Development and Application of Quantitative Polymerase Chain Reaction Assay Based on the ABI 7700 System (TaqMan) for Detection and Quantification of Mycobacterium Avium Subsp. Paratuberculosis

Abstract

Abstract. Numerous reports have described diagnostic methods based on the polymerase chain reaction (PCR) used to detect Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease. The result of conventional PCR tests has been only qualitative, either positive or negative; it does not present any quantitative information about the number of the agents in the specimen. A quantitative PCR method (IS900 TaqMan) zooepidemicus).Even at high cell numbers (10 5 CFU/reaction), most of the organisms tested negative for the IS900 insertion element except M. marinum and M. scrofulaceum. This finding for M. scrofulaceum was consistent with previous reports that several M. scrofulaceum-like isolates were positive for IS900. Those isolates had 71-79% homology with M. a. paratuberculosis in the region of IS900. When used in conjunction with the new liquid medium-based ESP culture system II for bovine clinical fecal samples, IS900 TaqMan confirmed that the ESP II-positive samples contained 10 5 -10 6 CFU/ml of M. a. paratuberculosis. All of the 222 ESP II-positive and acid-fast bacilli-positive samples tested in this study were positive by IS900 TaqMan. IS900 TaqMan was also useful in the study of growth characteristics of 3 groups of M. a. paratuberculosis strains in bovine fecal samples from 3 shedding levels (heavy, medium, and low) based on cell numbers measured by Herrold egg yolk (HEY) agar culture. When cultured in ESP medium, M. a. paratuberculosis reached 10 5 -10 6 CFU/ml within 2 weeks for heavy shedders, 3-4 weeks for medium, and 6-8 weeks for low shedders. No significant growth was observed after up to 5 weeks of incubation for some of low shedders. No or extremely slow growth characteristic of low shedders might be a possible explanation for frequent false-negative results by HEY. The detection time was dependent on the inoculum size and the growth rate of M. a. paratuberculosis. Generation times were inversely proportional to the shedding level: 1-2 days for medium and heavy shedders and Ͼ4 days for low shedders. IS900 TaqMan could be a useful tool for determining viable cell counts by measuring changes in cell numbers over the incubation period.

Comparison of Milk and Serum Enzyme–Linked Immunosorbent Assays for Diagnosis ofMycobacterium AviumSubspeciesParatuberculosisInfection in Dairy Cattle

“…15,29 Briefly, 2 g of specimen was resuspended in 35 ml of distilled water and shaken to disperse visible clumps. After 30 min, a 5-ml aliquot was removed and transferred to a solution containing 0.9% (w/v) hexadecylpyridium chloride in half-strength brain heart infusion (BHI) broth.…”

“…15 Each specimen was tested in duplicate wells of a 96-well plate, using a total of 5 ml of extracted DNA in a 25-ml reaction, prepared with a universal master mix as supplied by the manufacturer. k Thermocycling profiles consisted of an initial 10-min denaturation step at 95uC, followed by 40 cycles of a 25-sec denaturation step at 94uC, plus a 1-min annealing/extension step at 66uC.…”

Effect of Egg Yolk on the Detection ofMycobacterium AviumSubsp.ParatuberculosisUsing the ESP II Liquid Culture System

“…For example, some comparative culture studies have classified specimens based on the severity of histopathologic lesions and the number of mycobacteria observed in acid-faststained tissue sections, 3,25 whereas others have utilized a more direct approach and categorized animals as high, moderate, and low shedders based on bacterial counts from cultured specimens. 9 Even among studies that directly quantitate bacterial counts, cutoff points vary for the various shedding status levels. 9,21 Thus, comparison of results from different laboratories and culture methods need to be interpreted with caution.…”

“…All acid-fast-positive cultures were confirmed as M. paratuberculosis using a previously described quantitative PCR method targeting the IS900 insertion element. 9 For all positive cultures, the time to detection was considered to be the earliest positive signal recorded by the instrument that correlated with both a positive acid-fast stain and positive IS900 PCR result.…”

“…All specimens were processed for decontamination using a previously published method. 9 Briefly, 2 g of feces were resuspended in 35 ml of distilled water and shaken to disperse visible clumps. After 30 min, a 5-ml aliquot was removed and transferred to a solution containing 0.9% (w/v) hexadecylpyridipium chloride (HPC) b in half-strength Brain Heart Infusion broth.…”