Culture phenotypes and molecular characterization of Mycobacterium avium subsp. paratuberculosis isolates from small ruminants.

In this study the suitability of different solid media was investigated for the isolation of Mycobacterium avium subsp. paratuberculosis (Map) in order to identify the optimum single or combination of media to permit the isolation of all strain types from small ruminants. A subset of these Map strains was then further characterized by molecular typing methods to assess the genetic diversity of Map strains in the study area (Northern Greece). Map strains were isolated from tissues and faeces of infected goats (n=52) and sheep (n=8) and were analysed for polymorphisms in IS1311 to classify the strain type as Type C or S. The study found that M7H11 supplemented with mycobactin j, OADC and new born calf serum (M7H11+Mj) is the best single choice of medium for the primary isolation of Map of both Type C and S from small ruminants. The combination of M7H11+Mj and Herrolds egg yolk medium supplemented with mycobactin j and sodium pyruvate allowed the detection of all Map isolates in this study. Nineteen Map isolates were characterised by pulsed-field gel electrophoresis and the isolates demonstrated significant genetic diversity. Twelve different SnaBI and 16 distinct SpeI profiles were detected of which 25 have not been described previously and are new profiles. The combination of both enzyme profiles gave 13 different multiplex profiles. Ten different multiplex profiles were detected in goats and three in sheep. One ovine isolate gave the same multiplex profile as a caprine isolate and two different profiles were found within a single goat herd.

Study of microbiological and molecular typing aspects of paratuberculosis in sheep and goats in Northern Greece.

Mycobacterium avium subsp. paratuberculosis, an acid-fast bacterium is the agent of Johne's disease, an intestinal disease that causes poor nutrient intake in ruminants. During the period 1987-2003, 322 of 777 (41.4%) goat herds and 97 of 458 (21.1%) sheep flocks were found to be infected with M.a.paratuberculosis in Northern Greece. From goats, mycobacteria were isolated from 238 of 652 (36.5%) of intestinal tissues, 14 of 119 (11.8%) of lymph nodes and five of 369 (1.4%) of faecal samples. From sheep, mycobacteria were isolated from 25 of 162 (15.4%) of intestinal tissues, three of 41 (7.3%) of lymph nodes and two of 322 (0.6%) of faecal samples. Isolates were typed by pulsed-field gel electrophoresis (PFGE) and restriction fragment length polymorphism followed by hybridization to IS900 [IS900- restriction fragment-length polymorphism (RFLP)]. IS900-RFLP BstEII profiles C1 and C5 and PFGE profiles [2-19] and [29-15] were identified. These PFGE profiles have not been found outside Greece to date.

Comparison of DNA probe test and cultivation methods for detection of Mycobacterium avium subsp paratuberculosis in caprine and ovine faeces.

OBJECTIVE: To compare a DNA probe test with two cultivation methods for the detection of Mycobacterium avium subsp paratuberculosis in goat and sheep faeces. DESIGN: Comparison of the results of the three methods with histological examination as the reference standard. PROCEDURE: Faecal specimens were obtained from goats and sheep originating from flocks known to be affected with paratuberculosis and tested for Mycobacterium avium subsp paratuberculosis with a DNA probe test and two cultivation methods (old conventional culture and new double incubation method in Herrold's and Lowenstein-Jensen medium). RESULTS: In goats, the sensitivity of the various tests were for the DNA probe test 17.2%, for the double incubation culture method 25.4% and for the old conventional culture method 22.8% using the histopathological results as reference. In sheep the sensitivity of the various tests were for the DNA probe test 13.2%, for the double incubation culture method 8.8% and for the old conventional culture method 5.9% using the histopathological results as reference. The specificity of the above tests was 100% in goats and sheep and the specificity of the double incubation culture method in goats was 91%. CONCLUSIONS: The DNA probe test is a rapid and specific test that could be used in a control program if the sensitivity of the test were improved and possibly in combination with another test.