bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of a Digoxigenin-Labeled RNA Probe to Detect All 24 Serotypes of Bluetongue Virus in Cell Culture
Authors: Brown Corrie C. , Meyer Richard F. , Grubman Marvin J.
Published: May 2011

A digoxigenin-labeled RNA probe, corresponding to the section of the bluetongue virus (BTV) serotype 17 genome coding for nonstructural protein-1 (NS1), was applied to noninfected cell cultures and cell cultures infected with 24 different serotypes of BTV, 2 serotypes of epizootic hemorrhagic disease virus, and African horse sickness virus type 4. The probe hybridized to all cell cultures infected with the various BTV serotypes but did not hybridize to noninfected cell cultures or cell cultures infected with any of the other orbiviruses.
Comparison of Porcine Alveolar Macrophages and CL 2621 for the Detection of Porcine Reproductive and Respiratory Syndrome (PRRS) Virus and Anti-PRRS Antibody
Authors: Bautista Elida M. , Goyal Sagar M. , Yoon In J. , Joo Han S. , Collins James E.
Published: May 2011

The American and European strains of porcine reproductive and respiratory syndrome (PRRS) virus were initially isolated in an established cell line (CL 2621) and porcine alveolar macrophages (PAM), respectively. Subsequent isolation of American strains of this virus in PAM has also been reported. To determine their relative sensitivity for virus isolation, both PAM and CL 2621 cells were inoculated with 98 tissue specimens and 73 serum samples from animals suspected of having PRRS. Four of the 98 tissue samples yielded virus in both cell types, whereas 7 samples were positive only in PAM and 4 samples only in CL 2621. Of the 73 serum samples tested, 18 were positive in PAM of which only 2 were positive in CL 2621. Additionally, 82 isolates obtained initially in CL 2621 were inoculated in PAM cells, and 18 strains isolated originally in PAM were inoculated in CL 2621. Of the 82 CL 2621 isolates, 25 could not be propagated on PAM. Of the 57 that replicated in PAM, as detected by a positive test on indirect fluorescent antibody test, only 28 produced cytopathic effects and 29 did not. Of the 18 PAM isolates, 5 did not grow on CL 2621. Although PAM were relatively more sensitive for virus isolation, their failure to support the growth of certain strains of PRRS virus indicates the existence of variants among PRRS virus strains, and both PAM and CL 2621 should be used for virus isolation from clinical samples. In addition, the sensitivity of these 2 cell types was compared for the detection of fluorescent antibodies to PRRS virus using 179 serum samples from PRRS-infected animals. The results were comparable in both cell systems.
Evaluation of an ELISA for Detection of Ovine Progressive Pneumonia Antibodies using a Recombinant Transmembrane Envelope Protein
Authors: Kwang Jimmy , Keen Jim , Cutlip Randall C. , Littledike E. Travis
Published: May 2011

An enzyme-linked immunosorbent assay (ELISA) was developed using a recombinant protein corresponding to the N'-terminal hydrophilic region of transmembrane glycoprotein (TM) of ovine lentivirus. This assay reproducibly detected antibodies in sera from 207 of 212 ovine progressive pneumonia (OPP) virus-infected sheep, and the recombinant TM ELISA accurately identified 26% (35 vs. 9) more seropositive samples than did the agar gel immunodiffusion test when applied to 100 sera from an infected flock. This assay also yielded no false-positive results in 14 true negative sera. Results of these experiments were further confirmed by the recombinant TM and recombinant p25 Western blot assay. A single recombinant TM antigen, as the coating antigen in ELISA, can be used successfully for the detection of OPP virus-infected animals and can improve the sensitivity and specificity for OPP diagnosis.
Erysipelas in Caged Laying Chickens and Suspected Erysipeloid in Animal Caretakers
Authors: Mutalib Ahmed A. , King John M. , McDonough Patrick L.
Published: May 2011

Erysipelas was diagnosed in 2 succeeding caged layer flocks housed in the same building and was characterized clinically by sudden death. At necropsy, lesions comprised generalized congestion, hemorrhages in the skeletal muscles and visceral organs, and swelling of liver, spleen, and kidney with or without large irregular zones of necrosis. Focal to massive hepatic necrosis was seen histologically with minimal or no inflammatory reaction. Erysipelothrix rhusiopathiae was cultured from internal organs of affected birds in both outbreaks. Medication with penicillin in the feed controlled the mortality, but relapse occurred after cessation of medication. Two attendants who handled the dead birds in this building developed a painful localized infection of the fingers that later spread to the regional lymph node. The infection responded when antibiotic therapy for erysipeloid was initiated.
Hemorrhagic Diathesis Associated with a Hereditary Platelet Disorder in Simmental Cattle
Authors: Steficek Barbara A. , Thomas Jennifer S. , Baker John C. , Bell Thomas G.
Published: May 2011

A severe bleeding disorder in Simmental cattle has been described in widespread locations in the USA and Canada. The clinical findings are consistent with a hemophilia-like disease or, more precisely, a hereditary hemorrhagic diathesis and include spontaneous epistaxis, hematuria, and excessive bleeding associated with trauma or standard management procedures such as tattooing, ear tagging, and castration. A preliminary investigation of this defect showed that blood-platelet numbers and coagulation profiles of affected cattle were normal. Affected animals have a marked dysfunction of platelets (thrombopathy), termed Simmental hereditary thrombopathy. The defect is very similar or identical to that described in the same breed by 2 other laboratories.
Serum Triiodothyronine and Thyroxine Concentrations in Neonatal Llamas
Authors: McLaughlin Bruce G. , Evans C. Norman , Colton Ron L.
Published: May 2011

