bluuebunny/crossref_metadata_2025 huggingface dataset:
A Multiplex Real-Time Reverse Transcription Polymerase Chain Reaction Assay for Detection and Differentiation of Bluetongue Virus and Epizootic Hemorrhagic Disease Virus Serogroups
Authors: Wilson William C. , Hindson Benjamin J. , O'Hearn Emily S. , Hall Sara , Tellgren-Roth Christian , Torres Clinton , Naraghi-Arani Pejman , Mecham James O. , Lenhoff Raymond J.
Published: May 2011

Bluetongue virus (BTV) causes disease in domestic and wild ruminants and results in significant economic loss. The closely related Epizootic hemorrhagic disease virus (EHDV) has been associated with bluetongue-like disease in cattle. Although U.S. EHDV strains have not been experimentally proven to cause disease in cattle, there is serologic evidence of infection in cattle. Therefore, rapid diagnosis and differentiation of BTV and EHDV is required. The genetic sequence information and bioinformatic analysis necessary to design a real-time reverse transcription polymerase chain reaction (RT-PCR) assay for the early detection of indigenous and exotic BTV and EHDV is described. This sequence data foundation focused on 2 conserved target genes: one that is highly expressed in infected mammalian cells, and the other is highly expressed in infected insect cells. The analysis of all BTV and EHDV prototype strains indicated that a complex primer design was necessary for both a virus group-comprehensive and virus group-specific gene amplification diagnostic test. This information has been used as the basis for the development of a rapid multiplex BTV-EHDV real-time RT-PCR that detects all known serotypes of both viruses and distinguishes between BTV and EHDV serogroups. The sensitivity of this rapid, single-tube, real-time RT-PCR assay is sufficient for diagnostic application, without the contamination problems associated with standard gel-based RT-PCR, especially nested RT-PCR tests.
Removal of Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR) Inhibitors Associated with Cloacal Swab Samples and Tissues for Improved Diagnosis of Avian Influenza Virus by RT-PCR
Authors: Das Amaresh , Spackman Erica , Pantin-Jackwood Mary J. , Suarez David L.
Published: May 2011

Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) is routinely used for the rapid detection of Avian influenza virus (AIV) in clinical samples, but inhibitory substances present in some clinical specimens can reduce or block PCR amplification. Most commercial RNA extraction kits have limited capacity to remove inhibitors from clinical samples, but using a modified commercial protocol (Ambion® MagMAX™, Applied Biosystems, Foster City, CA) with an added high-salt wash of 2 M NaCl and 2 mM ethylenediamine tetra-acetic acid was shown to improve the ability of the kit to remove inhibitors from cloacal swabs and some tissues. Real-time RT-PCR was carried out in the presence of an internal positive control to detect inhibitors present in the purified RNA. Cloacal swabs from wild birds were analyzed by real-time RT-PCR comparing RNA extracted with the standard (MagMAX-S) and modified (MagMAX-M) protocols. Using the standard protocol on 2,668 samples, 18.4% of the samples had evidence of inhibitor(s) in the samples, but the modified protocol removed inhibitors from all but 21 (4.8%) of the problem samples. The modified protocol was also tested for RNA extraction from tissues using a TRIzol-MagMAX-M hybrid protocol. Tissues from chickens and ducks experimentally infected with high-pathogenicity Asian H5N1 AIV were analyzed by real-time RT-PCR, and the limit of detection of the virus was improved by 0.5–3.0 threshold cycle units with the RNA extracted by the MagMAX-M protocol. The MagMAX-M protocol reported in the present study can be useful in extracting high-quality RNA for accurate detection of AIV from cloacal swabs and tissues by real-time RT-PCR.
Optimization of Immunohistochemical and Fluorescent Antibody Techniques for Localization of Foot-and-Mouth Disease Virus in Animal Tissues
Authors: Arzt Jonathan , Gregg Douglas A. , Clavijo Alfonso , Rodriguez Luis L.
Published: May 2011

Immunohistochemical (IHC) and fluorescent antibody (FA) techniques were optimized for the detection of Foot-and-mouth disease virus (FMDV) structural and nonstructural proteins in frozen and paraformaldehyde-fixed, paraffin-embedded (PFPE) tissues of bovine and porcine origin. Immunohisto-chemical localization of FMDV was compared with 7 detection systems, 8 primary antibodies, and 11 epitope retrieval techniques. All serotypes tested (O, A, Asia, C [cryosection]; O, A, Asia [PFPE]) were localized in association with mature vesicles. Multi-label FA was used in conjunction with IHC and conventional histopathology to characterize vesicle maturation in 4 steers and 2 pigs experimentally infected with FMDV. At the edge of advancing vesicles, a consistent finding was acantholytic degeneration of basal keratinocytes surrounding dermal papillae with suprabasilar clefts and microvesiculation. Progression of microvesiculation led to coalescence with the expanding vesicle. Cells at the leading edge of vesicles were positive for FMDV antigens by IHC and FA. Cell marker profile of these cells by FA was consistent with keratinocytes (i.e., cytokeratin [CK]-positive, S100-negative, MHC-II-negative). In rare instances, CK-negative, MHC-II-positive, and FMDV-positive cells (presumptive dendritic cells or macrophages) were identified within dermis subjacent to vesicles.
Reference Intervals and Method Optimization for Variables Reflecting Hypocoagulatory and Hypercoagulatory States in Dogs using the STA Compact® Automated Analyzer
Authors: Bauer Natali , Eralp Oya , Moritz Andreas
Published: May 2011

