bluuebunny/crossref_metadata_2025 huggingface dataset:
The Diagnostic Sensitivity of Immunohistochemistry for the Detection of Porcine Reproductive and Respiratory Syndrome Virus in the Lung of Vaccinated and Unvaccinated Swine
Authors: Yaeger Michael J.
Published: May 2011

Immunohistochemistry (IHC) is used routinely to detect porcine reproductive and respiratory syndrome virus (PRRSV) in the lung of nursery and grow/finish pigs with respiratory disease and has been reported to be highly specific (100%) but only moderately sensitive (67%). When multiple sections of lung are examined from field cases of porcine pneumonia, it is common to detect PRRSV antigen in only 1 or 2 of the sections. This study was undertaken to determine the impact of the number of lung sections evaluated on the diagnostic sensitivity of IHC for the detection of PRRSV in vaccinated and unvaccinated swine. Five anterioventral sections of lung from animals experimentally challenged with PRRSV were evaluated on a single IHC slide. Utilizing a beta binomial model, observed results were used to calculate the probability of detecting PRRSV with IHC as a function of the number of lung sections assessed. Results demonstrate that the diagnostic sensitivity of PRRSV IHC is dependent on the number of lung sections examined. In unvaccinated pigs, a beta binomial model predicts that if a single lung section were evaluated, PRRSV would likely be confirmed in only 48% of infected animals, and at least 5 sections of anterioventral lung would need to be assessed to detect >90% of PRRSV-infected pigs. Vaccination resulted in significantly lower gross and microscopic lung lesion scores and significantly fewer antigen-positive cells. In vaccinated swine, the calculated probability of detecting a PRRSV-infected pig with IHC when a single lung section is evaluated was only 14%. If PRRSV is a primary concern, diagnosticians should collect at least 5 anterioventral sections of lung from each pig to be evaluated on a single IHC slide. This approach will diminish the number of false-negative results obtained with this method of antigen detection.
Effect of Transport Enrichment Medium, Transport Time, and Growth Medium on the Detection of Campylobacter Fetus subsp. Venerealis
Authors: Monke Holly J. , Love Brenda C. , Wittum Thomas E. , Monke Donald R. , Byrum Beverly A.
Published: May 2011

The combination of medium and growth conditions, including transport enrichment medium (TEM), transport time, TEM incubation time, and growth medium, that best support Campylobacter fetus subsp. venerealis while inhibiting contaminants was studied. The 3 TEMs evaluated, Weybridge, Cary-Blair, and 0.85% saline solution, were inoculated with preputial smegma spiked with C. fetus subsp. venerealis and held in the laboratory for 4 or 24 hours before inoculation onto growth medium. The effect of overnight incubation at 37 C of the TEM was also evaluated. Median scores of C. fetus subsp. venerealis growth and microbial contaminant inhibition were compared within TEM, transport time, overnight incubation, and growth medium groups using the Mann–Whitney U-test and the Kruskal–Wallis test. The proportion of samples with any growth or contamination within each group was also compared using the chi-square test. Results suggest that the growth of C. fetus subsp. venerealis was influenced by 3 of the 4 criteria evaluated. Weybridge TEM more effectively maintained the organism than did either Cary–Blair or 0.85% saline solution ( P < 0.001). Transport time of 4 hours rather than 24 hours ( P < 0.001) and avoiding overnight incubation of TEM at 37 C ( P < 0.001) were associated with improved growth. Significant differences were not identified among growth media; however, Skirrow Campylobacter agar appeared to yield slightly better growth than did either blood agar or Greenbriar Plus agar. Contaminant growth was also influenced by 3 of the 4 variables. Weybridge TEM inhibited contaminant growth more effectively than did either Cary–Blair or 0.85% saline solution ( P < 0.001). Transport time was not associated with contaminant growth. Eliminating overnight incubation of TEM reduced contamination ( P < 0.01). Skirrow agar was preferred to both blood agar and Greenbriar Plus agar for suppression of contaminants on solid medium ( P < 0.001). These results suggest that the detection of C. fetus subsp. venerealis is enhanced when preputial smegma samples arrive at the diagnostic laboratory within 4 hours after inoculation into Weybridge TEM and are transferred to Skirrow agar the same day they arrive in the laboratory.
Development of a Semi-Nested Reverse Transcription Polymerase Chain Reaction Assay for the Retrospective Diagnosis of Canine Distemper Virus Infection
Authors: Stanton James B. , Poet Steven , Frasca Salvatore , Bienzle Dorothee , Brown Corrie C.
Published: May 2011

Formalin-fixed, paraffin-embedded tissue blocks from 14 dogs were used to test the utility of a newly developed semi-nested reverse transcription polymerase chain reaction (RT-PCR) assay for canine distemper virus (CDV). The results from this new test were compared with those of histopathologic examination, fluorescent antibody detection (FA), and immunohistochemistry (IHC). The semi-nested RT-PCR protocol was used to detect CDV RNA in 9 of the 10 cases that were positive by at least 1 of the immunologic (FA and IHC) methods. Sequence data indicated that the amplified strains of CDV are more closely related to a naturally occurring strain than to a vaccine strain. Thus, the semi-nested RT-PCR assay is a useful diagnostic method applicable to the retrospective diagnosis of CDV infection. Sequence determination may yield molecular epidemiologic information regarding vaccine efficacy.
Detection and Differentiation of North American and European Genotypes of Porcine Reproductive and Respiratory Syndrome Virus in Formalin-Fixed, Paraffin-Embedded Tissues by Multiplex Reverse Transcription-Nested Polymerase Chain Reaction
Authors: Chung Han-Kook , Choi Changsun , Kim Junghyun , Chae Chanhee
Published: May 2011

