bluuebunny/crossref_metadata_2025 huggingface dataset:
Fatal Bluetongue Virus Infection in an Alpaca (Vicugna Pacos) in California
Authors: Ortega Joaquín , Crossley Beate , Dechant Julie E. , Drew Clifton P. , MacLachlan N. James
Published: May 2011

In October 2008, a 15-year-old female alpaca ( Vicugna pacos) housed at a breeding farm in northern California died after a brief illness characterized by sudden onset of weakness, recumbency, and respiratory distress. Postmortem examination revealed severe hydrothorax and hydropericardium, marked pulmonary edema, and acute superficial myocardial hemorrhage affecting the left ventricle. Bluetongue virus (BTV) was detected in the spleen by quantitative real-time reverse transcription polymerase chain reaction and confirmed by sequence analysis. No antibodies against BTV were detected in the serum using a competitive enzyme-linked immunosorbent assay, confirming acute, fulminant BTV infection.
Molecular Characterization of Ovine Zygomycosis in Central Western Brazil
Authors: de Paula Daphine Ariadne Jesus , de Oliveira Filho João Xavier , da Silva Maria Cristina , Colodel Edson Moleta , Broetto Leonardo , Pinto Paulo Marcos , Schrank Augusto , Nakazato Luciano , Dutra Valéria
Published: May 2011

Zygomycosis is an important granulomatous disease that affects humans and animals, particularly sheep in tropical regions. Rhinofacial and nasopharyngeal zygomycosis were described in sheep in association with Conidiobolus spp. The present study characterized 5 samples of Conidiobolus isolated from 3 herds with clinical disease in Mato Grosso State, Brazil. The clinical and pathological findings were similar to nasopharyngeal zygomycosis. Based on morphological features, isolates were classified as Conidiobolus spp., and molecular phylogenetic analyses based on 18S ribosomal DNA grouped all isolates in a Conidiobolus lamprauges cluster. The current report describes the molecular characterization of ovine nasopharyngeal zygomycosis associated with C. lamprauges.
Development of Enzyme-Linked Immunosorbent Assay for Detection of Antibody against Caprine Arthritis Encephalitis Virus Using Recombinant Protein of the Precursor of the Major Core Protein, p55gag
Authors: Konishi Misako , Yamamoto Takehisa , Shimada Takako , Shirafuji Hiroaki , Kameyama Ken-Ichiro , Sentsui Hiroshi , Murakami Kenji
Published: May 2011

An indirect enzyme-linked immunosorbent assay (ELISA) by using recombinant Caprine arthritis encephalitis virus (CAEV) p55gag antigen (rELISA), an indirect ELISA by using whole CAEV (wELISA), and Western blot analysis by using the recombinant p55gag antigen (rWB) were developed for detection of CAEV-specific antibodies in goats. Seven hundred and forty-five sera from goats were tested by rELISA, wELISA, rWB, and agar gel immunodiffusion test (AGID), and the results were compared with those of WB analysis by using the whole CAEV antigen (wWB). The AGID test and rWB had similar sensitivities of 93.3% (95% confidence interval [CI]) and 93% (95% CI), respectively, and similar specificities of 96.0% (95% CI) and 96.3% (95% CI), respectively, compared with wWB. The wELISA had substantially lower sensitivity (80.4%) and specificity (78.0%) compared with wWB, and rELISA had the lowest sensitivity (78.2%) and specificity (61.1%) compared with wWB. The lack of adequate sensitivity and specificity for rELISA and wELISA suggests that these assays need considerable modification. However, the results for rWB show that this assay has excellent agreement with wWB and that it can be used as a confirmatory test for the presence of anti-CAEV antibodies.
Development of a Panel of Multiplex Real-Time Polymerase Chain Reaction Assays for Simultaneous Detection of Major Agents Causing Calf Diarrhea in Feces
Authors: Cho Yong-Il , Kim Won-Il , Liu Siyuan , Kinyon Joann M. , Yoon Kyoungjin J.
Published: May 2011

Calf diarrhea is a major economic burden to the bovine industry. Since multiple infectious agents can be involved in calf diarrhea, and the detection of each of the causative agents by traditional methods is laborious and expensive, a panel of 2 multiplex real-time polymerase chain reaction (PCR) assays was developed for rapid and simultaneous detection of the 5 major bovine enteric pathogens (i.e., Bovine coronavirus [BCoV; formally known as Betacoronavirus 1], group A Bovine rotavirus [BRV], Salmonella spp., Escherichia coli K99+, and Cryptosporidium parvum). The estimated detection limit (i.e., analytic sensitivity) of the panel was 0.1 TCID50 (50% tissue culture infective dose) for BCoV and group A BRV; 5 and 0.5 colony-forming units for E. coli K99+ and Salmonella, respectively; and 50 oocysts for Cryptosporidium per reaction. In testing 243 fecal samples obtained from submissions to the Iowa State University Veterinary Diagnostic Laboratory or from experimental animals with known infection status, the newly developed multiplex realtime PCR panel simultaneously detected all 5 pathogens directly from fecal samples and was more rapid and sensitive than the traditional diagnostic tests. The PCR panel showed 89%–97% agreement with those conventional diagnostic tests, demonstrating diagnostic sensitivity equal to or better than that of the conventional tests. In conclusion, the multiplex real-time PCR panel can be a tool for a timely and accurate diagnosis of calf diarrhea associated with BCoV, group A BRV, E. coli K99+, Salmonella, and/or Cryptosporidium.
Genotyping of Porcine Teschovirus from Nervous Tissue of Pigs with and without Polioencephalomyelitis in Indiana
Authors: Bangari Dinesh S. , Pogranichniy Roman M. , Gillespie Tom , Stevenson Gregory W.
Published: May 2011

