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At least 19 recordsLinked to original sources

A novel herpesvirus detected in 3 different species of chelonians

Herpesviruses are found in free-living and captive chelonian populations, often in association with morbidity and mortality. To date, all known chelonian herpesviruses fall within the subfamily Alphaherpesvirinae . We detected a novel herpesvirus in 3 species of chelonians: a captive leopard tortoise ( Stigmochelys pardalis ) in western TX, USA; a steppe tortoise ( Testudo [ Agrionemys ] horsfieldii ) found near Fort Irwin, CA, USA; and 2 free-living, three-toed box turtles ( Terrapene mexicana triunguis ) found in Forest Park, St. Louis, MO. The leopard tortoise was coinfected with the tortoise intranuclear coccidian and had clinical signs of upper respiratory tract disease. The steppe tortoise had mucopurulent nasal discharge and lethargy. One of the three-toed box turtles had no clinical signs; the other was found dead with signs of trauma after being observed with blepharedema, tympanic membrane swelling, cervical edema, and other clinical signs several weeks prior to death. Generally, the branching order of the turtle herpesviruses mirrors the divergence patterns of their hosts, consistent with codivergence. Based on phylogenetic analysis, this novel herpesvirus clusters with a clade of viruses that infect emydid hosts and is likely of box turtle origin. Therefore, we suggest the name terrapene alphaherpesvirus 3 (TerAHV3) for the novel virus. This virus also has the ability to host-jump to tortoises, and previously documented herpesviral morbidity tends to be more common in aberrant hosts. The relationship between clinical signs and infection with TerAHV3 in these animals is unclear, and further investigation is merited.

Journal of Veterinary Diagnostic Investigation

Postmortem diagnostic investigation of disease in free-ranging marine turtle populations: A review of common pathologic findings and protocols

Over the past few decades, there have been increasing numbers of reports of diseases in marine turtles. Furthermore, in recent years, there have been documented instances of apparently new diseases emerging in these species of which the etiology and/or pathogenesis remain unknown. These instances i) raise concern for the survival of marine turtles, and ii) question the health and stability of the benthic marine environments in which turtles live. Knowledge of common disease processes and pathologic changes in lesions, along with a standardized approach to postmortem and sample collection are required to document and understand the host-agent-environment interactions in marine turtle health. This review combines, for the first time, a standardized approach to the postmortem of marine turtles for veterinary clinicians, with a concurrent descriptive review of the gross and microscopic pathologic changes in lesions commonly seen.

Journal of Veterinary Diagnostic Investigation

Second round of an interlaboratory comparison of SARS-CoV2 molecular detection assays used by 45 veterinary diagnostic laboratories in the United States

The COVID-19 pandemic presents a continued public health challenge. Veterinary diagnostic laboratories in the United States use RT-rtPCR for animal testing, and many laboratories are certified for testing human samples; hence, ensuring that laboratories have sensitive and specific SARS-CoV2 testing methods is a critical component of the pandemic response. In 2020, the FDA Veterinary Laboratory Investigation and Response Network (Vet-LIRN) led an interlaboratory comparison (ILC1) to help laboratories evaluate their existing RT-rtPCR methods for detecting SARS-CoV2. All participating laboratories were able to detect the viral RNA spiked in buffer and PrimeStore molecular transport medium (MTM). With ILC2, Vet-LIRN extended ILC1 by evaluating analytical sensitivity and specificity of the methods used by participating laboratories to detect 3 SARS-CoV2 variants (B.1; B.1.1.7 [Alpha]; B.1.351 [Beta]) at various copy levels. We analyzed 57 sets of results from 45 laboratories qualitatively and quantitatively according to the principles of ISO 16140-2:2016. More than 95% of analysts detected the SARS-CoV2 RNA in MTM at ≥500 copies for all 3 variants. In addition, for nucleocapsid markers N1 and N2, 81% and 92% of the analysts detected ≤20 copies in the assays, respectively. The analytical specificity of the evaluated methods was >99%. Participating laboratories were able to assess their current method performance, identify possible limitations, and recognize method strengths as part of a continuous learning environment to support the critical need for the reliable diagnosis of COVID-19 in potentially infected animals and humans.