North American and European genotypes of porcine reproductive and respiratory syndrome virus (PRRSV) were distinguished in formalin-fixed, paraffin-embedded tissues from PRRSV-infected pigs by multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR). A method based on xylene deparaffinization followed by proteinase K digestion yielded RNA of quality for reliable and consistent RT-nPCR analyses. The PRRSV nucleic acid was detected in lung, mediastinal lymph node, tonsil, and liver samples from pigs inoculated with North American strain virus, European strain virus, or both North American and European strains. All 30 archival formalin-fixed, paraffin-embedded lung tissues from pigs naturally infected with PRRSV were positive for PRRSV by RT-nPCR amplication and gave a pattern that corresponded to the North American genotype. Multiplex RT-nPCR could be applied successfully to formalin-fixed, paraffin-embedded tissues for the detection and differentiation between North American and European genotypes of PRRSV.
A Simple Field Method for Spinal Cord Removal Demonstrated in the Cheetah (Acinonyx Jubatus)
Authors: Walzer Christian , Kübber-Heiss Anna , Robert Nadia
Published: May 2011

Removal of the spinal cord is considered time consuming and difficult. A delay in the necropsy procedure, especially in the central nervous system, can result in significant tissue autolysis and subsequent diagnostic difficulties. In the field, where many necropsies are performed, suitable electric saws are mostly unavailable. A technically simple and rapid method for spinal cord removal, requiring only a straightforward tool, has been devised. No necropsy-induced structural damage has been noted on histopathologic examination.
Ulcerative Pododermatitis in a Cat Associated with Anatrichosoma sp.
Authors: Ramiro-Ibañez F. , Winston J. , O'Donnell E. , Mansell J.
Published: May 2011

A 9-year-old castrated male Chartreaux cat was presented for an ulcerative pododermatitis of all 4 paws. A clinical exam was inconclusive and supportive therapy did not improve the condition. Histologic examination revealed an ulcerative and eosinophilic dermatitis associated with epidermal and dermal nematodes and ova consistent with the aphasmid Anatrichosoma sp. Treatment with ivermectin completely resolved the skin lesions. Anatrichosomiasis should be included in the differential diagnosis of ulcerative pododermatitis in cats, at least in the southwestern United States.
Preliminary Study on Differentiation of Leptospira Grippotyphosa and Leptospira Sejroe from Other Common Pathogenic Leptospiral Serovars in Canine Urine by Polymerase Chain Reaction Assay
Authors: Cai Hugh Y. , Hornby Geoff , Key Douglas W. , Osuch Maria R. , Maxie M. Grant
Published: May 2011

A multiplex polymerase chain reaction (PCR) method using primer sets of G1/G2 and B64-I/B64-II was validated to detect pathogenic leptospira serovars from canine urine samples. The PCR method was found to be specific and sensitive with a detection limit of 100 cells of Leptospira icterohaemorrhagiae per milliliter of urine. The primer set previously designated and erroneously transcribed B64-I/B64-II amplified a DNA fragment of 352 base pairs from Leptospira grippotyphosa and Leptospira sejroe but not from Leptospira autumnalis, Leptospira bratislava, Leptospira canicola, Leptospira hardjo, Leptospira icterohaemorrhagiae, and Leptospira pomona. From 100 diagnostic canine urine samples, 5 were found positive for Leptospira grippotyphosa/ sejroe with a PCR product of 352 base pairs and 6 were positive for other pathogenic leptospira serovars with a PCR product of 285 base pairs. One 285-base pair product was sequenced and found to be 99.3% homologous to the G1/G2 PCR fragment sequence reported previously. All 352-base pair PCR products of clinical samples and pure cultures of L. grippotyphosa and L. sejroe were sequenced. The 352-base pair fragment sequences of L. grippotyphosa and L. sejroe were identical. Only 2 base pairs were found different between the sequences from pure cultures and those from clinical samples. Serum samples from 3 positive cases that generated a PCR product of 352 base pairs were tested by the microscopic agglutination test, and 2 were found to be positive for L. grippotyphosa (1:10,240 and 1:5,120), 1 was positive for L. grippotyphosa (1:320) or L. icterohaemorrhagiae (1:320). The results of this study suggest that the multiplex PCR with the primer set G1/G2 and the erroneously transcribed B64-I/B64-II may be able to differentiate L. grippotyphosa or L. sejroe from other pathogenic leptospira serovars commonly tested for in Canadian diagnostic laboratories.
Rapid Detection of Avian Pneumovirus in Tissue Culture by Microindirect Immunofluorescence Test
Authors: Jirjis Faris F. , Noll Sally L. , Halvorson David A. , Nagaraja Kakambi V. , Townsend Evelyn L. , Goyal Sagar M. , Shaw Daniel P.
Published: May 2011

An indirect immunofluorescence (IFA) test with a 96-well, flat-bottomed microplate was developed to detect avian pneumovirus (APV) antigen in Vero cell cultures. Samples of nasal turbinates and swabs from infraorbital sinuses and trachea were collected from 4-week-old poults experimentally inoculated with APV. The APV titers by tissue culture IFA staining were compared with that of visual reading of cytopathic effect (CPE). The ability of IFA staining to detect APV antigen correlated well with visualizing CPE. The use of IFA staining of Vero cell cultures allowed detection of APV in substantially less time than the use of visualizing CPE. In addition, the use of IFA allowed specific identification of the virus in cell culture.
Paranasal Meningioma in a Horse
Authors: Kreeger John M. , Templer Alison , Turnquist Susan E. , Bailey Keith L. , Johnson Philip J. , Wilson David A.
Published: May 2011

Paranasal meningioma was diagnosed in a 5-year-old Appaloosa gelding. The mass occupied the right maxillary, frontal, and sphenopalatine sinuses but did not invade the calvarium. The diagnosis was based on histologic evaluation, positive immunohistochemical staining for vimentin and cytokeratin, and ultrastructural features including the presence of interdigitating spindle cells with numerous desmosomes.
Metastatic Pulmonary Adenocarcinoma in a Llama (Lama Glama)
Authors: Ramos-Vara José A. , Miller Margaret A.
Published: May 2011

