Journal of Veterinary Diagnostic Investigation

Bead-based multiplex assays (BBMAs) are applicable for high throughput, simultaneous detection of multiple analytes in solution (from several to 50–500 analytes within a single, small sample volume). Currently, few assays are commercially available for veterinary applications, but they are available to identify and measure various cytokines, growth factors and their receptors, inflammatory proteins, kinases and inhibitors, neurobiology proteins, and pathogens and antibodies in human beings, nonhuman primates, and rodent species. In veterinary medicine, various nucleic acid and protein-coupled beads can be used in, or for the development of, antigen and antibody BBMAs, with the advantage that more data can be collected using approximately the same amount of labor as used for other antigen and antibody assays. Veterinary-related BBMAs could be used for detection of pathogens, genotyping, measurement of hormone levels, and in disease surveillance and vaccine assessment. It will be important to evaluate whether BBMAs are “fit for purpose,” how costs and efficiencies compare between assays, which assays are published or commercially available for specific veterinary applications, and what procedures are involved in the development of the assays. It is expected that many veterinary-related BBMAs will be published and/or become commercially available in the next few years. The current review summarizes the BBMA technology and some of the currently available BBMAs developed for veterinary settings. Some of the human diagnostic BBMAs are also described, providing an example of possible templates for future development of new veterinary-related BBMAs.

In the current study, mycotoxicoses of ruminants and horses are reviewed, with an emphasis on the occurrence of these diseases in South America. The main mycotoxicoses observed in grazing cattle include intoxications by indole-diterpenoid mycotoxins (Paspalum spp. contaminated by Claviceps paspali, Lolium perenne infected by Neotyphodium lolii, Cynodon dactylon infected by Claviceps cynodontis, and Poa huecu), gangrenous ergotism and dysthermic syndrome (hyperthermia) caused by Festuca arundinacea (syn. Festuca elatior) infected by Neotyphodium coenophialum (syn. Acremonium coenophialum), and photosensitization in pastures contaminated by toxigenic Pithomyces chartarum. Other mycotoxicoses in grazing cattle include slaframine toxicity in clover pastures infected by Rhizoctonia leguminicola and diplodiosis in cattle grazing in corn stubbles. The mycotoxicoses caused by contaminated concentrated food or byproducts in cattle include poisoning by toxins of Aspergillus clavatus, which contaminate barley or sugar beetroot by-products, gangrenous ergotism or dysthermic syndrome caused by wheat bran or wheat screenings contaminated with Claviceps purpurea, and acute respiratory distress caused by damaged sweet potatoes (Ipomoea batatas). The main mycotoxicosis of horses is leukoencephalomalacia caused by the fumonisins B1 and B2 produced by Fusarium spp. Poisoning by C. purpurea and F. elatior infected by N. coenophialum has also been reported as a cause of agalactia and neonatal mortality in mares. Slaframine toxicosis caused by the ingestion of alfalfa hay contaminated by R. leguminicola has also been reported in horses.

Bluetongue virus (BTV) and Epizootic hemorrhagic disease virus (EHDV) possess similar structural and molecular features, are transmitted by biting midges (genus Culicoides), and cause similar diseases in some susceptible ruminants. Generally, BTV causes subclinical disease in cattle, characterized by a prolonged viremia. EHDV-associated disease in cattle is less prominent; however, it has emerged as a major economic threat to the white-tailed deer (Odocoileus virginianus) industry in many areas of the United States. The recent emergence of multiple BTV and EHDV serotypes previously undetected in the United States demonstrates the need for robust detection of all known serotypes and differential diagnosis. For this purpose, a streamlined workflow consisting of an automated nucleic acid purification and denaturation method and a multiplex one-step reverse transcription quantitative polymerase chain reaction for the simultaneous detection of BTV serotypes 1–24 and EHDV serotypes 1–7 was developed using previously published BTV and EHDV assays. The denaturation of double-stranded (ds) BTV and EHDV RNA was incorporated into the automated nucleic acid purification process thus eliminating the commonly used separate step of dsRNA denaturation. The performance of this workflow was compared with the World Organization of Animal Health BTV reference laboratory (National Veterinary Services Laboratory, Ames, Iowa) workflow for BTV and EHDV detection, and high agreement was observed. Implementation of the workflow in routine diagnostic testing enables the detection of, and differentiation between, BTV and EHDV, and coinfections in bovine blood and cervine tissues, offering significant benefits in terms of differential disease diagnosis, herd health monitoring, and regulated testing.

