Detection of Crimean-Congo haemorrhagic fever virus in Hyalomma marginatum ticks, southern France, May 2022 and April 2023.

Crimean-Congo haemorrhagic fever (CCHF), a potentially severe zoonotic viral disease causing fever and haemorrhagic manifestations in humans. As the Crimean-Congo haemorrhagic fever virus (CCHFV) has been detected in ticks in Spain and antibodies against the virus in ruminant sera in Corsica, it was necessary to know more about the situation in France. In 2022-2023, CCHFV was detected in 155 ticks collected from horses and cattle in southern France.

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.

The Crimean-Congo haemorrhagic fever tick vector Hyalomma marginatum in the south of France: Modelling its distribution and determination of factors influencing its establishment in a newly invaded area.

We developed a correlative model at high resolution for predicting the distribution of one of the main vectors of Crimean-Congo haemorrhagic fever virus (CCHFV), Hyalomma marginatum, in a recently colonised area, namely southern France. About 931 H. marginatum adult ticks were sampled on horses from 2016 to 2019 and 2021 in 14 southern French departments, which resulted in the first H. marginatum detection map on a large portion of the national territory. Such updated presence/absence data, as well as the mean number of H. marginatum per examined animal (mean parasitic load) as a proxy of tick abundance, were correlated to multiple parameters describing the climate and habitats characterising each collection site, as well as movements of horses as possible factors influencing tick exposure. In southern France, H. marginatum was likely detected in areas characterised by year-long warm temperatures and low precipitation, especially in summer and mostly concentrated in autumn, as well as moderate annual humidity, compared to other sampled areas. It confirms that even in newly invaded areas this tick remains exclusively Mediterranean and cannot expand outside this climatic range. Regarding the environment, a predominance of open natural habitats, such as sclerophyllous vegetated and sparsely vegetated areas, were also identified as a favourable factor, in opposition to urban or peri-urban and humid habitats, such as continuous urban areas and inland marshes, respectively, which were revealed to be unsuitable. Based on this model, we predicted the areas currently suitable for the establishment of the tick H. marginatum in the South of France, with relatively good accuracy using internal (AUC = 0.66) and external validation methods (AUC = 0.76 and 0.83). Concerning tick abundance, some correlative relationships were similar to the occurrence model, as well as the type of horse movements being highlighted as an important factor explaining mean parasitic load. However, the limitations of estimating and modelling H. marginatum abundance in a correlative model are discussed.

Where to find questing Ixodes frontalis ticks? Under bamboo bushes!

Tick-borne diseases have a complex epidemiology that depends on different ecological communities, associating several species of vertebrate hosts, vectors and pathogens. While most studies in Europe are focused on Ixodes ricinus, other Ixodes species may also be involved in the transmission or maintenance of pathogens. This is the case of Ixodes frontalis, a poorly known species associated with different bird species such as blackbirds, thrushes and robins, with a wide distribution covering most European countries. In a previous study, high densities of questing I. frontalis larvae were found during autumn-winter at a site close to Nantes (western France) where a long-term survey focused on I. ricinus was conducted. These I. frontalis were mostly observed under bamboo bushes. In the present study, we investigated the presence of I. frontalis under bamboo bushes at various locations. With that aim in mind, a systematic search for questing I. frontalis was undertaken by the flagging method in public urban parks and private gardens presenting bamboo bushes (32 sites). This survey was carried out during autumn-winter to maximize the probability of finding the most abundant stage, i.e. larvae. We searched for I. frontalis first in the area of Nantes (10 sites), then in other regions of France (21 sites) and at one site in northern Italy. A single visit to each site revealed the presence of I. frontalis at 29 out of 32 sites: larvae were always present, nymphs were frequent (59 % of the positive sites), while adults were found at only 14 % of the sites. Questing stages of this understudied species are thus easy to find, by dragging or flagging under bamboo bushes in autumn or winter. We make the assumption that bamboo offers a favourable place for birds to roost overnight outside their breeding period (i.e. spring), sheltered from both predators and wind. This would explain higher densities of I. frontalis under bamboo, relative to other biotopes. As I. frontalis is known to harbour zoonotic pathogens, the consequences of this discovery on the epidemiology of tick-borne diseases are discussed.