An adult female llama with a comminuted fracture of the left femoral head was necropsied. A firm multinodular mass infiltrated skeletal muscle adjacent to the fracture. Multiple, firm, white nodules were in the pulmonary parenchyma and pleura. A single nodule was in the liver. Microscopically, transition from nonneoplastic bronchiolar epithelium to neoplastic epithelium that formed acinar structures was evident at bronchioloalveolar junctions. A diagnosis of pulmonary adenocarcinoma was made. Similar neoplastic tissue was in the liver and in the perifemoral mass. Immunohistochemically, neoplastic cells were positive for pan-cytokeratin, cytokeratin 7, and cytokeratin 5/6 antibodies and negative for vimentin and cytokeratins 8/18 and 20.
An Outbreak of Adenoviral Infection in Inland Bearded Dragons (Pogona Vitticeps) Coinfected with Dependovirus and Coccidial Protozoa (Isospora Sp.)
Authors: Kim Dae Young , Mitchell Mark A. , Bauer Rudy W. , Poston Rob , Cho Doo-Youn
Published: May 2011

Thirty of 200 (15%) hatchling inland bearded dragons were found dead after a short period (48 hours) of weakness and lethargy. The most common clinical signs were head tilt and circling. Six bearded dragons with neurological signs were euthanized, and postmortem examination revealed no gross abnormalities. Microscopically, severe, randomly distributed hepatocellular necrosis with large basophilic intranuclear inclusion bodies in numerous hepatocytes was noted. Small-intestinal enterocytes contained intracytoplasmic coccidial protozoa ( Isospora sp.) and occasional enterocytes had basophilic intranuclear inclusion bodies. Transmission electron microscopy revealed both 80- and 20-nm-diameter viral particles, which were consistent with adenoviruses and dependoviruses, respectively. Adenoviral outbreaks in groups of animals are uncommon. An adverse synergistic effect of the coccidiosis with the adenoviral infection may have played a critical role in the high morbidity and mortality in this case.
Minimum Inhibitory Concentration Breakpoints and Disk Diffusion Inhibitory Zone Interpretive Criteria for Tilmicosin Susceptibility Testing against Pasteurella Multocida and Actinobacillus Pleuropneumoniae Associated with Porcine Respiratory Disease
Authors: Shryock Thomas R. , Staples J. Mitchell , DeRosa David C.
Published: May 2011

Tilmicosin is a novel macrolide antibiotic developed for exclusive use in veterinary medicine. Tilmicosin has been approved as a feed premix to control porcine respiratory disease associated with Pasteurella multocida and Actinobacillus pleuropneumoniae. The development of antimicrobial susceptibility testing guidelines for tilmicosin was predicated on the relationship of clinical efficacy studies that demonstrated a favorable therapeutic outcome, on pharmacokinetic data, and on in vitro test data, as recommended by the National Committee for Clinical Laboratory Standards (NCCLS). The approved breakpoints for the minimum inhibitory concentration dilution testing for both species are resistant, ≥32 μg/ml, and susceptible, ≤16 μg/ml. The zone of inhibition interpretive criteria for disk diffusion testing with a 15-μg tilmicosin disk are resistant, ≤10 mm, and susceptible, ≥11mm.
Neutralizing Antibodies against Vesicular Stomatitis Viruses (Serotypes New Jersey and Indiana) in Horses in Costa Rica
Authors: Blickwede Maren , Dolz Gaby , Herrero Marco V. , Tomlinson Sarah M. , Salman Mo
Published: May 2011

Serum samples were collected from domestic horses in 4 different regions of Costa Rica to detect antibodies against vesicular stomatitis viruses, serotypes New Jersey (VSV-NJ) and Indiana (VSV-IN). A total of 214 samples were tested by the virus neutralization test. The sampling regions were identified as low North Pacific dry area (1), low Middle Atlantic humid area (2), low South Pacific humid area (3), and the highlands (4). In region 1, 97.1% of horses were positive for VSV-NJ and 16.5% were positive for VSV-IN. The mean antibody titer and its standard deviation after logarithmic transformation were 5.86 ± 0.9 for VSV-NJ and 3.55 ± 1.66 for VSV-IN for region 1. In region 2, 40.7% of horses were positive for VSV-NJ and 32.2% were positive for VSV-IN. The mean antibody titer in region 2 was 4.33 ± 1.82 for VSV-NJ and 3.47 ± 1.73 for VSV-IN. In region 3, 20.79% of horses were positive for VSV-NJ and 27.6% were positive for VSV-IN. The mean antibody titer in region 3 was 4.39 ± 1.89 for VSV-NJ and 3.47 ± 1.82 for VSV-IN. In region 4, 91.3% of horses were positive for VSV-NJ and 73.9% were positive for VSV-IN. The mean antibody titer in region 4 was 5.77 ±1.10 for VSV-NJ and 4.85 ± 1.63 for VSV-IN. This is the first published report of the detection of virus-neutralizing antibodies against VSV-NJ and VSV-IN in horses in Costa Rica.
Mycobacterium Bovis–Infected White-Tailed Deer (Odocoileus Virginianus): Detection of Immunoglobulin Specific to Crude Mycobacterial Antigens by ELISA
Authors: Waters W. Ray , Palmer Mitchell V. , Whipple Diana L.
Published: May 2011