Reference intervals for coagulation parameters have been rarely determined in dogs for the STA Compact® automated coagulation analyzer, so it is the aim of the current study to validate assays and establish reference ranges for its use in canine specimens. Coagulation parameters were assessed in 56 healthy dogs with a median age of 2 years and evenly distributed sex. The 95% reference intervals were as follows: 1-stage prothrombin time = 5.7–8.0 sec; activated partial thromboplastin time (APTT) = 10.0–14.3 sec; thrombin time (TT) = 11.9–18.3 sec; fibrinogen = 1.3–3.1 g/1; antithrombin (AT) = 107.9–128.0%; D-dimer = 0.023–0.65 μg/ml; anti-factor Xa = 0.04–0.26 IU/1; and activated protein C (APC) ratio = 2.0–3.0. Protein C and S activity was markedly below (<-20%) and factor VIII was 2- to 11-fold above the human calibration standard, so a standard curve had to be prepared from canine pooled plasma. Reference intervals for protein C, protein S, and factor VIII were 75.5–118.9%, 74.4–160.5%, and 70.9–136.4%, respectively, compared with a canine standard curve. Streptokinase-activated plasminogen assay was not suitable for dogs. There was no significant impact of sex on hemostasis test results. Factor VIII activity, AT, protein C, protein S, and APC ratio were overestimated in hemolytic plasma, whereas fibrinogen, TT, and APTT were underestimated. Lipemia resulted only in false-high D-dimers. This study provided useful reference intervals for dogs, but some human tests (i.e., protein C, protein S, factor VIII, and plasminogen) required modification.
Nuclear Morphometry in Cytopathology: A Prognostic Indicator for Canine Cutaneous Mast Cell Tumors
Authors: De Strefezzi Ricardo Francisco , Xavier José Guilherme , Kleeb Silvia Regina , Catão-Dias José Luiz
Published: May 2011

Twenty-nine canine cutaneous mast cell tumors (MCTs) were morphometrically analyzed with regard to mean nuclear area (MNA) using cytopathology smears. The results showed a correlation between MNA and survival. When graded into 2 morphometrically different groups, there were statistically significant differences among high- and low-grade MCTs, regarding both Romanowsky-type stain and hematoxylin and eosin. Cytomorphometry could also separate histologic grade II tumors with better prognosis from the more aggressive MCTs. The results indicated that nuclear morphometry on cytopathology preparations can predict the biological behavior of cutaneous MCTs in dogs in an independent manner, yielding a rapid and reproducible diagnosis, which renders the method useful for veterinary oncology.
Impact of Age and Sex of Rocky Mountain Elk (Cervus Elaphus Nelsoni) on Follicle Counts from Rectal Mucosal Biopsies for Preclinical Detection of Chronic Wasting Disease
Authors: Spraker Terry R. , VerCauteren Kurt C. , Gidlewski Tom L. , Munger Randy D. , Walter W. David , Balachandran Aru
Published: May 2011

To determine if the number of rectal lymphoid follicles decreases with respect to age and sex relative to diagnosis of chronic wasting disease (CWD), rectal biopsies ( n = 1,361) were taken from captive Rocky Mountain elk ( Cervus elaphus nelsoni) at 4 ranches in the western United States between 2005 and 2008. Rectal tissues were stained with a monoclonal antibody (F99/97.6.1), which selectively stains the abnormal isoform of the prion protein associated with CWD of elk. The number of lymphoid follicles obtained from typical biopsy tissues decreased with the age of the animal. The acceptable number of lymphoid follicles for detection of CWD was not considered to be a problem in elk up to 8.5 years of age, but in elk over 8.5 years of age, the follicle count was considered to be low. Sex of the animal had no effect on the number of lymphoid follicles observed in each age group. Rectal biopsies were an accurate test to diagnose preclinical stages of CWD in elk but may be best suited to elk that are less then 8.5 years of age.
Serum Thymidine Kinase Activity as a Useful Marker for Bovine Leukosis
Authors: Sakamoto Leo , Ohbayashi Tetsu , Matsumoto Kotaro , Kobayashi Yoshiyasu , Inokuma Hisashi
Published: May 2011

Serum thymidine kinase (TK) activity has recently been evaluated as a serum marker for human and canine hematopoietic neoplasms. The purpose of the current study was to establish the significance of serum TK activity in the diagnosis of bovine leukosis. The discrimination value for TK activity was set at 5.4 U/l based on the receiver operating characteristic curve. In the group of clinically healthy cows, only 2 out of 83 cows (2.4%) had serum TK activity above the discrimination value. In contrast, 19 out of 20 cows (95.0%) with bovine leukosis showed serum TK activity above the discrimination value, although only 7 of 79 (8.9%) cows diagnosed with diseases other than bovine leukosis showed elevated serum TK activity. Thymidine kinase activities of all Bovine leukemia virus-positive cows with or without lymphocytosis were below the discrimination value. Sensitivity and specificity of measuring serum TK activity as a diagnostic tool for bovine leukosis was 95.0% and 95.9%, respectively. Results indicate that serum TK activity may be a marker for bovine leukosis.
Coinfection of a Cow with Bovine Leukemia Virus and Mycobacterium Bovis
Authors: Sledge Dodd G. , Maes Roger , Wise Annabel , Kiupel Matti , Fitzgerald Scott D.
Published: May 2011