A competitive liquid-phase–blocking concanavalin A enzyme-linked immunosorbent assay (LPB-ConA-ELISA) was developed in the current study. The assay used ConA as a capture reagent, and the sera of specific pathogen–free chickens immunized with nonpurified Newcastle disease virus (NDV) suspension as detector antibodies, to detect and quantify specific antiviral antibodies in serum samples from free-ranging pigeons. The comparison between the LPB-ConA-ELISA and the hemagglutination inhibition (HI) test for the detection of antibodies in serum samples from 107 pigeons showed significant correlation between the assays (r = 0.875), a high sensitivity (100%), specificity (95.8%), accuracy (96.3%) for the ELISA, and good agreement (κ = 0.83) between the 2 assays. The results of this study suggest that the LPB-ConA-ELISA could be a useful alternative to HI test in the serodiagnosis of NDV in pigeons, or other species of birds.

The objective of the present study was to validate a previously described competitive enzyme-linked immunosorbent assay (cELISA) to detect antibody to Equine arteritis virus (EAV) based on GP5-specific nonneutralizing monoclonal antibody (mAb) 17B7⁹ using the World Organization for Animal Health (OIE)–recommended protocol, which includes the following 5 in-house analyses. 1) The assay was calibrated with the OIE-designated reference serum panel for EAV; 2) repeatability was evaluated within and between assay runs; 3) analytical specificity was evaluated using sera specific to related viruses; 4) analytical sensitivity was evaluated with sera from horses vaccinated with an EAV modified live virus (MLV) vaccine; and 5) the duration of cELISA antibody detection following EAV vaccination was determined. The positive cELISA cutoff of ≥35% inhibition (%I) was confirmed by receiver operating characteristic plot analysis. Analytical sensitivity of the cELISA was comparable to the serum neutralization (SN) assay in that it detected EAV-specific antibody as early as 8 days postvaccination. The duration of EAV-specific antibody detected by cELISA was over 5 years after the last vaccination. This cELISA could detect EAV-specific antibody in serum samples collected from horses infected with various EAV strains. In the field trial performed by American Association of Veterinary Laboratory Diagnosticians–accredited state laboratories and OIE laboratory, the diagnostic specificity of the cELISA was 99.5% and the diagnostic sensitivity was 98.2%. The data using various serum panels also had consistently significant positive correlation between SN titers and cELISA %I results. The results further confirm that the EAV antibody cELISA is a reliable, simple alternative to the SN assay for detecting EAV-specific antibodies in equine sera.

Anticoagulant rodenticides are frequently a cause of poisoning of domestic animals, wildlife, and human beings. A toxicosis in 6,000 laying hens caused by the malicious addition of unknown amounts of coumatetralyl bait as well as the insecticides aldicarb, methomyl, and imidacloprid in the drinking water, was investigated in the current study. In order to determine a possible carryover of coumatetralyl into eggs, a rapid and reliable analytical method was developed and fully validated for the simultaneous detection of 6 anticoagulant rodenticides (warfarin, coumatetralyl, coumachlor, bromadiolone, difenacoum, and brodifacoum) in yolk and albumen using high-performance liquid chromatography (HPLC) with fluorescence detection. The method developed was reproducible, sensitive, accurate, and linear within the range of 0.01–1 mg/kg, which is the concentration range of bromadiolone and warfarin found in yolk in previously reported studies. The coefficient of variations of within and between days was 1.0–8.5% for yolk and 0.6–3.8% for albumen, while recoveries from spiked albumen and yolk samples were all in the range of 79–99% and 51–95%, respectively. Limits of detection in yolk were 0.01 mg/kg for warfarin and 0.003 mg/kg for the remaining compounds; in albumen, the limit of detection was 0.003 mg/kg for warfarin, coumatetralyl, and coumachlor, and 0.0015 mg/kg for difenacoum and brodifacoum. The application of the validated method revealed the presence of coumatetralyl in the yolk only at levels of 0.0057 mg/kg and 0.0052 mg/kg on the second and fourth day of the poisoning. In conclusion, the HPLC method demonstrated suitability for application in official analysis of anticoagulants in hen eggs.