White-tailed deer ( Odocoileus virginianus) have recently emerged as a source of Mycobacterium bovis infection for cattle within North America. The objective of this study was to evaluate the antibody response of M. bovis–infected deer to crude mycobacterial antigens. Deer were experimentally inoculated with M. bovis strain 1315 either by intratonsilar instillation or by exposure to M. bovis-infected (i.e., in contact) deer. To determine the time course of the response, including the effects of antigen administration for comparative cervical skin testing, serum was collected periodically and evaluated by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin (i.e., IgG heavy and light chains) reactivity to mycobacterial antigens. The reactivity to M. bovis purified protein derivative (PPDb) exceeded ( P < 0.05) the reactivity to M. avium PPD (PPDa) only after in vivo administration of PPDa and PPDb for comparative cervical testing of the infected deer. The mean immunoglobulin response, as measured by ELISA, of intratonsilar-inoculated deer to a proteinase K-digested whole-cell sonicate (WCS-PK) of M. bovis strain 1315 exceeded ( P < 0.05) the mean of the prechallenge responses to this antigen at approximately 1 month after inoculation and throughout the remainder of the study (i.e., ∼11 months). This response also exceeded ( P < 0.05) that of the uninfected deer. Although this is encouraging, further studies are necessary to validate the use of the proteinase K-digested M. bovis antigens in the antibody-based assays of tuberculosis.
Detection of Porcine Teschovirus and Enterovirus Type II by Reverse Transcription–Polymerase Chain Reaction
Authors: Palmquist Joseph M. , Munir Shirin , Taku Anil , Kapur Vivek , Goyal Sagar M.
Published: May 2011

Porcine enteroviruses (PEVs) have been recognized as the causative agents of various clinical manifestations such as fertility disorders, neurological defects, and dermal lesions in pigs. Currently, the diagnosis of PEV infection is carried out by virus isolation, which although useful, is labor- and time-intensive. The present investigation describes the development of a reverse transcription–polymerase chain reaction (RT-PCR) assay for the rapid and sensitive detection of PEVs of cytopathic effect groups I (now known as porcine teschoviruses [PTVs]) and II. The assay described not only detects the PTVs and CPE group II of PEVs but also allows them to be differentiated on the basis of the size of the amplification product, using the same set of oligonucleotide primers. The availability of specific and sensitive molecular diagnostic tools such as the RT-PCR assay described herein should facilitate efficient diagnosis of PTV and CPE group II infections in pigs.
An Estimated Prevalence of Johne's Disease in a Subpopulation of Alabama Beef Cattle
Authors: Hill B. B. , West M. , Brock K. V.
Published: May 2011

The objective of this study was to estimate the overall prevalence of animals that were infected with Mycobacterium avium ssp. paratuberculosis in a subpopulation of Alabama beef cattle. This was determined using a commercial enzyme-linked immunosorbent assay (ELISA) for the detection of M. avium ssp. paratuberculosis–specific antibodies in serum. Serum was collected from 79 herds that were participating in the Alabama Brucellosis Certification program. A total of 2,073 beef cattle were randomly tested by selecting 30 animals per herd in herds greater than 30 and selecting all animals in herds 30 and less for testing. It has been estimated that the commercial ELISA test used has a 60% sensitivity and a 97% specificity. Of the 79 herds tested, 29 herds were seronegative, 24 herds had 1–2 positive animals, and 26 herds had 3 or more seropositive animals. The average number of infected animals per positive herd was 3.3. In addition, a calculated minimum of 53.5% of the herds were identified as Johne's positive herds with a 95% confidence level. Of the total number of animals tested, 8.0% (166/2,073) of them were positive by the ELISA. After adjustments for test sensitivity and specificity and the proportion of animals sampled per herd, the true prevalence was calculated to be 8.75%. These data suggest that approximately 50% of the herds are infected with M. avium ssp. Paratuberculosis, and the overall prevalence of infection in Alabama beef cattle is approximately 8%, which correlates with other previously published regional estimates.
Antimicrobial Susceptibility of Riemerella Anatipestifer Isolated from Ducks and the Efficacy of Ceftiofur Treatment
Authors: Chang Chao-Fu , Lin Wen-Hwa , Yeh Tung-Mao , Chiang Tai-Sheng , Chang Yung-Fu
Published: May 2011

The in vitro susceptibilities of 50 field isolates of Riemerella anatipestifer from ducks to ceftiofur and 16 other commonly used antimicrobials were determined. The MIC90 values (MIC refers to minimum inhibitory concentrations) for the antimicrobials used in this study are as follows: penicillin was 16 μg/ml; ceftiofur was 32 μg/ml; cephalothin, chloramphenicol, flumequine, and kanamycin were 64 μg/ml; nalidixic acid, nitrofurantoin, and sulfamethoxazole were 128 μg/ml; amikacin, ampicillin, gentamicin, lincomycin, spec-tinomycin, streptomycin, tetracycline, and trimethoprim were ≥256 μg/ml. The therapeutic efficacy of ceftiofur against a highly lethal experimental R. anatipestifer infection in ducks was also evaluated. All experimental ducks were infected through the infraorbital sinus with 1 ml of 9 × 109 CFU of R. anatipestifer. Ceftiofur (0, 0.25, 0.5, 1, and 2 mg/kg) was injected subcutaneously 5 hours after infection. A single dose of 2 mg/kg resulted in 73% survival as compared with 10% survival in the infected, but untreated controls.
Detection of Classical Swine Fever Virus in Boar Semen by Reverse Transcription–Polymerase Chain Reaction
Authors: Choi Changsun , Chae Chanhee
Published: May 2011