Bovine leukosis associated with infection with the delta retrovirus Bovine leukemia virus (BLV) is endemic in many cattle herds in the United States. Infection has been associated with immunosuppression and decreased productivity. Cases of tuberculosis in cows due to infection with Mycobacterium bovis reemerged in Michigan in 1998, and despite intensive eradication attempts, new cases of bovine tuberculosis are sporadically identified. The present report details a coinfection with BLV and M. bovis in a Holstein cow from Michigan that presented as part of a bovine tuberculosis screening program. Peripheral and visceral lymph nodes of this animal were markedly enlarged, homogeneously pale white, and bulged on the cut surface. The submandibular, mesenteric, and caudal mediastinal lymph nodes contained multifocal to coalescing caseogranulomas that ranged from 1 to 5 cm in diameter. Histologically, dense sheets of monomorphic populations of neoplastic lymphocytes obliterated the normal architecture of all lymph nodes. Caseogranulomas were characterized by central pools of amorphous degenerate eosinophilic and occasionally mineralized granular debris surrounded by thick rims of epithelioid macrophages, occasional Langhan's type giant cells, and fibrosis. Polymerase chain reaction assay was positive for BLV. Cultures of affected lymph nodes yielded growth of M. bovis.
Colic Caused by Panicum Maximum Toxicosis in Equidae in Northern Brazil
Authors: Cerqueira Valíria D. , Riet-Correa Gabriela , Barbosa José D. , Duarte Marcos D. , Oliveira Carlos M. C. , de Oliveira Carlos A. , Tokarnia Carlos , Lee Stephen T. , Riet-Correa Franklin
Published: May 2011

In the Amazon region of northern Brazil, Panicum maximum cultivars Mombaça, Tanzânia, and Massai cause severe colic and death in horses and mules. The disease occurs in the rainy season, when sprouting pastures are grazed by equidae. In the 8 separate disease outbreaks studied, a total of 52 out of 153 equidae were affected, including 19 that died (10 mules and 9 horses). Clinical signs were colic and abdominal dilatation, with a clinical manifestation period of 12 hr to 4 days. Serum activities of gamma-glutamyl transferase and aspartate aminotransferase were within reference intervals; however, serum urea nitrogen and creatinine concentrations were occasionally elevated. The primary gross and histologic lesions were observed in the digestive system. The stomach, small intestine, and large intestine had severe hemorrhages and occasional mucosal erosions and ulcerations. Ulceration and hemorrhage of the urinary bladder were rarely observed. Histologic examination revealed diffuse lymphoplasmacytic gastritis and enteritis with severe congestion, hemorrhage, and occasional epithelial necrosis and ulceration. Lymphocellular necrosis was occasionally observed within gut-associated lymphoid tissue. Tubular nephrosis occurred in some animals. Degeneration and occasional necrosis of bile duct epithelial cells and degeneration of hepatocytes were observed in the liver. Toxic pastures were negative for diosgenin- and yamogenin-based saponins, and oxalate concentrations were within reference intervals for the species. The toxin or toxins causing disease and the reason for the toxicity of the plant in the northern region are unknown.
Hypertrophic Cardiomyopathy in Two Captive Bennett's Wallabies (Macropus Rufogriseus Rufogriseus)
Authors: Stern Adam W. , Smith Stephen , Snider Timothy A.
Published: May 2011

Hypertrophic cardiomyopathy (HCM) was diagnosed during postmortem examination of 2 captive adult Bennett's wallabies ( Macropus rufogriseus rufogriseus). The wallabies were members of a mob (herd) of 3 wallabies, and 2 died spontaneously without clinical signs of heart failure being detected. Gross lesions in both cases included marked concentric hypertrophy of the left ventricle, pulmonary edema, and multifocal hemorrhage and subcutaneous edema of the hind limbs. Histologic lesions of the heart were limited to mild cardiac myofiber disarray and marked cardiac myofiber hypertrophy. A specific etiology for the HCM was not determined in either animal. The cardiac changes are similar to the left ventricular hypertrophy previously described in kangaroos.
Oral Squamous Cell Carcinoma in a Vietnamese Pot-Bellied Pig (Sus Scrofa)
Authors: Swenson Julie , Carpenter James W. , Ragsdale John , Kuroki Kei , Ketz-Riley Cornelia , Brinkman Erin , Cole Gretchen
Published: May 2011

An 18-year-old, neutered, male Vietnamese pot-bellied pig ( Sus scrofa) was treated for chronic, intermittent nasal discharge and sneezing. The animal was diagnosed with severe periodontal disease (grade IV), an oronasal fistula, and multiple tooth root abscesses via dental examination and computed tomography of the skull. Dentistry was performed, including multiple tooth extractions, and antibiotic therapy was initiated. Eighteen months later, the animal was evaluated for lethargy, anorexia, and a firm, 12 cm × 12 cm mass between the 2 rami of the mandible. Laboratory testing revealed moderate anemia, severe leukocytosis, and hyperglobulinemia. Skull radiographs indicated osteomyelitis of the mandible and soft-tissue swelling. A fine-needle aspirate and biopsy were taken, and results were consistent with squamous cell carcinoma. Treatment with piroxicam and antibiotics was initiated as needed to control signs of pain and secondary infection, respectively. Three months after diagnosis, the pig was euthanized due to cachexia and severe depression secondary to squamous cell carcinoma. On postmortem examination, the right mandibular area contained multiple, coalescing, irregular masses extending from the ramus rostrally to the mandibular canine teeth and ventrally within the intermandibular space, completely obliterating the normal anatomy. An open midshaft fracture was present on the right mandible. On histopathology, the masses were confirmed as locally invasive and destructive squamous cell carcinoma. No evidence of metastasis was noted in regional lymph nodes or in any of the distant sites evaluated.
A KIT-Positive Gastrointestinal Stromal Tumor in a Ferret (Mustela Putovius Furo)
Authors: Girard-Luc Anne , Prata Daniela , Hélène Huet , Lagadic Marie , Bernex Florence
Published: May 2011

A 1.5-year-old, neutered, male ferret ( Mustela putorius furo) was presented with sudden lethargy, anorexia, and diarrhea. Clinical and radiographic examinations revealed an intra-abdominal mass. An explorative laparotomy was performed. A neoplasm, located in the ileum wall, was submitted for histopathologic examination. The tumor consisted of weakly eosinophilic spindle cells arranged in a compact pattern with haphazardly interlacing bundles. Neoplastic cells labeled positively for KIT (cluster of differentiation 117, stem cell factor receptor) and vimentin. Based on histologic and immunohistologic results, this tumor was diagnosed as a gastrointestinal stromal tumor. Results suggest that this ferret tumor shares strong similarities with the canine and human counterparts.
Respiratory Epithelial Adenomatoid Hamartoma in a Dog
Authors: LeRoith Tanya , Binder Ellen M. , Graham A. Heather , Duncan Robert B.
Published: May 2011

