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New molecular high throughput methods for Ehrlichia ruminantium tick screening and characterization of strain genetic structure in Mozambique and at worldwide scale

Cangi N.. 2017. Pointe-à-Pitre : Université des Antilles, 166 p.. Thesis Ph.D. -- Life science. Molecular biology and Genetic.

Ehrlichia ruminantium is the causal agent of heartwater, a ruminant tropical fatal disease transmitted by Amblyomma ticks. Up to now, no effective vaccine is available due to a limited cross protection of vaccinal strains on field isolates mainly associated to a high genetic diversity of E. ruminantium within geographical locations. Thus, both characterization of E. ruminantium genetic population structure at worldwide and regional scale and estimation of E. ruminantium tick prevalence are important to delimitate better control strategies and improve heartwater monitoring strategies. In Section I, we developed two new qPCR s, pCS20 Sol1 T q M and Sol1 SG, to scree n E. ruminantium in Amblyomma ticks, which are powerful tools for: 1) heartwater epidemiological studies, 2) diagnosis in the context of heartwater clinical cases and 3) follow - up of experimental infections, both in ticks and hosts. The pCS20 Sol1 T q M qPCR was found as sensitive (up to 30 copies/ reaction ) and specific as the gold standard pCS20 nested PCR but less prone to sample contamination and less time - consuming. The whole method including the automated DNA extraction and pCS20 Sol1 T q M qPCR demonstrate d to be sensitive, specific and reproducible. It displayed the same limit of detection of the manual DNA extraction and pCS20 nested PCR, (60 copies/ reaction). Moreover, the development of a high - throughput automated DNA/RNA extraction makes the screen of any tick - borne pathogen in several tick species possible. The development of this new method allowed processing of a high number of tick samples collected in Mozambique that were then typed by Multi Locus Sequence Typing (MLST) and included into a worldwide E. ruminantium strain genetic structure study ( Section II ). Our study reveals the repeated occurrence of recombination between E. ruminantium genotypes and its important role in E. ruminantium genetic div ersity and evolution. Despite the unclear phylogeny and phylogeography due to recombination events, E. ruminantium isolates are clustered into two main groups: Group 1 (West Africa) and a Group 2 (worldwide) which is represented by West, East and South Afr ica, Indian Ocean and Caribbean strains. Common genotypes between West Africa and Caribbean and Southern Africa and Indian Ocean allow to identify two possible ways of E. ruminantium introduction in these regions, associated with cattle movement. In S ecti on III, we focused mainly on E. ruminantium tick prevalence and genetic diversity and structure of Mozambican isolates from A. variegatum and A. hebraeum ticks collected in cattle and wildlife. Sampling was performed in 30 localities for Mozambique and in Kruger National Park (KNP, South Africa). E. ruminantium tick prevalence in cattle was between 0% [0 - 23.2 %] and 26.7% [12 - 45 %], with no infected ticks in 7 localities. In wildlif e, tick prevalence was 8.2 [4 - 14.6 %] % in the KNP and 6.2% [0.2 - 30.2 %] in hunting concessions of Sofala province. However, no significant difference in prevalence was found between sampling sites and tick species, as well as no linear correlation between E. ruminantium prevalence and tick abundance was observed. There was a high genetic diversity of E. ruminantium, with 39 different genotypes detected and distribution of identical genotypes in several distant localities. Most genotypes from Mozambique clu stered in genetic subgroup G2C (strictly clustering Zimbabwe and Mozambican isolates) and G2E. Interestingly, genotypes from group G1 and G2D associated mainly with West Africa and Caribbean strains were in minority , probably highlighting a recent introduction. (Résumé d'auteur)...

Mots-clés : ehrlichia; agent pathogène; maladie transmissible par tiques; méthodologie; diagnostic; test biologique; biologie moléculaire; amblyomma; amblyomma hebraeum; amblyomma variegatum; distribution géographique; variation génétique; génétique des populations; Épidémiologie; pcr; ehrlichia ruminantium; mozambique; afrique; caraïbes; océan indien

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