The potential of Rhipicephalus microplus as a vector of Ehrlichia ruminantium in West Africa.

Heartwater, or cowdriosis, is a virulent tick-borne rickettsial disease of ruminants caused by Ehrlichia ruminantium, biologically transmitted by Amblyomma species (A. variegatum in West Africa). In West Africa, this bacterium was recently reported to naturally infect the invasive cattle tick, Rhipicephalus microplus (Rm) through trans-ovarian transmission from replete adult females to offspring. A 'sheep-tick-sheep' cycle was set up to determine whether feeding the progeny of these ticks on naïve sheep could lead to infection, and to compare clinical outcomes resulting from this transmission with those observed following infection by the natural A. variegatum (Av) vector. Using local strains of ticks (KIMINI-Rm and KIMINI-Av) and of E. ruminantium (BK242), we recorded, using the PCR technique, the presence of bacterial DNA in ticks (larvae for Av and females for Rm) engorged on sheep inoculated by BK242-infected blood. The bacterial DNA was also detected in the next stages of the lifecycle of R. microplus (eggs and larvae), and in sheep infested either by those R. microplus larvae or by A. variegatum nymphs moulted from larvae engorged on blood-inoculated sheep. Bacterial infection in these sheep was demonstrated by detecting antibodies to E. ruminantium using the MAP1-B ELISA and by isolation of the bacterium on cell culture from blood. The sequences of PCS20 gene detected in ticks and sheep were identical to that of the BK242 strain. Our results confirm that R. microplus can acquire and transmit E. ruminantium to the next stage. However, this transmission resulted in a mild subclinical disease whereas severe clinical disease was observed in sheep infested by A. variegatum infected nymphs, suggesting differences in the tick/bacteria relationship. Future studies will focus on replicating these findings with ticks of different isolates and life stages to determine if R. microplus is playing a role in the epidemiology of heartwater in West Africa. Additionally, studies will investigate whether sheep that are seropositive due to infestation by E. ruminantium-infected R. microplus are subsequently protected against heartwater. Such data will add to our understanding of the possible impact of R. microplus in areas where it has become recently established.

First digital characterization of the transhumance corridors through Benin used by cattle herds from Burkina Faso and associated risk scoring regarding the invasion of Rhipicephalus (Boophilus) microplus.

BACKGROUND: Livestock plays a key role in the macro-economy of West Africa and provides livelihoods for millions of people. The main cattle rearing strategy in West Africa is pastoralism, including transhumance, that is a seasonal migration of cattle with their herders. This adaptive strategy aims to optimize livestock access to water and pastures. However, it can favour pathogens and vectors transboundary spread. The aim of this study was to highlight firstly, the corridors and grazing areas used by Burkina Faso transhumant cattle herds going to Benin; secondly, the characteristics of departure and arrival areas of transhumance; and thirdly, the risk score related to introduction and spread of the invasive tick species, Rhipicephalus (Boophilus) microplus, in free areas. METHODS AND PRINCIPAL FINDINGS: Therefore, GPS devices were given to 27 herders to monitor a full transhumance season between East Burkina Faso and North Benin. The analysis of 14,966 spots generated by the GPS devices revealed four main corridors and five main grazing areas used by cattle herds during transhumance. Statistical analysis of normalized difference vegetation index (NDVI), rainfall and temperature data, highlighted significant differences between departure and arrival areas. NDVI and rainfall are significantly higher in Benin than Burkina Faso, whereas temperature is significantly lower. Additionally, using biotic and abiotic parameters, a risk scoring was developed to predict the presence of Rhipicephalus (Boophilus) microplus at municipality level. CONCLUSIONS AND SIGNIFICANCE: The better vegetation, temperature and rainfall conditions during the dry seasons in Benin attract cattle herds from Burkina Faso. The invasiveness and adaptability of Rhipicephalus (Boophilus) microplus added to the frequent stays of transhumant herds in infested areas suggest its potential introduction and establishment in free areas soon. Moreover, frequent intrusions of the transhumant cattle in the wildlife reserves are another risk of vectors and pathogen exchange between domestic and wild animals.

West African Cattle Farmers' Perception of Tick-Borne Diseases.

Worldwide, cattle production is struggling to face the negative impacts caused by ticks and Rhipicephalus (Boophilus) microplus is one of the most harmful ticks for livestock. Most of the people in West Africa depend on cattle farming and subsistence agriculture. The presence of ticks on cattle is a major problem faced by smallholder farmers who fight for their livelihood. National and regional tick control programs could assist these rural communities in protecting their livelihoods against ticks and tick-borne diseases, but only if they take into account the targeted herders and their perception on cattle management and tick control. This paper aims to provide a better insight in the socio-economic characteristics of Beninese cattle farmers, and their perception on tick burden, as well as to document common tick control strategies. Different tick species and their seasonality are well understood by cattle herders. For tick control, many still use manual tick removal, especially in the north of the country. The high cost of acaricides, the lack of financial means of African farmers, and of the local stockbreeders in particular, limits the use of acaricides in livestock breeding in Benin. While aiming to increase the meat or milk production of their animals, stockbreeders who can afford it sometimes turn to an abusive use of acaricides, which might in time lead to an increase in tick resistance. This study remains one of the rare studies to report extensively on the perceptions of West African cattle herders.