A 6-month-old, intact, male Weimaraner dog presented to the veterinary teaching hospital for bilateral mucopurulent ocular and nasal discharge that began at approximately 10 weeks of age. A computed tomography scan showed an expansile soft-tissue mass involving both frontal sinuses, the ethmoid regions, and nasal cavities with lysis of the maxillary turbinates and hyperostosis of the walls of the frontal sinus. The dog was euthanized after complications during a trephination and biopsy procedure. At necropsy, a large, tan, papillary, gelatinous mass filled the entire nasal cavity and frontal sinus. The mass was composed of large fronds of loose fibrovascular stroma covered by a single layer of pseudostratified, columnar, ciliated epithelium and intermixed goblet cells. The cells occasionally formed glandular structures that were continuous with the surface epithelium. The mass was diagnosed as a respiratory epithelial adenomatoid hamartoma based on the morphologic appearance.
Malignant Edema in Postpartum Dairy Cattle
Authors: Odani Jenee S. , Blanchard Patricia C. , Adaska John M. , Moeller Robert B. , Uzal Francisco A.
Published: May 2011

Five cases of postparturient vulvovaginitis and metritis in cattle caused by Clostridium septicum (malignant edema) are described in the current report. The diagnosis was established based on detection of C. septicum by culture and fluorescent antibody test. All animals were Holsteins, and 4 were primiparous (the parity of 1 animal was not reported). All animals developed clinical signs 1–3 days after calving, consisting of swelling of perineal and perivulvar areas, fever, and depression. Perineal, perivulvar, and perivaginal gelatinous and often hemorrhagic edema was consistently observed on gross examination. Longitudinal vulvar, vaginal, cervical, and uterine body tears, covered by fibrinous exudates, were also present. Microscopically, vulvar, vaginal, and uterine mucosae were multifocally necrotic and ulcerated. Large Gram-positive rods, some with subterminal spores, were present within the edematous subcutaneous and submucosal tissues. Clostridium septicum was demonstrated by culture and/or fluorescent antibody test in tissues of most animals. These cases of malignant edema were considered to be produced by C. septicum and predisposed by the trauma occurring during parturition.
Improved Polymerase Chain Reaction–Based Method to Detect Early-Stage Epitheliotropic T-Cell Lymphoma (Mycosis Fungoides) in Formalin-Fixed, Paraffin-Embedded Skin Biopsy Specimens of the Dog
Authors: Chaubert Pascal , Chaubert Audrey S. Baur , Sattler Ursula , Forster Ursula , Bornand Valérie , Suter Maja , Welle Monika
Published: May 2011

In the dog, early-stage epitheliotropic T-cell lymphoma (ETCL) can clinically and histologically mimic a large range of inflammatory dermatoses and often progresses rapidly to a more aggressive tumor stage. Early diagnosis of ETCL is essential to proceed with a specific oncologic therapy that is favorable for the prognosis. In the present study, an improved method for the detection of T-cell receptor gamma (TCRγ) rearrangement was developed by designing a new set of consensus primers to amplify the different forms of rearranged canine TCRγ gene sequences by polymerase chain reaction. The amplicons were analyzed by conventional polyacrylamide gel electrophoresis, which requires minimal specific equipment and may be performed in almost every pathology laboratory at low costs. The method proved to be highly specific and sensitive to detect early ETCL in formalin-fixed, paraffin-embedded biopsy specimens, providing an efficient tool for veterinary pathologists to distinguish early neoplastic from reactive cutaneous T-cell infiltrates (tumor-specific marker) or to discriminate T-cell lymphoma from B-cell lymphomas or nonlymphoid neoplasms (T-cell lineage marker). By direct sequencing analysis of amplified TCRγ gene sequences, ETCL was found to rearrange exclusively the joining (J) 4 region, which suggests specific biology for primary cutaneous T-cell lymphomas. Also, a novel (seventh) functional J region in the TCRγ gene, localized approximately 2.3 kb upstream of J5, was identified.
Loop-Mediated Isothermal Amplification Assays for Detection of Equid Herpesvirus 1 and 4 and Differentiating a Gene-Deleted Candidate Vaccine Strain from Wild-Type Equid Herpesvirus 1 Strains
Authors: Nemoto Manabu , Tsujimura Koji , Yamanaka Takashi , Kondo Takashi , Matsumura Tomio
Published: May 2011

Loop-mediated isothermal amplification (LAMP) is a novel method for the rapid and sensitive detection of DNA without the need for expensive equipment. In the present study, LAMP assays were developed for the specific detection of Equid herpesvirus 1 and 4 (EHV-1 and EHV-4, respectively) and for the differentiation of glycoprotein E (gE)-deleted EHV-1 (ΔgE) strain, a candidate strain for a live vaccine, from field EHV-1 strains. Specific primer sets were designed for the gC and gE genes of EHV-1 and for the gC gene of EHV-4. The analytical sensitivities of the LAMP assays were compared with those of polymerase chain reaction (PCR). The detection limits of LAMP for EHV-1 gC and gE and PCR for EHV-1 gC were 1 plaque-forming unit (PFU)/tube, and those of LAMP and PCR for EHV-4 gC were 0.1 PFU/tube. The DgE strain could be differentiated from wild-type EHV-1 strains based on the reactivity in the LAMP for EHV-1 gC in combination with the LAMP for EHV-1 gE. The analytical specificities of the LAMP for EHV-1 and EHV-4 were examined by using several equine pathogens, and no cross-reactions were observed. The LAMP detection abilities for EHV-1 and EHV-4 on nasal swab samples collected from experimentally infected horses were in good agreement with that of PCR for EHV-1 and EHV-4, respectively. The LAMP assays developed in the current study were sensitive and specific for EHV-1 and EHV-4, and should provide a valuable alternative to PCR for use in clinical laboratories in the field.
Comparison of Fluorescent Antibody and Microscopic Agglutination Testing for Leptospira in Pregnant and Nonpregnant Cows
Authors: Rajeev Sreekumari , Berghaus Roy D. , Overton Michael W. , Pence Mel E. , Baldwin Charles A.
Published: May 2011