Porcine teschovirus (PTV) was isolated in cell culture and/or demonstrated by polymerase chain reaction in samples of brain and/or spinal cord in pigs in Indiana during the 2002–2007 period. Testing was initiated on pigs originating from populations exhibiting nervous clinical disease and/or pigs with microscopic lesions in central nervous tissues, indicating viral encephalitis and/or myelitis. Virus was demonstrated in pigs with and without lesions as well as with and without nervous clinical disease. Nucleotide sequence analysis of the 5′-nontranslated region of the viral genome revealed that these isolates had low-level genetic heterogeneity but were homologous to porcine PTV serotype 1 (PTV-1). These findings indicate that low-to-moderate virulence strains of PTV with some homology to PTV-1 are endemic in many swineherds of Indiana and are associated with subclinical and clinical nervous disease in weaned pigs.
Idiopathic Eosinophilic Meningoencephalomyelitis in a Rottweiler Dog
Authors: Olivier Alicia K. , Parkes Jennifer D. , Flaherty Heather A. , Kline Karen L. , Haynes Joseph S.
Published: May 2011

A 6-month-old, female, intact Rottweiler dog was presented to the Iowa State University Veterinary Teaching Hospital for a progressive history of abnormal behavior and generalized ataxia. At necropsy, there was eosinophilic infiltration of the brain and spinal cord, most severe in the medulla oblongata, cerebellum, and cervical spinal cord. Infiltrates of eosinophils were also present in the liver and small intestines. The dog was diagnosed with idiopathic eosinophilic meningoencephalomyelitis based on cerebrospinal fluid analysis, histopathology, and special stains to exclude etiologic agents.
The Effect of Inoculum Volume on the Microbiologic Detection of Naturally Occurring Staphylococcus Aureus Intramammary Infections
Authors: Walker Jennifer B. , Rajala-Schultz Päivi J. , DeGraves Fred J.
Published: May 2011

Currently no standard definitions for the diagnosis of Staphylococcus aureus intramammary infection (IMI) exist. As a result, criteria applied in research to diagnose S. aureus IMIs have varied making comparisons between published works difficult. The goal of the current study was to define the optimal inoculum volume used in the diagnosis of naturally occurring S. aureus IMIs. Microbiologic results from 2 field studies examining S. aureus IMIs were used to examine the effects of inoculum volume on the microbiologic detection of S. aureus. A total of 1,583 milk samples were included in the analysis, and the results of using a 0.01-ml and a 0.1-ml inoculum are presented. Using a 0.01-ml inoculum resulted in a sensitivity of 91% (95% confidence interval [CI]: 88.6–93%) and a specificity of 99.4% (95% CI: 98.6–99.8%). Using the larger 0.1-ml inoculum resulted in a sensitivity of 96.8% (95% CI: 95.2–97.9%) and a specificity of 99.3% (95% CI: 98.4–99.7%). All false-positive samples were from S. aureus–negative quarters in S. aureus–positive cows. There were no false-positive cultures from S. aureus–negative cows. Of the false-negative samples, the majority (77%) were from 6 of the 34 S. aureus–positive quarters. Results from the current study of naturally occurring S. aureus IMIs support the hypothesis that, when using quarter level milk samples, a S. aureus IMI is most accurately diagnosed using a 0.1-ml inoculum. Regardless of inoculum volume, a single quarter sample culture that is positive with S. aureus (≥1 colony-forming unit) is sufficient to diagnose a S. aureus IMI.
Two New Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction Assays with Unique Target Sites for the Specific and Sensitive Detection of Lineages 1 and 2 West Nile Virus Strains
Authors: Eiden Martin , Vina-Rodriguez Ariel , Hoffmann Bernd , Ziegler Ute , Groschup Martin H.
Published: May 2011

Two novel 1-step real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays for the simultaneous detection of West Nile virus (WNV) lineage 1 and 2 strains were developed. Primers and the probe of assay 1 target the 5′-untranslated region (UTR), whereas the amplicon of assay 2 is located in the nonstructural region NS2A, which enables an unambiguous and independent WNV diagnosis based on 2 different amplicons. Both assays allow the detection of as few as 2–4 genome copies of WNV strains NY99, Uganda B956, Kunjin, and Sarafend (all cultured on Vero cells). A new synthetic RNA mutant of the 5′-UTR amplicon, which contains 6 twist inverted base-pair changes at the probe attachment site, was used as external calibrator control.
Detection of Infectious Pancreatic Necrosis Virus in Subclinically Infected Atlantic Salmon by Virus Isolation in Cell Culture or Real-Time Reverse Transcription Polymerase Chain Reaction: Influence of Sample Preservation and Storage
Authors: Ørpetveit Irene , Mikalsen Aase B. , Sindre Hilde , Evensen Øystein , Dannevig Birgit H. , Midtlyng Paul J.
Published: May 2011

