Journal of Veterinary Diagnostic Investigation最新文献

Canine astrovirus (mamastrovirus 5 [MAstV5], formerly CaAstV; family Astroviridae, taxon species Mamastrovirus canis) and canine kobuvirus (aichivirus A2 [AiV-A2], formerly CaKoV; Picornaviridae, Kobuvirus aichi) are emerging enteric viruses increasingly detected in both diarrheic and subclinical dogs. Although primarily associated with gastrointestinal illness, recent evidence suggests a potential for systemic dissemination, which remains insufficiently explored. To improve detection, we developed and validated a duplex quantitative real-time PCR (dqPCR) assay for simultaneous identification of MAstV5 and AiV-A2. Primers and TaqMan probes were designed based on conserved regions of the MAstV5 ORF1b and AiV-A2 3D polymerase genes. Assay optimization included primer-probe concentration titration and thermal gradient analysis. Analytical performance was assessed using synthetic plasmid standards and RNA from non-target viruses, including canine parvovirus (CPV), canine morbillivirus (CDV), and canine enteric coronavirus (CCoV). Our dqPCR assay had high linearity (R² > 0.99) and sensitivity, with limits of detection of 10 copies/μL for MAstV5 and 100 copies/μL for AiV-A2. No cross-reactivity or interference was observed in coinfection simulations across various target ratios. Intra- and inter-assay CV values were <2.2%, indicating excellent reproducibility. Validation using 50 clinical rectal swabs and tissue samples from 25 autopsied dogs revealed 1 additional AiV-A2-positive case undetected by RT-PCR but confirmed by sequencing. Importantly, viral RNA was also found in extraintestinal tissues-spleen, liver, trachea, and mesenteric lymph nodes-suggesting systemic distribution in naturally infected dogs. Our dqPCR assay provides a sensitive, specific, and efficient tool for the detection of MAstV5 and AiV-A2, supporting both clinical testing and epidemiologic studies.

Diagnosis of lymphoid hyperplasia and neoplasia due to lymphoproliferative disease virus (LPDV; Retroviridae, Alpharetrovirus) infection in turkeys is challenging due to histopathologic similarities with nonspecific inflammation and cellular responses to other retroviral infections. Localization of LPDV RNA or protein antigens within affected tissues, which has previously not been shown, would allow for more definitive diagnoses. We evaluated formalin-fixed paraffin-embedded tissues from 32 turkeys, including 15 naturally infected wild turkeys, 11 experimentally infected domestic turkeys, and 6 uninfected wild and domestic turkeys, using RNAscope in situ hybridization (ISH) and immunohistochemistry (IHC). ISH probes targeted a segment of the gag gene, and IHC antibodies were designed to recognize the capsid protein. Tissues from 4 infected turkeys were subjected to concurrent ISH and IHC labeling. All infected turkeys had ISH and IHC cytoplasmic and/or nuclear labeling of lymphocytes in at least one tissue, including within lymphoid tumors. ISH labeling was widely scattered in lymphocytes, whereas IHC labeling distribution was more limited. Spleen consistently exhibited the strongest and most widespread ISH and IHC labeling in both wild and domestic turkeys. Labeled lymphocytes typically were localized to splenic germinal centers and around periarteriolar lymphoid sheaths in the thymic cortex. Lymphocytes occasionally had simultaneous ISH and IHC labeling in selected cases. No uninfected turkeys had ISH or IHC labeling. Our 2 methods of LPDV detection in formalin-fixed paraffin-embedded tissues can aid in distinguishing lymphoid proliferation due to LPDV from other etiologies and further characterize pathogenesis.

Urinary cystatin B (uCysB) is a biomarker of kidney injury in dogs and cats. A high-throughput agglutination immunoassay (Idexx Laboratories) was developed for widespread commercial availability of uCysB testing in a reference laboratory setting. We evaluated immunoassay performance and included analyses of precision, accuracy, linearity, interference, analytical specificity, lot-to-lot variation, and stability. CVs from precision studies on the range of 50-500 ng/mL were 0.38-2.53% (canine) and 0.44-3.5% (feline) for within-run precision, and 1.49-5.09% (canine) and 0.65-5.05% (feline) for between-run precision. Accuracy was measured by recovery percentage and was 89-101% (canine) and 91-112% (feline). Amoxicillin, ciprofloxacin, low concentrations of doxycycline, bilirubin, glucose, ketones, RBCs, hemoglobin, cloudiness, lipids, protein, and pH did not affect results. Urinary cystatin A did not cross-react with the uCysB immunoassay. Results of lot-to-lot linear regressions were 0.90-1.07 (slopes) and 0.97-1.00 (coefficient of determination). One or more freeze-thaw cycles and storage at 30°C impacted the immunoassay stability of canine samples but not feline samples under the same conditions. Our results validate this novel agglutination immunoassay for accurate and precise measurement of uCysB in canine and feline urine samples. For optimal immunoassay performance, samples should be kept at 4°C for a maximum of 1 wk. Our uCysB immunoassay is a useful and practical tool to be used in assessing kidney injury in canine and feline patients in the clinical setting.

