Enhanced radiometric detection of Mycobacterium paratuberculosis by using filter-concentrated bovine fecal specimens

Abstract

A commercial radiometric medium, BACTEC 12B, was modified by addition of mycobactin, egg yolk suspension, and antibiotics (vancomycin, amphotericin B, and nalidixic acid). Decontaminated bovine fecal specimens were filter concentrated by using 3-,um-pore-size, 13-mm-diameter polycarbonate filters, and the entire filter was placed into the radiometric broth. Comparison of the radiometric technique with conventional methods on 603 cattle from 9 Mycobacterium paratuberculosis-infected herds found that of 75 positive specimens, the radiometric technique detected 92% while conventional methods detected 60% (P < 0.0005). Only 3.9% of radiometric cultures were contaminated. To measure the effect of filter concentration of specimens on the detection rate, 5 cattle with minimal and 5 with moderate ileum histopathology were sampled weekly for 3 weeks. M. paratuberculosis was detected in 33.3% of nonfiltered specimens and 76.7% of filtered specimens (P < 0.005). Detection rates were directly correlated with the severity of disease, and the advantage of specimen concentration was greatest on fecal specimens from cattle with low-grade infections. Detection times were also correlated with infection severity: 13.4 5.9 days with smear-positive specimens, 27.9 8.7 days with feces from cows with typical subclinical infections, and 38.7 ± 3.8 days with fecal specimens from cows with low-grade infections. Use of a cocktail of vancomycin, amphotericin B, and nalidixic acid for selective suppression of nonmycobacterial contaminants was better than the commercial product PANTA (Becton Dickinson Microbiologic Systems, Towson, Md.) only when specimens contained very low numbers of M. paratuberculosis. Radiometric culture of filter-concentrated specimens generally doubled the number of positive fecal specimens detected over conventional methods, making it a useful tool for diagnosis and control of bovine paratuberculosis. Bovine paratuberculosis (Johne's disease) affects at least 2.9% of the 10.3 million dairy cattle in the United States (23) and is equally prevalent in most other countries where laboratory diagnosis of the infection is possible (34). The prevalence of paratuberculosis in some states exceeds 15% (3, 4, 41). The national economic impact has been estimated to be as high as $1.5 billion annually (12). The etiological agent, Mycobacterium paratuberculosis, is among the slowest growing of the cultivable mycobacteria, normally requiring 8 to 16 weeks to produce visible colonies on conventional agar media (40). It infects the terminal ileum of most ruminants and is excreted in the feces. Control of the disease in a dairy cattle herd involves hygienic measures to avoid exposure of calves to the organism or removal of infected animals from the herd (particularly those excreting M. paratuberculosis) or both (5, 10, 20, 29, 36). Damato and Collins reported detection of M. paratuber

Infection rates in reactors to an absorbed ELISA used in a test and cull program for bovine Johne's disease

“…M a paratuberculosis is relatively difficult to culture and the organisms might be too few in number in the tissues collected. 8 One possible criticism of this study is the potential for cross-contamination to cause culture confirmation rates to be artificially high.…”

Whole-Genome association analysis of susceptibility to paratuberculosis in Holstein cattle

“…Diagnosis of MAP infection was based on faecal culture of MAP (Collins et al 1990) or evidence of antibody titre to MAP as based on an ELISA test (Shin et al 2008) of blood samples. Results from the ELISA were categorized as strongpositive, positive, low-positive, suspect or negative for sample:positive ratios of ‡1.00, 0.40 to <1.00, 0.25 to <0.40, 0.10 to <0.25, 0 < 0.10, respectively.…”

Heat inactivation data for Mycobacterium avium subsp. paratuberculosis: implications for interpretation

“…Hence, recovery of injured MAP cells in an appropriate medium after heat treatment is very important and may in¯uence MAP survival counts (Damato and Collins 1990;Keswani and Frank 1998). Liquid media seem to be more advantageous to the recovery of sublethally injured cells than solid media (Collins et al 1988;Sockett et al 1992). The resuscitation of MAP cells after heat treatment in a liquid medium followed by plating on a solid medium may, therefore, result in higher colony counts.…”