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 <= 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.