Infectious pancreatic necrosis, an important problem of the salmon industry worldwide, is caused by Infectious pancreatic necrosis virus (IPNV). Fish surviving an IPNV infection become virus carriers, and the identification of infected fish is highly relevant to disease control. The target organ for IPNV diagnosis is the kidney, where the virus persists, usually with low virus loads. The current study documents a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assay that proved 100 times more sensitive than a conventional RT-PCR. Cell culture and real-time RT-PCR were compared for their ability to detect IPNV in carrier Atlantic salmon kidney samples after different preservation and storage procedures. Storage of whole tissue at −80°C for 1 month and storage of tissue homogenized in transport medium (TM) at +4°C for 1 week before investigation in cell cultures resulted in a marked reduction of virus infectivity. For detection by real-time RT-PCR, storage of whole tissue was suboptimal, whereas storage of tissue homogenized in TM did not affect virus detection. The results of the present study demonstrate that both cell culture and real-time RT-PCR are reliable tests for the detection of low amounts of IPNV in kidneys of carrier Atlantic salmon, and both methods are relatively robust against minor preservation and storage deviations, or both. Preservation of tissues in RNA stabilization solution seems only necessary when samples are to be shipped at ambient temperatures or when laboratory testing might be delayed. Independent of detection method, these results indicate that for long-term storage, samples are best kept at −80°C after homogenization in TM.
Ameloblastic Fibro-Odontoma Associated with a Retained Molar in an Oldenburg Mare
Authors: Knowles Susan , Blas-Machado Uriel , Butler Abigail M. , Gomez-Ibañez Sara E. , Lowder Michael Q. , Fayrer-Hosken Richard A.
Published: May 2011

An 11-year-old Oldenburg mare presented with a 3-month history of weight loss and swelling of the posterior right mandible. Physical examination and radiographs showed a soft-tissue mass in the right mandible with foci of mineralization, periosteal proliferation, and a retained molar. The tumor increased in size over several weeks, and the mare was euthanized. On necropsy, a 24 cm × 15 cm × 15 cm firm, white mass had obliterated the posterior right mandible. The mass was multinodular with discrete clusters of blood-filled cystic spaces. Histologically, the mass was composed of neoplastic odontogenic epithelium and pulpal mesenchyme with an accumulation of eosinophilic material resembling dentin or enamel. Microscopic and immunohistochemical staining features of the neoplasm were most consistent with an ameloblastic fibro-odontoma.
Disseminated T-cell Lymphoma in a Brazilian Porcupine (Coendou Prehensilis)
Authors: de Araújo Marina Rios , Luppi Marcela Miranda , de Campos Cordeiro Malta Marcelo , Facchetti Vinhaes Assumpção Anna Luiza , Langohr Ingeborg Maria , Ecco Roselene
Published: May 2011

The current study describes the clinical, gross, histopathologic, and immunohistochemical findings of a T-cell lymphoma in a captive porcupine ( Coendou prehensilis), a species typically seen in the tropical forests of Brazil. At necropsy, extensive neoplastic involvement was observed in the cervical lymph nodes, with extension into the salivary gland. The spleen was mildly enlarged, and neoplastic nodules were grossly evident in the liver and right kidney. Histologically, sheets of large and markedly pleomorphic round cells were observed in the cervical lymph nodes, lung, liver, spleen, and kidney. The neoplastic cells were positive for cluster of differentiation (CD)3 and negative for CD79a by immunohistochemistry.
Canine Parvovirus Epidemiology in Bulgaria
Authors: Filipov Chavdar , Decaro Nicola , Desario Costantina , Amorisco Francesca , Sciarretta Rossana , Buonavoglia Canio
Published: May 2011

Canine parvovirus 2 (CPV-2) emerged in 1978 as one of the most pathogenic etiologic agents in dogs. Under the influence of evolution, the original CPV-2 was replaced, a few years later, by 2 variants, CPV-2a and CPV-2b. In 2000, a new variant, CPV-2c, was detected first in Italy and later in other countries. The current study was conducted to provide data about the CPV types circulating in Bulgaria. Forty-two fecal samples from dogs with clinical signs of parvovirosis, collected between June 2009 and February 2010, were tested for CPV using a rapid test based on detection of CPV antigens and a real-time polymerase chain reaction (PCR) for detection of viral DNA. Positive samples were characterized by means of minor groove binder probe PCR assays. Forty samples were positive, of which 30 were identified as CPV-2a, 9 as CPV-2b, and 1 as CPV-2c. The results from this molecular investigation of CPV show the prevalence of type 2a and occurrence of type 2c for the first time in Bulgaria.
Modification of a Commercial Toxoplasma Gondii Immunoglobulin G Enzyme-Linked Immunosorbent Assay for use in Multiple Animal Species
Authors: Schaefer John J. , White Holly A. , Schaaf Stephanie L. , Mohammed Hussni O. , Wade Susan E.
Published: May 2011

A challenge faced by veterinary diagnosticians in serologic analysis for exposure to pathogens is the need for a protein conjugate capable of antibody attachment in many animal species. The advent of protein conjugates that are less specific in nature allows diagnosis across many species with little or no modification of technique. Toxoplasma gondii is an organism of veterinary interest that has been demonstrated to infect a plethora of warm-blooded animals. However, the serologic tests available for simultaneous diagnosis in this broad range are limited in number. The current study examined the use of an immunoglobulin G enzyme-linked immunosorbent assay (ELISA) modified by the use of non-species-specific protein conjugates in domestic animal species commonly submitted to diagnostic laboratories for evaluation of Toxoplasma exposure status. Comparison with results from an established indirect hemagglutination technique revealed very good agreement between the 2 test methods. This modification of the ELISA provides a useful method for veterinary diagnosticians to perform rapid and accurate evaluation of multiple animal species for Toxoplasma exposure using a single test.
The Effect of Environmental Storage Conditions on Bone Marrow Fat Determination in Three Species
Authors: Lamoureux Jennifer L. , Fitzgerald Scott D. , Church Michael K. , Agnew Dalen W.
Published: May 2011