A seminested reverse transcription–polymerase chain reaction (RT-PCR) was developed for the detection of classical swine fever virus (CSFV) in semen. Five boars were inoculated intranasally with CSFV isolate propagated in PK15 cells. Two boars inoculated with the supernatant of noninfected PK15 cells were kept as controls. Semen and serum samples were collected twice weekly for 63 days postinoculation (dpi). Samples were tested for the presence of antibodies to CSFV by an enzyme-linked immunosorbent assay and for the presence of CSFV nucleic acid by seminested RT-PCR. Antibodies to CSFV could be detected as early as 7 dpi in 1 boar, and all 5 infected boars were found positive by 14 dpi. CSFV from boar semen was infrequently identified by virus isolation compared with seminested RT-PCR. CSFV nucleic acid was detected in semen by seminested RT-PCR as early as 7 dpi in 3 infected boars and persistently thereafter in all 5 infected boars until 63 dpi. When separated fractions of CSFV-contaminated semen were analyzed by the seminested RT-PCR, the CSFV nucleic acid was detected mainly in seminal fluid and occasionally in nonsperm cells. CSFV antigen was also detected in nonsperm cells from semen smear by immunohistochemistry. Thus, infection via semen, specially through CSFV-infected seminal fluid, seems to be a major route of transmission of CSFV.
Dermal Malignant Melanoma in a Horse with Multifocal Pancytokeratin Expression
Authors: Patterson-Kane Janet C. , Ginn Pamela E.
Published: May 2011

The current report describes a malignant melanoma in the dermis of a 13-year-old bay Thoroughbred mare. Microscopic examination revealed that tumor cells were arranged in cords and packets within an abundant collagenous stroma containing scattered myxomatous foci. Tumor cells stained positively for S-100, neuron-specific enolase, and vimentin and some contained melanin granules. Some clusters of tumor cells were also positive for pancytokeratin. Expression of epithelial cell markers has been described in small numbers of human melanomas but has not been reported previously in equine melanomas.
Chronic Hyperplastic and Neoplastic Cutaneous Lesions (Marjolin's Ulcer) in Hot-Brand Sites in Adult Beef Cattle
Authors: O'Toole D. , Fox J. D.
Published: May 2011

Linear exophytic cutaneous lesions (brand keratomas) are a chronic sequel to hot-iron branding in a small proportion of beef cattle in the western United States. Rarely, brand keratomas progress to form large ulcerated masses. Samples of chronically thickened skin were collected from 8 adult cattle with hot-brand lesions and from 2 cattle with ulcerated masses at brand sites. Cutaneous thickening was attributable to abrupt transition from normal haired skin to regular epidermal hyperplasia with marked orthokeratotic hyperkeratosis, acanthosis, hypopigmentation, and loss of adnexae. Epithelial atypia was absent. Normal dermal collagen was replaced by mature granulation tissue containing islands of dense hyalinized collagen. Two cows, aged 5 and 13 years, developed large, slow-growing squamous cell carcinomas at brand sites. Malignancy in branded skin is a rare complication of hot-iron branding in cattle and may arise because of malignant transformation of brand keratomas.
Detection of Bovine Leukemia Virus in Blood and Milk by Nested and Real-Time Polymerase Chain Reactions
Authors: Kuckleburg Christopher J. , Chase Christopher C. , Nelson Eric A. , Marras Salvatore A. E. , Dammen Matthew A. , Christopher-Hennings Jane
Published: May 2011

Concerns about retroviruses in livestock and products derived from them have necessitated the development of tests to detect the bovine leukemia virus (BLV) in blood and milk from cattle. Dairy cattle ( n = 101) from 5 different geographical areas were used for this study. A nested polymerase chain reaction (PCR) identified 98% of BLV seropositive cattle ( n = 80) from blood and 65% from milk, whereas real-time PCR detected 94% of BLV seropositive cattle from blood and 59% from milk. Bovine leukemia virus was also detected by PCR in approximately 10% of seronegative cattle ( n = 21), most likely because of early detection before seroconversion.
Comparison of ELISA and RT-PCR versus Immune Electron Microscopy for Detection of Bovine Torovirus (Breda Virus) in Calf Fecal Specimens
Authors: Hoet Armando E. , Chang Kyeong-Ok , Saif Linda J.
Published: May 2011

Bovine Torovirus (BoTV) is an uncultivable enteric pathogen of cattle. Its failure to grow in vitro limits epidemiological studies, characterization of the virus, and development of diagnostic techniques. The objectives of this study were to develop and standardize an antigen-capture enzyme-linked immunosorbent assay (ELISA) and a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the detection of BoTV in fecal specimens. These assays were compared with immunoelectron microscopy (IEM) to evaluate their sensitivity, specificity, and efficiency as well as their advantages and limitations. Additionally, several methods to calculate ELISA cutoff values were used and compared using a statistical approach to obtain the optimal cutoff value for the ELISA. A plate cutoff ELISA value was determined to be the best method to calculate the cutoff value. The ELISA and RT-PCR assays developed in this study identified BoTV antigen and viral nucleic acids in feces without cross-reactions with the other calf enteric viruses examined. Both assays showed good agreement with IEM, with a Kappa value of 0.86 for ELISA and 0.85 for RT-PCR. The latter exhibited the higher analytical sensitivity. On the basis of the results obtained in this study, it is recommended that no single test should be used alone in an epidemiological survey because of the observed limitations of each assay. The fast and inexpensive ELISA combined with the highly specific and sensitive RT-PCR are a practical approach for future epidemiological studies of BoTV. These results should provide other researchers with the information needed to develop similar diagnostic assays for the study of BoTV.
Malignant Peripheral Nerve Sheath Tumor with Divergent Mesenchymal Differentiations in a Dog
Authors: Kim D.-Y. , Cho D.-Y. , Kim D. Y. , Lee J. , Taylor H. W.
Published: May 2011

A malignant peripheral nerve sheath tumor with divergent osteogenic, myxomatous, and myoblastic differentiations is described in a 10.5-year-old male Labrador Retriever dog. The tumor involved the sixth and seventh cervical spinal cord segments and nerves. The diagnosis was based on the results of histopathology, electron microscopy, and immunohistochemistry.
Detection of Bovine Torovirus and other Enteric Pathogens in Feces from Diarrhea Cases in Cattle
Authors: Hoet Armando E. , Nielsen Paul R. , Hasoksuz Mustafa , Thomas Christopher , Wittum Thomas E. , Saif Linda J.
Published: May 2011

