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°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 × 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 × 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.