Tick-borne pathogens in ticks (Acari: Ixodidae) collected from various domestic and wild hosts in Corsica (France), a Mediterranean island environment.

Corsica is a mountainous French island in the north-west of the Mediterranean Sea presenting a large diversity of natural environments where many interactions between humans, domestic animals and wild fauna occur. Despite this favourable context, tick-borne pathogens (TBPs) have not systematically been investigated. In this study, a large number of TBPs were screened in ticks collected over a period of one year from domestic and wild hosts in Corsica. More than 1,500 ticks belonging to nine species and five genera (Rhipicephalus, Hyalomma, Dermacentor, Ixodes and Haemaphysalis) were analysed individually or pooled (by species, gender, host and locality). A real-time microfluidic PCR was used for high-throughput screening of TBP DNA. This advanced methodology enabled the simultaneous detection of 29 bacterial and 12 parasitic species (including Borrelia, Anaplasma, Ehrlichia, Rickettsia, Bartonella, Candidatus Neoehrlichia, Coxiella, Francisella, Babesia and Theileria). The Crimean-Congo haemorrhagic fever (CCHF) virus was investigated individually in tick species known to be vectors or carriers of this virus. In almost half of the tick pools (48%), DNA from at least one pathogen was detected and eleven species of TBPs from six genera were reported. TBPs were found in ticks from all collected hosts and were present in more than 80% of the investigated area. The detection of DNA of certain species confirmed the previous identification of these pathogens in Corsica, such as Rickettsia aeschlimannii (23% of pools), Rickettsia slovaca (5%), Anaplasma marginale (4%) and Theileria equi (0.4%), but most TBP DNA identified had not previously been reported in Corsican ticks. This included Anaplasma phagocytophilum (16%), Rickettsia helvetica (1%), Borrelia afzelii (0.7%), Borrelia miyamotoi (1%), Bartonella henselae (2%), Babesia bigemina (2%) and Babesia ovis (0.5%). The high tick infection rate and the diversity of TBPs reported in this study highlight the probable role of animals as reservoir hosts of zoonotic pathogens and human exposure to TBPs in Corsica.

Immunomodulatory Effects of Amblyomma variegatum Saliva on Bovine Cells: Characterization of Cellular Responses and Identification of Molecular Determinants.

The tropical bont tick, Amblyomma variegatum, is a tick species of veterinary importance and is considered as one of major pest of ruminants in Africa and in the Caribbean. It causes direct skin lesions, transmits heartwater, and reactivates bovine dermatophilosis. Tick saliva is reported to affect overall host responses through immunomodulatory and anti-inflammatory molecules, among other bioactive molecules. The general objective of this study was to better understand the role of saliva in interaction between the Amblyomma tick and the host using cellular biology approaches and proteomics, and to discuss its impact on disease transmission and/or activation. We first focused on the immuno-modulating effects of semi-fed A. variegatum female saliva on bovine peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages in vitro. We analyzed its immuno-suppressive properties by measuring the effect of saliva on PBMC proliferation, and observed a significant decrease in ConA-stimulated PBMC lymphoproliferation. We then studied the effect of saliva on bovine macrophages using flow cytometry to analyze the expression of MHC-II and co-stimulation molecules (CD40, CD80, and CD86) and by measuring the production of nitric oxide (NO) and pro- or anti-inflammatory cytokines. We observed a significant decrease in the expression of MHC-II, CD40, and CD80 molecules, associated with decreased levels of IL-12-p40 and TNF-α and increased level of IL-10, which could explain the saliva-induced modulation of NO. To elucidate these immunomodulatory effects, crude saliva proteins were analyzed using proteomics with an Orbitrap Elite mass spectrometer. Among the 336 proteins identified in A. variegatum saliva, we evidenced bioactive molecules exhibiting anti-inflammatory, immuno-modulatory, and anti-oxidant properties (e.g., serpins, phospholipases A2, heme lipoprotein). We also characterized an intriguing ubiquitination complex that could be involved in saliva-induced immune modulation of the host. We propose a model for the interaction between A. variegatum saliva and host immune cells that could have an effect during tick feeding by favoring pathogen dissemination or activation by reducing the efficiency of host immune response to the corresponding tick-borne diseases.

