles

ABORTION AND NEONATAL MORTALITY DUE TO TOXOPLASMA GONDII IN BIGHORN SHEEP (OVIS CANADENSIS).

Low lamb recruitment can be an obstacle to bighorn sheep (Ovis canadensis) conservation and restoration. Causes of abortion and neonate loss in bighorn sheep, which may affect recruitment, are poorly understood. Toxoplasma gondii is a major cause of abortion and stillbirth in domestic small ruminants worldwide, but no reports exist documenting abortion or neonatal death in bighorn sheep attributable to toxoplasmosis. Between March 2019 and May 2021, eight fetal and neonatal bighorn lamb cadavers from four western US states (Idaho, Montana, Nebraska, and Washington) were submitted to the Washington Animal Disease Diagnostic Laboratory for postmortem examination, histologic examination, and ancillary testing to determine the cause of abortion or neonatal death. Necrotizing encephalitis characteristic of toxoplasmosis was identified histologically in six of eight cases, and T. gondii infection was confirmed by PCR in five cases with characteristic lesions. Other lesions attributable to toxoplasmosis were pneumonia (3/5 cases) and myocarditis (2/5 cases). Protozoal cysts were identified histologically within brain, lung, heart, skeletal muscle, adipose tissue, or a combination of samples in all five sheep with PCR-confirmed T. gondii infections. Seroprevalence of T. gondii ranged from 40-81% of adult females sampled in the Washington population in October and November 2018-2021, confirming high rates of exposure before detection of Toxoplasma abortions in this study. Of 1,149 bighorn sheep postmortem samples submitted to Washington Animal Disease Diagnostic Laboratory between January 2000 and May 2021, 21 of which were from fetuses or neonates, a single case of chronic toxoplasmosis was diagnosed in one adult ewe. Recent identification of Toxoplasma abortions in bighorn sheep suggests that toxoplasmosis is an underappreciated cause of reproductive loss. Abortions and neonatal mortalities should be investigated through postmortem and histologic examination, particularly in herds that are chronically small, demographically stagnant, or exhibit reproductive rates lower than expected.

Accessory Tragus: A Rare Congenital Anomaly in a Dog

Accessory tragus is a congenital malformation of part of the external ear, commonly reported in humans. Clinically, it is a benign, cutaneous mass located anywhere between the tragus of the ear and the angle of the mouth, along the migratory path of the first branchial arch. An accessory tragus was diagnosed in an otherwise healthy six-month-old male castrated American pit bull terrier that had a haired, pedunculated cutaneous mass on the left maxillary region from birth. Histologically, the mass was a polypoid extension of histologically normal haired skin with a central core of subcutaneous adipose tissue and well-differentiated elastic cartilage. To the authors' knowledge, this is the first report of this lesion in a non-human species. Retrospective examination of records from 2008 to 2018 at the Washington Animal Disease Diagnostic Laboratory failed to identify any other case. As accessory tragus in humans is commonly linked with other congenital anomalies and syndromes, recognition of this lesion in animals may aid in early discovery of other congenital defects and inform adequate excision of the lesion to prevent chondrodermatitis.

Clarifying dairy calf mortality phenotypes through postmortem analysis

Health problems can be thought of as phenotypic expressions of the complex relationships between genes, environments, and phenomes as a whole. Detailed evaluations of phenotypic expressions of illness are required to characterize important biological outcomes. We hypothesized that classifying dairy calf mortality phenotypes via a systematic postmortem analysis would identify different cause-of-death diagnoses than those derived from treatments alone. This cross-sectional study was carried out on a dairy calf ranch in the northwestern United States from June to September 2017 and focused on calves ≤90 d of age. Comparisons were made between causes of death based on 3 levels of information: on-farm treatment records alone, necropsy-based postmortem analyses in addition to treatment records, and Washington Animal Disease Diagnostic Laboratory (WADDL) results in addition to all other information. A total of 210 dairy calves were necropsied during this study, of which 122 cases were submitted to WADDL. Necropsy- and WADDL-derived mortality phenotypes were in almost perfect agreement (Cohen's κ = 0.86) when broadly categorized as diarrhea, respiratory, diarrhea and respiratory combined, or other causes. The level of agreement between on-farm treatment records and postmortem-derived results was low and varied by the level of diagnostic detail provided. There was just fair agreement (κ = 0.22) between treatment-based and necropsy-based phenotypes without WADDL input and only slight agreement (κ = 0.13) between treatment-based and corresponding necropsy-based phenotypes with WADDL input. Even for those cases in which causes of death aligned along a comparable pathologic spectrum, the lack of detail inherent to standard treatment-based causes of death failed to identify meaningful target areas for intervention. This was especially apparent for numerous cases of necrotizing enteritis and typhlitis (cecal inflammation) that were variously categorized as diarrhea and pneumonia by treatment-based diagnoses. The specificity of these lesions stood in stark contrast to the otherwise generic cause of death diagnoses derived from treatments. The findings from this study supported the hypothesis and highlighted the value of on-farm necropsies and laboratory-based diagnostics to (1) detect antemortem disease misclassifications, (2) provide detail regarding disease processes and mortality phenotypes, and (3) direct disease mitigation strategies.

