Molecular detection of Rickettsia africae in Amblyomma ticks collected in cattle from Southern and Central Mozambique.

INTRODUCTION: Rickettsia are Gram-negative and obligate intracellular bacteria, which cause typhus and spotted fever-like diseases in humans. In Africa, Rickettsia africae of the Spotted Fever Group Rickettsia (SFGR) is the etiologic agent of the African Tick-Bite Fever. The disease is transmitted by ticks of the genus Amblyomma, which serve as vectors and reservoirs of Rickettsia. In this study, we aimed to detect Rickettsia species in ticks collected from cattle in south and central Mozambique. METHODOLOGY: DNA from 412 adult ticks and 22 pools of larvae were extracted and tested for the presence of Rickettsia genes gltA, ompA and ompB by PCR, followed by sequencing and phylogenetic analysis. RESULTS: Our results showed that in adult ticks, 79.5% (n = 330), 66% (n = 274) and 67% (n = 275) samples were positive for gltA, ompA and ompB genes, respectively. Among the 22 pools of larvae analysed, 77.2% (n = 17) were positive for the three genes tested. The infection rates ranged from 43% to 100% for Rickettsia by gltA in all locations studied, with maximum values of 100% observed in the districts of Maputo province namely Changalane, Boane and Matutuine district. The phylogenetic analysis of amplified sequences revealed that samples under study grouped with R. africae for the 3 genes. CONCLUSION: The study showed that Spotted Fever Group Rickettsia represented by R. africae widely circulate in Amblyomma ticks collected in south and central regions of Mozambique.

Efficient high-throughput molecular method to detect Ehrlichia ruminantium in ticks.

BACKGROUND: Ehrlichia 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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.

The influence of interspecific competition and host preference on the phylogeography of two African ixodid tick species.

A comparative phylogeographic study on two economically important African tick species, Amblyomma hebraeum and Hyalomma rufipes was performed to test the influence of host specificity and host movement on dispersion. Pairwise AMOVA analyses of 277 mtDNA COI sequences supported significant population differentiation among the majority of sampling sites. The geographic mitochondrial structure was not supported by nuclear ITS-2 sequencing, probably attributed to a recent divergence. The three-host generalist, A. hebraeum, showed less mtDNA geographic structure, and a lower level of genetic diversity, while the more host-specific H. rufipes displayed higher levels of population differentiation and two distinct mtDNA assemblages (one predominantly confined to South Africa/Namibia and the other to Mozambique and East Africa). A zone of overlap is present in southern Mozambique. A mechanistic climate model suggests that climate alone cannot be responsible for the disruption in female gene flow. Our findings furthermore suggest that female gene dispersal of ticks is more dependent on the presence of juvenile hosts in the environment than on the ability of adult hosts to disperse across the landscape. Documented interspecific competition between the juvenile stages of H. rufipes and H. truncatum is implicated as a contributing factor towards disrupting gene flow between the two southern African H. rufipes genetic assemblages.