Total serum triiodothyronine (T3) and thyroxine (T,) concentrations were assayed in neonatal llamas from birth to 90 days of age. T3 concentrations were high at birth and increased from birth to approximately 6 days, then gradually decreased. T4 concentrations were highest at time of birth and decreased gradually over the 90-day period.
Eosinophilic Mediastinitis, Myositis, Pleuritis, and Pneumonia of Cattle Associated with Migration of First-Instar Larvae of Hypoderma Zineatum
Authors: Panciera R. J. , Ewing S. A. , Johnson E. M. , Johnson B. J. , Whitenack D. L.
Published: May 2011

Migrating first-instar larvae of Hypoderma Zineatum are a frequent cause of focal inflammatory lesions in connective tissues of the mediastinum, parietal and visceral pleura, peritoneum, lungs, diaphragm, and other loci. The lesions are characterized grossly by foci of yellowish or greenish gelatinous edema and microscopically by infiltration of the edematous tissue by a dense array of eosinophils. Lesions were recognized during a period of several weeks in late spring; the timing was attributable to events in the life cycle of the fly. The larvae, which were small (≍ 1 × 4.5 mm), transparent, and unobtrusive, were recovered from lesions in 12 of 20 cattle in which careful parasitologic examination was made.
Immunohistochemical Detection of Prion Protein in Sheep with Scrapie
Authors: Miller Janice M. , Jenny Allen L. , Taylor William D. , Marsh Richard F. , Rubenstein Richard , Race Richard E.
Published: May 2011

Prion protein (PrP), which is involved in the pathogenesis of scrapie, occurs in 2 forms. The form extracted from scrapie brain is protease resistant (PrP-res), whereas PrP from normal brain is protease sensitive (PrP-sen). This study examined whether PrP-res could be detected in brains of sheep with scrapie by immunohistochemistry (IHC). A suitable IHC procedure was developed using brain tissue from hamsters that had been inoculated with the transmissible mink encephalopathy agent. Tissue samples were fixed in PLP (periodate, lysine, paraformaldehyde) that contained paraformaldehyde at a concentration of 0.125%. Before application of the IHC technique, tissue sections were deparaffinized and treated with formic acid to simultaneously enhance PrP-res immunoreactivity and degrade PrP-sen. Primary antibody was obtained from a rabbit immunized to PrP-res extracted from brains of mice with experimentally induced scrapie. Brains from 21 sheep with histopathologically confirmed scrapie were examined by IHC. In all 21 brains, PrP-res was widely distributed throughout the brain stem. Staining was particularly intense in neuronal cell bodies and around blood vessels. The IHC technique successfully detected PrP-res in brain samples that had been frozen or that were severely autolyzed before fixation in PLP. Brains from 11 scrapie-suspect sheep that were not considered histologically positive were also examined by IHC. PrP-res was found in 4 of these brains. Sections of brains from 14 clinically normal sheep did not have detectable PrP-res. Results of this study indicate that IHC detection of PrP-res is equivalent, and perhaps superior, to histopathology for the diagnosis of scrapie in sheep. Furthermore, IHC is applicable to tissues that have autolytic changes or processing artifacts that prevent satisfactory histopathologic evaluation for lesions of scrapie.
Characteristic Differences in Reverse Transcription-Polymerase Chain Reaction Products of Ovine, Bovine, and Human Respiratory Syncytial Viruses
Authors: Oberst Richard D. , Hays Michael P. , Evermann Jim F. , Kelling Clayton L.
Published: May 2011

In reverse transcription-polymerase chain reactions (RT-PCR) and DNA hybridizations using primers and an oligonucleotide probe to the fusion (F) protein mRNA of bovine respiratory syncytial virus (BRSV), all the BRSV isolates and a goat isolate could be distinguished from prototype isolates of human respiratory syncytial viruses (HRSV) and ovine (sheep and bighorn sheep) respiratory syncytial viruses (RSV). However, RT-PCR amplifications with primers to sequences of the HRSV F protein mRNA resulted in amplified products of ≍ 243 bp if mRNA templates of subgroup A HRSV strains were present and slightly larger amplified products with subgroup B HRSV strains. No amplified products were observed in HRSV-primed RT-PCR with BRSV or goat or ovine RSV mRNA templates. Although the ovine RSV isolates were antigenically cross-reactive with the goat RSV, HRSV and BRSV isolates, they were not amplified with either HRSV- or BRSV-specific primers in RT-PCR. These results confirm previous immunological comparisons suggesting that some ovine RSV isolates should be considered as distinct respiratory syncytial viruses.
Evaluation of a Commercial Competitive ELISA Test Kit for the Detection of Group-Specific Antibodies to Bluetongue Virus
Authors: Afshar A. , Trotter H. C. , Dulac G. C. , Reddington J. J.
Published: May 2011

The performance of a competitive (c) enzyme-linked immunosorbent assay (ELISA) test kit, Blueplate Special@ (BPS), commercially produced by DiagXotics (Wilton, CT) for detection of group-specific antibodies to bluetongue virus (BTV) was compared with that of an internationally endorsed cELISA-I. A total of 1,026 serum samples were tested in this study: 133 samples from 23 calves and 3 sheep experimentally infected with South African isolates of 19 BTV serotypes and US isolates of 5 BTV serotypes and 7 calves infected with US isolates of 2 epizootic haemorrhagic disease of deer virus (EHDV); 102 paired sera from cattle, sheep, and goats experimentally infected with the Australian isolates of BTV, EHDV, and Palyam virus; 229 bovine and ovine samples of Canadian origin (BTV free); and 562 bovine and ovine field samples from the USA and Barbados (BTV endemic). Seroconversion was demonstrable by the BPS cELISA 10 days postinfection in all experimental animals inoculated with BTV, with the exception of 4 calves in which there was a delay of 10–20 days. Similar to the cELISA-I, none of the sera from calves inoculated with US and Australian isolates of EHDV and Palyam viruses cross-reacted with the BTV antigen in the BPS cELISA. The total agreement between the two assays for all the total bovine and ovine field sera was 98.1%. The overall results substantiate the usefulness of the BPS cELISA test kit for monitoring animal sera for group-specific antibodies to BTV. The slightly lower analytical sensitivity associated with the detection of antibody during early phase of infection in some animals would not be significant in the context of herd testing or any regulatory program.
Diagnostic Compatibility of a Thymidine Kinase, Inverted Repeat, gI, and gpX Modified Live Gene-Deleted PRV Vaccine with Three Differential ELISAs
Authors: Swenson Sabrina L. , McMillen Janis , Hill Howard T.
Published: May 2011