Antimicrobial Resistance Profiles and Diversity in Salmonella from Humans and Cattle, 2004-2011.

Analysis of long-term anti-microbial resistance (AMR) data is useful to understand source and transmission dynamics of AMR. We analysed 5124 human clinical isolates from Washington State Department of Health, 391 cattle clinical isolates from the Washington Animal Disease Diagnostic Laboratory and 1864 non-clinical isolates from foodborne disease research on dairies in the Pacific Northwest. Isolates were assigned profiles based on phenotypic resistance to 11 anti-microbials belonging to eight classes. Salmonella Typhimurium (ST), Salmonella Newport (SN) and Salmonella Montevideo (SM) were the most common serovars in both humans and cattle. Multinomial logistic regression showed ST and SN from cattle had greater probability of resistance to multiple classes of anti-microbials than ST and SN from humans (P < 0.0001). While these findings could be consistent with the belief that cattle are a source of resistant ST and SN for people, occurrence of profiles unique to cattle and not observed in temporally related human isolates indicates these profiles are circulating in cattle only. We used various measures to assess AMR diversity, conditional on the weighting of rare versus abundant profiles. AMR profile richness was greater in the common serovars from humans, although both source data sets were dominated by relatively few profiles. The greater profile richness in human Salmonella may be due to greater diversity of sources entering the human population compared to cattle or due to continuous evolution in the human environment. Also, AMR diversity was greater in clinical compared to non-clinical cattle Salmonella, and this could be due to anti-microbial selection pressure in diseased cattle that received treatment. The use of bootstrapping techniques showed that although there were shared profiles between humans and cattle, the expected and observed number of profiles was different, suggesting Salmonella and associated resistance from humans and cattle may not be wholly derived from a common population.

Francisella tularensis infection without lesions in gray tree squirrels (Sciurus griseus): a diagnostic challenge

Fifteen cases of Francisella tularensis infection (tularemia) were identified in western gray (Sciurus griseus) and eastern gray (Sciurus carolinensis) squirrels submitted to the Washington Animal Disease Diagnostic Laboratory between 2008 and 2011. All of the squirrels originated in Washington State, a geographical area with endemic tularemia in wildlife. Nine of the 15 squirrels with F. tularensis infection had gross (2/15) or microscopic (9/15) multifocal necrotizing lesions in the spleen, liver, or lymph nodes, typical of tularemia. Special stains did not reliably identify intralesional bacteria microscopically. Six of the 15 squirrels infected with F. tularensis lacked gross and microscopic lesions typical of tularemia. All 15 squirrels with F. tularensis infection were identified by polymerase chain reaction tests on the spleen, liver, or lymph node (including all 6 squirrels without typical tularemia lesions); 8 out of 9 squirrels were positive by direct fluorescent antibody test of tissues, and 5 out of 15 squirrels were positive by culture of tissues. The findings underscore the importance of considering tularemia as a possible cause of death when no lesions of tularemia can be identified at necropsy. Furthermore, the findings suggest the possibility of subclinical infections in gray squirrels, and the importance of molecular diagnostics for definitive diagnosis of F. tularensis infection in wild squirrels.

Seroprevalence of Coxiella burnetii in Washington State domestic goat herds.

A caprine herd seroprevalence of Coxiella burnetii infection was determined by passive surveillance of domestic goat herds in Washington State. Serum samples (n=1794) from 105 herds in 31 counties were analyzed for C. burnetii antibodies using a commercially available Q fever antibody enzyme-linked immunosorbent assay (ELISA) test kit. The sera were submitted to the Washington Animal Disease Diagnostic Laboratory for routine serologic screening over an approximate 1-year period from November, 2010, through November, 2011. To avoid bias introduced by testing samples from ill animals, only accessions for routine screening of nonclinical animals were included in the study. A standard cluster sampling approach to investigate seroprevalence at the herd level was used to determine optimal study sample size. The results identified C. burnetii antibodies in 8.0% of samples tested (144/1794), 8.6% of goat herds tested (9/105), and 25.8% of counties tested (8/31). Within-herd seroprevalence in positive counties ranged from 2.9% to 75.8%. Counties with seropositive goats were represented in the western, eastern, southeastern, and Columbia basin agricultural districts of the state. To our knowledge this is the first county-specific, statewide study of C. burnetii seroprevalence in Washington State goat herds. The findings provide baseline information for future epidemiologic, herd management and public health investigations of Q fever.

