Contribution of qPCR in detection of infections of ruminants with Anaplasmataceae: case study in the Basque country and in Corsica

Les Anaplasmataceae sont des bactéries intracellulaires transmises aux animaux et à l’homme, principalement par les tiques. Les Anaplasma spp. (A. marginale, A. phagocytophilum…) et les Ehrlichia spp. (E. canis, E. ruminantium...) en sont les principaux représentants d’importance économique et sanitaire. Les outils de diagnostic disponibles jouent un rôle déterminant dans leur identification. Nous proposons une approche globale par un système permettant d’identifier la plupart des espèces d’Anaplasmataceae. Il est basé sur le gène codant l’ARNr 23S ; il couple une qPCR et une PCR standard avec deux jeux d’amorces suivie d’un séquençage. Il est testé pour en vérifier la spécificité, puis mis en oeuvre à partir de prélèvements de sang de ruminants et de tiques : dans les Pyrénées-Atlantiques, des brebis suspectes d’infection sont porteuses d’A. ovis et leurs tiques Rhipicephalus bursa, d’A. ovis, d’A. phagocytophilum et d’une nouvelle ehrlichia. En Haute-Corse, une forte prévalence d’infections par les Anaplasmataceae est détectée dans des élevages (bovins, ovins, caprins) où la symptomatologie rétrocède lors de traitements à l’oxytétracycline. La spécificité et la sensibilité du système de diagnostic utilisé et sa capacité à identifier de nouvelles espèces offrent des perspectives pour l’étude de l’épidémiologie des Anaplasmataceae et permettront de mettre en évidence de nouveaux réservoirs.The intracelullar bacteria Anaplasmataceae are transmitted by ticks both to animals and to man. The most important species for the economy and for health are Anaplasma spp. (A. marginale, A. phagocytophilum…) and Ehrlichia spp. (E. canis and E. ruminantium...). Whilst current means of diagnosis are indispensible they could be improved. We therefore propose a global approach which will identify most of the species of Anaplasmataceae. It is based on the genetic code ARNr 23S and involves dividing a qPCR into a standard PCR and sequencing. This specificity has been tested, verified and demonstrated in blood samples from ruminants and ticks. Sheep (from Atlantic-Pyrenees) suspected of being infected, were shown to be carriers of A. ovis and their ticks (Rhipicephalus bursa) had A. ovis, A. phagocytophilum and also a new species of ehrlichia. In Corsica, a strong incidence of Anaplasmataceae was detected in the blood of cattle, sheep and goats in which symptoms antedated treatment with oxytetracycline. The specificity and sensibility of this method of diagnosis, as well as its ability to detect new species, are advantageous for further studies of Anaplasmataceae, and facilitate the search for new sources of infection

Detecting zoonotic and non‐zoonotic pathogens in livestock and their ticks in Corsican wetlands

Abstract Background Corsica is a large French island in the Mediterranean Sea with high human and animal migration rates, especially near wetlands where these migrations are particularly frequent. Among the livestock populations, cattle and sheep are widely present all across the entire Mediterranean region. Trade can be responsible for the circulation of numerous pathogens and their vectors, thereby representing a health and economic threat for the livestock industry. Objectives The objective of our study was to investigate the presence of pathogens in cattle and sheep farms in the wetlands of Corsica using a high‐throughput screening technique. Methods In our study, blood samples and ticks were collected from cattle and sheep in 20 municipalities near Corsican wetlands to screen for the presence of various types of pathogens. The samples were processed using a high‐throughput screening technique based on real‐time microfluidic PCR: 45 pathogens were screened in 47 samples simultaneously. Results A total of 372 cattle and 74 sheep were sampled, and 444 ticks were collected from cattle. Out of the eight tick species detected, the main one was Rhipicephalus bursa (38.7% of the ticks collected). From cattle blood samples, one species and two genera were found: Anaplasma marginale, Trypanosoma sp. and Babesia sp. in respectively 61.5%, 58.3% and 12.2% of the cattle blood samples. From sheep blood samples, 74.3% were positive for Anaplasma sp, 2.7% for Anaplasma ovis and 1.4% for Anaplasma capra. This is the first report of A. ovis DNA in blood samples from sheep in Corsica. Out of 444 the tick samples, 114 were positive: 77.2% for Rickettsia aeschlimannii, 20.2% for Rickettsia sp., 3.5% for Babesia sp. and 1.8% for Anaplasma sp. Among them, 2.7% were co‐infected with R. aeschlimannii and Babesia sp. Conclusions Our results confirm the extent of possible circulation of different pathogens near Corsican wetlands, not only in ticks collected from livestock but also directly in cattle and sheep, with two (Trypanosoma sp. and Babesia sp.) being detected for the first time in cattle, one for the first time in sheep (A. ovis) and one for the first time in Corsica (A. capra