Journal of Veterinary Diagnostic Investigation

A review of episodes of zinc phosphide toxicosis in wild geese (Branta spp.) in Oregon (2004−2011)

Epizootic mortality in several geese species, including cackling geese (Branta hutchinsii) and Canada geese (Branta canadensis), has been recognized in the Willamette Valley of Oregon for over a decade. Birds are generally found dead on a body of water or are occasionally observed displaying neurologic clinical signs such as an inability to raise or control the head prior to death. Investigation of these epizootic mortality events has revealed the etiology to be accidental poisoning with the rodenticide zinc phosphide (Zn 3 P 2 ). Gross and histologic changes are restricted to acute pulmonary congestion and edema, sometimes accompanied by distension of the upper alimentary tract by fresh grass. Geese are unusually susceptible to this pesticide; when combined with an epidemiologic confluence of depredation of specific agricultural crops by rodents and seasonal avian migration pathways, epizootic toxicosis may occur. Diagnosis requires a high index of suspicion, appropriate sample collection and handling, plus specific test calibration for this toxicant. Interagency cooperation, education of farmers regarding pesticide use, and enforcement of regulations has been successful in greatly decreasing these mortality events since 2009.

Oregon

Type C botulism in dairy cattle from feed contaminated with a dead cat

Four hundred twenty-seven of 441 adult Holstein dairy cattle from a 1,200-cow dairy died over a 1-week period during early spring 1998. Affected animals were from 4 late lactation pens, one of which included the bull string. Signs included weakness, recumbency, watery diarrhea, and death. Eighty animals from the 4 pens were dead approximately 8 hours after the first ill cows were noted. Affected cows would collapse on stimulation and extend all 4 limbs with moderate rigidity. Several lacked lingual tonus and had abdominal breathing patterns. The animals had been fed a load of total mixed ration that included a rotten bale of oat hay containing a dead cat. No common toxicants were identified, and pathologic examination revealed no consistent lesions. Testing of tissue from the cat carcass found in the feed sample using mouse protection bioassay identified the presence of type C botulinum toxin. Samples of feed, tissue from affected animals, cat tissue from feed, milk, and serum were also tested using an enzyme-linked immunosorbent assay (ELISA) specific for type C botulinum. Two samples of rumen contents were tested and found to be positive for botulism by ELISA, and 1 of 3 liver samples had a weak positive finding. No botulinum toxin was found in milk or sera using the ELISA.

Journal of Veterinary Diagnostic Investigation

Single-dilution enzyme-linked immunosorbent assay for quantification of antigen-specific salmonid antibody

An enzyme-linked immunosorbent assay (ELISA) was developed on the basis of testing a single dilution of serum to quantify the level of antibody to the p57 protein of Renibacterium salmoninarum in sockeye salmon ( Oncorhynchus nerka ). The levels of antibody were interpolated from a standard curve constructed by relating the optical densities (OD) produced by several dilutions of a high-titer rainbow trout ( O. mykiss ) antiserum to the p57 protein. The ELISA OD values produced by as many as 36 test sera on each microplate were compared with the standard curve to calculate the antigen-specific antibody activity. Repeated measurements of 36 samples on 3 microplates on each of 6 assay dates indicated that the mean intraassay coefficient of variation (CV) was 6.68% (range, 0–23%) and the mean interassay CV was 8.29% (range, 4–16%). The antibody levels determined for the serum sample from 24 sockeye salmon vaccinated with a recombinant p57 protein generally were correlated with the levels determined by endpoint titration ( r 2 = 0.936) and with results from another ELISA that was based on extrapolation of antibody levels from a standard curve ( r 2 = 0.956). The single-dilution antibody ELISA described here increases the number of samples that can be tested on each microplate compared with immunoassays based on analysis of several dilutions of each test serum. It includes controls for interassay standardization and can be used to test fish weighing <3 g.

Journal of Veterinary Diagnostic Investigation

Determination of the median toxic dose of type C botulism in lactating dairy cows

Because of the difficulty in identifying botulinum toxin in cattle, it is hypothesized that cattle are sensitive to levels of toxin below the detection limits of current diagnostic techniques (the mouse protection bioassay and the immunostick enzyme-linked immunosorbent assay [ELISA] for type C botulinum toxin). Using an up-down method for toxicologic testing, the median toxic dose (MTD50) for cattle was determined. Four lactating Holstein cows were dosed at 0.125 or 0.25 ng/kg with Clostridium botulinum type C toxin and failed to develop clinical signs of botulism during the 7-day observation period. Three cows given 0.50 ng/kg of toxin developed clinical signs of botulism. From these results, the MTD50 was calculated at 0.388 ng/kg (3.88 mouse lethal doses/kg) using the trim-logit method. These results suggest that cattle are 12.88 times more sensitive to type C botulinum toxin than a mouse on a per kilogram weight basis. The mouse protection bioassay and the immunostick ELISA for type C botulinum toxin failed to identify the presence of the toxin in the serum, blood, and milk samples taken from all 7 animals.