Serological Survey for Antibodies toLeptospirain Dogs and Raccoons in Washington State

A high number of reported canine leptospirosis cases occurred in Washington State from 2004 to 2006. This prompted a serosurvey of healthy dogs from around the state to determine the distribution of exposure risk and to provide insight into serovar epidemiology in the region. In addition, a convenience sample of sera from injured raccoons was also tested, and clinical serological data from the Washington Animal Disease Diagnostic Laboratory were examined. The proportion of dogs with an antibody titre (>or=1:100) to any serovar was 27/158 (17.1%, 95% CI 11.6-23.9), and that proportion among raccoons was 22/115 (19.1%, 95% CI 12.4-27.5) suggesting that the potential for exposure in Washington state is not uncommon. The most frequently detected serovars in healthy dogs were Autumnalis, Icterohemorrhagiae and Canicola, in clinical canine samples Autumnalis, Bratislava and Pomona were more frequent and in sick or injured raccoons Autumnalis, and Pomona were most frequently detected. Clinical canine serology demonstrated a late summer-fall seasonality that was consistent with other reports. An outbreak of canine leptospirosis occurred during 2004-2006 and was located primarily in western Washington counties, as were three reported human cases in 2005. Canine leptospirosis surveillance is an important tool for detecting human risk of exposure and may provide insights into which serovars are currently of clinical importance.

Prevalence of vesivirus in a laboratory-based set of serum samples obtained from dairy and beef cattle

To examine sera obtained from dairy and beef cattle to detect antibodies against vesivirus and compare seroprevalence among cattle within the sample population. Cattle sera from 8 western states and Maryland submitted to the Washington Animal Disease Diagnostic Laboratory during 1999 and 2000. Sera were analyzed for vesivirus-specific antibodies by use of a recombinant vesivirus-San Miguel sea lion virus serotype 5-capsid peptide antigen in an indirect ELISA. Overall, 693 sera were tested and 105 (15.2%) had positive results. Seropositive cattle were from 7 states (all cattle from Montana and Maryland 10 and 4, respectively were seronegative). Overall seroprevalence for antivesivirus antibody in herds ranged between 0% and 80% (median, 14%). Higher antibody prevalence was significantly associated with older age, dairy rather than beef cattle, and reasons for submission. Logistic regression of factors (abortion, respiratory tract disease, and all other reasons for sample submission) revealed that older age and other reasons were independently associated with higher seroprevalence. Higher seropositive optical density values for the ELISA were observed among older cattle and cattle that aborted, compared with values for cattle with respiratory tract disease or other reasons for submission. This laboratory-based surveillance sample provided a point estimate of seroprevalence against vesivirus among cattle in 9 US states. This suggests that vesivirus infection is widespread with high prevalence in some herds. Risk factors associated with vesivirus seroprevalence in beef and dairy cattle should be confirmed in population-based studies.

Results of a new serologic test suggest an association of Waddlia chondrophila with bovine abortion.

Waddlia chondrophila is a little-known intracellular organism belonging to the order Chlamydiales that has twice been isolated from aborted bovine fetuses. To initiate an investigation of the possibility that W. chondrophila may be an abortifacient pathogen, a serologic test was developed and used to screen bovine sera that were submitted to the Washington Animal Disease Diagnostic Laboratory (Pullman, WA). A highly significant statistical association (P < 0.00001) was observed when comparing antibody titers in cows that had aborted with those in other classes of cattle. Although this result is consistent with the possibility that infection with W. chondrophila may be a cause of bovine abortion, it is also possible that seroprevalence simply increases with age or that exposure rates differ between cows and other classes of cattle. Future serologic studies should specifically compare antibody titers between aborting cows and carefully matched nonaborting cohorts.