Serum and urine samples from 30 cows (15 pregnant and 15 nonpregnant) from each of 10 Georgia dairy herds (total cows = 300) were examined by microscopic agglutination testing (MAT) and direct fluorescent antibody testing (FAT), respectively. Seven of the 10 herds had at least 1 cow with a positive FAT, and all of the herds had at least 1 cow with a reciprocal MAT titer ≥100 for 1 or more serovars. Serological testing was not helpful in identifying the infecting serovar for cows with a positive FAT result. The MAT titers for all 7 of the serovars evaluated were significantly correlated with one another, with 17 (81%) of the 21 Spearman rank correlation coefficients ≥0.4 in magnitude. Twenty (56%) of 36 FAT-positive cows did not have a titer that was highest for any particular serovar. Four of the 7 herds that reported using a Leptospira borgpetersenii serovar Hardjo-bovis vaccine had one or more FAT-positive cows compared with 3 out of 3 herds that reported they were not using this type of vaccine, although this difference was not statistically significant.
A TaqMan Quantitative Polymerase Chain Reaction Assay for the Detection of Lawsonia Intracellularis in Fecal and Tissue Samples from Pigs
Authors: Richter Barbara , Ladinig Andrea , Nedorost Nora , Herbert Weissenboöck
Published: May 2011

In the present study, a TaqMan quantitative polymerase chain reaction (qPCR) assay for detecting the 16S ribosomal RNA gene of Lawsonia intracellularis in porcine native ileal mucosal scrapings, fecal samples, and formalin-fixed and paraffin-embedded (FFPE) ileal samples is described. Samples from 62 pigs were examined. The results of the qPCR were compared with results obtained with conventional detection methods (PCR, immunohistochemistry, in situ hybridization, and silver staining) from a previous study and correlated well. The qPCR assay proved to be very sensitive and specific. In particular, the sensitivity of TaqMan PCR was significantly higher than conventional PCR on FFPE tissues because of a much shorter amplicon. A higher number of copies per gram of sample material was detected in native mucosa and FFPE tissue compared with feces, especially in highly positive animals. The detection limit for the qPCR was at 4 copies per well in native mucosal scrapings and 18 copies per well in feces and FFPE tissue, respectively. Inhibition of the qPCR reaction was checked by simultaneous detection of a recombinant beta-actin plasmid using a second fluorescent probe. A decreased signal from this internal control plasmid revealed inhibition of the PCR reaction in 21% of native mucosal samples and 1.6% of fecal samples. With a 10-fold dilution of template, the inhibition could be overcome.
Methicillin-Resistant Staphylococci Isolated from Healthy Horses and Horse Personnel in Italy
Authors: De Martino Luisa , Lucido Maria , Mallardo Karina , Facello Bruna , Mallardo Michelina , Iovane Giuseppe , Pagnini Ugo , Tufano Maria Antonietta , Catalanotti Piergiorgio
Published: May 2011

Methicillin-resistant staphylococci (MRS) were isolated from nasal swabs of 56 of 159 (35.2%; 95% confidence interval [CI]: 27.9–43.2%) healthy horses. Two nasal swabs were collected from each horse; 43 of 159 (27%; 95% CI: 20.5–34.8%) of the cohort were colonized by MRS strains in 1 nostril, while in the remaining 13 of 159 (8.2%; 95% CI: 4.6–13.9%), different or identical MRS strains were isolated in both nostrils. Of the 29 humans in close contact with the horses tested, 4 (13.8%; 95% CI: 4.5–32.6%) were found to be carriers of MRS. All isolates were coagulase negative with the exception of 2 coagulase-positive MRS strains, Staphylococcus aureus and Staphylococcus pseudintermedius, both isolated from horses. To assay the methicillin resistance, a susceptibility test to oxacillin with standardized disk diffusion method, a PBP-2a latex agglutination test, and a methicillin resistance gene ( mecA) polymerase chain reaction assay were performed. Pulsed-field gel electrophoresis patterns of isolates from horses and humans in close contact with the horses revealed similarity. The results suggest evidence of transmission between animals, from animals to humans, and vice versa.
Neuropathologic Features of Aleutian Disease in Farmed Mink in Ireland and Molecular Characterization of Aleutian Mink Disease Virus Detected in Brain Tissues
Authors: Jahns Hanne , Daly Paul , McElroy Maire C. , Sammin Donal J. , Bassett Hugh F. , Callanan John J.
Published: May 2011

A neuropathologic survey was conducted on mink brains from the 5 licensed mink farms in Ireland. The survey was part of a transmissible spongiform encephalopathy surveillance study. Aleutian disease (AD) was present on 4 of the 5 farms (80%). Neuropathologic features of nonsuppurative meningoencephalitis were common in mink from the 4 affected farms but were absent in the mink from the fifth farm, which was free of AD. The meningoencephalitis was characterized by infiltrates of lymphocytes and plasma cells, which were present in meninges, perivascular spaces, and the brain parenchyma. Fibrinoid necrotizing arteritis was seen in 11 mink brains, all of which were obtained from a single farm. Aleutian mink disease virus (AMDV) sequences for the capsid protein VP2 were obtained from brain samples from all affected farms. Although containing previously unreported amino acid residues, similarities with European and North American isolates were observed in the hypervariable regions within VP2, suggesting Irish AMDV is related to those isolates. The predicted amino acid residues, suspected of conferring pathogenicity at certain positions of the VP2 sequence, were present in the viral nucleic acid sequences.
Bovine Abortion Associated with Nocardia Farcinica
Authors: Bawa Bhupinder , Bai Jianfa , Whitehair Mike , Purvis Tanya , DeBey Brad M.
Published: May 2011