The objectives of this study were to determine the prevalence of bovine torovirus (BoTV) in bovine fecal samples from diarrhea cases submitted to the Ohio Animal Disease Diagnostic Laboratory (ADDL) and to assess if a relationship exists between BoTV and the other enteric pathogens detected. From November 1999 to May 2001, 259 specimens from 53 calves (≤6 months old), 27 young adults (≤2 years), 125 adults (≥2 years), and 54 animals of unknown age were examined by an antigen-capture enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR) assay developed to detect BoTV Testing for other enteric pathogens was performed by ADDL, and the results were analyzed with the BoTV data. The BoTV was detected using ELISA or RT-PCR in 9.7% (25/259) of the clinical samples, 56% (14/25) of which were from calves ( P < 0.001) representing 26.4% (14/53) of the calves tested. Of the BoTV-positive calves, 71% (10/14) were less than 3 weeks of age. In 11/25 positive specimens, BoTV was the only pathogen detected among those examined. Other enteric organisms detected alone or in combination with BoTV in calf samples were rotavirus, coronavirus, Salmonella spp., Cryptosporidium spp., and Giardia spp.; but no consistent association between BoTV and these organisms was observed. In summary, BoTV was detected in fecal samples from cattle with diarrhea, principally in young calves less than 3 weeks of age. Future studies of infectious diarrhea in cattle should also include assays for this etiologic agent.
Protein G Binding to Enriched Serum Immunoglobulin from Nondomestic Hoofstock Species
Authors: Kramsky Joely A. , Manning Elizabeth J. B. , Collins Michael T.
Published: May 2011

Quick and cost-effective serologic assays, such as those based on enzyme-linked immunosorbent assay (ELISA) technology, are useful for screening animal populations for infectious diseases. Recombinant protein G is described as an almost universal ELISA conjugate for the detection of antibodies from a wide range of animal species. However, there is limited data documenting the ability of protein G to bind immunoglobulin (Ig) from many captive and free-ranging nondomestic hoofstock (Order Artiodactyla, e.g., elk, antelope, bison). Protein G binding to Ig from 11 species within this taxonomic order (addax, antelope, bison, bontebok, elk, impala, kudu/nyala, muntjac, oryx, sheep, and white-tailed deer) and 2 control species (bovine and chicken) was assessed. A serum Ig enrichment protocol, using high-performance liquid chromatography (HPLC), was optimized in bovids ( Bos taurus) and then applied to the other study species. Binding assays were performed by adding protein G to microtiter wells coated with titrated dilutions of enriched artiodactyl Ig. Optical densities were measured and binding curves generated. Differences in protein G binding were observed, both within and among species, as well as within taxonomic families. Significant intraspecies binding variation was observed for 7 species tested (antelope, oryx, sheep, muntjac, impala, bontebok, and addax). No statistically significant intraspecies differences in protein G binding were found for Ig from bison, elk, kudu/nyala, white-tailed deer, plus control species (cattle and chicken). Binding of protein G to Ig from impala, muntjac, and elk was statistically different from the positive control (cattle), with muntjac binding curves statistically comparable with the negative control (chicken). For the other 7 species tested, binding curves illustrated the ability of protein G to bind Ig as well as, or better than, the positive control. These findings expand the list of animal species whose Ig is capable of being detected using recombinant protein G, with the caveat that protein G does not bind Ig uniformly in closely related species. It is concluded that recombinant protein G conjugates may serve as useful reagents for serodiagnosis by ELISA in nondomestic hoofstock, although different assay interpretation algorithms and assay protocols may need to be developed on a per species basis for maximum diagnostic effectiveness.
Comparison of Three Enzyme-Linked Immunosorbent Assays for Serologic Diagnosis of Contagious Agalactia in Sheep
Authors: Pépin Michel , Dufour Philippe , Lambert Maurice , Aubert Michel , Valognes Aurèle , Rotis Thierry , Van de Wiele Anne , Bergonier Dominique
Published: May 2011

Serologic diagnosis of ovine contagious agalactia ( Mycoplasma agalactiae) with the enzyme-linked immunosorbent assay (ELISA) developed by Agence Française de Séceurité Sanitaire des Aliments (AFS-SA) may produce a few false-positive (FP) and false-negative (FN) results. When the prevalence of disease is low, these erroneous results may generate problems for eradication schemes. To prevent this, 2 commercial ELISAs were compared with the AFSSA ELISA. Flocks of known status were selected and classified into 4 categories: true positive (TP), FP, true negative (TN), and FN; 20 sheep per flock were submitted for blood sampling. A flock was considered positive when at least 1 out of 20 sera was positive or 2 sera were doubtful. In the flock, the diagnostic sensitivity of the 3 kits was very good (100%), and the diagnostic specificity showed an improvement from 46% (AFSSA test) to 88% and 92% (commercial tests). Considering individual animals, very few positive ewes were detected within TN or FP flocks; the proportion of positive ewes varied greatly from one kit to another (48% to 82%) within TP flocks. The kinetics of antibody response in sheep experimentally infected with various field strains of M. agalactiae were quite similar with all 3 ELISAs. The agreement between the 3 tests, assessed using the kappa value, varied from moderate to good (respective values of 0.56, 0.61, and 0.86). The 2 commercial ELISAs showed better performances, probably because of a superior analytical sensitivity, and are a good alternative for the serodiagnosis of contagious agalactia in sheep.
Coccygeal Chordoma in a Dog
Authors: Munday John S. , Brown Cathy A. , Weiss Raul
Published: May 2011