Development of a serum plate agglutination test to detect antibodies to Ornithobacterium rhinotracheale.

sistent with Elaeophora, a diagnosis of elaeophorosis, probably caused by E. schneideri, was made. Blindness, circling, and ataxia as seen in this moose are characteristic clinical signs of infection with E. schneideri in moose from Montana5 and Colorado.4 Histopathologic findings in moose elaeophorosis are similar to those of elk (Cervus elaphus)5 and include regions of cerebrocortical necrosis, intimal proliferation within leptomeningeal arteries, and thrombosis of cerebral vessels. The vasculitis reported in this moose has been described in elk with elaeophorosis.1 Both previous reports of elaeophorosis in moose contained descriptions of nematodes within histologic sections of brain, and nematodes were recovered from the carotid arteries.4,5 Despite examination of numerous brain sections and other tissues, only the nematode within an artery surrounding a mesenteric lymph node was observed; the carotid arteries were not examined grossly. The observation of the larval nematode within a mesenteric vessel is not surprising based on observations in elk with elaeophorosis in which parasites were observed within multiple organ systems. Postmortem migration of nematodes was also thought to occur.1 The cause of the observed eosinophilic dermatitis was not determined and although a localized reaction to circulating Elaeophora microfilariae was considered, no microfilariae were observed on histologic examination. This is the first report of elaeophorosis in a moose from Washington state and the first description of this parasite from the eastern portion of the state. The only previous report of elaeophorosis reported from Washington state was E. schneideri from a black-tailed deer (Odocoileus hemionus columbianus) from the western portion of the state.2 Although E. schneideri is considered rare in Washington,2 moose may be a sensitive indicator species for the presence of the parasite, especially in areas where the parasite has not been diagnosed previously. Acknowledgements. We thank Dr. Melinda Wilkerson, Washington Animal Disease Diagnostic Laboratory, for assistance with histopathology, Dr. Scott Lyell Gardner, University of Nebraska State Museum, for assistance in examination of the parasite, and Steve Judd, the Colville Indian Tribe, for bringing this moose to our attention.

An outbreak of listeriosis in a breeding colony of chinchillas.

Listeria monocytogenes is a facultatively anaerobic short gram-positive rod-shaped bacterium that infects a wide range of animals, including ruminants, monogastric animals, birds, fish, rodents, and humans. Infection with this obligate intracellular pathogen causes 3 distinct clinical entities, septicemia, encephalitis, and abortion, which rarely occur simultaneously in the same animal. The septicemic form affects the viscera with or without meningoencephalitis and is common in monogastric animals, whereas encephalitis and abortion occur principally in adult ruminants. Chinchillas are considered one of the species more susceptible to visceral listeriosis, especially when reared in confinement. Listeriosis in chinchillas has been described in many areas of the United States during a period (1949-1955) when chinchilla pelts were prized commodities in the fur industry. More recently, chinchillas have become a widely used animal model for studies of noise-induced hearing loss. An outbreak of disseminated visceral listeriosis occurred in a breeding colony of 130 chinchillas located in northern Idaho. The chinchillas in this operation were raised for pelts. Polygamous breeding was practiced; 1 male had access to 8 females. The feed, open sacks of commercially formulated chinchilla pellets and a bale of hay, was stored on a wood pallet. To promote maintenance of their hair coats, a dust bath pan was provided for the animals. During December 1995, there was an unusually large influx of mice into the chinchilla building, and on several occasions the owners noted mouse droppings in the feed. By January 1996, the colony experienced a 23% mortality (30 of 130) in breeding animals of various ages. Approximately 4 days prior to death, the animals were anorectic and hunched, and some had torticollis. Many animals were found dead without premonitory clinical signs. Four chinchillas (2 dead males, 1 dead female, 1 moribund female) were submitted to the Washington Animal Disease Diagnostic Laboratory for necropsy. All 4 chinchillas were emaciated and lacked subcutaneous and retroperitoneal fat. Consistent gross lesions in the 3 dead chinchillas included multifocal 0.1-0.3-cm-diameter whitetan foci affecting the capsular surfaces and parenchyma of the liver, mesenteric lymph nodes, and the serosa of the small and large intestines, particularly the cecum and colon. Individual differences in postmortem lesions are listed in Table 1. Animal A, a dead female, had a 2-cm rectal prolapse and colonic intussusception in addition to the visceral foci. The intussusception consisted of a 3-cm segment of dark red prox-

Canine Fatalities Associated with the use of a Modified Live Vaccine Administered during Late Stages of Pregnancy

