Efficient high-throughput molecular method to detect <em>Ehrlichia ruminantium</em> in ticks

International audienceEhrlichia ruminantium is the causal agent of heartwater, a fatal tropical disease affecting ruminants with important economic impacts. This bacterium is transmitted by Amblyomma ticks and is present in sub-Saharan Africa, islands in the Indian Ocean and the Caribbean, where it represents a threat to the American mainland. An automated DNA extraction method was adapted for Amblyomma ticks and a new qPCR targeting the pCS20 region was developed to improve E. ruminantium screening capacity and diagnosis. The first step in the preparation of tick samples, before extraction, was not automated but was considerably improved by using a Tissue Lyser. The new pCS20 Sol1 qPCR and a previously published pCS20 Cow qPCR were evaluated with the OIE standard pCS20 nested PCR. pCS20 Sol1 qPCR was found to be more specific than the nested PCR, with a 5-fold increase in sensitivity (3 copies/reaction vs 15 copies/reaction), was less prone to contamination and less time-consuming. As pCS20 Sol1 qPCR did not detect Rickettsia, Anasplasma and Babesia species or closely related species such as Panola Mountain Ehrlichia, E. chaffeensis and E. canis, its specificity was also better than Cow qPCR. In parallel, a tick 16S qPCR was developed for the quality control of DNA extraction that confirmed the good reproducibility of the automated extraction. The whole method, including the automated DNA extraction and pCS20 Sol1 qPCR, was shown to be sensitive, specific and highly reproducible with the same limit of detection as the combined manual DNA extraction and nested PCR, i.e. 6 copies/reaction. Finally, 96 samples can be tested in one day compared to the four days required for manual DNA extraction and nested PCR. The adaptation of an automated DNA extraction using a DNA/RNA viral extraction kit for tick samples and the development of a new qPCR increased the accuracy of E. ruminantium epidemiological studies, as well as the diagnostic capabilities and turn-over time for surveillance of heartwater. This new method paves the way for large-scale screening of other bacteria and viruses in ticks as well as genetic characterization of ticks and tick-pathogen coevolution studies

Vector-pathogen association: lack of congruence of phylogeograpical pattern of the vector tick <em>Amblyomma variegatum</em> (Acari: Ixodidae) and of the Ricketssiales <em>Ehrlichia ruminantium</em>

International audienc

Vector-pathogen association: lack of congruence of phylogeograpical pattern of the vector tick <em>Amblyomma variegatum</em> (Acari: Ixodidae) and of the Ricketssiales <em>Ehrlichia ruminantium</em>

International audienc

Recombination is a major driving force of genetic diversity in the Anaplasmataceae Ehrlichia ruminantium

The disease, Heartwater, caused by the Anaplasmataceae E. ruminantium, represents a major problem for tropical livestock and wild ruminants. Up to now, no effective vaccine has been available due to a limited cross protection of vaccinal strains on field strains and a high genetic diversity of Ehrlichia ruminantium within geographical locations. To address this issue, we inferred the genetic diversity and population structure of 194 E. ruminantium isolates circulating worldwide using Multilocus Sequence Typing based on lipA, lipB, secY, sodB, and sucA genes. Phylogenetic trees and networks were generated using BEAST and SplitsTree, respectively, and recombination between the different genetic groups was tested using the PHI test for recombination. Our study reveals the repeated occurrence of recombination between E. ruminantium strains, suggesting that it may occur frequently in the genome and has likely played an important role in the maintenance of genetic diversity and the evolution of E. ruminantium. Despite the unclear phylogeny and phylogeography, 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 Southern Africa, Indian Ocean, and Caribbean strains. Some sequence types are common between West Africa and Caribbean and between Southern Africa and Indian Ocean strains. These common sequence types highlight two main introduction events due to the movement of cattle: from West Africa to Caribbean and from Southern Africa to the Indian Ocean islands. Due to the long branch lengths between Group 1 and Group 2, and the propensity for recombination between these groups, it seems that the West African clusters of Subgroup 2 arrived there more recently than the original divergence of the two groups, possibly with the original waves of domesticated ruminants that spread across the African continent several thousand years ago

Additional file 1: of Efficient high-throughput molecular method to detect Ehrlichia ruminantium in ticks

Text. PCR protocol for the detection of E. ruminantium. Table S1. Primers and probes for pCS20 Sol1TqM, Sol1SG, Cow™ and 16SSG qPCRs. (DOC 48 kb

Additional file 2: of Efficient high-throughput molecular method to detect Ehrlichia ruminantium in ticks

Text. Protocol of extraction of tick DNA. (DOCX 12 kb

Assessment of Microbial Contamination in the Infulene River Basin, Mozambique

Water microbial contamination is one of the major threats to human health. The study focus is on Infulene River Basin, a urban catchment with mainly informal settlements, with limited water supply and sanitation. In the catchment there are two wastewater treatment plants, one hospital and beer factory located on the banks of the main stream; water from this stream is used for urban agriculture and domestic uses by some dwellers. These factors present a significant health risk from water-borne diseases. At the moment there is limited knowledge about the level of microbial contamination of the different sources of water at the disposal of the communities. Thus, a preliminary study on fecal microbial contamination was conducted targeting the Infulene River and the drainage system from the nearby Maputo city draining into the system, with additional investigation on the drinking water provided by the city water supply company. The quantification of Total Coliforms (TC) and Escherichia coli (EC) was conducted at several sampling locations. Results were compared with official drinking water standards. Eighty two percent (82%) and 61% of Infulene river water and drainage water samples were positive for TC (105 to 109 NPN/100 mL) and EC (105 to 107 NPN/100 mL), respectively. For drinking water samples, 63% and 23% were positive for TC (up to 6000 NPN/100 mL) and EC (up to 1000 NPN/100 mL), respectively. Higher microbial contamination was found in neighborhoods with the poorest sanitation and shallow groundwater, i.e., Chamanculo, Xipamanine, Mafalala, Aeroporto and Maxaquene, a situation that was more expressive during the rainy season. Overall, the study confirmed the high vulnerability to microbial contamination of all sources investigated due to poor sanitation and lack of drainage infrastructure. The risks to human health might be even higher considering that contaminated water is used for gardening of vegetable watering and domestic use

Haplotype network of the 19 ITS-2 haplotypes detected for <i>H. rufipes.</i>

<p>The size of the circles corresponds with the number of individuals represented by the haplotype. Each line separating haplotypes represents one mutational step and missing/intermediate haplotypes are shown by an white circle. For visual comparison between data sets, localities were color coded based on the outcome of the mtDNA analyses (southern clade = red; northern clade = blue) and these are also similarly indicated on the map inset.</p

Climate suitability of <i>H. rufipes</i> in Africa based on temperature and the Normalized Derived Vegetation Index (NDVI), at 0.1° spatial resolution.

<p>Suitability are indicated in a sliding window where red are most suitable and blue are least suitable. Sampling localities are indicated by black circles.</p