Nocardia spp. are recognized as a cause of bovine mastitis, cutaneous or subcutaneous abscesses, pneumonia, and disseminated disease. Abortion caused by Nocardia spp. is uncommon, and only a few sporadic cases have been reported in horses, pigs, and cattle. In all previous reports, of nocardial abortion, the causative agent was identified as Nocardia asteroides. The current report describes an aborted bovine fetus that was infected with Nocardia farcinica. Placenta, abomasal fluid, lung, liver, and kidney specimens from a lateterm bovine abortion were submitted to the Kansas State Veterinary Diagnostic Laboratory. The gross findings included purulent exudate in the placenta and numerous abscesses in lung. Histologically, there was necrotizing and suppurative placentitis, pyogranulomatous pneumonia, and nephritis with numerous intralesional branching and filamentous, Gram-positive bacteria. Nocardia farcinica was isolated by bacteriology, and the bacteriology result was confirmed by 2 established polymerase chain reaction protocols and by DNA sequencing.
High Serum and Urine Lysozyme Levels in a Dog with Acute Myeloid Leukemia
Authors: Tasca Silvia , Furlanello Tommaso , Caldin Marco
Published: May 2011

A 2-year-old, female German Shepherd Dog with facial nerve paralysis was diagnosed with acute myelomonocytic leukemia based on clinical, cytologic, and immunologic findings. Proteinuria (urine protein:creatinine ratio = 1.5) occurred in the absence of renal failure. Qualitative assessment of proteinuria by sodium dodecyl sulfate–agarose gel electrophoresis revealed a broad band with a molecular weight of approximately 15 kDa that was compatible with lysozyme (LZM). A diagnosis of tubular proteinuria was made, and a chemical evaluation of LZM in serum and urine samples was performed using a turbidimetric assay. The LZM concentrations were 24.5 mg/l (reference interval: 2.5–8.0 mg/l) and 274.5 mg/l (reference interval: <2 mg/l) in serum and urine, respectively.
Necrotizing Pneumonia and Pleuritis Associated with Extraintestinal Pathogenic Escherichia Coli in a Tiger (Panthera Tigris) Cub
Authors: Carvallo Francisco R. , DebRoy Chitrita , Baeza Esteban , Hinckley Lynn , Gilbert Kelly , Choi Soo Jeon , Risatti Guillermo , Smyth Joan A.
Published: May 2011

Extraintestinal pathogenic Escherichia coli (ExPEC) cause diseases in humans and animals, affecting organs outside the alimentary canal. In recent years, ExPEC have been reported as a cause of fatal pneumonia in dogs, cats, and in a horse. In the current report, a fatal case of pneumonia and pleuritis is described in a 4-week-old tiger ( Panthera tigris) cub associated with ExPEC. The cub was presented with a sudden-onset respiratory illness and died after a few hours. Postmortem examination of the cub revealed an acute necrotizing pneumonia. The alveolar spaces were filled with large numbers of inflammatory cells (predominantly macrophages), edema, fibrin strands, and short bacillary bacteria. Escherichia coli O6:H31 was isolated in pure culture from the affected lung. It carried virulence genes cnf-1, sfa, fim, hlyD, and papG allele III, which are known to be associated with ExPEC strains. No evidence of infection by any other agent was detected. This is the first report, to the authors' knowledge, in which ExPEC has been associated with pneumonia in tigers.
Prevalence and Antigenic Differences Observed between Bovine Viral Diarrhea Virus Subgenotypes Isolated from Cattle in Australia and Feedlots in the Southwestern United States
Authors: Ridpath Julia F. , Fulton Robert W. , Kirkland Peter D. , Neill John D.
Published: May 2011

Bovine viral diarrhea virus (BVDV) is divided into 2 different species within the Pestivirus genus, BVDV type 1 (BVDV-1) and BVDV type 2 (BVDV-2). Further phylogenetic analysis has revealed subgenotype groupings within the 2 types. Thus far, 12 BVDV-1 subgenotypes (a–l) and 2 BVDV-2 subgenotypes (a and b) have been identified. The purpose of the current study was to determine the prevalence of BVDV subgenotypes in the United States and Australia and to determine if there are detectable antigenic differences between the prevalent subgenotypes. To determine prevalence, phylogenetic analysis was performed on 2 blinded panels of isolates consisting of 351 viral isolates provided by the Elizabeth Macarthur Laboratory, New South Wales, and 514 viral isolates provided by Oklahoma State University. Differences were observed in the prevalence of BVDV subgenotypes between the United States (BVDV-1b most prevalent subgenotype) and Australia (BVDV-1c most prevalent subgenotype). To examine antigenic differences between the subgenotypes identified in samples from the United States and Australia, polyclonal antisera was produced in goats by exposing them at 3-week intervals to 2 noncytopathic and 1 cytopathic strain of either BVDV-1a, BVDV-1b, BVDV-1c, BVDV-2a, or Border disease virus (BDV). Virus neutralization (VN) assays were then performed against 3 viruses from each of the 5 subgenotypes. Comparison of VN results suggests that there are antigenic differences between BVDV strains belonging to different subgenotypes. The present study establishes a foundation for further studies examining whether vaccine protection can be improved by basing vaccines on the BVDV subgenotypes prevalent in the region in which the vaccine is to be used.
Diagnostic Accuracy of Methods for Detecting Anaplasma Marginale Infection in Lactating Dairy Cattle of Puerto Rico
Authors: Fosgate Geoffrey T. , Urdaz-Rodríguez José H. , Dunbar Mark D. , Rae D. Owen , Donovan G. Arthur , Melendez Pedro , Dobek Georgina L. , Alleman A. Rick
Published: May 2011