The differential pseudorabies virus (PRV) vaccines currently in use in the USA have deletions of the genes coding for the glycoprotein I (gl) and/or glycoprotein X (gpX). The absence of gI and/or gpX allow for the serologic differentiation of vaccinated swine from PRV-infected swine using differential enzyme-linked immunosorbent assays (ELISAs). A newly developed pseudorabies vaccine virus has 4 deletions of the viral genome: the genes coding for gI, gpX, and thymidine kinase and a portion of the repeat region to attenuate the virus. The purpose of this work was to evaluate the diagnostic compatibility of the gI/gpX gene-deleted vaccine with 3 differential vaccines and 3 differential ELISAs currently in use. Pigs vaccinated 3 times with the gI/gpX gene-deleted vaccine remained seronegative on the 3 differential ELISAs tested. Pigs previously vaccinated with either a gI or gpX gene-deleted vaccine and then vaccinated with the gI/gpX gene-deleted vaccine remained seronegative on the respective gI or gpX differential assay.
Development and Evaluation of a Mixed-Antigen ELISA for Serodiagnosis of Actinobacillus Pleuropneumoniae Serotypes 1, 5, and 7 Infections in Commercial Swine Herds
Authors: Bossé Janine T. , Friendship Robert , Rosendal Soren , Fenwick Brad W.
Published: May 2011

A mixed-antigen enzyme-linked immunosorbent assay (ELISA) containing serotype-specific polysaccharide antigens from serotypes 1, 5, and 7 of Actinobacillus pleuropneumoniae was developed. This ELISA was evaluated using sera from experimentally infected pigs. With a negative cutoff value of 0.250 (optical density at 405 nm), sensitivity and specificity were determined to be 96% and 99.5%, respectively. The ELISA was further evaluated using sera from commercial swine. Sows and suckling piglets were found to be the best age groups for detection of positive reactors to A. pleuropneumoniae. The mixed-antigen ELISA could be used in a herd health monitoring program for detection of A. pleuropneumoniae infections.
Streptococcus Suis Infection in Swine: A Retrospective Study of 256 Cases. Part I. Epidemiologic Factors and Antibiotic Susceptibility Patterns
Authors: Reams Rachel Y. , Glickman Lawrence T. , Harrington Daniel D. , Bowersock Terry L. , Thacker H. Leon
Published: May 2011

A retrospective study of 256 cases of naturally acquired Streptococcus suis infections in swine submitted to the Indiana Animal Disease Diagnostic Laboratory from 1985 to 1989 was performed to determine the epidemiologic factors and antibiotic susceptibility patterns associated with S. suis serotypes 1–8 and 1/2. A standardized computer form was used to record the history, signalment, and clinical signs obtained from the records of selected cases and the microscopic lesions identified after review of the histopathology slides for each case. A computer statistics package (SAP) was used to evaluate the data. Although the number of recovered S. suis isolates increased in the fall and winter months, most serotypes were readily isolated throughout the year; only serotypes 1, 4, 7, and 1/2 increased in frequency of isolation in the fall, winter, and spring months. The majority (6 1.1%) of infected pigs in this study were < 12 weeks of age. More than 75% of pigs infected with serotypes 1, 6, 7, and 1/2 were < 12 weeks of age. There was extensive overlap in the age distributions for pigs with each serotype, and statistically significant differences for most serotypes were not observed. Fifty percent of pigs infected with S. suis serotypes 1 and 1/2 were 3–10 weeks of age, 50% of pigs infected with serotype 2 were 6–14 weeks of age, and 50% of pigs infected with serotypes 3, 4, 5, 7, and 8 were 2–16 weeks of age. Isolates of S. suis were not uniformly susceptible to penicillin, and a large percentage of isolates were resistant to many antibiotics in common usage. The results of this study indicated that the various serotypes of S. suis could not be readily separated based on antibiograms, epidemiologic factors (herd size, breed, etc.), or geographic location.
Genus-Specific Detection of Salmonellae using the Polymerase Chain Reaction (PCR)
Authors: Cohen Noah D. , Neibergs Holly L. , McGruder Edward D. , Whitford Howard W. , Behle Robert W. , Ray Pamela M. , Hargis B. M.
Published: May 2011

Oligonucleotide primers for the polymerase chain reaction (PCR) that enable genus-specific detection of members of the genus Salmonella were developed. The primers amplify a 496-bp genetic sequence of members of the genus Salmonella. Amplification of DNA extracted from all other genera of the family Enterobacteriaceae and various other gram-positive aerobic and anaerobic bacteria yielded negative results. Applications of the PCR using these genus-specific primers are discussed.
Application of Polymerase Chain Reaction for the Correlation of Salmonella Serovars Recovered from Greyhound Feces with Their Diet
Authors: Stone Gregory G. , Chengappa M. M. , Oberst Richard D. , Gabbert Nathan H. , McVey Scott , Hennessy Kristina J. , Muenzenberger Margaret , Staats Jacque
Published: May 2011