Commencing in November 1992, tissue samples from 10 cases of canine abortion were submitted to the Washington Animal Disease Diagnostic Laboratory for analyses. A common feature was a history of vaccination of the pregnant dam, 34 weeks prior to whelping, with a modified live virus (MLV) multi-component canine vaccine (MLV canine distemper, adenovirus type 2, Parainfluenza , and parvovirus) reconstituted with a killed canine coronavirus vaccine. A cytopathic agent was identified in Vero cells and Crandell feline kidney (CrFK) cells (after repeated passage) inoculated with tissues originating from the dams. No evidence of the usual abortigenic agents of dogs, such as Brucella canis and canine herpesvirus, was found from histopathology, virology, or bacteriology. 6,9 Because the common link among the cases was the use of the MLV vaccine during late-term pregnancy, it was speculated that disease resulted from a virulent component of the vaccine, such as canine parvovirus (CPV). Although isolation attempts were negative for CPV, multiple other viruses isolated had properties consistent with members of the family Reoviridae. 1 In this paper, we present the laboratory findings from 3 naturally occurring cases of canine disease associated with the administration of an MLV canine vaccine during late stages of gestation. This study was a collaborative project with investigators at the Veterinary Biologics Laboratory (National Veterinary Services, Ames, IA). An adjunct report on the properties of the viral isolate and its subsequent identification as bluetongue virus are the subject of a separate report. 15

Serological Survey for Heartworm (Dirofilaria immitis) in Dogs in Washington

The serologic prevalence of Dirofilaria immitis infection in 601 dogs in Washington was investigated in 1989-1990. Blood samples for serum were obtained from dogs approximately 2 yr of age or older in humane society shelters (n = 392) or veterinary clinics (n = 209). Serum samples were tested for heartworm infection using an enzyme-linked immunosorbent assay antigen test. Three (0.5%) dogs were positive for D. immitis infection, all of which were born and lived several years in states other than Washington. Heartworms have not been detected during the last 9 yr in the 1,203 dogs examined at the Washington Animal Disease Diagnostic Laboratory, Pullman, Washington. Consequently, the likelihood of indigenous heartworm infections in dogs in Washington remains low at this time.

Applications of a sensitive, GC-ECD analysis for anatoxin-A: Douglas K. Stevens1 and Robert I. Krieger2 ( 1University of Idaho, WOI Regional Program in Veterinary Medical Education, Washington Animal Disease Diagnostic Laboratory and Pharmacology/Toxicology Program, Moscow, Idaho 83843; and 2Department of Food and Agriculture, Worker Health and Safety Unit, 1220 N Street, Sacramento, California 95814, U.S.A.)

Applications of a sensitive, GC-ECD analysis for anatoxin-A: Douglas K. Stevens1 and Robert I. Krieger2 ( 1University of Idaho, WOI Regional Program in Veterinary Medical Education, Washington Animal Disease Diagnostic Laboratory and Pharmacology/Toxicology Program, Moscow, Idaho 83843; and 2Department of Food and Agriculture, Worker Health and Safety Unit, 1220 N Street, Sacramento, California 95814, U.S.A.)

Canine granular cell tumour (myoblastoma): a report of four cases and review of the literature

ABSTRACTFour cases of canine granular cell tumour (myoblastoma) were diagnosed at the Washington Animal Disease Diagnostic Laboratory between 1977 and 1981. Two of the tumours occurred in the tongue, one in the lip and one in the larynx. Three cell types were found in the tumours. Granular cells, with abundant periodic acid‐Schiff (PAS)‐positive cytoplasmic granules, were most numerous. Interstitial cells resembled fibroblasts and were often adjacent to collagen fibrils. Angulate body cells contained PAS‐positive, ovoid cytoplasmic inclusions composed of microtubular subunits. Review of these and reported cases indicates no breed or sex predisposition for this rare tumour. The tongue is the most common site and all but one tumour have occurred in or adjacent to the oral cavity. There have been no reports of recurrence or metastasis of granular cell tumours except the recurrence of a disputed case in the subcutis of a dog.

Nonhematopoietic gastrointestinal neoplasia in cats: a retrospective study of 44 cases.

Gastrointestinal neoplasms other than lymphosarcomas and mast cell tumors were diagnosed in 44 cats during a 14-year period at the Washington Animal Disease Diagnostic Laboratory. All the tumors were malignant; 31 metastasized or recurred. One cat had fibrosarcoma; another, leiomyosarcoma. The other 42 cats had adenocarcinomas, which were subclassified into three histologic patterns: tubular adenocarcinoma; undifferentiated carcinoma; and mucinous adenocarcinoma. The cats averaged 10.6 years of age. There was no sex predisposition. Siamese cats had a higher frequency of adenocarcinomas than other breeds. Osseous and chondroid metaplasia occurred in nine adenocarcinomas.