Ehrlichia ruminantium infects Rhipicephalus microplus in West Africa.

BACKGROUND: The invasion of West Africa by Rhipicephalus microplus during the past decade has changed the ecological situation of the agent of heartwater Ehrlichia ruminantium in this area. Before, its local vector, Amblyomma variegatum, was the most abundant tick species found on livestock. Today, the abundance of the R. microplus is one magnitude higher than that of A. variegatum in many west-African localities. We investigated the potential of this new ecological situation to impact the circulation of E. ruminantium in West Africa. METHODS: Ehrlichia ruminantium infections were assessed with the specific PCR-diagnosis targeting the PCS20 region. This screening was applied on field samples of 24 R. microplus adults, on four females from a laboratory strain that had been blood-fed since larvae on one E. ruminantium-infected steer as well as on the offspring of these females at egg and larval stages. RESULTS: The PCR detected E. ruminantium in 29 % of the field-collected R. microplus, i.e. twice as much as reported for A. variegatum with the same protocol. Regarding the laboratory strain, the PCR-diagnosis performed showed that all females were infected and passed the rickettsia to their progeny. Sequencing of the PCR product confirmed that the maternally inherited rickettsia was E. ruminantium. CONCLUSION: According to the present findings, the invasive dynamic of R. microplus in West Africa is currently impacting the local evolutionary conditions of E. ruminantium since it offers new transmission roads such as maternal transmission in R. microplus.

Community structure, seasonal variations and interactions between native and invasive cattle tick species in Benin and Burkina Faso.

BACKGROUND: The variation of tick abundance on ruminants had received little attention in West Africa before Rhipicephalus (Boophilus) microplus started to invade this region in the early 2000s. Ten years later, R. microplus was suspected to have replaced the native ticks. In addition to testing this hypothesis, this study investigated the interactions between native and invasive ticks and the relative role of climatic and geographical variables in the variations of tick community composition (beta diversity) on cattle herds. METHODS: A one-year-long survey was performed in Benin and Burkina Faso during which adult ticks were collected from 144 steers from 12 localities in four different areas once a month. Morphological features were used to assign the collected ticks to different species (A. variegatum, R. annulatus, R. decoloratus, R. microplus and R. geigyi). Beta diversity analyses and generalized linear models allowed characterizing the geographical variations in species assemblage and the effect of co-infestation patterns on the seasonal variations in the abundance and incidence rates of each taxon. RESULTS: About 68% (22,491/32,148) of all the adult ticks collected in one year were R. microplus. The most heterogeneously distributed taxa were Hyalomma spp and R. microplus and the lowest specific diversity was found in Central Burkina Faso. Although climatic variables did not provide any additional information on the variation in species assemblages compared with the sampling geography, adult tick abundance tended to peak during the late (Boophilus subgenus) or early (other taxa) rainy season. In most taxon-per-locality analyses, the abundance and incidence rate of a given tick taxon significantly increased when the host was co-infested by other taxa. The comparison with previous estimates (when possible) did not support the hypothesis that R. microplus invasion led to a decrease in native tick species abundance. CONCLUSIONS: The co-infestation patterns among native and invasive tick species are key factors for the determination of the community structure and the infestation dynamics of each tick taxon in West African cattle.

The Recent Evolution of a Maternally-Inherited Endosymbiont of Ticks Led to the Emergence of the Q Fever Pathogen, Coxiella burnetii.

Q fever is a highly infectious disease with a worldwide distribution. Its causative agent, the intracellular bacterium Coxiella burnetii, infects a variety of vertebrate species, including humans. Its evolutionary origin remains almost entirely unknown and uncertainty persists regarding the identity and lifestyle of its ancestors. A few tick species were recently found to harbor maternally-inherited Coxiella-like organisms engaged in symbiotic interactions, but their relationships to the Q fever pathogen remain unclear. Here, we extensively sampled ticks, identifying new and atypical Coxiella strains from 40 of 58 examined species, and used this data to infer the evolutionary processes leading to the emergence of C. burnetii. Phylogenetic analyses of multi-locus typing and whole-genome sequencing data revealed that Coxiella-like organisms represent an ancient and monophyletic group allied to ticks. Remarkably, all known C. burnetii strains originate within this group and are the descendants of a Coxiella-like progenitor hosted by ticks. Using both colony-reared and field-collected gravid females, we further establish the presence of highly efficient maternal transmission of these Coxiella-like organisms in four examined tick species, a pattern coherent with an endosymbiotic lifestyle. Our laboratory culture assays also showed that these Coxiella-like organisms were not amenable to culture in the vertebrate cell environment, suggesting different metabolic requirements compared to C. burnetii. Altogether, this corpus of data demonstrates that C. burnetii recently evolved from an inherited symbiont of ticks which succeeded in infecting vertebrate cells, likely by the acquisition of novel virulence factors.