Journal of Veterinary Diagnostic Investigation

Combined use of the ASK and SHK-1 cell lines to enhance the detection of infectious salmon anemia virus

Infectious salmon anemia (ISA) is a severe disease primarily affecting commercially farmed Atlantic salmon (Salmo salar) in seawater. The disease has been reported in portions of Canada, the United Kingdom, the Faroe Islands, and the United States. Infectious salmon anemia virus (ISAV), the causative agent of ISA, has also been isolated from several asymptomatic marine and salmonid fish species. Diagnostic assays for the detection of ISAV include virus isolation in cell culture, a reverse transcriptase-PCR, an enzyme-linked immunosorbent assay, and an indirect fluorescent antibody test. Virus isolation is considered the gold standard, and 5 salmonid cell lines are known to support growth of ISAV. In this study, the relative performance of the salmon head kidney 1 (SHK-1), Atlantic salmon kidney (ASK), and CHSE-214 cell lines in detecting ISAV was evaluated using samples from both experimentally and naturally infected Atlantic salmon. Interlaboratory comparisons were conducted using a quality control-quality assurance ring test. Both the ASK and SHK-1 cell lines performed well in detecting ISAV, although the SHK-1 line was more variable in its sensitivity to infection and somewhat slower in the appearance of cytopathic effect. Relative to the SHK-1 and ASK lines, the CHSE-214 cell line performed poorly. Although the ASK line appeared to represent a good alternative to the more commonly used SHK-1 line, use of a single cell line for diagnostic assays may increase the potential for false-negative results. Thus, the SHK-1 and ASK cell lines can be used in combination to provide enhanced ability to detect ISAV.

Journal of Veterinary Diagnostic Investigation

Detection and quantification of Renibacterium salmoninarum DNA in salmonid tissues by real-time quantitative polymerase chain reaction analysis

Renibacterium salmoninarum is an important salmonid pathogen that is difficult to culture. We developed and assessed a real-time, quantitative, polymerase chain reaction (qPCR) assay for the detection and enumeration of R. salmoninarum. The qPCR is based on TaqMan technology and amplifies a 69-base pair (bp) region of the gene encoding the major soluble antigen (MSA) of R. salmoninarum. The qPCR assay consistently detected as few as 5 R. salmoninarum cells per reaction in kidney tissue. The specificity of the qPCR was confirmed by testing the DNA extracts from a panel of microorganisms that were either common fish pathogens or reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA). Kidney samples from 38 juvenile Chinook salmon (Oncorhynchus tshawytscha) in a naturally infected population were examined by real-time qPCR, a nested PCR, and ELISA, and prevalences of R. salmoninarum detected were 71, 66, and 71%, respectively. The qPCR should be a valuable tool for evaluating the R. salmoninarum infection status of salmonids.

Journal of Veterinary Diagnostic Investigation

Analytical validation of a real-time reverse transcription polymerase chain reaction test for Pan-American lineage H7 subtype Avian influenza viruses

A real-time reverse transcription polymerase chain reaction test for the identification of the H7 subtype in North American Avian influenza viruses (AIVs) was first reported in 2002; however, recent AIV surveillance efforts in wild birds and H7 outbreaks in poultry demonstrated that the 2002 test did not detect all H7 AIVs present in North and South America. Therefore, a new test, the 2008 Pan-American H7 test, was developed by using recently available H7 nucleotide sequences. The analytical specificity of the new assay was characterized with an RNA panel composed of 19 H7 viruses from around the world and RNA from all hemagglutinin subtypes except H16. Specificity for North and South American lineage H7 viruses was observed. Assay limits of detection were determined to be between 103 and 104 gene copies per reaction with in vitro transcribed RNA, and 100.0 and 10 0.8 50% egg infectious doses per reaction. The 2008 Pan-American H7 test also was shown to perform similarly to the 2002 test with specimens from chickens experimentally exposed to A/Chicken/BritishColumbia/314514-2/04 H7N3 highly pathogenic AIV. Furthermore, the 2008 test was able to detect 100% (n = 27) of the H7 AIV isolates recovered from North American wild birds in a 2006-2007 sample set (none of which were detected by the 2002 H7 test).