Diagnostic laboratories are frequently required to assess the antemortem nutritional condition of deceased animals. The percentage of fat in the bone marrow is used to diagnose starvation because this fat depot is typically the last in the body to be depleted. Diagnosticians rely on measurement of bone marrow adipose content using fat solvent–extraction methods; however, the effects of tissue storage conditions before processing have not been fully assessed. The current study focuses on evaluating the effects of 3 storage conditions (refrigeration [4°C], freezing [–20°C], and ambient temperature [9.9–34.4°C]) on the percentage of fat in the bone marrow from 3 species. Equine, bovine, and canine humeri and femurs were removed within 24 hr of death from adult animals in adequate body condition and then stored as described for a minimum of 30–60 days. Bone marrow was harvested from these tissues at the time of necropsy and after 30–60 days. Percentage of fat was measured using an automated solvent extractor. Mean percentage of fat in the bone marrow in initial equine, bovine, and canine samples was 81.75%, 86.33%, and 59.96%, respectively. The results indicate that bovine and equine percentage of fat in bone marrow does not change after 30–60 days, regardless of the storage condition, whereas the fat content from canine tissues varies when stored at ambient temperatures. Results suggest that postmortem interval and environmental conditions of samples must be considered in the postmortem evaluation of bone marrow fat concentration in at least some species.
Experimental interspecies transmission studies of the transmissible spongiform encephalopathies to cattle
Authors: Hamir Amir N. , Kehrli Marcus E. , Kunkle Robert A. , Greenlee Justin J. , Nicholson Eric M. , Richt Jürgen A. , Miller Janice M. , Cutlip Randall C.
Published: May 2011

Prion diseases or transmissible spongiform encephalopathies (TSEs) of animals include scrapie of sheep and goats; transmissible mink encephalopathy (TME); chronic wasting disease (CWD) of deer, elk and moose; and bovine spongiform encephalopathy (BSE) of cattle. The emergence of BSE and its spread to human beings in the form of variant Creutzfeldt-Jakob disease (vCJD) resulted in interest in susceptibility of cattle to CWD, TME and scrapie. Experimental cross-species transmission of TSE agents provides valuable information for potential host ranges of known TSEs. Some interspecies transmission studies have been conducted by inoculating disease-causing prions intracerebrally (IC) rather than orally; the latter is generally effective in intraspecies transmission studies and is considered a natural route by which animals acquire TSEs. The “species barrier” concept for TSEs resulted from unsuccessful interspecies oral transmission attempts. Oral inoculation of prions mimics the natural disease pathogenesis route whereas IC inoculation is rather artificial; however, it is very efficient since it requires smaller dosage of inoculum, and typically results in higher attack rates and reduces incubation time compared to oral transmission. A species resistant to a TSE by IC inoculation would have negligible potential for successful oral transmission. To date, results indicate that cattle are susceptible to IC inoculation of scrapie, TME, and CWD but it is only when inoculated with TME do they develop spongiform lesions or clinical disease similar to BSE. Importantly, cattle are resistant to oral transmission of scrapie or CWD; susceptibility of cattle to oral transmission of TME is not yet determined.
Polymerase chain reaction–based method for the typing of F18 fimbriae and distribution of F18 fimbrial subtypes among porcine Shiga toxin–encoding Escherichia coli in Germany
Authors: Barth Stefanie , Schwanitz Anja , Bauerfeind Rolf
Published: May 2011

Edema disease is an enterotoxemic disorder of weaned piglets that represents a significant threat to pig husbandry worldwide. The causative Escherichia coli strains are highly adapted to the porcine host and characterized by the production of Shiga toxin type 2e (Stx2e) and adhesive F18 fimbria. The current study assessed the occurrence of F18 fimbrial subtypes in 241 porcine stx2e+ fedA+ E. coli strains in Germany, including 116 Shiga toxin–encoding E. coli (STEC) and 125 Shiga toxin E. coli/enterotoxigenic E. coli (STEC/ETEC) isolates. In addition, a novel multiplex polymerase chain reaction (PCR) was developed in order to improve the typing system in terms of costs, time, and discriminative power. Utilizing the novel F18 typing PCR, 93 E. coli strains (38.5%) tested positive for the F18ab fimbrial subtype and 147 strains (61.0%) for the F18ac fimbrial subtype, while 1 strain remained nontypeable. Six strains were classified as F18ac using the F18 typing PCR, but were classified as F18ab using the F18-restriction fragment length polymorphism assay. Nucleotide sequencing of the FedA gene revealed that 5 of these strains encoded F18ac fimbriae, while the FedA of 1 strain did not cluster with F18ab or with F18ac amino acid sequences. The F18 fimbrial subtype was significantly associated with the pathovar of the E. coli strains, as 73.2% of the STEC isolates harbored F18ab genes whereas 93.6% of the STEC/ETEC isolates proved F18ac positive. In conclusion, the novel F18 typing PCR allows a specific identification of the F18 fimbrial subtype. The genetic and phenotypic heterogeneity of F18 fimbriae in porcine E. coli strains should be considered in the development of new vaccines and diagnostic tools.
Case Studies on Second-Generation Anticoagulant Rodenticide Toxicities in Nontarget Species
Authors: DuVall Michael D. , Murphy Michael J. , Ray Allen C. , Reagor John C.
Published: May 2011