Abnormal sudden mortality of 2 goats and 10 sheep occurred in a 27-head flock in Verona province (northeastern Italy). In the 24 h before death, animals had acute ataxia, lateral recumbency, spastic convulsions, and dyspnea. Autopsy of 4 animals was performed at the Verona Veterinary Diagnostic Laboratory of the Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe; Italy). In the forestomachs, abundant fibrous material and numerous seeds were observed. Bacteriologic, parasitologic, and histologic investigations were carried out; samples of rumen contents and liver were analyzed by direct analysis real-time high-resolution mass spectrometry (DART-HRMS), which allows rapid screening of toxic substances. The combination of DART-HRMS and liquid chromatography high-resolution tandem MS (LC-HRMS/MS) confirmed the acute intoxication and provided insights into the clinicopathologic findings due to the ingestion of Chimonanthus praecox (wintersweet), a plant species that contains alkaloids including calycanthine, which is known to be toxic in several domestic species, including small ruminants.

Six 11-wk-old, commercial, Broad-Breasted White, meat turkeys were submitted to the Turlock branch of the California Animal Health & Food Safety (CAHFS) laboratory for autopsy and diagnostic work-up. Clinical signs in the turkeys of the affected flock included depression, ruffled feathers, swollen periorbital areas, rales, and sneezing. A mortality of 50% (5,000 of 10,000) was reported at the time of case submission. Flock morbidity was 100% by 12 wk of age, and mortality eventually exceeded 90%. Fibrinous pleuropneumonia, airsacculitis, increased luminal mucoid exudate in the nasal cavities and tracheas, mottled and enlarged spleens, and hepatomegaly were the most remarkable gross findings. Microscopically, fibrinoheterophilic pneumonia and epicarditis with intralesional bacterial colonies, and necrotizing hepatitis and splenitis, were noted. Mycoplasmoides (Mycoplasma) gallisepticum (MG) was detected in tracheal and sinus pools by quantitative real-time PCR. Multilocus sequence analysis of the mgc2 gene and IGSR segment of MG differentiated our strain from MG vaccine strains, but were similar to MG isolates detected previously in other commercial turkey operations in California. Pasteurella multocida was isolated from air sacs, lungs, tracheas, hearts, and livers, and classified as profile HhaI 0001, strain X-73, by restriction enzyme analysis DNA fingerprinting. Coinfection with P. multocida and MG in a susceptible flock resulted in rapid elevation of mortality and significant economic losses in this commercial meat turkey operation.

Cystoisospora species that infect dogs and cats are ubiquitous, and oocysts can be found in the feces of both subclinically infected and sick animals. Most clinical cases are diagnosed in puppies and kittens <4-mo-old. We validated a high-throughput immunoassay (Fecal Dx immunoassay; Idexx), which uses 2 monoclonal antibodies to detect Cystoisospora spp. coproantigen, using zinc sulfate centrifugal flotation (ZCF) as the reference method and samples from experimentally infected animals. This new genus-level coproantigen assay had equivalent sensitivity for at least 4 relevant Cystoisospora spp.-C. canis, C. ohioensis-like, C. felis, and C. rivolta-with no observed cross-reactivity to other protozoal parasites (Giardia, Cryptosporidium, Tritrichomonas, Eimeria) of companion animals. In comparison to ZCF, the immunoassay had a positive percent agreement of 88.5% (95% CI [86.1, 91.6]) and a negative percent agreement of 98.2% (95% CI [98.1, 98.2]). The prevalence of Cystoisospora spp. detected by ZCF was 1%; the prevalence identified through coproantigen testing was 2.7% (95% CI [2.6, 2.8]). Among puppies and kittens <1-y-old, the prevalence according to coproantigen testing was 7.4% (95% CI [6.6, 8.2]) for puppies and 8.2% (95% CI [7.7, 8.6%]) for kittens; the prevalence detected by ZCF was 3.6% (95% CI [3.0, 3.9]) for puppies and 2.9% (95% CI [2.4, 3.4]) for kittens. Our results validate our Cystoisospora coproantigen immunoassay as specific, precise, and sensitive.

Rotavirus A (RVA; family Sedoreoviridae, taxon species Rotavirus alphagastroenteritidis) is a non-enveloped double-stranded RNA virus that has been reported from both diarrheic and non-diarrheic pigs worldwide. With significant morbidity and mortality rates in neonatal piglets, rotavirus-associated illness adds enormous economic losses to the pig industry. Furthermore, the proximity of humans and pigs facilitates cross-species infection, which results in the formation of novel strains through genetic recombination. We aimed to detect and characterize porcine RVA (PRVA) in Haryana, India, using reverse-transcription PCR targeting the VP6, VP4, and VP7 genes. We detected 46 of 137 (34%) rectal swab samples as positive for PRVA, including 27 of 63 (43%) from diarrheic pigs and 19 of 74 (26%) from non-diarrheic pigs. In addition, phylogenetic analysis revealed the presence of genotypes I1, I5, P[13], P[6], G11, G4, and combinations of G4P[6], G4P[13], and G11P[13] in the pig population of Haryana. G4P[6] was the most common combination found, followed by G11P[13] and G4P[13]. Genotype G11 and the combinations G4P[13] and G11P[13] have not been reported previously in pigs, in India, to our knowledge. Our finding of various genotypes, and their genetic proximity to human RVA, indicates their potential zoonotic importance.

An adult male western grey kangaroo (Macropus fuliginosus) developed lameness, a stiff gait, and weight loss, and deteriorated despite medical treatment. Postmortem examination revealed a primary gastric squamous cell carcinoma (SCC) with associated cardiac, pulmonary, diaphragmatic, hepatic, and vertebral metastases with lytic bone lesions. Before histologic examination, the macroscopic appearance of the liver lesions had raised concerns about mycobacteriosis. Metastatic gastric SCC has not been reported previously in a western grey kangaroo, to our knowledge.