Study of epidemiology and pathogenesis of paratuberculosis using RFLP (Restriction Fragment Length Polymorphism)

RFLP analysis (Restriction Fragment Length Polymorphism) has been used for the study of epidemiology of paratuberculosis In the present study, DNA of 336 M. paratuberculosis strains were examined by RFLP using restriction endonuclease PstI (in selected 306 strains, restriction endonuclease BstEII was used in parallel) and a specific probe IS900. After digestion with the restriction endonuclease PstI the strains were differentiated into 9 different RFLP types named A (n = 90), B (n = 164), C (n = 4), D (n = 16), E (n = 53), F (n = I), G (n = 1), H (n = 6), I (n = 1). After digestion with the restriction endonuclease BstEII, the strains were differentiated into 14 different RFLP types named according previously presented results by Collins et al., 1990 IJ. Clin. Microbiol., 28:1591-1596). CI (n = 173), C2 (n = 2), C3 (n = 8), C5 (n = 1), SI (n = 4) and II (n = 2). Other strains of 8 RFLP types were provisionally named C8-C15 (n = 116). In the group of RFLP strains type A, C8, CIO and C12 were found after the digestion with the restriction endonuclease BstEII. In the group of RFLP type B, highest heterogeneity was found after digestion with the restriction endonuclease BstEII (RFLP types C1-3, C5, C9, CII, C12, C14 and 15). The strains of RFLP type C were of RFLP type SI, the strains of RFLP type D were of RFLP type C12, the strains of RFLP type E were RFLP type CI, the strains of RFLP types F and G were of RFLP type II, RFLP type H was of RFLP type CI and RFLP type I was RFLP type C13. Systematic long-term investigation allowed us to perform a regressive epidemiological study, on spreading of the infection in cattle herds of the Czech Republic during the recent nine year. In 5 farms located in one region the isolated strains were of the same RFLP type during 3 years. High regional homogeneity can be explained by the same infection source in pastured cattle. Since 1988 RFLP types A-E, I and C1-2, C8-10, C12-13 and SI of M. paratuberculosis have been isolated from ruminants in the Czech Republic. This variety of the isolated RFLP types indicates different infection sources in ruminants kept in the Czech Republic. RFLP types of strains isolated from imported ruminants including beef cattle breeds (Blonde-d'Aquitaine ,Limousine, Charolais, Hereford) and dairy, cows (Jersey, Holstein, Mont Belliarde) from various European countries, e.g. Holland, Denmark, France etc., and from goats and capricorns (Capra cylindricornis) in the Zoo were determined Strains isolated from ruminants and obtained from different laboratories throughout Europe, the USA, Australia and New Zealand were also analysed as well as strains isolated from patients with Crohns disease (USA, Holland, Australia and the Czech Republic). Identification of different RFLP types allowed to monitor both vertical and horizontal spreading of the infection in herds with imported cattle infected with strains of different RFLP types and in infection-free cattle bred ipl the Czech Republic. It was found, using the same methods, that calves up the age of 1-2 months after birth being in contact with infected cow are the most susceptible.