The poisoning of wildlife with fly-bait containing the active ingredient methomyl is an intentional and illegal act in many jurisdictions. A case of 2 animals poisoned by methomyl through consumption of tainted bait at multiple stations is described. Although thermally and ultraviolet-labile, methomyl can be identified by gas chromatography–mass spectrometry and is detected in abundance in bait samples; however, it is not readily observed in tissues, owing to its rapid metabolism and elimination. The application of derivatizing functionalities, such as trimethylsilyl groups, stabilizes the methomyl-oxime metabolite to facilitate its detectability during exposure to the relatively harsh conditions of gas chromatography. This brief communication reports on the analytical detection of methomyl in baits and biological samples from poisoned wildlife. Essential to the case were the added determination of a fly-bait coactive ingredient, (Z)-9-tricosene, and identification of a chemical indicator, caffeine, to confirm both the type of pesticide product involved in the poisoning incident and the vehicle used to perpetrate its delivery.

A survey was conducted to investigate viral infection in 253 wild marine fishes harvested in the southern coastal area of Korea from 2010 to 2012. The fish that were captured by local anglers were randomly bought and sampled for virus examination. The samples were tested for presence of virus by virus isolation with FHM, FSP, and BF-2 cells and molecular methods (polymerase chain reaction and sequencing). Of the 253 fish sampled, 9 fish were infected with virus. Aquabirnaviruses (ABVs), Viral hemorrhagic septicemia virus (VHSV), and Red seabream iridovirus (RSIV) were detected in 7, 1, and 1 fish, respectively. Molecular phylogenies demonstrated the detected viruses (ABV, VHSV, and RSIV) were more closely related to viruses reported of the same type from Korea and Japan than from other countries, suggesting these viruses may be indigenous to Korean and Japanese coastal waters.

A study was performed to assess the validity of the BAX automated polymerase chain reaction system (DuPont Nutrition & Health, Wilmington, Delaware) to detect the shedding of Salmonella species in bovine fecal samples. A total of 133 bovine fecal samples that were submitted to the Cornell University Animal Health Diagnostic Laboratory for Salmonella culture were also tested in the BAX system with a modified version of the manufacturer’s enrichment protocol. Using culture as the gold standard test, the BAX system was found to have a sensitivity of 85.7% and a specificity of 90.5%. There was excellent agreement (κ = 0.71, standard error = 0.072) and no significant differences between the 2 methods (McNemar χ² = 0.180).

A Bayesian latent class model was used to estimate the sensitivity and specificity of an immunoglobulin G1 serum enzyme-linked immunosorbent assay (Paralisa) and individual fecal culture to detect young deer infected with Mycobacterium avium subsp. paratuberculosis. Paired fecal and serum samples were collected, between July 2009 and April 2010, from 20 individual yearling (12–24-month-old) deer in each of 20 South Island and 18 North Island herds in New Zealand and subjected to culture and Paralisa, respectively. Two fecal samples and 16 serum samples from 356 North Island deer, and 55 fecal and 37 serum samples from 401 South Island deer, were positive. The estimate of individual fecal culture sensitivity was 77% (95% credible interval [CI] = 61–92%) with specificity of 99% (95% CI = 98–99.7%). The Paralisa sensitivity estimate was 19% (95% CI = 10–30%), with specificity of 94% (95% CI = 93–96%). All estimates were robust to variation of priors and assumptions tested in a sensitivity analysis. The data informs the use of the tests in determining infection status at the individual and herd level.

A 2-stage algorithmic framework was developed to automatically classify digitized photomicrographs of tissues obtained from bovine liver, lung, spleen, and kidney into different histologic categories. The categories included normal tissue, acute necrosis, and inflammation (acute suppurative; chronic). In the current study, a total of 60 images per category (normal; acute necrosis; acute suppurative inflammation) were obtained from liver samples, 60 images per category (normal; acute suppurative inflammation) were obtained from spleen and lung samples, and 60 images per category (normal; chronic inflammation) were obtained from kidney samples. An automated support vector machine (SVM) classifier was trained to assign each test image to a specific category. Using 10 training images/category/organ, 40 test images/category/organ were examined. Employing confusion matrices to represent category-specific classification accuracy, the classifier-attained accuracies were found to be in the 74–90% range. The same set of test images was evaluated using a SVM classifier trained on 20 images/category/organ. The average classification accuracies were noted to be in the 84–95% range. The accuracy in correctly identifying normal tissue and specific tissue lesions was markedly improved by a small increase in the number of training images. The preliminary results from the study indicate the importance and potential use of automated image classification systems in the histologic identification of normal tissues and specific tissue lesions.