The polymerase chain reaction was employed to correlate Salmonella serovars isolated from fecal material of greyhounds suffering from gastroenteritis with those isolated from the diet fed to the greyhounds prior to onset of diarrhea. Kennels around the Abilene, Kansas, area were contacted and supplied with materials needed to collect a portion of the diet each day. With t e onset of diarrhea, the kennels were instructed to ship the fecal material and diet from the previous 10 days to the laboratory for testing. Forty-one fecal samples and corresponding diets were screened for Salmonella, Clostridium perfringens, Campylobacterjejuni, Staphylococcus aureus, Staphylococcus intermedius, and pathogenic (piliated) Escherichia coli by direct culture using standard procedures. The fecal material was also screened for coronavirus and parvovirus using electron microscopy. Thirty-five “normal” fecal samples were screened for all of the above mentioned microorganisms as a control. In addition, the fecal material was screened for E. coli verotoxins I and II and clostridial enterotoxins. A total of 61 Salmonella isolates were recovered from the 41 samples of feces and diet submitted for testing; 31 were recovered from the feces and 30 from the diet. Four Salmonella isolates were recovered from the normal fecal samples. Results obtained by PCR, plasmid profiles, antigenic analysis, and antibiogram profiles indicated that 16 of the 31 isolates recovered from the fecal material were the same strain as that recovered from the diet.
Gross and Microscopic Lesions of 230 Kansas Greyhounds
Authors: Schoning Polly , Cowan Laine A.
Published: May 2011

Two hundred thirty greyhounds from Kansas were submitted to Kansas State University for necropsy to identify and document their diseases. Sex distribution was 124 females (I spayed) and 106 males (1 castrated). Age range was 5 days-150 months. The mean body, heart, and liver weights for adult dogs (> 12 months) were 30 kg, 367 g, and 1,019 g, respectively. Greyhounds were divided into six groups by histories: skeletal injuries, 24 (10%); nonskeletal injuries, 23 (10%); old age (x = 89 months), 25 (11%); poor performers, 83 (36%); sick, 68 (30%); and no history, 7 (3%). Gross lesions by system were as follows: skin, 85 (37%); lung, 13 (6%); skeletal, 36 (16%); gastrointestinal, 55 (23.9%); central nervous system, 3 (1%); thyroid, 5 (2%); cardiovascular and hemopoietic, 36 (16%); spleen, 30 (13%); male reproductive, 13 (12%); kidney, 9 (4%); liver, 7 (3%); and no gross lesions, 23 (10%). Correlation of the histories to the gross lesions showed that poor performers had the largest number with no gross lesions, the skeletal injury and sick groups had more gastrointestinal changes, and the nonskeletal injury and sick groups had most of the male reproductive abnormalities. Microscopic lesions of the 6 tissues examined were as follows: lung, 21 (9%); small intestine, 15 (6.5%); brain, 6 (3%); heart, 9 (4%); kidney, 57 (24.3%); liver, 26 (11.3%); and no microscopic lesions, 131 (57%). Correlation of the histories to microscopic lesions showed that poor performers had the largest number with no microscopic lesions, the sick group had the largest number of lesions of the lungs, small intestine, brain, heart, kidney, and liver, however, the old group had renal lesions almost as often as the sick group.
Diagnoses in 1,784 Ovine Abortions and Stillbirths
Authors: Kirkbride Clyde A.
Published: May 2011

Of 1,799 accessions of ovine abortions and stillbirths by the South Dakota Animal Disease Research and Diagnostic Laboratory during a IO-year period, 1,784 were suitable for examination. Etiologic diagnoses were made in 786 (44%). Infectious agents were found responsible in 702 accessions (39%), and noninfectious causes were involved in 84 (5%). No diagnosis was made in 998 accessions (56%), 61 of which were mummified fetuses. Lesions, most of which indicated the presence of an infectious agent, were found in 274 cases (15%) in which no agent could be identified. Together, Toxoplasma gondii, Campylobacter sp., and Chlamydia psittaci caused −25% of all abortions and stillbirths examined.
Chemical Study of the Hepatotoxins from Microcystis Aeruginosa Collected in California
Authors: DeVries S. E. , Namikoshi M. , Galey F. D. , Merritt J. E. , Rinehart K. L. , Beasley V. R.
Published: May 2011

Four cyclic peptide toxins were purified and quantified from the aqueous extract of algal cell material utilizing high performance liquid chromatography, thin layer chromatography, and fast atom bombardment mass spectrometry. The cyclic peptide toxins appear to be similar structurally to hepatotoxins Iron previously identified blooms of the blue-green alga Microcystis aeruginosa.
Immunohistochemical Study to Demonstrate Sarcocystis Neurona in Equine Protozoal Myeloencephalitis
Authors: Hamir A. N. , Moser G. , Galligan D. T. , Davis S. W. , Granstrom D. E. , Dubey J. P.
Published: May 2011

A 5-year (1985–1989) retrospective immunohistochemical study was conducted using an avidinbiotin complex (ABC) immunoperoxidase method to demonstrate Sarcocystis neurona in histologically suspect cases of equine protozoal myeloencephalitis (EPM). Primary antibodies against S. neurona and S. cruzi were utilized for the ABC technique. The findings were compared with those from cases in which the organisms were detected by examination of hematoxylin and eosin (HE)-stained neuronal sections. HE-stained sections detected the presence of the organisms in 20% of the suspect cases; whereas the ABC technique confirmed the presence of S. neurona in 51% and 67% of the cases by S. neurona and S. cruzi antibodies, respectively. A review of clinical case histories showed that 21/47 (45%) of the EPM horses with parasites in the tissue sections had prior treatment with antiprotozoal drugs and/or steroids. Using the test results of S. neurona and S. cruzi as a standard reference, HE test sensitivity based on examination of up to 30 neuronal sections per case was only 25%, and test specificity was 91%.
Examination of Frozen Cross Sections of Cervical Spinal Intersegments in Nine Horses with Cervical Vertebral Malformation: Lesions Associated with Spinal Cord Compression
Authors: Trostle S. S. , Dubielzig R. R. , Beck K. A.
Published: May 2011