Journal of Veterinary Diagnostic Investigation

Histopathologic criteria to confirm white-nose syndrome in bats

White-nose syndrome (WNS) is a cutaneous fungal disease of hibernating bats associated with a novel Geomyces sp. fungus. Currently, confirmation of WNS requires histopathologic examination. Invasion of living tissue distinguishes this fungal infection from those caused by conventional transmissible dermatophytes. Although fungal hyphae penetrate the connective tissue of glabrous skin and muzzle, there is typically no cellular inflammatory response in hibernating bats. Preferred tissue samples to diagnose this fungal infection are rostral muzzle with nose and wing membrane fixed in 10% neutral buffered formalin. To optimize detection, the muzzle is trimmed longitudinally, the wing membrane is rolled, and multiple cross-sections are embedded to increase the surface area examined. Periodic acid-Schiff stain is essential to discriminate the nonpigmented fungal hyphae and conidia. Fungal hyphae form cup-like epidermal erosions and ulcers in the wing membrane and pinna with involvement of underlying connective tissue. In addition, fungal hyphae are present in hair follicles and in sebaceous and apocrine glands of the muzzle with invasion of tissue surrounding adnexa. Fungal hyphae in tissues are branching and septate, but the diameter and shape of the hyphae may vary from parallel walls measuring 2 ??m in diameter to irregular walls measuring 3-5 ??m in diameter. When present on short aerial hyphae, curved conidia are approximately 2.5 ??m wide and 7.5 ??m in curved length. Conidia have a more deeply basophilic center, and one or both ends are usually blunt. Although WNS is a disease of hibernating bats, severe wing damage due to fungal hyphae may be seen in bats that have recently emerged from hibernation. These recently emerged bats also have a robust suppurative inflammatory response.

Journal of Veterinary Diagnostic Investigation

Optimized methods for total nucleic acid extraction and quantification of the bat white-nose syndrome fungus, Pseudogymnoascus destructans , from swab and environmental samples

The continued spread of white-nose syndrome and its impacts on hibernating bat populations across North America has prompted nationwide surveillance efforts and the need for high-throughput, noninvasive diagnostic tools. Quantitative real-time polymerase chain reaction (qPCR) analysis has been increasingly used for detection of the causative fungus, Pseudogymnoascus destructans , in both bat- and environment-associated samples and provides a tool for quantification of fungal DNA useful for research and monitoring purposes. However, precise quantification of nucleic acid from P. destructans is dependent on effective and standardized methods for extracting nucleic acid from various relevant sample types. We describe optimized methodologies for extracting fungal nucleic acids from sediment, guano, and swab-based samples using commercial kits together with a combination of chemical, enzymatic, and mechanical modifications. Additionally, we define modifications to a previously published intergenic spacer&ndash;based qPCR test for P. destructans to refine quantification capabilities of this assay.

Journal of Veterinary Diagnostic Investigation

Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA

Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii . The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

Journal of Veterinary Diagnostic Investigation

Cyanide poisoning of a Cooper’s hawk ( Accipiter cooperii )

A Cooper’s hawk ( Accipiter cooperii ) was found dead in a ditch leading from a heap leach pad at a gold mine in Nevada. Observations at autopsy included an absence of external lesions, traces of subcutaneous and coronary fat, no food in the upper gastrointestinal tract, and no lesions in the viscera. Cyanide concentrations (µg/g ww) were 5.04 in blood, 3.88 in liver, and 1.79 in brain. No bacteria or viruses were isolated from tissues, and brain cholinesterase activity was within the normal range for a Cooper’s hawk.

Journal of Veterinary Diagnostic Investigation

Validation of laboratory tests for infectious diseases in wild mammals: Review and recommendations

Evaluation of the diagnostic sensitivity (DSe) and specificity (DSp) of tests for infectious diseases in wild animals is challenging, and some of the limitations may affect compliance with the OIE-recommended test validation pathway. We conducted a methodologic review of test validation studies for OIE-listed diseases in wild mammals published between 2008 and 2017 and focused on study design, statistical analysis, and reporting of results. Most published papers addressed Mycobacterium bovis infection in one or more wildlife species. Our review revealed limitations or missing information about sampled animals, identification criteria for positive and negative samples (case definition), representativeness of source and target populations, and species in the study, as well as information identifying animals sampled for calculations of DSe and DSp as naturally infected captive, free-ranging, or experimentally challenged animals. The deficiencies may have reflected omissions in reporting rather than design flaws, although lack of random sampling might have induced bias in estimates of DSe and DSp. We used case studies of validation of tests for hemorrhagic diseases in deer and white-nose syndrome in hibernating bats to demonstrate approaches for validation when new pathogen serotypes or genotypes are detected and diagnostic algorithms are changed, and how purposes of tests evolve together with the evolution of the pathogen after identification. We describe potential benefits of experimental challenge studies for obtaining DSe and DSp estimates, methods to maintain sample integrity, and Bayesian latent class models for statistical analysis. We make recommendations for improvements in future studies of detection test accuracy in wild mammals.