Ticks (Acari: Ixodidae) infesting cattle and some other domestic and wild hosts on the French Mediterranean island of Corsica.

BACKGROUND: Corsica is a mountainous French island in the north-western Mediterranean presenting a large diversity of natural environments where many interactions between domestic animals and wild fauna occur. Despite a favourable context for ticks and tick-borne diseases (TBDs), the tick fauna of Corsica has not systematically been investigated. METHODS: For one year (May 2014-May 2015), a survey of ticks infesting cattle was performed in the three Corsican cattle slaughterhouses. Two of these were visited monthly and one quarterly; the ticks were manually collected, just after flaying. Ticks were identified on their morphology; when necessary, some specimens were also molecularly identified by sequencing mitochondrial cox1 (cytochrome c oxidase subunit 1) and 16S ribosomal RNA genes and ITS2 (internal transcribed spacer 2). During the same period, ticks from other domestic animals (small ruminants, horses, domestic carnivores) and wild animals (wild boars, mouflons, deer) were occasionally collected. RESULTS: A total of 1,938 ticks was collected from 264 of 418 cattle examined, reared in 86 different localities. Eight tick species were found infesting cattle: Rhipicephalus bursa (56.1 %), Hyalomma marginatum (21.5 %), Hy. scupense (8.7 %), Ixodes ricinus (5.7 %), Haemaphysalis punctata (4.8 %), Rh. sanguineus (sensu lato) (2.3 %), Rh. (Boophilus) annulatus (0.7 %) and Dermacentor marginatus (0.2 %). The cattle infestation rate remained high all year (more than 50 %). Several tick species showed seasonal variation of their activity. From other Corsican animals 1,196 ticks were collected. Comparing ticks collected from cattle with those found on other animals, several host preferences were shown. A noteworthy record is that of a few Ha. sulcata on mouflons which were mainly infested by Rh. bursa. CONCLUSION: The Corsican tick fauna is characterized by typical Mediterranean species (Rh. bursa and Hy. marginatum), but the mild climate and diversified environment provide satisfactory habitats both for species usually found in dry areas (Hy. scupense) and species usually collected in humid areas (I. ricinus).

Experiences in Tick Control by Acaricide in the Traditional Cattle Sector in Zambia and Burkina Faso: Possible Environmental and Public Health Implications.

Livestock, especially cattle, play a paramount role in agriculture production systems, particularly in poor countries throughout the world. Ticks and tick-borne diseases (TBDs) have an important impact on livestock and agriculture production in sub-Saharan Africa. The authors review the most common methods used for the control of ticks and TBDs. Special emphasis is given to the direct application of acaricides to the host animals. The possible environmental and public health adverse effects (i.e., risks for the workers, residues in the environment and in food products of animal origin) are mentioned. The authors present two case studies, describing different field experiences in controlling ticks in two African countries. In Zambia (Southern Africa), a strategic dipping regime was used to control Rhipicephalus appendiculatus ticks, vectors of theileriosis, a deadly disease affecting cattle in the traditional livestock sector in Southern Province. The dipping regime adopted allowed to reduce the tick challenge and cattle mortally rate and, at the same time, to employ less acaricide as compared to the intensive dipping used so far, without disrupting the building-up of enzootic stability. In Burkina Faso (West Africa), where dipping was never used for tick control, an acaricide footbath was employed as an alternative method to the traditional technique used locally (portable manual sprayers). This was developed from field observations on the invasion/attachment process of the Amblyomma variegatum ticks - vector of cowdriosis - on the animal hosts, leading to a control method aimed to kill ticks temporarily attached to the interdigital areas before their permanent attachment to the predilection sites. This innovative method has been overall accepted by the local farmers. It has the advantage of greatly reducing costs of treatments and has a minimal environmental impact, making footbath a sustainable and replicable method, adoptable also in other West African countries. Although the two methods described, developed in very different contexts, are not comparable - if public health and environmental implications are taken into account, if a balance among efficacy of the control method(s), cost-effectiveness and sustainability is reached - a way forward for the implementation of a One Health strategy can be set.

Strong evidence for the presence of the tick Hyalomma marginatum Koch, 1844 in southern continental France.