Specimens from 10 cases of second-generation anticoagulant rodenticide poisoning in dogs and cats were submitted to the Texas Veterinary Medical Diagnostic Laboratory during 1986 and 1987. The clinical signs most frequently observed were lethargy, dyspnea, and ventral hematomas; common necropsy findings included hemoperitoneum, hemothorax, and pulmonary hemorrhage. In the instances when histopathological examination of the tissue was done, it supported a diagnosis of coagulopathy. The presence of anticoagulants in serum or liver was confirmed by high pressure liquid chromatography, gas chromatography/mass spectrometry, or a combination of the two. Five cases of brodifacoum poisoning, 2 of bromadiolone, and 3 of diphacinone toxicity were verified. Concentrations of these rodenticides ranged from approximately 0.001 to 12 ppm.
Neosporosis-Like Abortions in a Herd of Dairy Cattle
Authors: Thilsted John P. , Dubey J. P.
Published: May 2011

Twenty-nine cows from a 240-cow drylot Holstein dairy in north-central New Mexico aborted over a period of approximately 5 months. Nine aborted fetuses were necropsied, and all but 1 of the fetuses were between 5 and 7 months of gestation. Microscopic examination of fetal tissues revealed focal necrotizing encephalitis and nonsuppurative myocarditis in 7 of the 9 fetuses. Additional lesions observed in some fetuses were focal hepatic necrosis, nonsuppurative myositis, focal necrotizing placentitis, focal nonsuppurative pneumonia, and focal nonsuppurative nephritis. A few groups of Neospora caninum -like protozoan organisms were observed adjacent to necrotic foci in the brain of 2 fetuses and in the kidney of a third fetus. No other cause of abortion was detected.
Comparison of Three Culture Techniques for the Isolation of Brucella Abortus from Bovine Supramammary Lymph Nodes
Authors: Ewalt Darla R.
Published: May 2011

Three different culturing techniques were compared and evaluated to determine the most effective method for isolating Brucella abortus from bovine supramammary lymph nodes (SM's). In method I, the SM was sliced in half, and the inner surface was minced finely with a sterile scalpel. The minced surface was spread onto the agar surface of 4 selective media. In method II, the SM was cut into small pieces and placed in a bag with a volume of phosphate-buffered saline equal to the volume of the lymph node. The bag was placed in a laboratory blender and the SM was macerated for 5 min. The tissue suspension was spread with a sterile cotton swab onto the agar surface of 4 selective media. In method III, the SM was processed in the laboratory blender. One milliliter of the suspension was pipetted into a flask of biphasic medium, and 2 ml of the suspension was pipetted into another flask of biphasic medium. A total of 626 SM's from 285 cows were cultured. Brucella abortus was isolated from 149 (52.3%) cows by 1 or more methods. Brucella abortus was isolated from 136 cows by method I, 137 cows by method II, and 86 cows by method III. Nine (3.2%) cows were positive by method I only, 11 (3.9%) cows by method II only, and 2 (0.7%) cows by method III only. The isolation rate for method III was significantly lower than for method I or II. There was no significant difference between methods I and II.
A Comparison of Six Monoclonal Antibodies for Detection of Cytokeratins in Normal and Neoplastic Canine Tissues
Authors: Cardona Aglaia , Madewell Bruce Raymond , Naydan Diane Kathy , Lund Judi Kay
Published: May 2011

Twenty normal canine tissue specimens, both fetal and adult; 19 epithelial neoplasms; and 18 nonepithelial neoplasms were examined using 6 commercially available monoclonal antibodies differing in their recognition of various molecular weight cytokeratins in human tissues. Fresh tissue samples were fixed in 100% ethanol and paraffin embedded prior to sectioning. The intermediate filament proteins were identified by an avidin-biotin-immunoperoxidase method. Primary antisera used included AE1/AE3, CAM-5.2, 35BH11, 34BE12, PKK1, MAK-6 cytokeratins, and vimentin. Monoclonal antibodies detected cytokeratins in a wide variety of canine epithelial tissues and neoplasms. Normal mesenchymal tissues and neoplasms, and stromal elements of epithelial tissues, showed no reactivity with anti-cytokeratins, but reacted positively with vimentin. Although PKK1, CAM-5.2, and MAK-6 were the most consistently reactive anti-cytokeratins, the full panel of monoclonals was required to detect cytokeratins in all of the epithelia evaluated.
Hexachlorophene Toxicosis in a Litter of Doberman Pinschers
Authors: Poppenga Robert H. , Trapp Allan L. , Braselton W. Emmett , Louden Calvert G. , Gumbs John M. , Dalley James B.
Published: May 2011

A litter of 5-week-old Doberman Pinschers with pustular dermatitis was treated dermally with a hexachlorophene-containing emulsion. Shortly after a second treatment, all of the puppies developed neurologic signs consisting of muscle tremors, ataxia, and apparent muscle weakness. The clinical history and signs, histologic lesions within the central nervous system, and measurement of hexachlorophene in liver and kidney tissue confirmed a diagnosis of hexachlorophene toxicosis.
Comparison of a Veterinary Breakpoint Minimal Inhibitory Concentration System and a Standardized Disk Agar Diffusion Procedure for Antimicrobic Susceptibility Testing
Authors: Watson Cindy K. , Cole John R. , Purse Alfred R.
Published: May 2011

The correlation between the antimicrobic standard disk agar diffusion procedure and a veterinary antimicrobic breakpoint minimal inhibitory concentration system was evaluated. Bacterial isolates representing 5 different genera were tested against 15 antimicrobics. There were 3,795 tests performed; 77.3% of the test results were in agreement, 22.6% were in disagreement. Thirty-one percent of the conflicting results were due to the organism being intermediate on the standard disk agar diffusion procedure. Results suggest that the breakpoint system needs more than 1 antimicrobic dilution per antimicrobic, or a change in some of the single dilutions in the breakpoint system would eliminate some of these discrepancies. Another possibility would be to utilize more than 1 panel design, i.e., 1 for gram-positive organisms, 1 for gram-negative organisms, and 1 for fastidious organisms.
Detection of Bluetongue Virus Serogroup by Polymerase Chain Reaction
Authors: Akita Geoffrey Y. , Chinsangaram Jarasvech , Osburn Bennie I. , Ianconescu Marius , Kaufman Rozalia
Published: May 2011