A 4-cm diameter spherical mass was removed from the region of the second coccygeal vertebra of a 6-year-old female Doberman Pinscher dog. The mass had been present for 7 months and was described as multilobular and smooth. Histologically, the neoplasm consisted of small spindle-shaped cells surrounding lobules of large vacuolated polygonal cells. Mucin was present within most lobules. The large polygonal cells stained positively with antibodies against vimentin, cytokeratin, and S-100 protein. Although the spindle-shaped cells stained positively with antibodies against vimentin, they only occasionally contained cytokeratin. The histology and immunohistochemistry are consistent with that reported for chordoma. Although 4 chordomas have previously been reported in dogs, this is the first case in which immunohistochemistry has been used to support the histological diagnosis. Neither recurrence nor metastasis was reported 10 months after surgical excision.
Pulmonary Sarcocystosis in a Puppy with Canine Distemper in Costa Rica
Authors: Berrocal Alexis , López Alfonso
Published: May 2011

Canine distemper and pulmonary sarcocystosis were diagnosed in a 10-week-old Rottweiler with 4-day history of diarrhea, vomiting, and weakness. Microscopic examination of the lung revealed bronchointerstitial pneumonia typical of morbillivirus infection. Also, numerous apicomplexan parasites were scattered in the alveolar walls. This protozoan infection was first thought to be toxoplasmosis but immunoperoxidase staining revealed large numbers of Sarcocystis canis. This is the first case of canine sarcocystosis reported from Latin America that further emphasizes the importance of immunohistochemistry in the differential diagnoses of apicomplexan infections in dogs.
Valid Methods: The Quality Assurance of Test Method Development, Validation, Approval, and Transfer for Veterinary Testing Laboratories
Authors: Wiegers Ann L.
Published: May 2011

Third-party accreditation is a valuable tool to demonstrate a laboratory's competence to conduct testing. Accreditation, internationally and in the United States, has been discussed previously. However, accreditation is only 1 part of establishing data credibility. A validated test method is the first component of a valid measurement system. Validation is defined as confirmation by examination and the provision of objective evidence that the particular requirements for a specific intended use are fulfilled. The international and national standard ISO/IEC 17025 recognizes the importance of validated methods and requires that laboratory-developed methods or methods adopted by the laboratory be appropriate for the intended use. Validated methods are therefore required and their use agreed to by the client (i.e., end users of the test results such as veterinarians, animal health programs, and owners). ISO/IEC 17025 also requires that the introduction of methods developed by the laboratory for its own use be a planned activity conducted by qualified personnel with adequate resources. This article discusses considerations and recommendations for the conduct of veterinary diagnostic test method development, validation, evaluation, approval, and transfer to the user laboratory in the ISO/IEC 17025 environment. These recommendations are based on those of nationally and internationally accepted standards and guidelines, as well as those of reputable and experienced technical bodies. They are also based on the author's experience in the evaluation of method development and transfer projects, validation data, and the implementation of quality management systems in the area of method development.
Field Validation and Assessment of an Enzyme-Linked Immunosorbent Assay for Detecting Chronic Wasting Disease in Mule Deer (Odocoileus Hemionus), White-Tailed Deer (Odocoileus Virginianus), and Rocky Mountain Elk (Cervus Elaphus Nelsoni)
Authors: Hibler Charles P. , Wilson Kathi L. , Spraker Terry R. , Miller Michael W. , Zink Robert R. , DeBuse Linda L. , Andersen Elaine , Schweitzer Darrell , Kennedy James A. , Baeten Laurie A. , Smeltzer John F. , Salman Mo D. , Powers Barbara E.
Published: May 2011

Tissue samples ( n = 25,050 total) from 23,256 mule deer ( Odocoileus hemionus), Rocky Mountain elk ( Cervus elaphus nelsoni), and white-tailed deer ( Odocoileus virginianus) collected statewide in Colorado were examined for chronic wasting disease (CWD) using an enzyme-linked immunosorbent assay developed by Bio-Rad Laboratories, Inc. (brELISA), in a 2-phase study. In the validation phase of this study, a total of 4,175 retropharyngeal lymph nodes (RLN) or obex (OB) tissue samples were examined independently by brELISA and immunohistochemistry (IHC). There were 137 IHC-positive samples and 4,038 IHC-negative samples. Optical density (OD) values from brELISA were classified as “not detected” or “suspect” based on recommended cutoff values during the validation phase. Using IHC-positive cases as known CWD-infected individuals and assuming IHC-negative cases as uninfected, the relative sensitivity of brELISA depending on species ranged from 98.3% to 100% for RLN samples and 92.1% to 93.3% for OB samples; the relative specificity of brELISA depending on species ranged from 99.9% to 100% for RLN samples and was 100% for OB samples. Overall agreement between brELISA and IHC was ≥97.6% in RLN samples and ≥95.7% in OB samples of all species where values could be calculated; moreover, mean brELISA OD values were ≥46× higher in IHC-positive samples than in IHC-negative samples. Discrepancies were observed only in early-stage cases of CWD. Based on the validation phase data, only RLN samples were collected for the field application phase of this study and only samples with brELISA OD values ≤0.1 were examined by IHC. Among 20,875 RLN samples screened with brELISA during this second testing phase, 155 of 8,877 mule deer, 33 of 11,731 elk, and 9 of 267 white-tailed deer samples (197 total) had OD values ≤0.1 and were further evaluated by IHC to confirm evidence of CWD infection. Of cases flagged for IHC follow-up, 143 of 155 mule deer, 29 of 33 elk, and all 9 white-tailed deer were confirmed positive. Mean (±SE) OD values for IHC-positive cases detected during the field application phase were comparable with those measured in RLN tissues during the validation phase. Based on these data, brELISA was determined to be an excellent rapid test for screening large numbers of samples in surveys designed to detect CWD infections in deer and elk populations.
Abundant PrPCWD in Tonsil from Mule Deer with Preclinical Chronic Wasting Disease
Authors: O'Rourke Katherine I. , Zhuang Dongyue , Lyda Amy , Gomez Gabriel , Williams Elizabeth S. , Tuo Wenbin , Miller Michael W.
Published: May 2011