Journal of Veterinary Diagnostic Investigation

Interlaboratory comparison of SARS-CoV2 molecular detection assays in use by U.S. veterinary diagnostic laboratories

The continued search for intermediate hosts and potential reservoirs for SARS-CoV2 makes it clear that animal surveillance is critical in outbreak response and prevention. Real-time RT-PCR assays for SARS-CoV2 detection can easily be adapted to different host species. U.S. veterinary diagnostic laboratories have used the CDC assays or other national reference laboratory methods to test animal samples. However, these methods have only been evaluated using internal validation protocols. To help the laboratories evaluate their SARS-CoV2 test methods, an interlaboratory comparison (ILC) was performed in collaboration with multiple organizations. Forty-four sets of 19 blind-coded RNA samples in Tris-EDTA (TE) buffer or PrimeStore transport medium were shipped to 42 laboratories. Results were analyzed according to the principles of the International Organization for Standardization (ISO) 16140-2:2016 standard. Qualitative assessment of PrimeStore samples revealed that, in approximately two-thirds of the laboratories, the limit of detection with a probability of 0.95 (LOD95) for detecting the RNA was ≤20 copies per PCR reaction, close to the theoretical LOD of 3 copies per reaction. This level of sensitivity is not expected in clinical samples because of additional factors, such as sample collection, transport, and extraction of RNA from the clinical matrix. Quantitative assessment of Ct values indicated that reproducibility standard deviations for testing the RNA with assays reported as N1 were slightly lower than those for N2, and they were higher for the RNA in PrimeStore medium than those in TE buffer. Analyst experience and the use of either a singleplex or multiplex PCR also affected the quantitative ILC test results.

Journal of Veterinary Diagnostic Investigation

Fatal infection with Versteria sp. in a muskrat, with implications for human health

The Taeniidae tapeworms are a family of helminths that have a similar life cycle, with intermediate hosts developing characteristic cysts in visceral organs. We describe here a case in Pennsylvania, USA, of fatal Versteria infection in a muskrat ( Ondatra zibethicus ), which, to our knowledge, has not been reported to develop disease associated with infection. Postmortem examination revealed widespread tissue loss and replacement by solid-bodied cestode larvae with minimal adjacent inflammation in many visceral organs, most severe in the lungs, liver, and brain. Key morphologic features via histology included cephalic structures and short rostellar hooklets, which are characteristic for the genus. Genetic characterization confirmed the cestode as being an undescribed lineage of Versteria that has been implicated as the cause of severe morbidity and mortality in humans and nonhuman primates in North America. Considering the zoonotic significance of this pathogen, our report expands on the limited literature regarding disease caused by Versteria and emphasizes the need to identify the causative tapeworm more accurately, especially in rodent intermediate hosts given that previous reports do not have molecular confirmation of species.

Pennsylvania

Retrospective review of the pathology of American pikas

American pikas ( Ochotona princeps ) are small lagomorphs that live in mountainous talus areas of western North America. Studies on the histopathology of American pikas are limited. We summarize here the clinical histories, and gross and histologic findings of 12 American pikas, including 9 captive (wild-caught) and 3 wild animals. Death was often attributed to stress (transport, handling, anesthesia) with few-to-no premonitory clinical signs. Infection was the cause of death in 2 cases: 1 had bacterial pyogranulomatous dermatitis, cellulitis, and lymphadenitis with sepsis; the other case had oomycete-induced necrotizing colitis. Incidental parasitic infections included sarcocystosis, nematodosis (oxyurids), and ectoparasitism. Most animals with adequate nutritional status had periportal hepatic lipidosis; this finding was absent in all animals with adipose atrophy, and it is possible that periportal hepatic lipidosis is non-pathologic in American pikas. Three cases had myocardial necrosis that was considered the cause of death; the cause of necrosis was not determined, but it may have been caused by stress or vitamin E–selenium deficiency. Esophageal hyperkeratosis was noted in animals with a history of anorexia and negative energy balance; accumulation of esophageal keratin can result from lack of mucosal abrasion by ingesta. Several histologic findings that are likely normal in American pikas include splenic extramedullary hematopoiesis, thymic tissue in adults, and Clostridium sp. in the enteric lumen.

Journal of Veterinary Diagnostic Investigation