The gold standard for paratuberculosis diagnosis in ruminants is culture. This technique is 100% specific but occasionally the sensitivity is low due to: 1) certain M. a. paratuberculosis strains that are difficult to grow (sheep type); 2) biological sample; 3) clinical stage of the animal; and 4) intermittent shedding of M. a. paratuberculosis in faeces.The objective of this study was to determine the usefulness of a complementary diagnostic technique to improve the sensitivity of the diagnosis. The protocol is based on a direct DNA extraction from tissues (ileocecal valve and lymph node, and mesenteric lymph node), and specific PCRs aimed at the IS900 and f57 sequences. Positive and negative controls were included in all the tests to evaluate the performance of the method. A total of two hundred and thirty-six tissue samples from two different farms, where paratuberculosis was confirmed by cultured, were tested: 105 samples from a caprine flock, and 131 tissue samples from a cattle herd. The selection of this group of animals was made on basis of the type of M. a. paratuberculosis strains (Collins et al. 2002), since they were infected with M. a. paratuberculosis isolates belonging to the cattle and sheep type, respectively.The application of this new technique based on direct DNA extraction allowed the detection of animals both culture positive and negative. This fact is really important mainly in farms with animals infected with M. a. paratuberculosis strains belonging to the sheep type, since isolation of the agent is difficult and in several occasions false negative results are obtained. An extra advantage to this protocol is that results are obtained in two days when compared with the long incubation period for culture. Moreover, the DNA obtained could be subjected to a classification by PCR for epidemiological purposes, allowing a prompt implementation of control measures.

Sponsorship

Attendance to this Congress was sponsored by EU-funded project SSPE-CT-2004-501903.