The availability of the fetal goat tongue cell line ZZ-R 127 for the isolation of Foot-and-mouth disease virus (FMDV) has not been evaluated using clinical samples other than epithelial suspensions. Therefore, in the current study, the availability of ZZ-R 127 cells for the isolation of FMDV was evaluated using clinical samples (e.g., sera, nasal swabs, saliva, feces, and oropharyngeal fluids) collected from animals experimentally infected with an FMDV isolate. Virus isolation rates for the ZZ-R 127 cells were statistically higher than those for the porcine kidney cell line (IB-RS-2) in experimental infections using cattle, goats, and pigs (P < 0.01). Virus titers in the ZZ-R 127 cells were also statistically higher than those in the IB-RS-2 cells. The availability of ZZ-R 127 cells for the isolation of FMDV not only from epithelial suspensions but also from other clinical samples was confirmed in the current study.

A total of 5 psittacine birds in an enclosed zoological exhibit, including 2 princess parrots and 3 cockatoos of 2 different species, developed severe central nervous system clinical signs over a 2–3-month period and died or were euthanized. Histologically, all birds had a lymphoplasmacytic and histiocytic encephalitis with intralesional protozoa consistent with a Sarcocystis species in addition to intramuscular tissue sarcocysts. By immunohistochemical staining, merozoites in brain and tissue cysts in muscle did not react with polyclonal antisera against Sarcocystis falcatula, Sarcocystis neurona, Toxoplasma gondii, and Neospora caninum, or with a monoclonal antibody to S. neurona. Transmission electron microscopy on sarcocyst tissue cyst walls from 2 birds was morphologically consistent with Sarcocystis calchasi. Polymerase chain reaction (PCR) amplification and sequencing of partial 18S ribosomal RNA from muscle tissue cysts and brain schizonts from 3 birds was consistent with a clade containing S. calchasi and Sarcocystis columbae but could not distinguish these closely related Sarcocystis species. However, PCR amplification and sequencing of the internal transcribed spacer 1 RNA segment in the brain from 2 birds and muscle from 2 birds specifically identified the isolates as S. calchasi. The current report documents that multiple psittacine species are susceptible intermediate hosts of S. calchasi, and that infection can cause encephalitis resulting in significant morbidity and mortality in psittacine aviaries.

Influenza A virus subtype H1N1 A(H1N1)pdm09 was first confirmed in pigs in the United States in October 2009. In November 2010, lungs and intestines from 2 York piglets from a small, privately owned herd were submitted to the Colorado State University Veterinary Diagnostic Laboratory. The submitting veterinarian reported rapid weight loss and signs of pneumonia in the piglets. Gross lesions included caudoventral pneumonia in both piglets, and histologic lesions in the lungs showed characteristics consistent with influenza virus and bacterial infection. Ribonucleic acid extracted from fresh lung homogenates from both piglets was positive for influenza A(H1N1)pdm09 by a real-time reverse transcription polymerase chain reaction. Virus was isolated from lung homogenates from both piglets in Madin–Darby canine kidney cells, as well as in 10-day-old specific pathogen–free embryonated chicken eggs. Sequence analysis showed 98% homology with 2009 H1N1 human isolates from across the United States and 98% homology against two 2009 and 2010 swine isolates from Nebraska and Minnesota. The current report documents the possible transmission of pandemic influenza A(H1N1)2009 virus [A(H1N1)pdm09] from a human being to a small, privately owned backyard swine herd. The owner was employed as a pharmacist, making occupational exposure to the pandemic influenza A(H1N1)pdm09 a possibility.