Moulting and survival of Amblyomma variegatum (Acari: Ixodidae) nymphs in quasi-natural conditions in Burkina Faso; tick predators as an important limiting factor.

The survival rate of Amblyomma variegatum engorged nymphs was studied in 9 cohorts released during the first part of the dry season (i.e. the nymph infestation period in the region) and placed on a natural savannah, either in plastic containers or in cages driven into the soil and open at the bottom. All nymphs put in the containers placed in the sun died rapidly, while a high moulting rate (94%) was observed in containers placed in the shade. This rate reached 63% for the ticks released in the cages. The moulting period lasted from less than 4 to more than 6 weeks, depending on the release period: unfed adults are therefore present in the pasture 4-5 months before the onset of the adult infestation period. The survival rate of the unfed adults did not vary according to the release period of the nymphs: at the beginning of the rainy season, 20-25% of the adult ticks were still alive in the cages. Very important variations in survival and disappearance rates were observed according to the place where the cages had been installed: within a few metres, the survival rate could vary from 0 to 90%, presumably because of the activity of predators which removed also dead tick from the plastic containers. Predators may then have a great but highly variable and unpredictable impact on the natural population of A. variegatum adults. Engorged nymphs released on pasture were able to find suitable micro-habitats allowing survival, covered up to 2.8 metres to reach them, and grouped together at the neck or root network of bushes and grass clumps, sometimes burrowing to a depth of 10 cm.

Clonal origin of emerging populations of Ehrlichia ruminantium in Burkina Faso.

Cowdriosis or heartwater is a major tick-borne disease on ruminants in Africa and the Caribbean. The causative agent is Ehrlichia ruminantium, an intracellular bacterium. Development of vaccines against heartwater has been hampered the limited efficiency of vaccine in the field, thought to be a consequence of the high genetic diversity of strains circulating in a same area. A sampling scheme was set to collect ticks over 2 years in a delimited area and well identified flock. Prevalence was low at about 3%. A set of 37 strains was considered for MLST analysis along with two reference strains, i.e. ERGA and ERWO, for which full-length genome was available, using a previously described scheme based on the genes gltA, groEL, lepA, lipA, lipB, secY, sodB and sucA. Two populations were identified both with limited genetic variability but with differing evolutionary patterns. Population 1 is in genomic stasis, in agreement with the paradigm for intracellular bacteria. The two reference strains, one from the Caribbean separated from West African strains three centuries ago and another one isolated in South Africa, belong to Population 1. Population 2 is on expansion following a recent clonal emergence from Population 1. The founder strain was identified as strain 395. Strain 623 displays a particularly high rate of mutations in groEL. Owing to the chaperone function of GroEL, this might indicate another clonal emergence under way. This work brings further insight in the genomic plasticity of E. ruminantium and its impact on vaccine strategy.

Efficiency of inactivated vaccines against heartwater in Burkina Faso: impact of Ehrlichia ruminantium genetic diversity.

In order to identify the appropriate strains to use in vaccination trials against heartwater in Burkina Faso, the protective effect of Gardel and Welgevonden strains was assessed against local strains on sheep vaccinated by infection-and-treatment method: Gardel protected significantly against Burkina Faso strains tested (survival rate 59% for immunised sheep vs 13% for control sheep) while Welgevonden did not (survival rate 45% for immunised sheep vs 25% for control sheep). The efficacy of the ISA50 inactivated vaccine, produced under industrial process, was evaluated in sheep during field challenges in two successive years. During year 1, there was a limited protective effect of the Gardel vaccine with 65% of survival rate for the vaccinated group compared to 49% for the control group (N=153, p=0.053). During year 2, the vaccine containing Gardel and a local strain gave an increased protective effect compared to the first trial: 72% of the vaccinated animals survived compared to 47% of the naïve animals (N=173, p<0.001). There was an important genetic diversity of strains in the field with detection of 11 different map1 genotypes in brains from control and vaccinated sheep post mortem. Map1 genotyping of strains detected in brains from control sheep showed that genotype distribution varied according to time and study areas, which could explain the difference in efficacy of the vaccine.