Development and validation of an inexpensive and efficient method for the extraction of Theileria orientalis DNA from blood.

Theileria orientalis is an emerging bovine pathogen in Australasia. PCR-based detection methods for this parasite are sensitive but relatively expensive, partly due to costs associated with DNA extraction. An inexpensive and efficient technique was developed for the extraction of T. orientalis DNA from blood based on hypotonic erythrocyte lysis and detergent-proteinase K treatment (DPK method). The DPK method compares favourably to a commercial extraction kit when paired with a T. orientalis multiplex qPCR.

Isolation of Mycobacterium avium subspecies paratuberculosis from muscle and peripheral lymph nodes using acid-pepsin digest prior to BACTEC culture.

Meat has received little attention regarding human exposure to Mycobacterium avium subsp. paratuberculosis, a possible infectious trigger of Crohn's disease. Meat has less contamination with other organisms than gut tissues, facilitating modifications to existing decontamination protocols prior to BACTEC culture that could increase analytical sensitivity. Using spiked meat samples we trialled enzymatic and chemical digestion techniques to concentrate larger starting samples, and modifications to existing clinical mycobacteriological decontamination protocols. An acid-pepsin digestion method using a 20 g sample was considerably more sensitive (detection limit 0.88 log(10) viable organisms per gram) than previous techniques. However, it was cumbersome for routine use, and subject to frequent contamination. Modifications to an existing centrifugation protocol yielded a simple, robust technique with slightly improved sensitivity (detection limit 1.77 log(10) per gram). Use of these sensitive tests in parallel identified M. a. paratuberculosis in the muscle of 59% and peripheral lymph nodes (PLN) of 85% of clinically infected sheep. The numbers of M. a. paratuberculosis in these infected tissues were low (1.67+/-0.92 log(10) per gram in muscle and 2.06+/-0.69 log(10) per gram in PLN), such that many would not have been detected by routine methods. Fewer subclinically infected animals with gross lesions harboured M. a. paratuberculosis in meat (4.5%) or PLN (32%), and the numbers of organisms in such infected animals were lower. Because most animals raised specifically for meat production are young and unlikely to be heavily infected, and because meat is usually consumed cooked, the risk of human exposure to viable M. a. paratuberculosis via meat may be small. Measures to prevent heavily infected animals, especially those with clinical signs, from entering the human food chain would further reduce this risk.

Radiometric pooled faecal culture for the detection of Mycobacterium avium subsp paratuberculosis in low-shedder cattle.

OBJECTIVE: To identify the optimum pooling rate for pooled faecal culture (PFC) as a diagnostic tool in bovine Johne's disease control, for detection of cattle shedding low concentrations of Mycobacterium avium subsp paratuberculosis (Map). METHOD: Thirteen target animals were selected by delayed growth of Map from initial individual radiometric faecal cultures (first growth index at 5 weeks or later). A procedure based on radiometric culture and IS900 polymerase chain reaction and restriction endonuclease analysis confirmation was then used for PFC. RESULTS: Eight samples (stored for up to 17 months at -80 degrees C) yielded Map on subsequent culture, either from undiluted faeces or those mixed with normal cattle faeces at dilution rates from 1 in 5 to 1 in 50. From a regression equation, culture-positive animals were considered to be shedding relatively low levels of Map (< 6 x 10(4)/g of faeces). Pooling dilutions of more than 1 in 5 reduced PFC sensitivity. A minimum incubation period of 10 weeks at a dilution of 1 in 5 is recommended to detect such infected cattle. This pooling rate in radiometric culture is probably capable of detecting cattle shedding < or = 5 x 10(3) Map organisms/g of faeces, representing an estimated inoculum per culture vial of fewer than 20 viable organisms. CONCLUSION: Map was detected in more than 50% of the stored faecal samples from cattle shedding low concentrations of the organism. A pooling rate of 5 samples per pool is required to reliably detect infected low-shedder cattle using PFC based on radiometric culture.

Pooled faecal culture for the detection of Mycobacterium avium subsp paratuberculosis in goats.

OBJECTIVE: To evaluate pooled faecal culture for herd diagnosis of caprine Johne's disease and relate these findings to faecal shedding rates of Mycobacterium avium subsp paratuberculosis (Map). DESIGN: Radiometric broth culture was applied to several pooling dilutions, and shedding rates were estimated from a regression equation based on bacterial growth rates and known processing losses during radiometric culture. PROCEDURE: Sixteen faecal samples from goats naturally infected with sheep (n = 3) or cattle (n = 13) strains of Map, were diluted in normal goat faeces from 1 in 5 to 1 in 50. Cultures were confirmed by IS900 polymerase chain reaction and restriction endonuclease analysis, and mycobactin dependency. The numbers of viable Map in the culture inocula were determined by endpoint titration (most probable number) of nine samples and related to a cumulative growth index. RESULTS: A pooling dilution of 1 in 25 with an incubation period of 10 weeks detected 13 of 16 culture positive goats, all shedding > or = 2 x 10(4) Map per gram of faeces. Two samples containing very low numbers of Map (< 2 x 10(3)/g) were only culture positive from undiluted faeces. Thirteen of 16 goats were considered to be shedding low to moderate concentrations of Map (< 2 x 10(5)/g faeces). CONCLUSIONS: These data support a pooling dilution of 1 in 25 for application of pooled faecal culture as a diagnostic tool in caprine Johne's disease control. A test based on this dilution would reduce laboratory costs of whole herd testing in goats by approximately 40% relative to serology and 75 to 90% relative to individual faecal culture.