A commercial facility producing hamsters with a history of infection by dwarf tapeworm (Hymenolepis nana) submitted 15 animals for necropsy and postmortem parasitological and microscopic examination. No tapeworms were detected grossly or microscopically. Fecal examination including gastrointestinal mucosal smears demonstrated mixed intestinal bacteria and low numbers of Giardia sp. Histologic examination of small intestine demonstrated filling of the small intestinal crypts by large numbers of 7–9 µm × 3 µm, rod to crescent or teardrop-shaped flagellates consistent with Spironucleus sp. These organisms had two 1-µm, basophilic, oval nuclei and multiple superficial flagella-like structures. Much larger 10–15 µm × 8–10 µm, oval to pear-shaped organisms were also present in lower numbers and usually located with the crypts. These larger flagellates had multiple flagella and a basophilic rod-shaped nucleus. The larger flagellates included Giardia sp., which had an intimate interface with the surface of the mucosal epithelium, bilaterally symmetry, and binucleation. Lower numbers of trichomonads were also present and were distinguished by an undulating surface membrane and a single nucleus. The mucosa was hyperplastic and moderately inflamed. Although the tapeworm infection was resolved, diagnosis of multiple intestinal flagellates by fecal examination is complicated by the varying sensitivity and diagnostic accuracy of different types of fecal analysis for different flagellate types. Key differences in the morphology and location of the different types of flagellates as observed by histology of intestinal tissues provide important additional diagnostic information to distinguish trichomonads, Spironucleus sp., and Giardia sp.

Pasteurella multocida is a common pathogen of swine that causes specific diseases with great economic impact. However, the importance of this pathogen in wild boar is still unknown. In the current work, an outbreak of systemic pasteurellosis in wild boar with a high mortality rate is described. A total of 23 wild boar of all ages were found dead over a 5-day period on a game estate in southwest Spain (11.11% mortality). Three animals were necropsied and showed subcutaneous edema, a generalized congestion, and fibrin deposits in the peritoneal cavity. Hemorrhages, general congestion, and intravascular thrombosis were microscopically observed. Pasteurella multocida type B was isolated from all of the studied organs. Outbreaks of systemic pasteurellosis have been described in domestic pigs from Asia and Australia, but not to date in Europe. This outbreak suggests that systemic pasteurellosis affecting wild boar populations may be an important cause of mortality.

Geotrichum spp. are ubiquitous, saprotrophic fungi found in soil, organic matter, and silage, as a contaminant in food products and in the digestive tracts of mammals. The current study reports a case of Geotrichum candidum infection with dermatitis in an aborted bovine fetus with skin and lung lesions. A 6-month-old aborted male Holstein Friesian fetus displayed unusual lesions on the skin of the abdomen, thorax, and head, which was excessively thickened and wrinkled. These changes corresponded to orthokeratotic hyperkeratosis, neutrophil accumulation in the stratum corneum, a pyogranulomatous inflammatory infiltrate, and superficial dermal necrosis. Moderate suppurative multifocal pneumonia was observed. Large numbers of mononuclear cells and occasional fibrin thrombi within blood vessels were found in the lungs, brain, and cerebellum. Gridley staining revealed fungal structures within the skin lesions. The mycological exam demonstrated the growth of G. candidum, and phase contrast microscopy conducted on the abomasal fluid revealed hyphae compatible with this agent. The skin lesions observed, in association with the fungus isolated, indicated that the abortion was due to G. candidum infection of the bovine fetus.

Pulmonary alveolar microlithiasis (PAM) is a rare pulmonary disorder characterized by the accumulation of calcium phosphate microliths within the alveoli, with only a few cases described in animals. A 10-year-old female Bulldog was euthanized due to history of dyspnea and recurrent pleural and pericardial effusions. At necropsy, numerous multifocal to coalescent protruding nodules of 1–5 mm in diameter were scattered throughout the thoracic serosal surfaces. Moreover, lungs showed a diffuse pale gray color and had a generalized fine grainy consistency. Histological investigations revealed abundant intra-alveolar laminated microliths that stained positive with periodic acid–Schiff and von Kossa stains. The pulmonary interstitium showed multifocal, mild to moderate thickening, due to collagen deposition and mild hyperplasia of type 2 pneumocytes. The pulmonary lesion was not associated with any inflammatory response, and mineral deposition was not observed in any other organ or tissue. In addition, pulmonary, pericardial, and pleural surfaces were extensively infiltrated by an epithelioid mesothelioma. Immunohistochemical staining revealed neoplastic cells that strongly coexpressed vimentin and cytokeratin, supporting the diagnosis of mesothelioma. An overview of PAM, including pathogenesis and histological characteristics, are discussed in relation to the concurrent pleural mesothelioma. The potential cause and effect relationship between the 2 conditions could neither be established nor ruled out.

