Evaluation of radiometric faecal culture and direct PCR on pooled faeces for detection of Mycobacterium avium subsp. paratuberculosis in cattle

Abstract

Dilution rates for pooled faecal culture (PFC) and direct IS900 polymerase chain reaction (D-PCR) tests were evaluated on faecal samples from infected cows mixed with uninfected faeces in dilutions from 1 in 5 to 1 in 50. PFC was performed by radiometric culture, with confirmation by IS900 PCR and restriction endonuclease analysis (PCR/REA) on growth, and by mycobactin dependency testing on solid medium. Using 37 culture positive faecal samples from 12 subclinical cows, 83.8% and 94.6% of samples were confirmed positive in the PFC assay at dilutions of 1 in 50 and 1 in 30, respectively. Lower dilutions (1 in 5 to 1 in 20) provided only marginally better sensitivity, and confirmation of PFC growth by PCR/REA was significantly more sensitive than mycobactin dependency. D-PCR had significantly lower sensitivity than PFC confirmed by PCR/REA, with pools of 1 in 50, 30, 10 and 5 yielding positive results in 64.9%, 70.3%, 78.4% and 83.8% of samples, respectively. Cattle considered to be shedding 1.5 × 10⁶ viable M. avium subsp. paratuberculosis (Map)/g faeces (on the basis of estimated losses in processing and growth rates in radiometric broth) were positive at dilutions up to 1 in 50 in the PFC and D-PCR. Those shedding 5 × 10⁵ viable Map/g were positive in the PFC at dilutions up to 1 in 40, but required a 1 in 10 dilution or less for D-PCR. The results suggest that for cattle shedding relatively high concentrations of Map in faeces (>2 × 10⁵ viable Map/g), maximal dilutions of 1 in 30 for PFC and 1 in 10 for D-PCR would be applicable.

Introduction

Strategies that reduce the cost of testing for Johne's disease or the time taken to identify infected animals and certify herds are key objectives of control programs for this disease. In Australia, pooled faecal culture (PFC) of sheep, based on radiometric culture detection of as few as one shedder animal among 49 cohorts, is currently used in disease control and certification testing (Sergeant et al., 2002). However, this technique has not been well documented in cattle, and to date most investigations of PFC have been based on culture on solid media such as Herrold's egg yolk (HEY) with varying decontamination regimes on the faecal samples (Kalis et al., 2000, Wells et al., 2002, Wells et al., 2003, Kalis et al., 2004). Non-radiometric pooled faecal culture has also been examined, but did not demonstrate improved sensitivity compared to HEY (Tavornpanich et al., 2004). Another limitation of these studies was their low pooling rates of 5–10 samples per pool.

Culture incubation times of 12–20 weeks (conventional) or 8 weeks (radiometric) are commonly used to detect Mycobacterium avium subsp. paratuberculosis (Map) in bovine faecal samples, and growth then requires confirmation by mycobactin dependency, growth rate, and/or molecular-based tests such as IS900 PCR and restriction endonuclease (REA) analysis (Eamens et al., 2000, Whitlock et al., 2000, Cousins et al., 2002). To reduce the time taken to detect infection and certify herds, several diagnostic serological tests based on ELISA have been developed (Cousins et al., 2002). These tests are often less sensitive and specific in individual animals than culture-based systems (Whitlock et al., 2000). Efforts to develop specific direct PCR tests on faeces have resulted in a range of assays with variable sensitivities compared to culture. In some studies, conventional or nested PCR procedures have been less sensitive than culture in faeces (Collins et al., 1993, Stabel et al., 2004, Wells et al., 2006) or tissues (Erume et al., 2001), while in others, nested and real time PCR have been considered more sensitive for faeces than culture (Bögli-Stuber et al., 2005, Christopher-Hennings et al., 2003). Several reports confirm that PCR sensitivity, like culture sensitivity, is related to the shedding rate of the individual cattle under test (Collins et al., 1993, Christopher-Hennings et al., 2003, Stabel et al., 2004, Taddei et al., 2004, Wells et al., 2006). However, comparisons of direct PCR and culture in cattle faecal samples have previously been based on culture using solid media with varying protocols to concentrate and decontaminate the Map inoculum by sedimentation (Collins et al., 1993, Taddei et al., 2004), double centrifugation and decontamination (Barrington et al., 2003, Stabel et al., 2004) or by a range of sedimentation, or centrifugation, or double centrifugation methods (Christopher-Hennings et al., 2003).

Earlier radiometric culture studies in our laboratory of 16 cattle found to be shedding Map showed that sub-samples of faecal samples collected rectally were uniform in Map concentration for individual animals, although the concentrations varied between days over 2 weeks (Eamens et al., 2007). This information is important in selecting a sampling strategy suitable for culture or DNA-based diagnostic tests on small amounts of bovine faeces. Faecal samples that had been stored from the earlier study were used to assess the radiometric culture detection rate of dilutions of selected samples in normal bovine faeces, to determine appropriate dilutions for detection of Map in infected herds.