To facilitate detection of active bluetongue virus (BTV) infection, a polymerase chain reaction (PCR) protocol was developed. The BTV reverse transcriptase PCR (RT-PCR) is a 1-tube reaction and involves chemical denaturation of the double-stranded viral RNA target, a complementary DNA (cDNA) synthesis step, and PCR amplification of the cDNA. BTV RT-PCR using primers derived from highly conserved genome segment 10 results in a 251–base pair (bp) product. BTV RNA from all USA prototype serotypes 2, 10, 11, 13, and 17; a wide spectrum of USA BTV field isolates including serotypes 10, 11, 13, and 17; and a spectrum of Israeli field isolates including serotypes 2, 4, 6, 10, and 16 were detected by BTV RT-PCR. With agarose gels, the 251–bp product was detected from as little as 100 fg-1 pg of BTV RNA, which is equivalent to 5 × 103-5 × 104 viral particles or 5 × 102-5 × 103 infectious units. With dot blot hybridization, specific PCR product was detected from as little as 1 fg of BTV RNA, which is equivalent to 50 viral particles, or 5 infectious units. This level of sensitivity is comparable to that of virus isolation. The BTV RT-PCR using primers derived from genome segment 10 can detect a wide spectrum of USA and Israeli BTV serotypes and has potential for detection of infection by the BTV serogroup. Application of this BTV PCR to clinical samples is in progress.
Evaluation of Serological Pseudorabies Tests for the Detection of Antibodies during Early Infection
Authors: Oren Shelley L. , Swenson Sabrina L. , Kinker David R. , Hill Howard T. , Hu Hui-Lin , Zimmerman Jeff
Published: May 2011

Six enzyme-linked immunosorbent assays, a latex agglutination test, and the standard microtitration serum virus neutralization test were compared for their ability to detect antibodies against pseudorabies virus (PRV) during the early stages of infection. Thirty-five pigs were infected intranasally with 105-107 TCID50 of either the Iowa 4892 pneumotropic or the Becker strain of PRV. Blood samples were drawn from experimentally inoculated animals on days 4–10, 14, and 21 postchallenge. Test sensitivity estimates and comparisons among tests were made for each sampling day over the 21-day monitoring period. Results of this study demonstrated differences among tests in 1) the time from inoculation to initial antibody detection, 2) the time to detect ≥ 95% of the infected pigs, and 3) the time from initial antibody detection to determination of ≥ 95% as positive. By day 10 postchallenge, no statistically significant difference in diagnostic sensitivity was observed among the 8 tests compared in the study.
Fibrosing Granulomas in the Equine Liver and Peritoneum: A Retrospective Morphologic Study
Authors: Buergelt Claus D. , Greiner Ellis C.
Published: May 2011

Fibrosing granulomas were found in the liver and occasionally on the intestinal and diaphragmatic serosae and in the lung of 11 horses submitted for necropsy. Although these granulomas were considered incidental findings in most of the horses, they had caused liver failure in 1 horse. The granulomas typically were characterized by a dense collagenous core that frequently was mineralized. The periphery contained a rim of inflammatory cells. Only in 1 of the 11 horses was there evidence of an egg shell suggestive of schistosomes. Schistosomal eggs were not detected in the feces of the horses. The typical architecture of the granulomas combined with the occasional finding of a residual egg led to the circumstantial conclusion that the fibrosing granulomas were the result of chronic schistosomiasis of undeterminable origin.
Rotavirus Shedding in Feces of Gnotobiotic Calves Orally Inoculated with a Commercial Rotavirus-Coronavirus Vaccine
Authors: Theil K. W. , McCloskey C. M.
Published: May 2011

The purpose of this study was to monitor by negative stain electron microscopy the shedding of rotavirus in the feces of gnotobiotic calves orally inoculated with a commercial modified live bovine rotavirus-bovine coronavirus vaccine. Negative stain electron microscopic examination detected vaccine rotavirus in only 1 of 41 daily fecal specimens collected from 3 gnotobiotic calves during the 2 weeks following oral inoculation with a US Department of Agriculture-licensed modified live bovine rotavirus-bovine coronavirus vaccine. In contrast, rotavirus was demonstrable by the same negative stain electron microscopic examination procedure in 17 of 19 fecal specimens collected from diarrheic gnotobiotic or colostrum-deprived calves during the first 8 days after inoculation with virulent bovine rotavirus field strains. Rotavirus was also detected by this procedure in 4 enzyme-linked immunosorbent assay positive fecal specimens collected from naturally-infected diarrheic dairy calves. These results suggest that fecal shedding of vaccine rotavirus demonstrable by electron microscopic examination is uncommon following oral inoculation of calves with the bovine rotavirus-bovine coronavirus vaccine.
Comparison of Four Methods for Forage Nitrate Analysis
Authors: Bedwell Cathy L. , Hamar Dwayne W. , Hoesterey Martha L. , Sonderman John P. , Odde Kenneth G.
Published: May 2011

