Isolation of Mycobacterium avium subsp. paratuberculosis(Map) by faecal culture is the gold standard tests to in vivo diagnose paratuberculosis in infected animals. Culture is supposed to be able of detecting around 50-100 cells/g of faeces. But Map shedding varies according to the immuno-pathological status of animals. The presence of sub-clinically infected animals and other factors including infection with strains difficult to grow reduce considerably the sensitivity of this method. In addition, Map culture is expensive, slow and laborious. In this study preliminary results of a strategy based on cross-pooled faecal culture and individual faecal PCR for herd follow up are presented.

The cross-pooled culture method lies in crossing pools of 5 samples in 5x5 squares of samples. This allows direct identification of shedder animals in the pool without subsequent individual culture for confirmation. During the setting up of the method results of pooled and individual cultures were compared. A reduction of 43% in the number of cultures needed to detect all positive animals was observed in a herd with a prevalence of 10.2%. Once the cross-pooled culture method was adopted 699 bovine faecal samples were used to inoculate 2 Herrold´s egg yolk (HEY) and 2 Lowenstein-Jensen (LJ) slants. Individual PCR (conventional PCR in the beginning and real-time PCR later; Adiavet kits) was used for confirmation of inconclusive results of cross-pooled cultures. Later all individual faecal samples were tested by PCR and compared with the previous culture results.

Six-hundred and ten samples were directly diagnosed by cross-pooled culture, 21 classified as positive and 589 as negative. Eighty-nine samples needed individual PCR to be correctly identified, 19 were finally classified as positive and 70 as negative. Agreement between definitive culture qualifications and individual PCR general results was observed in 639 samples. Thirty-three out of those 639 were identified as positive, while 606 were identified as negative. Surprisingly, the individual PCR detected 53 positive samples not identified by pooled culture.

The reduction in culture number using cross-pooled culture compared to individual culture was of 59.9%. But it should be considered that PCR was needed to confirm the final culture result in 89 samples. Our results also indicate that individual PCR alone can perform better than the cross-pooled culture in herd monitoring strategies. Nevertheless, this pooled culture method is still interesting when culture is the selected test for paratuberculosis screening of herds.