Validation of new diagnostic tests for Caprine Johne's Disease: pooled f aecal culture and direct PCR on pooled goat faeces

Summary

Whole herd faecal culture, based on individual culture of samples, is recognised as a sensitive, but expensive diagnostic tool to evaluate herd infection rates of M. avium subsp. paratuberculosis (Map) in goats. Pooled faecal culture (PFC), based on radiometric (Bactec) culture procedures with confirmation by IS900 PCR and REA, has been proven to offer cost savings in detecting and evaluating infection rates in sheep flocks at a 1:50 pooling dilution, but this technology has not been validated in goats. 
This study evaluated PFC at dilutions ranging from 1:5 to 1:50 in goats shedding a wide range of concentrations of Map. Since prior work with sheep samples by Reddacliff et al (2003a) showed Map concentrations in inocula for Bactec culture correlate with their growth rate in the culture media, this approach was adopted to quantify the Map shedding rate of the animals under test. Faeces from 17 goats naturally infected with the cattle (C) strain and four goats naturally infected with the sheep (S) strain of Map were evaluated. Of 21 faeces from goats previously confirmed culture positive for Map, and stored at -80oC for up to 4 years, 14 were found to yield Map on subsequent culture, including evaluation studies when samples were mixed with normal goat faeces at pooling rates of 1:5, 1:10, 1:20, 1:25. 1:30 and 1:50. 
An additional two samples yielded Map only on undiluted samples, and both were only intermittently positive in undiluted faeces. All samples were processed using procedures similar to those employed for OJD PFC, including a 12 week incubation period. Depending on the infecting strain, subcultures were made on Herrold's egg yolk medium (for C strain) or modified 7H10 media (for S strain), as the former is more suitable for growth of cattle strains of Map, and S strains only grow on modified 7H10 medium and not Herrold's.