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Abstract

Objective—To determine the sensitivity of a real-time PCR assay for the detection of Tritrichomonas foetus in protozoal cultures of preputial scraping samples pooled from up to 25 bulls and to determine the specificity of that assay for detection of T foetus in cultures for individual animals.

Design—Cross-sectional study.

Animals—188 bulls and 150 steers.

Procedures—Preputial scraping samples were collected, placed in a culture kit, and incubated at 37°C for 7 days. Cultures for individual animals were tested for T foetus by means of a real-time PCR assay. Pools of protozoal cultures were made by including fixed aliquots of samples with known positive and negative results in ratios of 1:2, 1:3, 1:5, 1:10, 1:15, 1:20, and 1:25. Specificities of the real-time PCR assay and culture for detection of T foetus in samples obtained from individual animals and sensitivity of real-time PCR assay for each evaluated pool ratio were determined.

Results—Specificity estimates for culture and the real-time PCR assay for detection of T foetus in preputial scraping samples for individual animals were not significantly different (98.8% and 100%, respectively). Sensitivities of the real-time PCR assay for the various pooled samples with known positive and negative T foetus results were not significantly different; overall sensitivity of the assay was 94%.

Conclusions and Clinical Relevance—Results indicated the evaluated real-time PCR assay had high specificity and good sensitivity for the detection of T foetus in pooled protozoal cultures of preputial scraping samples obtained from up to 25 animals.

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in Journal of the American Veterinary Medical Association

Abstract

Objective—To determine clinical sensitivity and specificity of a quantitative real-time PCR (qRT-PCR) assay for Campylobacter fetus subsp venerealis (Cfv) in preputial samples of bulls.

Animals—313 beef bulls.

Procedures—Preputial samples were collected from 300 virgin bulls and 13 Cfv-infected bulls. Specificity of the qRT-PCR assay, determined on the basis of results for samples collected from virgin bulls, was compared with specificity of bacteriologic culture performed with transport enrichment medium (TEM). Sensitivity of the qRT-PCR assay, determined on the basis of results for multiple samples collected at weekly intervals from infected bulls, was compared with sensitivity of the direct fluorescent antibody test (DFAT), bacteriologic culture, and bacteriologic culture with TEM.

Results—Specificity was 85% for the qRT-PCR assay and 100% for bacteriologic culture; results were significantly different. Mean sensitivity was 85.4% for the qRT-PCR assay, 82.3% for direct culture in blood agar, 72.1% for the DFAT, 32.7% for direct culture in Skirrow agar, 30% for bacteriologic culture with TEM and blood agar, and 38.1% for bacteriologic culture with TEM and Skirrow agar. Differences in sensitivity among tests varied with ambient outdoor temperature. Repeated sampling significantly increased sensitivity of the qRT-PCR assay.

Conclusions and Clinical Relevance—Use of the qRT-PCR assay as a screening test on direct preputial samples had comparable sensitivity to bacteriologic culture, and repeated sampling improved sensitivity. Although improved performance of the qRT-PCR assay, compared with direct bacteriologic culture, was dependent on temperature, transport times that allow direct culture are unlikely under field conditions. The qRT-PCR assay would provide a fast and sensitive screening method for Cfv in bulls.

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in American Journal of Veterinary Research

Abstract

Objective—To compare the recovery rates of Campylobacter fetus subsp venerealis (Cfv) from preputial scrapings of infected bulls with passive filtration on selective medium versus nonselective medium, with and without transport medium.

Samples—217 preputial scrapings from 12 bulls (4 naturally and 8 artificially infected with Cfv).

Procedures—Preputial scrapings were collected in 2 mL of PBS solution and bacteriologically cultured directly on Skirrow medium or passively filtered through 0.65-μm filters onto blood agar, with or without 24 hour preincubation in modified Weybridge transport enrichment medium (TEM). After 72 hours, plates were examined for Cfv and bacterial and fungal contamination or overgrowth.

Results—Passive filtration of fresh preputial scrapings onto blood agar yielded significantly higher recovery rates of Cfv (86%) than direct plating on Skirrow medium (32%), whereas recovery from TEM was poor for both media (35% and 40%, respectively). Skirrow cultures without TEM were significantly more likely to have fungal contamination than were cultures performed with any other technique, and fungal contamination was virtually eliminated by passive filtration onto blood agar. Bacterial contamination by Pseudomonas spp was significantly more common with Skirrow medium versus passive filtration on blood agar, regardless of TEM use.

Conclusions and Clinical Relevance—The use of transport medium and the choice of culture medium had significant effects on Cfv recovery and culture contamination rates from clinical samples. Both factors should be considered when animals are tested for this pathogen.

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in American Journal of Veterinary Research

Abstract

Objective—To determine the effect of paratuberculosis on culling, milk production, and milk quality in infected dairy herds.

Design—Cross-sectional study.

Animals—689 lactating dairy cows in 9 herds.

Procedure—Milk, blood, and fecal samples were obtained from all cows. Fecal samples were evaluated via mycobacterial culture. Serum samples were tested with a commercially available ELISA for antibodies against Mycobacterium avium subsp paratuberculosis, and preserved milk samples were tested with an ELISA for antibodies against M paratuberculosis. Mixed effect and proportional hazards models were used to determine the effect of paratuberculosis on 305-day milk, fat, and protein production; somatic cell count linear score; and the risk of culling.

Results—Cows with positive results of bacteriologic culture of feces and milk ELISA produced less milk, fat, and protein, compared with herdmates with negative results. No difference in 305-day milk or fat production was detected in cows with positive results of serum ELISA, compared with seronegative cows. The 3 survival analyses revealed that cows with positive results of each test were at higher risk of being culled than cows with negative results. Paratuberculosis status, as determined by use of all 3 diagnostic tests, was not associated with milk somatic cell count linear score.

Conclusions and Clinical Relevance—Results suggest that for the 9 herds in this study, paratuberculosis significantly decreased milk production and cow longevity. (J Am Vet Med Assoc 2005;227:1302–1308)

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in Journal of the American Veterinary Medical Association

Abstract

Objective—To determine whether results obtained for milk and serum samples with ELISAs intended for diagnosis of paratuberculosis in dairy cows were comparable to results obtained by means of mycobacterial culture of fecal samples.

Design—Cross-sectional study.

Animals—689 lactating dairy cows in 9 Ontario herds.

Procedure—Milk, serum, and fecal samples were obtained from all cows. Fecal samples were submitted for mycobacterial culture. Serum samples were tested with a commercially available ELISA for antibodies against Mycobacterium paratuberculosis, and preserved milk samples were tested with an indirect ELISA for antibodies against M paratuberculosis.

Results—Results were positive for 130 of the 689 (18.9%) serum samples, 77 of the 689 (11.1%) milk samples, and 72 of the 689 (10.4%) fecal samples. The level of agreement between results for milk and serum samples was only moderate. Proportions of positive results for serum and fecal samples were significantly different, but proportions of positive results for milk and fecal samples were not significantly different. In addition, results for milk samples had a higher level of agreement with results of mycobacterial culture than did results for serum samples.

Conclusions and Clinical Relevance—Results suggest that the indirect ELISA used on milk samples may be a convenient method of detecting paratuberculosis in dairy herds. (J Am Vet Med Assoc 2005; 226:424–428)

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in Journal of the American Veterinary Medical Association