Twenty forage samples were collected and selected for variation in nitrate content. Each forage sample was analyzed 4 times by 4 different methods: diphenylamine spot plate, spectrophotometric, nitrate-selective electrode, and high-performance liquid chromatographic. Five feed extracts were spiked with 2 different amounts of nitrate and analyzed by each method. The spectrophotometric and nitrate-selective electrode had similar percent recoveries, which were close to 100%. The nitrate-selective electrode method had the least variation of the 4 methods. The diphenylamine spot plate method had the poorest average recovery, greatest variation, and was the least accurate. The average coefficients of variation for all samples within a method were 15%, 12%, 6.4%, and 16% for the diphenylamine spot plate, spectrophotometric, nitrate-selective electrode, and high-performance liquid chromatographic methods, respectively. The variation in the nitrate-selective electrode method was lower ( P < 0.05) than the other methods. The results from this study suggest that the nitrate-selective electrode method is more accurate and precise than the other methods of analysis tested.
Detection of Porcine Reproductive and Respiratory Syndrome Virus in Cell Cultures and Formalin-Fixed Tissues by in Situ Hybridization using a Digoxigenin-Labeled Probe
Authors: Larochelle Renée , Mardassi Helmi , Dea Serge , Magar Ronald
Published: May 2011

A nonradioactive in situ hybridization method is described for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) in cell cultures and in formalin-fixed paraffin-embedded tissue sections originating from experimentally infected pigs and from 1 field case. A 174 bp cDNA probe targeting the viral RNA encoding the nucleocapsid protein of a Canadian PRRSV isolate was generated by polymerase chain reaction. The cDNA probe was labeled by random priming with digoxigenin-dUTP using a commercially available kit. The ability of the digoxigenin-labeled probe to specifically detect PRRSV RNA was tested on cultured cells infected with 6 Canadian PRRSV isolates, a US PRRSV isolate and the European Lelystad isolate. The probe detected all Canadian PRRSV isolates tested as well as the US PRRSV isolate but did not detect the Lelystad isolate. In addition, when tested on formalin-fixed paraffin-embedded tissue sections from pigs experimentally infected with several Canadian isolates and from a field case, a strong signal without background staining was obtained. Our results indicate that nonradioactive in situ hybridization could represent a useful tool for the detection of PRRSV in routinely fixed and processed tissues. In situ hybridization could also be used to differentiate infection by North American and European Lelystad-like PRRSV isolates.
Polyomavirus Encephalopathy in a Ducorps Cockatoo (Cacatua Ducorpsii) with Psittacine Beak and Feather Disease
Authors: Latimer Kenneth S. , Niagro Frank D. , Steffens W. L. , Ritchie Branson W. , Campagnoli Raymond P.
Published: May 2011

Necropsy tissues were examined from an adult wild-caught Ducorps cockatoo (Cacatua ducorpsii) with progressive neurologic signs. Of the tissue specimens selected for histologic evaluation, only the brain contained rare amphophilic, glassy intranuclear inclusions within astrocytes and some neurons. Astrocyte and neuronal degeneration and necrosis also were observed. Scattered astrocytes, with and without discernable inclusions, contained avian polyomavirus (APV) nucleic acid, as determined by DNA in situ hybridization. In addition, endothelial cells and intravascular leukocytes contained psittacine beak and feather disease viral nucleic acid, as determined by DNA in situ hybridization, indicating dual viral infection. Electron microscopic examination of formalin-fixed brain tissue revealed typical intranuclear APV particles in some astrocytes. Encephalopathy ultimately was attributed to APV infection.
A Specific and Sensitive PCR Assay Suitable for Large-Scale Detection of Toxigenic Pasteurella Multocida in Nasal and Tonsillar Swabs Specimens of Pigs
Authors: Kamp Elbarte M. , Bokken Gertie C. A. M. , Vermeulen Thea M. M. , de Jong Marten F. , Buys Herma E. C. M. , Reek Frans H. , Smits Mari A.
Published: May 2011

A polymerase chain reaction (PCR) assay for the detection of toxigenic Pasteurella multocida in nasal and tonsillar swab specimens collected from pigs was developed. Target DNA was isolated with guanidine thiocyanate and diatomite, and 2 primer sets derived from sequences in the gene that encodes the dermonecrotic toxin-of P. multocida were used simultaneously. The method was adapted to microtiter plate format allowing large-scale use of the PCR assay. To identify false-negative test results caused by failure of amplification, a positive control template was constructed that was spiked to each DNA sample. The PCR assay was evaluated with clinical samples and compared with 2 routinely used methods for detection of toxigenic P. multocida: isolation from a selective agar and direct detection of the toxin in extracts of primary cultures by an enzyme-linked immunosorbent assay (ELISA). The sensitivity of the PCR assay was tested with 346 nasal and tonsillar swabs specimens collected from pigs of 9 herds known to be infected with toxigenic P. multocida. Toxigenic P. multocida was isolated from 22 specimens, only 28 specimens tested positive in ELISA, but 40 tested positive in the PCR assay; thus the PCR assay is the most sensitive of the 3 methods. The specificity of the PCR assay was tested with 372 swab specimens collected from pigs of 6 herds certificated to be free from toxigenic P. multocida. Toxigenic P. multocida was not isolated from any of these specimens, all tested negative in ELISA, and 370 tested negative in PCR. The 2 positive specimens came from 2 pigs of 1 litter and tested only weakly positive in the PCR assay. From these results, it was concluded that the PCR assay is not only highly sensitive but also highly specific.
Production of γ-Interferon by Peripheral Blood Mononuclear Cells: An Important Diagnostic Tool for Detection of Subclinical Paratuberculosis
Authors: Stabel Judith R.
Published: May 2011