An extra-adrenal retroperitoneal paraganglioma was observed in a 10.5-year-old male Boxer dog. Additionally, the dog had an aortic base tumor, multiple thyroid adenomas, multiple testicular interstitial cell tumors, bilateral nodular adrenal cortical hyperplasia, and parathyroid gland hyperplasia. The hypothesis that the retroperitoneal mass represents a primary extra-adrenal paraganglioma rather than metastatic mass from the aortic body tumor is considered. Either primary or metastatic extra-adrenal retroperitoneal paragangliomas are rarely reported in dogs.

A 4-year-old, female, spayed Border Collie dog was presented for progressive lethargy, inappetence, and weakness of 4 days duration. The animal had been diagnosed with pemphigus foliaceus 3 months prior and was receiving combination immunosuppressive therapy. Serum biochemistry revealed severely elevated liver enzymes and bilirubin, and humane euthanasia was elected. Gross postmortem examination revealed a diffusely pale tan to slightly yellow, enlarged, markedly friable liver with an enhanced reticular pattern. Histologically, the hepatic changes consisted of multifocal to coalescing areas of severe vacuolar degeneration, numerous coalescing foci of hepatocellular necrosis, and myriad intra- and extracellular protozoa that reacted immunohistochemically with polyclonal antibodies to Neospora caninum, and not Toxoplasma gondii. Neosporosis in the current case is thought to be due to reactivation of latent N. caninum occurring with the administration of glucocorticoid therapy. The severe complication in the present case highlights the importance of early detection and mitigation of common infections in immunosuppressed animals.

Yolk sac tumors (YSTs) are rare neoplasms of germ cell origin. In humans, the tumors primarily occur in the testes or ovaries, but occasionally develop at other sites. The neoplastic cells of YSTs form many histological patterns resembling embryonal structures, and the World Health Organization classification lists 11 such patterns: reticular, macrocystic, endodermal sinus, papillary, solid, glandular-alveolar, myxomatous, sarcomatoid, polyvesicular vitelline, hepatoid, and parietal. Among domestic animals, only 2 cases of YST, which were of testicular and abdominal cavity origin, have been reported in calves. In both cases, neoplastic cells had epithelial properties and disseminated metastases in the abdomen. In the present study, the enlarged testis of a newborn calf, which was subsequently diagnosed as YST and exhibited myxomatous, reticular, and polyvesicular vitelline histological patterns, is described. There was no metastasis in this case, and histological and immunohistochemical features varied from previous cases of YST.

Five 1-month-old kid goats from a local herd in Kozani (northwest Greece) developed neurological disorders characterized by decreased appetite, ataxia, and head pressing. The animals received a 3-day course of treatment with intramuscular administration of enrofloxacin and ketoprofen. However, no significant clinical improvement was achieved, and 2 kids died. The remaining 3 animals were euthanized, and a necropsy was performed within 1 hr. Macroscopic lesions were confined to the central nervous system, with congestion and petechiae in the meninges. Microscopic lesions in all 3 animals revealed multifocal acute meningoencephalitis characterized by infiltrations composed of mononuclear inflammatory cells, lesser numbers of lymphocytes, and occasionally neutrophils and eosinophils. Additionally, in the kidney, there was multifocal expansion of the glomerular tufts by eosinophilic amorphous material, multifocal interstitial hemorrhages, and multifocal glomerular hypercellularity. The above noted lesions are consisted with an acute ongoing nephropathy indicative of a septicemic–toxemic procedure at its primary stages. Small, gray bacterial colonies, 3–4 mm in diameter, were obtained in pure culture from the brain of all 3 necropsied animals and were confirmed as Escherichia coli O157:H7 by use of phenotypic and genotypic methods. The isolates were sensitive to cefuroxime, ceftazidime, and gentamicin. In contrast, resistance to enrofloxacin, trimethoprim–sulfamethoxazole, and tetracycline was displayed. Additionally the bacterial isolates were found to carry a plasmid that harbored qnrS, sulII, and tetB genes that contribute to high-level resistance to fluoroquinolones, co-trimoxazole, and tetracycline, respectively.