Bovine anaplasmosis (BA) is a hemoparasitic disease of great importance in cattle within the tropical and subtropical regions of the world. Control programs for BA require accurate diagnostic assays but validation can be challenging because the true disease status of all animals is frequently not known with certainty. The objective of this study was to estimate the accuracy of assays for detection of Anaplasma marginale infection in lactating dairy cattle of Puerto Rico using Bayesian methods without a perfect reference test. There were 2,331 cattle with complete diagnostic results sampled from 79 herds, and the prevalence of BA was estimated as 22% (95% probability interval [PI]: 19–25%). The sensitivity (Se) and specificity (Sp) of a major surface protein 5 competitive enzyme-linked immunosorbent assay (MSP-5 cELISA) were estimated as 99% (95% PI: 96–100%) and 89% (95% PI: 87–92%), respectively. The Se and Sp of a quantitative polymerase chain reaction (qPCR) were 67% (95% PI: 60–74%) and 99% (95% PI: 99–100%). The Se and Sp of a card agglutination test were 34% (95% PI: 29–39%) and 99% (95% PI: 99–100%). Area under the receiver-operating characteristic curve for the MSP-5 cELISA was 0.748 (95% PI: 0.71–0.79). The MSP-5 cELISA appears to be the test of choice for screening cattle for subclinical BA based on the high estimated Se, rapidity of results, relative low cost, and ease of standardization.
Rapid Polymerase Chain Reaction Diagnosis of White-Nose Syndrome in Bats
Authors: Lorch Jeffrey M. , Gargas Andrea , Meteyer Carol Uphoff , Berlowski-Zier Brenda M. , Green D. Earl , Shearn-Bochsler Valerie , Thomas Nancy J. , Blehert David S.
Published: May 2011

A newly developed polymerase chain reaction (PCR)-based method to rapidly and specifically detect Geomyces destructans on the wings of infected bats from small quantities (1–2 mg) of tissue is described in the current study (methods for culturing and isolating G. destructans from bat skin are also described). The lower limits of detection for PCR were 5 fg of purified fungal DNA or 100 conidia per 2 mg of wing tissue. By using histology as the standard, the PCR had a diagnostic specificity of 100% and a diagnostic sensitivity of 96%, whereas the diagnostic sensitivity of culture techniques was only 54%. The accuracy and fast turnaround time of PCR provides field biologists with valuable information on infection status more rapidly than traditional methods, and the small amount of tissue required for the test would allow diagnosis of white-nose syndrome in live animals.
Detection of Caprine Herpesvirus 1–Specific Antibodies in Goat Sera Using an Enzyme-Linked Immunosorbent Assay and Serum Neutralization Test
Authors: Marinaro Mariarosaria , Bellacicco Anna Lucia , Tarsitano Elvira , Camero Michele , Colao Valeriana , Tempesta Maria , Buonavoglia Canio
Published: May 2011

An enzyme-linked immunosorbent assay (ELISA) was developed to detect immunoglobulin G (IgG) antibodies directed to whole Caprine herpesvirus 1 (CpHV-1). Sera from 248 goats were obtained from CpHV-1–free and CpHV-1–infected flocks and were subjected to both IgG ELISA and serum neutralization (SN) assays, with the latter considered the gold standard for the diagnosis of CpHV-1 infection. In flocks where CpHV-1 infection was detected, 57 sera were negative by the SN and the ELISA tests and 97 sera were positive with both tests. Thus, although based on biologically different principles, the ELISA was as sensitive as the SN assay in detecting seropositive animals and could be efficiently used as a faster and less expensive alternative to the SN test for the screening of many samples.
Evaluation of Formalin-Fixed Ileum as the Optimum Method to Diagnose Equine Dysautonomia (Grass Sickness) in Simulated Intestinal Biopsies
Authors: Milne Elspeth M. , Pirie R. Scott , McGorum Bruce C. , Shaw Darren J.
Published: May 2011

Equine dysautonomia, or grass sickness, is a frequently fatal disease of unknown etiology, manifested as poor gastrointestinal motility and colic as a result of degenerative changes in the autonomic nervous system. Examination of ileal biopsies collected at laparotomy is currently the best antemortem diagnostic method to distinguish equine dysautonomia from colic cases, which can present with similar signs, but their value has not been previously critically evaluated. Using simulated biopsies collected postmortem from 23 cases of equine dysautonomia and 11 of colic, the sensitivity and specificity of 1-cm long, formalin-fixed ileal biopsies was 100% for the diagnosis of equine dysautonomia. There was therefore no advantage to using larger biopsies or examining jejunum either in addition to or instead of ileal biopsies. Furthermore, although cryostat sections of ileum, 1-cm long, had a sensitivity of 100%, the specificity was only 73%, meaning that 27% of cases would have been misclassified, resulting in unnecessary euthanasia. Increasing the size of the cryostat or examining jejunum in addition to ileum cryostat sections did not significantly improve the specificity. Results of the current study indicate that in diagnostic practice, 1-cm long, formalin-fixed biopsies are likely to be the most suitable for accurate diagnosis, despite the slower turnaround time compared with cryostat sections.
Localized Pleural Mesothelioma Causing Cranial Vena Cava Syndrome in a Dog
Authors: Espino Luciano , Vazquez Sonia , Faílde Daniel , Barreiro Andres , Miño Natalia , Goicoa Ana
Published: May 2011