Nine horses with clinical and radiographic findings of cervical vertebral malformation that were necropsied and examined using frozen cervical spinal cord cross sections were reviewed. Only cases with actual distortion of the spinal cord due to compression were selected. The goal of the study was to determine the morphologic features responsible for narrowing of the spinal canal and compression of the spinal cord. In individual cases, bony changes are associated with osteochondrosis and osteomyelitis of the dorsal articular facets and osteosclerosis of the dorsal cervical lamina. Soft tissue pathology associated with spinal cord compression included ligamentum flavum hypertrophy, joint capsule swelling and hypertrophy, and synovial cysts. In most cases, a combination of abnormalities was found in horses with spinal cord compression.
ELISA Method for Detection of Influenza A Infection in Swine
Authors: Lee Brian W. , Bey Russell F. , Baarsch Mary J. , Simonson Randy R.
Published: May 2011

An antigen-capture enzyme-linked immunosorbent assay (ELISA) was developed to monitor virus shedding associated with experimental infection with a field strain of swine influenza in pigs. The assay consisted of a monoclonal anti-nucleoprotein capture antibody and a biotinylated rabbit anti-influenza A (H1N1) sandwich antibody. The antigen-capture system was capable of detecting as little as 1 ng/ml purified virus. The ELISA system surpassed egg cultivation procedures in the detection of low levels of shedding virus. Egg cultivation procedures indicated that most viral shedding had ceased by day 10 postinfection. In contrast, antigen-capture ELISA still showed an ongoing presence of viral antigen. A virus-capture ELISA, using this capture-sandwich antibody system, is equivalent in sensitivity to conventional egg inoculation procedures for the detection of the early phases of virus shedding. The automative potential of an ELISA-based system coupled with a substantially reduced assay time requirement give this virus-capture ELISA a distinct advantage over other cell culture or egg-based diagnostic techniques.
PCR Detection of Group A Bovine Rotaviruses in Feces
Authors: Chinsangaram Jarasvech , Akita Geoffrey Y. , Castro Anthony E. , Osburn Bennie I.
Published: May 2011

A polymerase chain reaction (PCR) protocol has been developed for identification of bovine group A rotavirus infection in feces. Primers (20mers) complementary to 3′ ends of double-stranded RNA genome segment 6 of bovine rotavirus NCDV strain were synthesized and used in PCR. Bovine rotavirus RNA from infected cell culture was employed to optimize the PCR protocol. Rotavirus-negative fecal samples were spiked with known quantities of bovine rotavirus, and the sensitivity of the PCR assay was determined. Fecal samples were extracted with phenol and treated to eliminate unidentified PCR inhibitor(s) in feces, and PCR was performed. PCR products were either visualized on ethidium bromide-stained agarose gels or detected by chemiluminescent hybridization. The sensitivity of the assay was 6 × 104 viral particles/ml of feces with ethidium bromide-stained agarose gel visualization or 6 × 102 viral particles/ml of feces with chemiluminescent hybridization. The PCR assay was applied to 18 fecal specimens from clinical cases. All 16 clinical samples that were positive for rotavirus by enzyme-linked immunosorbent assay (ELISA) or by ELISA and electron microscopy (EM) were positive by PCR. The 2 samples that were rotavirus negative by ELISA or by ELISA and EM were also negative on PCR analysis.
Microdilution Antimicrobial Susceptibilities of Selected Gram-Negative Veterinary Bacterial Isolates
Authors: Burrows G. E. , Morton R. J. , Fales W. H.
Published: May 2011

Gram-negative bacterial isolates (63 5) obtained from routine submissions to the Oklahoma Animal Disease Diagnostic Laboratory during 1983–1987 were tested for antimicrobial susceptibility. Minimal inhibitory concentrations (MICs) were determined for the following antimicrobials using commercially prepared microdilution assay materials: ampicillin, cephalothin, chloramphenicol, erythromycin, gentamicin, kanamycin, oxytetracycline, penicillin G, spectinomycin, sulfachlorpyridazine, sulfadimethoxine, and tylosin. Results for isolates from cattle, dogs, horses, and pigs are presented. In only a few instances were differences in MICs apparent among bacterial isolates from different tissues. Aminocyclitol MICs for equine uterine isolates of Klebsiella pneumoniae differed from MICs for isolates from other tissues, and ampicillin, kanamycin, and spectinomycin MICs for bovine fecal isolates of Escherichia coli differed from MICs for isolates obtained from other tissues. In several instances, bimodal distribution of susceptibilities was apparent for ampicillin, kanamycin, and/or oxytetracycline. There was also a bimodal distribution pattern for erythromycin against Pasteurella haemolytica of bovine origin.
Systemic Pasteurella Haemolytica Infection as a Rare Sequel to Avirulent Live Pasteurella Haemolytica Vaccination in Cattle
Authors: Zeman David , Neiger Regg , Nietfield Jerome , Miskimins Dale , Libal Melissa , Johnson Darrel , Janke Bruce , Gates Connie , Forbes Keith
Published: May 2011

Eleven cases of systemic Pasteurella haemolytica infection in cattle were identified from routine diagnostic laboratory submissions during the falls of 1988, 1989, and 1991. All cases came with a history of recent vaccination with an avirulent live culture P. haemolytica product. Nine of 11 cases involved cattle vaccinated between 2 and 18 days previously with this product. Ten of 11 cases involved 182-227-kg beef calves that were vaccinated between September and November during routine processing for entry into feedlots. The morbidity and mortality was generally low.

