Diagnosis, genotyping and epidemiology of mycobacterium avium subspecies paratuberculosis (MAP) in dairy cattle
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In the present dissertation four studies related to the diagnosis, genotyping, and epidemiology of MAP in dairy cattle were carried out. In the first study, IS900-RFLP, MIRU-VNTR, and MLSSR genotyping methods were evaluated for further application. From these methods, MIRU-VNTR and MLSSR were superior compared to RFLP-IS900 in terms of performance and convenience and were chosen as the primary option for further genotyping of MAP along this dissertation. However, some MIRU-VNTR loci showed no discriminability in MAP strains and were discarded and replaced with the MIRU-VNTR loci 1658, 292, 25, 47, 3, 7, 10, 32, and 259. In the second study, serum and fecal samples from asymptomatic cows (n=307) of 14 dairy herds from Colombia were screened for MAP by serum ELISA, fecal PCR and fecal culture. Fecal samples from animals of herds positive by ELISA and PCR (n=105) were inoculated onto 3 different culture media. ELISA-A produced positive results in 10.1% of the serum samples and 71% of the herds. ELISA-B and PCR results were positive in 2 and 6 serum and fecal samples from positive ELISA-A-animals, respectively. Fecal samples were negative for MAP on all culture media. Individual ELISA-A test results and collected information on individual animal features and management herd practices were analyzed for risk factors determination. The herd management factors Measures taken in the past with symptomatic animals, Feed type of calves before weaning, and Manure spread on pastures were significantly associated with the individual serum ELISA response on the univariate analysis. In the logistic regression, only the factor Manure spread on pastures was significantly associated with the individual serum ELISA response. In the second part of this study, serum and feces from animals of five suspicions dairy herds of the first part (i. e. simultaneously positive by ELISA and PCR) were tested by a different ELISA, pooled fecal culture, and PCR. In one herd, slurry and tissue samples from one animal were also taken and tested by PCR and culture. MAP isolates were genotyped by analysis of the MLSSR and the MIRU-VNTR methods. ELISA produced positive results in 1.8% (6/329) of the animals and 40% (2/5) of the herds. Four fecal, two tissue, and two slurry samples from a herd were positive for MAP by culture and PCR. Genotyping revealed two different strain profiles among the eight MAP isolates recovered. In the third study, MIRU-VNTR and MLSSR genotypes of MAP isolated from different hosts in Chile, Colombia, Argentina, and Venezuela were compared. So far, seven different MAP genotypes were produced by MIRU-VNTR and MLSSR in South American isolates, respectively. The combination of both methods produced 9 genotypes. Results revealed a predominant combined MIRU-VNTR and MLSSR profile, little differences in MAP genotypes among countries, and a similar MAP-genotype in livestock and in wild animals in one country. In the forth study, 91 MAP isolates from 71 dairy herds of Rhineland-Palatinate were genotyped by MIRU-VNTR and MLSSR. The combined analysis of both methods produced 25 genotypes with an index of discrimination (D) of 0.93 and the dominance of 2 genotypes. The results revealed the usefulness of genotyping methods in studies at regional scale, the high genetic diversity of MAP from cattle in Rhineland-Palatinate, and provided additional information for control programs currently carried out in the region.The main conclusion of the dissertation is that tools for diagnosis and genotyping of MAP were very useful to increase the knowledge of paratuberculosis in Colombia and Germany. In addition, all methods used in the present dissertation can be considered more or less imperfect and required strategic use and combination with other methods to increase accuracy.Verknüpfung zu Publikationen oder weiteren Datensätzen
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Giessen : VVB Laufersweiler