A monoclonal antibody dot-blot assay was used to evaluate detergent lysates of tonsil tissue from mule deer to detect PrPCWD, the marker for the cervid transmissible spongiform encephalopathy chronic wasting disease (CWD). Samples of formalin-fixed brain and tonsil tissues from mule deer were examined for PrPCWD using immunohistochemistry (IHC) with Mab F99/97.6.1, the gold standard for diagnosis of preclinical CWD. The contralateral tonsil from each of the 143 deer was prepared for confirmatory IHC and as a 10% (wt/vol) detergent lysate without purification or enrichment steps for monoclonal antibody dot-blot assay. PrPCWD was detected by dot-blot assay in 49 of 50 samples considered positive by IHC. Forty-eight of the positive samples were evaluated with a quantitative dot-blot assay calibrated with recombinant PrP. Tonsillar PrPCWD concentrations ranged from 34 to 1,188 ng per 0.5 mg starting wet weight of tissue. The abundant PrPCWD in mule deer tonsil will facilitate development and validation of high-throughput screening tests for CWD in large populations of free-ranging deer.
High-Grade Canine T-cell Lymphoma/Leukemia with Plasmacytoid Morphology: A Clinical Pathological Study of Nine Cases
Authors: Ponce F. , Magnol J. P. , Marchal T. , Chabanne L. , Ledieu D. , Bonnefont C. , Felman P. , Fournel-Fleury C.
Published: May 2011

The aim of this study is to determine the clinical, morphological, and immunophenotypical presentation of 9 cases of a particular type of canine T-cell lymphoma/leukemia. The morphological presentation was a diffuse infiltration of small, medium-sized, or large blast cells with eccentric nuclei, hyperbasophilic cytoplasm, and a juxtanuclear, pale cytoplasmic area, giving a plasmacytoid appearance and suggesting a B-cell morphology. Surprisingly, all 9 cases were of T-cell phenotype (CD3+). Among the 7 immunophenotyped cases, 4 were CD4–/CD8+, 2 CD8+/CD4+, and 1 CD4+/CD8–. The median Ki-67 index was 65.7%, which placed this lymphoma in the high-grade group. This type of lymphoma/leukemia was found in dogs between 1 and 11 years of age, with a median age of 5.8. The male-female ratio was 0.8 for a reference population of 1.04. The most significant clinical findings were lymphadenopathy either generalized or localized in all cases, a mediastinal mass in 4 cases, bone marrow involvement in 7 cases, hypercalcemia in 4 cases, along with an aggressive clinical course and a poor response to chemotherapy in all cases, with a median disease-free survival time of 3 months.
Infectious Salmon Anemia Virus RNA in Fish Cell Cultures and in Tissue Sections of Atlantic Salmon Experimentally Infected with Infectious Salmon Anemia Virus
Authors: Moneke Emeka E. , Kibenge Molly J. T. , Groman David , Johnson Gerald R. , Ikede Basil O. , Kibenge Frederick S. B.
Published: May 2011

Current understanding of the etiopathogenesis of infectious salmon anemia (ISA) virus (ISAV) infection in fish comes mostly from virus detection in homogenized tissues taken from ISA-suspected mortalities. This study combined in situ hybridization (ISH) and histology to demonstrate viral RNA transcripts in different fish cell lines infected with ISAV and in tissues collected during the clinical phase of ISAV infection in Atlantic salmon. For this, a riboprobe to mRNA transcripts of ISAV RNA segment 8 was shown to detect viral mRNA in ISAV-infected TO, CHSE-214, and SHK-1 cell cultures. Specific hybridization was initially detected exclusively in the nuclei of infected cells, which is consistent with the nuclear transcription of orthomyxoviruses. For use of the riboprobe on fish tissues fixed in paraformaldehyde or formalin, the conditions used to permeabilize tissues before ISH (Proteinase K or Tween 20) were first optimized. Tissues were collected 15—20 days after challenge from 7 fresh mortalities of Atlantic salmon parr (∼20 g) showing severe gross and microscopic lesions, consistent with ISAV infection. Reverse transcription-polymerase chain reaction on tissue pools confirmed the presence of ISAV in each of the 7 fish. Of the tissues examined in each fish, the heart and liver consistently showed the strongest hybridization signal and, therefore, the most in situ virus, which was located in the endothelium of small blood vessels and in macrophage-like cells.
Lead Contamination of Chicken Eggs and Tissues from a Small Farm Flock
Authors: Trampel Darrell W. , Imerman Paula M. , Carson Thomas L. , Kinker Julie A. , Ensley Steve M.
Published: May 2011

Twenty mixed-breed adult laying hens from a small farm flock in Iowa were clinically normal but had been exposed to chips of lead-based paint in their environment. These chickens were brought to the Iowa State University Veterinary Diagnostic Laboratory, Ames, Iowa, where the concentration of lead in blood, eggs (yolk, albumen, and shell), and tissues (liver, kidney, muscle, and ovary) from 5 selected chickens was determined over a period of 9 days. Blood lead levels ranged from less than 50 to 760 ppb. Lead contamination of the yolks varied from less than 20 to 400 ppb, and shells were found to contain up to 450 ppb lead. Albumen contained no detectable amount. Lead content of the egg yolks strongly correlated with blood lead levels. Deposition of lead in the shells did not correlate well with blood lead levels. Mean tissue lead accumulation was highest in kidneys (1,360 ppb), with livers ranking second (500 ppb) and ovarian tissue third (320 ppb). Muscle contained the lowest level of lead (280 ppb). Lead contamination of egg yolks and edible chicken tissues represents a potential public health hazard, especially to children repeatedly consuming eggs from contaminated family-owned flocks.