The major pathologic findings included meningitis, injection site abscessation and/or cellulitis, and polyarthritis. Systemic infection was indicated in all cases by the isolation of P. haemolytica from 2 or more organs or distinct anatomical sites. In 6 cases, the vaccine injection site was cultured, and in all 6 cases, P. haemolytica was isolated.

Three separate P. haemolytica isolates from 2 cases were further studied by restriction enzyme analysis (REA). These isolates were from tissues with suppurative inflammation, including the brain, joint, and injection site. The REA patterns of each of these 3 isolates were identical to the REA pattern of the vaccine masterseed, which strongly suggested that the organisms causing systemic infection were the same as the organism used to produce the vaccine. Because the overall incidence was quite low, other factors, such as stress, probably played a major role in the expression of this syndrome.
Detection of Serum Antibody Responses in Cattle with Natural or Experimental Neospora Infections
Authors: Conrad Patricia A. , Sverlow Karen , Anderson Mark , Rowe Joan , BonDurant Robert , Tuter Gwen , Breitmeyer Richard , Palmer Chuck , Thurmond Mark , Ardans Alex , Dubey J. P. , Duhamel Gerald , Barr Bradd
Published: May 2011

Parasite-specific antibody responses were detected using an indirect fluorescent antibody (IFA) test in cattle that were naturally or experimentally infected with Neospora parasites. The test was developed using Neospora tachyzoites isolated from an aborted bovine fetus and grown in bovine cell cultures (isolate BPA1). In all cases, infections were confirmed by the identification of Neospora tachyzoites and/or bradyzoite cysts in fetal or calf tissues using an immunoperoxidase test procedure. Fifty-five naturally infected cows that aborted Neospora-infected fetuses had titers of 320-5,120 at the time of abortion. The titer of 6 cows that were serologically monitored over a prolonged period decreased to 160–640 within 150 days after they aborted infected fetuses. Two of the cows showed an increase in their Neospora titers during their subsequent pregnancy, and they gave birth to congenitally infected calves that had precolostral titers of 10,240-20,480. Postcolostral titers of these calves and of 4 other calves with congenital Neospora infections were all 25,120, whereas calves with no detectable parasites had titers ≤ 160. Two pregnant heifers that were experimentally infected with the BPA1 isolate at approximately 120 days gestation seroconverted to Neospora antigens within 9 days and developed peak titers of 5,120 and 20,480 within 32 days of infection. The fetus taken by caesarian section 32 days postinfection from 1 heifer and the full-term calf born to the other had Neospora titers of 640 and 10,240, respectively. Nine cows that aborted uninfected fetuses and 61 adult cattle maintained under pasture or feedlot conditions, where risk of exposure to Neospora was considered to be low, had titers ≤ 320. Some of the feedlot cattle tested had serologic reactivity that was restricted to antigens at the apical end of both Neospora and Toxoplasma gondii tachyzoites. This type of reactivity, which may result from serologic cross-reactivity between conserved apical complex antigens of closely related sporozoan parasites, differed from the whole parasite fluorescence that was observed with sera from Neospora-infected animals. The significance of these results and the potential application of the IFA test for the diagnosis of Neospora infections in cattle are discussed.
Detection of Neospora Caninum in Tissue Sections Using a Murine Monoclonal Antibody
Authors: Cole R. A. , Lindsay D. S. , Dubey J. P. , Blagburn B. L.
Published: May 2011

A murine monoclonal antibody (MAb 6G7), isotype IgG2a, produced against tachyzoites of Neospora caninum (isolate NC-l) reacted specifically with tachyzoites of N. caninum in an indirect immunofluorescent antibody test. MAb 6G7 did not react with tachyzoites of Toxoplasma gondii, sporozoites of Isospora suis, Eimeria bovis, or E. tenella, or merozoites of E. bovis in the indirect immunofluorescent antibody test. MAb 6G7 reacted positively with both tachyzoites and bradyzoites of N. caninum in an avidin-biotin peroxidase complex immunohistochemical test on formalin-fixed paraffin-embedded tissues. No reaction was observed with the following: tachyzoites and bradyzoites of T. gondii, T. gondii-like parasites, or Hammondia hammondi; bradyzoites of Frenkelia microti; schizonts and merozoites of Sarcocystis-like organisms; schizonts, sarcocysts, and oocysts/sporocysts of S. cruzi; schizonts and merozoites of S. canis; schizonts of S. hirsuta, S. tenella, and S. capracanis; merozoites of S. neurona and S. neurona-like organisms, E. bovis, or Haemoproteus sp.; bradyzoites and merozoites of S. montanaensis; bradyzoites of S. odocoileocanis, S. cruzi, and S. tenella; meronts, sexual stages, and caryocysts of Caryospora sp. and C. bigenetica; micromerozoites, macromerozoites, and schizonts of Hepatozoon canis; sporozoites, sexual stages, and oocysts of Cryptosporidium parvum and C. baileyi; trophozoites of Monocystis Zumbrici, Tritrichomonas foetus, and Balantidium coli; tissue cysts and bradyzoites of Besnoitia sp. and B. jellisoni; amastigotes of Leishmania sp.; and trophic theronts of Ichthyopthirius multifilis. MAb 6G7 reacted with tachyzoites and bradyzoites of N. caninum in natural and experimental infections in dogs, cattle, mice, rats, sheep, and goats, indicating that host origin of the tissues did not affect the performance of the test.
Neuroaxonal Dystrophy in a Group of Related Cats
Authors: Carmichael K. Paige , Howerth Elizabeth W. , Oliver John E. , Klappenbach Kurt
Published: May 2011