Peripheral blood mononuclear cells were isolated from noninfected control cows and from cows with either subclinical or clinical paratuberculosis (Johne s disease). Cells were incubated for 6, 12, 24, and 48 hours in complete medium with the following mitogens: concanavalin A (ConA), phytohemagglutinin-P (PHAP), pokeweed mitogen (PWM), and Escherichia coli lipopolysaccharide. In addition, cells were incubated for the same time periods with a Mycobacterium paratuberculosis sonicate (MpS) and live and heat-killed M. paratuberculosis at 10:1 bacteria: cell ratio. After incubation, cell-free supernatants were analyzed for y-interferon (γ-IFN) production. Cells from subclinical cows produced significantly higher levels of γ-IFN than did cells from clinical animals after stimulation with mitogens ConA, PHAP, and PWM. Levels of γ-IFN produced by noninfected control animals generally followed the pattern of those of subclinical animals. After incubation with MpS, significantly greater quantities of γ-IFN were produced by cells isolated from subclinical animals than by cells from clinical cows and noninfected controls. Stimulation of cells with heat-killed or live M. paratuberculosis evoked a similar response. This study indicates that γ-IFN production by peripheral blood mononuclear cells in response to M. paratuberculosis antigen may be an important diagnostic tool for the detection of paratuberculosis in subclinically affected animals.
Immunoblot Analysis of the Immunoglobulin G Response to Ehrlichia Canis in Dogs: An International Survey
Authors: Hegarty Barbara C. , Levy Michael G. , Gager Robin F. , Breitschwerdt Edward B.
Published: May 2011

Historically, considerable variation has been reported in the type and severity of clinical and hematologic abnormalities associated with canine ehrlichiosis. Because of difficulties associated with the isolation of intracellular monocytic Ehrlichia species in tissue culture systems, few E. canis isolates are available for comparative microbiologic studies. To address the issue of potential E. canis antigenic diversity in different regions of the world, dog sera reactive by indirect fluorescent antibody testing to E. canis (Florida) antigen were obtained from France, Israel, Italy, the United States, the Virgin Islands, and Zimbabwe. Ehrlichia canis proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and at least 5 sera from each region were stained by western immunoblotting. Antibody immunodominance was scored based upon staining intensity. There was relative homogeneity in the immunogenic protein reactions to E. canis antigens. Of the 58 E. canis reactive sera, 54 samples resulted in immunoblot patterns indicative of chronic ehrlichiosis. Four reactive sera (reciprocal titers of 160–2,560) did not recognize any genus-specific antigens resulting in protein bands between 22 and 29 kD, indicating serologic cross-reactivity with other microorganisms. Relatively homogenous immunoblot patterns, consistent with the reported immunoblot response of dogs with experimental chronic ehrlichiosis, were observed with sera from Arizona, France, Israel, North Carolina, Texas, and the Virgin Islands. In contrast, unique major proteins were observed in dog sera from Italy and Zimbabwe. Our results indicate that although relatively homogeneous, antigenic diversity may exist among E. canis organisms in different regions of the world.
An Improved Method for the Recovery of Mycoplasmas from the External Ear Canal of Goats
Authors: Ribeiro Vinicius Rezende , do Nascimento Elmiro Rosendo , Faccini João Luiz Horácio , da Graça Fichel do Nascimento Maria , Lignon Gilberto Brasil
Published: May 2011

The efficacy of ear canal flushing and ear canal and mouth swabbing methods for the isolation of mycoplasmas was investigated in 39 goats. Of the 19 goats positive for Mycoplasma spp., 14 (73.7%) were positive with the ear canal flushing method, 4 (21.0%) were positive with both ear canal flushing and mouth swabbing methods, and 1(5.3%) was positive by the mouth swabbing method. Mycoplasma arginini, M. mycoides subsp. mycoides, and M. mycoides subsp. capri were identified by direct immunofluorescence and growth inhibition tests. Previous reports on the isolation of M. arginini from the ear canal of goats were not found in the literature.
Evaluation of an IgM-Specific Indirect Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Bovine Respiratory Syncytial Virus Infection: Influence of IgM Rheumatoid Factor on Test Results with Field Sera
Authors: Graham D. A. , Mawhinney K. A. , Elvander M. , Adair B. M. , Merza M.
Published: May 2011

A commercially available indirect enzyme-linked immunosorbent assay for measuring bovine respiratory syncytial virus (BRSV)-specific IgG was adapted to measure virus-specific IgM. Using this assay, the development of rapid IgM responses in experimentally infected calves was observed 7–9 days postinfection, with peak absorbance values ranging from 1.698 to 2.873. When absorbance values were expressed as a percentage of a positive reference serum, a positive/negative threshold of 22% was determined by testing serum samples from 59 healthy 3–5-month-old calves. Acute and convalescent serum samples collected from 151 calves during 38 outbreaks of respiratory disease were tested, and 130 sera were positive. To determine the number of false-positive results due to the presence of IgM rheumatoid factor, a method for depleting serum IgG by pretreatment of sera with a suspension of protein-G-agarose was developed. All sera that initially tested IgM positive were retested following depletion of serum IgG. False-positive IgM reactions were detected in 23 sera (17.7%). Specific IgM responses were confirmed in 107 sera from 84 calves. Evidence of BRSV infection was detected in 34 of 38 outbreaks. In contrast, seroconversion was detected in 69 calves from 24 outbreaks, confirming the diagnostic potential of the IgM assay. Overall correlation between IgM and seroconversion results was 74.2%. Intra- and interassay reproducibility were 12.50% and 17.48%, respectively (mean coefficients of variation).