A 9-year-old female crossbred dog was presented to the Hospital Universitario Veterinario Rof Codina (Universidad de Santiago de Compostela, Lugo, Spain) for acute onset of severe, progressive swelling of the head, neck, and cranial trunk. Survey radiographs and ultrasonography revealed a large, heterogeneous mass in the cranial mediastinum, compressing or growing into a large blood vessel within the cranial mediastinum and displacing the heart dorsocaudally. At postmortem examination, the mass was diagnosed as a large, localized mesothelioma. Localized mesotheliomas are rare neoplasms in dogs but should be considered as a possible differential diagnosis for cranial vena cava syndrome. The anatomic distribution and clinical features of mesothelioma in the present report are similar to other cases in humans.
Plasma and Brain Cholinesterase in Methomyl-Intoxicated Free-Ranging Pigeons (Columba Livia f. Domestica)
Authors: Villar David , Balvin Dubel , Giraldo Carlos , Motas Miguel , Olivera Marta
Published: May 2011

A mortality event caused by exposure to the carbamate insecticide methomyl was diagnosed in several hundred pigeons fed treated corn kernels in a city park. A cholinesterase inhibitor insecticide was initially suspected based on clinical signs and a significant inhibition ( P < 0.05) of brain cholinesterase (ChE) activity compared with normal values for the species. However, brain ChE activity was within the normal range in birds subsequently submitted in an advanced stage of autolysis. Two groups of 10 healthy pigeons were allocated into a control group and an experimental group, which was offered corn samples retrieved from the incident site. Within minutes of ingesting the contaminated corn, the birds became immobile, had transient wing fluttering, and developed profuse salivation immediately followed by death. Plasma ChE activity at death had declined by more than 95% of preexposure levels (0.04 ± 0.02 vs. 1.56 ± 0.23 μmol/min per milliliter). Brain activity in the sagittal brain sections that were immediately frozen after death was inhibited by ≥50% of control birds (13.5 ± 2.2 vs. 27.5 ± 1.8 μmol/min per gram). However, the sagittal sections left for 1.5 days at ambient temperature of 25°C had normal or higher activity, an effect that was attributed to a combination of spontaneous reactivation and dehydration. After incubation of both plasma and brain homogenates for 1 hr at 37°C, ChE activity recovered by 2- and 1.46-fold, respectively. An organophosphorus and carbamate screen conducted by 2 independent laboratories identified and quantified methomyl in treated kernels at 400 ppm. These results indicate that spontaneous reactivation and dehydration can mask previous reductions in ChE activity.
A Retrospective Study of Disease in Captive Kangaroos from Oklahoma State University, 1995–2009
Authors: Stern Adam W.
Published: May 2011

The antemortem biopsy records (5 cases) and necropsy records (34 cases) were reviewed from 39 kangaroo case submissions during a 14-year period to the Oklahoma State University and the Oklahoma Animal Disease Diagnostic Laboratory. The most common types of diseases in the kangaroos, based on necropsy examination, was disease of the alimentary tract (12 cases), pneumonia (6 cases), and disseminated toxoplasmosis (5 cases). The cause of disease was not determined in 6 case submissions. Based on histopathologic findings, clostridial enteritis/colitis was suspected in 2 kangaroos. Coccidian parasites were identified within histologic sections of the small intestine in 1 kangaroo. Pasteurella sp. (1 case) and Bacteroides sp. (1 case) were isolated from the lung in 2 cases of pneumonia. Most (77.3%) of disease in this study in the kangaroos with known ages occurred in animals older than 1 year. Two neoplasms were detected in the antemortem biopsy samples from 1 case.
Association of Vt1C with Verotoxin-Producing Escherichia Coli from Goats and Sheep
Authors: Horcajo Pilar , Domínguez-Bernal Gustavo , De La Fuente Ricardo , Ruiz-Santa-Quiterai José A. , Orden José A.
Published: May 2011

A total of 232 verotoxin 1 (VT1)-positive, VT-producing Escherichia coli (VTEC) strains isolated from goats, sheep, and cattle were analyzed for the presence of the vt1c gene by polymerase chain reaction. One hundred and forty of the 144 (97.2%) caprine strains and 55 of the 63 (87.3%) ovine strains possessed the vt1c gene. In contrast, the gene was not detected in any of the 25 bovine strains. These results show that the vt1c gene is found in caprine VTEC strains and confirm that gene is associated with ovine VTEC strains.
Experimental Infection of Pigs with Border Disease Virus Isolated from Pyrenean Chamois (Rupicapra Pyrenaica)
Authors: Oscar Cabezón , Rosell Rosa , Sibila Marina , Santiago Lavín , Marco Ignasi , Joaquim Segalés
Published: May 2011

Between 2001 and 2007, several outbreaks of disease associated with Border disease virus (BDV) infection were reported in the central Pyrenees (northeast Spain) and were associated with a major reduction in chamois ( Rupicapra pyrenaica) populations. At the same time, wild boars ( Sus scrofa) from the same area were found to be seropositive to this pestivirus, without showing clinical signs. The present study examines the susceptibility of domestic swine and the course of the infection with a BDV strain isolated from naturally infected chamois. Twenty pigs were inoculated with 1 × 107 TCID50 (50% tissue culture infective dose) by oronasal route, and 16 control pigs received Eagles sterile Minimal Essential Medium. Serologic (enzyme-linked immunosorbent assay and virus neutralization test) and reverse transcription polymerase chain reaction assays were performed on serum samples obtained at 0, 3, 7, 10, 14, 21, and 31 days postinoculation (dpi). All infected pigs were viremic from 3 to 14 dpi. After 14 dpi, all infected animals developed an antibody response against the homologous virus. Clinical signs or histologic lesions were not observed in inoculated pigs. The present work demonstrates the susceptibility of domestic swine to a BDV strain of chamois origin.