A syndrome resembling previously described feline hereditary neuroaxonal dystrophy (FHND) was diagnosed in a litter of cats. The disorder was characterized by a sudden onset of hind limb ataxia that slowly progressed to hind limb paresis and paralysis. The cats were between 6 and 9 months old when clinical signs were first noted. Histologically, there was marked ballooning of axonal processes, with spheroid formation and vacuolation in specific regions of the brain and spinal cord. Some dystrophic axons contained a central periodic acid-Schiff (PAS)-positive core. Neuronal loss and gliosis were seen in certain brain stem nuclei, spinal cord nuclei, and the cerebellum. Ultrastructurally, there was hypomyelination and dysmyelination of affected axons. The PAS-positive core in dystrophic axons corresponded ultrastructurally with accumulations of electrondense, flocculent, amorphous material. In addition, these axons contained membrane-bound osmiophilic bodies and large nonmembrane-bound vacuoles. The syndrome in this report differs from the previously described FHND in that no inner ear involvement was seen and onset of clinical signs occurred at a later age. In addition, although some of the affected cats did have diluted coat colors, abnormal coat color was not always associated with clinical disease. This disease is similar to juvenile neuroaxonal dystrophy in children and to neuroaxonal dystrophies described in horses, dogs, cattle, and sheep.
Molecular Characterization of Attenuated Vaccine Strains of Transmissible Gastroenteritis Virus
Authors: Register Karen B. , Wesley Ronald D.
Published: May 2011

Previous studies in our laboratory demonstrated that 2 attenuated strains of transmissible gastroenteritis virus (TGEV) contain deletions affecting messenger (m) RNAs 2, 3, or 4. In this report, we have compared mRNAs of four modified-live virus vaccines for TGEV with the virulent Miller PP3 isolate to determine whether any transcriptional patterns are shared among attenuated strains. Using northern blot analysis, all vaccine viruses expressed mRNAs indistinguishable in size from those of Miller PP3. However, using S1 nuclease protection experiments, alterations in the regions of the genome from which mRNAs 2 and 3 are transcribed were detected in 2 of the vaccine strains. When genomic cDNA fragments derived from the coding region for mRNA 2 were sequenced, a 6-nucleotide deletion, also found in the attenuated strain Purdue- 115, was discovered. The product of mRNA 2, a spike glycoprotein, was visualized by western blotting for each vaccine strain, and no profound differences in mobility were detected relative to Miller PP3. Alterations in the region of the genome from which mRNA 3 is transcribed appear to be identical or very similar to sequence alterations already described in this region for Purdue- 115, one of which is likely to alter the polypeptide product of mRNA 3. Insertions or deletions in mRNAs 2 or 3 may contribute to attenuation but are not a prerequisite for this phenotype. The S1 nuclease protection analysis is a sensitive tool for differentiating particular strains of TGEV.
Expression of Aleutian Mink Disease Parvovirus Capsid Proteins in a Baculovirus Expression System for Potential Diagnostic Use
Authors: Wu Wai-Hong , Bloom Marshall E. , Berry Bradley D. , McGinley Michael J. , Platt Kenneth B.
Published: May 2011

A 2.3-kb cDNA clone encoding Aleutian mink disease parvovirus (ADV) structural proteins VP1 and VP2 was inserted into the polyhedron gene of Autographa calijbmica nuclear polyhedrosis virus (AcNPV) and expressed by the recombinant virus, AcADV-1, in Spodoptera frugiperda-9 cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western immunoblot analysis (WIA) indicated that synthesis of both VP1 and VP2 was being directed by AcADV-1. Fluorescence microscopic examination of AcADV-1 -infected S. frugiperda-9 cells indicated that the recombinant protein was present within the nucleus of the cells, and electron microscopic examination of these cells revealed the presence of small particles 23–25 nm in diameter. Structures resembling empty ADV capsids could be purified on CsCl density gradients, thus indicating that the ADV proteins were self-assembling. The antigenicity of recombinant VP1 and VP2 was evaluated by WIA. Sera collected from 16 mink prior to infection with ADV did not react with VP1 and VP2. Ten sera collected from mink with counter current immunoelectrophoresis (CIE) titers greater than 4 (log2) reacted with VP1 and VP2 in WIA. Two of 6 sera with CIE titers of 4 and 1 of 14 sera with CIE titers <4 reacted with the recombinant proteins. These results suggest that baculovirus recombinant ADV capsid proteins may be useful as diagnostic antigens.
Diagnosis of Eastern Equine Encephalomyelitis Virus Infection in Horses by Immunoglobulin M and G Capture Enzyme-Linked Immunosorbent Assay
Authors: Sahu Sudhir P. , Alstad Arnold D. , Pedersen Douglas D. , Pearson James E.
Published: May 2011

