Cost-effectiveness of diagnostic strategies to identify Mycobacterium avium subspecies paratuberculosis super-shedder cows in a large dairy herd using antibody enzyme-linked immunosorbent assays, quantitative real-time polymerase chain reaction, and bacterial culture.

Diagnostic strategies to detect Mycobacterium avium subsp. paratuberculosis (MAP) super-shedder cows in dairy herds have been minimally studied. The objective of the current study was to compare the cost-effectiveness of strategies for identification of MAP super-shedders on a California dairy herd of 3,577 cows housed in free-stall pens. Eleven strategies that included serum or milk enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qPCR) or culture of environmental samples, pooled or individual cow fecal samples, or combinations thereof were compared. Nineteen super-shedders (0.5%) were identified by qPCR and confirmed by culture as cows shedding ≥ 10,000 colony forming units (CFU)/g feces (median of 30,000 CFU/g feces). A stratified random sample of the study herd based on qPCR results of fecal pools was the most sensitive (74%) strategy and had the highest cost ($5,398/super-shedder). The reference strategy with the lowest cost ($1,230/super-shedder) and sensitivity (47%) included qPCR testing of fecal samples from ELISA-positive lactating (milk) and nonlactating (serum) cows housed in pens with the highest MAP bioburden. The most cost-effective alternative to the reference was to perform qPCR testing of fecal samples from ELISA-positive cows (milk and serum for milking and dry cows, respectively) for a sensitivity of 68% and cost of $2,226/super-shedder. In conclusion, diagnostic strategies varied in their cost-effectiveness depending on the tests, specimen type, and labor costs. Initial qPCR testing of environmental samples from free-stall pens to target cows in pens with the highest MAP bioburden for further testing can improve the cost-effectiveness of strategies for super-shedder identification.

Quantitative real-time PCR for detection of the neurotoxin gene of Clostridium botulinum type B in equine and bovine samples.

Clostridium botulinum type B is estimated to cause more than 85% of cases of equine botulism in the United States, as well as many outbreaks in cattle. In this study, a quantitative real-time polymerase chain reaction for detection of the neurotoxin gene of C. botulinum type B was compared to the mouse bioassay using 45 positive and 43 negative samples of equine, bovine or associated environmental origin. The sensitivity of the qPCR assay was 96%, whereas the sensitivity of the mouse bioassay was 84%. The specificity of the qPCR assay was 95% and the specificity of the mouse bioassay was 100%.

Evaluation of the in vitro activity of gallium nitrate against Mycobacterium avium subsp paratuberculosis.

OBJECTIVE: To evaluate the in vitro susceptibility of various field isolates of Mycobacterium avium subsp paratuberculosis (MAP) to gallium nitrate. SAMPLE: 10 isolates of MAP, including 4 isolated from cattle, 2 isolated from bison, 1 isolated from an alpaca, and 3 isolated from humans. PROCEDURES: The in vitro susceptibility to gallium nitrate was tested by use of broth culture with detection of MAP growth by means of a nonradiometric automated detection method. For each MAP isolate, a series of 7 dilutions of gallium nitrate (concentrations ranging from 200 to 1,000 µM) were tested. Gallium nitrate was considered to have caused 90% and 99% inhibition of the MAP growth when the time to detection for culture of the MAP stock solution and a specific concentration of gallium nitrate was delayed and was similar to that obtained for culture of the MAP stock solution (without the addition of gallium nitrate) diluted 1:10 and 1:100, respectively. RESULTS: Gallium nitrate inhibited MAP growth in all 10 isolates. The susceptibility to gallium nitrate was variable among isolates, and all isolates of MAP were inhibited in a dose-dependent manner. Overall, the concentration that resulted in 90% inhibition ranged from < 200 µM for the most susceptible isolates to 743 µM for the least susceptible isolates. CONCLUSIONS AND CLINICAL RELEVANCE: Gallium nitrate had activity against all 10 isolates of MAP tested in vitro and could potentially be used as a prophylactic agent to aid in the control of MAP infections during the neonatal period.

Type A botulism in horses in the United States: a review of the past ten years (1998-2008).

The objective of the current retrospective study was to describe naturally occurring type A botulism in horses in the United States. In the past 10 years, the Botulism Laboratory at the University of Pennsylvania's School of Veterinary Medicine has identified 3 isolated cases and 8 outbreaks of type A botulism in horses via samples positive for Clostridium botulinum type A toxin or spores using the mouse bioassay test. Additional information was obtained by review of submission forms and by telephone or email interviews. Almost all type A cases and outbreaks occurred in the western United States, with Oregon and Idaho overrepresented. Type A toxin was identified in only 1 outbreak; all other identified cases and outbreaks were positive for spores but not preformed toxin. Reported clinical signs included progressive muscle weakness, recumbency, decreased tail and/or tongue tone, dysphagia, respiratory distress, and death. Isolated cases involved foals < or =1 month of age; outbreaks involved horses > or =11 months. One hundred and nineteen horses were potentially exposed to the toxin source; 54 out of 119 showed signs of botulism, and 49 out of 54 affected horses were confirmed dead. The number of horses affected per outbreak ranged from 2 to 24. The source of infection was confirmed to be hay or silage in 6 out of 8 outbreaks and was unknown in 2 out of 8 outbreaks. The present report is the first description of outbreaks of type A botulism in horses and has important implications for prevention and treatment. Based on these findings, type A botulism should be considered in suspect cases of equine botulism in the western United States.

Comparison of three DNA preparation methods for real-time polymerase chain reaction confirmation of Mycobacterium avium subsp. paratuberculosis growth in an automated broth culture system.

Three methods of harvesting DNA from broth culture tubes for quantitative real-time polymerase chain reaction (qrtPCR) confirmation of Mycobacterium avium subsp. paratuberculosis (MAP) were evaluated. A commercial DNA extraction kit, the boil method (boiling for 5 minutes), or direct addition of broth culture media to the PCR reaction mix were tested. Samples were evaluated at 8 or 11 days of incubation and at the time of instrument-signal culture-positive. In total, when tested at time to instrument signal positive, 10/10 (100%) of samples extracted by the commercial method were positive on qrtPCR, whereas 9/10 (90%) were positive after the boil method, and 6/10 (60%) were positive after the direct method. Increased volumes of egg-yolk emulsion added to the culture tubes prolonged the number of cycles to threshold positive for the samples that were not subjected to commercial extraction or boiling. Samples were not reliably positive when tested at 8 or 11 days of incubation. The boil method appears to represent a reasonable time- and money-saving method to harvest DNA for qrtPCR confirmation of MAP in broth culture at time to instrument signal positive.