Immunoglobulin M (IgM) and G (IgG) capture enzyme-linked immunosorbent assays (ELISAs) were used as possible adjuncts to hemagglutination inhibition (HI) and virus neutralization (VN) tests to differentiate between reaction to recent exposure to eastern equine encephalomyelitis (EEE) virus and those due to prior vaccination. Serum samples were evaluated by the IgM-capture ELISA, and the results were compared with those of HI and VN tests. Of 381 serum samples, 51% (195 samples) were positive by HI test (≥1:40) and 54% (205 samples) were positive by VN test (≥ 1:10), but only 35% (132 samples) were positive by IgM-capture ELISA (≥ 1:100). With only a few exceptions, the sera with IgG ELISA titers had a VN titer of ≥1: 100. When EEE virus isolation and serology were compared, the EEE cases were divided into three categories: 1) peracute cases- the serum was negative for EEE IgM and IgG by the ELISA, negative for VN antibody, but HI antibody positive; 2) acute cases-IgM and HI antibody positive but negative for IgG and VN antibody; and 3) transitional cases–positive for IgM and IgG antibodies, HI titers of 1:40–1:160, and VN titers of ≥ 1: 100. IgM antibodies of EEE virus were monospecific and did not cross-react with western or Venezuelan equine encephalomyelitis viral antigens by the ELISA. Isolation of EEE virus from tissue or detection of IgM antibodies to EEE virus in a single serum sample is evidence of recent EEE infection and differentiated serum from previously vaccinated horses. However, the presence of IgM in the absence of virus isolation could also be due to a recent viral vaccination.
Evaluation of PCR for Diagnosis of Bovine Viral Diarrhea Virus in Tissue Homogenates
Authors: Schmitt Beverly J. , Lopez Osvaldo J. , Ridpath Julia F. , Galeota-Wheeler Judith , Osorio Fernando A.
Published: May 2011

Tissue homogenates from 60 specimens submitted to the Veterinary Diagnostic Center were evaluated by polymerase chain reaction (PCR) for detection of bovine viral diarrhea virus (BVDV). Conventional virus isolation procedures showed the specimens contained BVDV. The BVDV RNA was extracted from the homogenates and subjected to a reverse transcription reaction followed by PCR amplification. The PCR product was blotted onto a nylon membrane and hybridized with a 30-base pair oligonucleotide probe labeled with 32P. One set of PCR primers detected BVDV in 46/60 (77%) of the tissue homogenates. An additional set of primers was used to detect 10/11 samples that had escaped detection with the first set of primers. The results indicate that BVDV can be detected by PCR directly out of tissue homogenates generated in a diagnostic setting.
Immunohistochemical Identification of Renibacterium Salmoninarum by Monoclonal Antibodies in Paraffin-Embedded Tissues of Atlantic Salmon (Salmo Salar L.), using Paired Immunoenzyme and Paired Immunofluorescence Techniques
Authors: Evensen Øystein , Dale Ole Bendik , Nilsen Arve
Published: May 2011

Renibacterium salmoninarum was identified in situ by immunoenzymatic and immunofluorescence techniques in paraffin-embedded tissue specimens collected during a natural outbreak of bacterial kidney disease (BKD) and from an experimental infection in Atlantic salmon ( Salmo salar L.). Monoclonal antibodies (MAbs) 4D3 and 2G5 were used in this study, both specific for the 57–58-kD outer membrane protein (p57) of the bacterium. Both MAbs revealed positive staining in ethanol-fixed tissue specimens, but only the epitope identified by MAb 4D3 was formalin resistant. Pretreatment with trypsin did not reestablish the antigenicity for the epitope identified by Mab 2G5. Paired immunoenzymatic staining for identification of the bacterium in sequential incubation steps on ethanol-fixed tissue specimens using an avidin-biotin-peroxidase system was obtained after serial dilution of the Mab (2G5) or the chromagen, amino ethyl carbazole, in the first sequence. Paired immunofluorescence staining with well-balanced color mixing was easily obtained on ethanol-fixed tissue specimens using sequential incubations. Single exposures gave blue (aminomethyl coumarin acetic acid) and green (fluorescein isothiocyanate) fluorescence for MAbs 2G5 and biotinylated 4D3, respectively. Color mixing was revealed as a turquoise staining. Studies on method sensitivity was performed by incorporating a known amount of a protein preparation of p57 into an inert matrix, creating an artificial test substrate. Antigen detection sensitivity based on an immunoenzymatic alkaline phosphatase method and Fast Red as chromagen and evaluated by an image-analysis system creating a relative grey-level score showed an 8-fold higher detection sensitivity on ethanol-fixed as compared with formalin-fixed specimens. Monoclonal antibodies specific for the outer membrane protein of Renibacterium salmoninarum are well suited for use in immunohistochemical techniques for in situ identification of the bacterium in paraffin-embedded tissue specimens. Serial evaluation of paired stained sections with a confirmatory diagnosis of BKD being dependent on both MAbs giving a positive reaction would increase the specificity of the test. Simultaneous identification of the two epitopes of p57 on the same section was dependent on ethanol-fixed tissue specimens.
Experimental Brucella Abortus Strain 19 Arthritis in Young Cattle
Authors: Johnson B. , Mosier D. A. , Morton R. J. , Confer A. W.
Published: May 2011

Several reports have shown an association between lameness in cattle and vaccination with Brucella abortus strain 19. Affected joints are culture negative for Brucella, but the synovial fluid is positive for B. abortus antibodies. The joints contain cloudy fluid, with villous proliferation of the synovium. Brucella abortus antigens are often found in the synovium with fluorescent antibody staining. This report describes the experimental reproduction of a chronic synovitis in 6 young Angus steers using intra-articular injections of B. abortus strain 19. The carpal and tibial joints were injected with 5 × 109 colony-forming units/ml of B. abortus strain 19 and regularly biopsied over a 28-day period. Steers started becoming serologically positive for B. abortus on post-inoculation day (PID) 5 and were all positive by PID 7. Joints were cultured and examined by fluorescent antibody staining, immunohistochemical methods, and light and transmission electron microscopy. Lesions typical of the field cases were present by PID 21. Brucella abortus was cultured more often during PID 1–5 (6 of 9 joints) than during PID 7–28 (3 of 15 joints). Brucella abortus was only found on PID 1, and 5 by fluorescent antibody staining and in only 2 joints immunohistochemically on PID 5 and 7. The reproduction of lesions typical of field cases but the inability to locate B. abortus antigens in the synovium raises the question of whether in field cases the synovium is continually or intermittently seeded with bacteria or if factors other than just the bacterium are needed to perpetuate the lesion.