Hyalomma ticks can transmit several human and animal pathogens in Eurasia and Africa. Interest in Hyalomma marginatum has increased since the recent (re)emergence of Crimean-Congo Hemorrhagic fever in the Palearctic region. Until now, continental France has been considered free of this tick species. Nevertheless, the existence of incomplete and occasionally incorrect records has maintained confusion about its status. Based on several tick sampling campaigns conducted on horses and birds from 2007 to 2016, we provided very strong evidence for the presence of reproducing populations of H. marginatum in parts of southern continental France. We also confirmed the introduction of immature developmental stages of H. marginatum, as well as H. rufipes, into France probably through trans-Mediterranean bird migrations.

First report of the tick Hyalomma scupense (natural vector of bovine tropical theileriosis) on the French Mediterranean island of Corsica.

Hyalomma scupense (Acari, Ixodidae) is a common tick species found in several areas in North Africa, Asia and South Europe and an efficient natural vector of bovine tropical theileriosis (Theileria annulata), a livestock disease with an important economic impact. For one year, 1938 ticks were collected on cattle in several Corsican slaughterhouses; 168 of them were morphologically identified as H. scupense. This result was confirmed by genetic identification using sequences of mitochondrial cytochrome c oxidase subunit I (COI) and ribosomal internal transcribed spacer 2 (ITS2) genes. The presence of 2 different stages (adults and nymphs), collected in various areas of the island, indicates that a population of H. scupense is established in Corsica. However, bovine tropical theileriosis has not been diagnosed on the island so far.

Survival and moulting of Amblyomma variegatum nymphs under cold conditions of the Malagasy highlands.

Although Amblyomma variegatum is now regularly recorded up to 1600 m in altitude in the Malagasy highlands, where it was previously reported not to persist without a constant supply of ticks introduced from lower infested regions, some parts of the highlands remain tick-free. Studies were carried out to verify whether the cold climate prevailing in these areas in June-September could prevent the survival and moulting of nymphs, the tick life stage present in the environment at this period. Cohorts of engorged A. variegatum nymphs were released from June to August in six different sites (three in 2010, altitudes 1200-1415 m; three in 2011, altitudes 1585-1960 m) which were reported to be either tick-infested (two in 2010, one in 2011) or tick-free. The ticks were placed in cages driven into the soil and open at the bottom so that they could hide in the soil or root network. Of the 1975 nymphs released in 2010 and the 1494 released in 2011, 86% and 85% were recovered, respectively. Twenty to 23% of the recovered ticks were dead, and some of them were obviously predated; predation also likely contributed to the disappearance of the non-recovered ticks. When the rainy season started in October, 59% of the newly moulted adults were still alive in the cages. The moulting period lasted up to 20 weeks, depending on the site and release period. As verified in 2011, unfed nymphs could also survive the cold season. Various A. variegatum life stages are thus able to survive the adverse cold and/or dry seasons: unfed nymphs, engorged nymphs in developmental diapause, moulted adults in behavioural diapause as observed previously. Strong variation in mortality and recovery rates was observed between cages, highlighting the importance of the micro-environment and micro-climate for tick survival. The minimum temperature recorded in the field sites varied from 1.1 °C to 6.8 °C, but the tick-free sites were not the coldest ones; they were, however, those for which the temperature remained below 10 °C for the longest time over the study period. Recovery and mortality rates in the tick-free sites were similar to those of the tick-infested sites: the temperatures recorded during the study periods did not prevent ticks from surviving and moulting although it did delay the metamorphosis. Low temperature alone can therefore not explain the persistence of tick-free areas in the highlands. To further monitor survival, cohorts of engorged nymphs were also maintained in an incubator at 3.6 °C, 6.2 °C or 12.8 °C. More than 50% mortality was observed after 6 days at 3.6 °C, and after 15 days at 6.2 °C, whereas 18 days at 12.8 °C only delayed moulting. The collected survival, moulting and climatic data presented in this study should help to develop a predictive model to assess the distribution of A. variegatum according to climate characteristics.

New data regarding distribution of cattle ticks in the south-western Indian Ocean islands.

Recent studies have produced new insight into the origin and distribution of some cattle ticks in the south-western Indian Ocean islands. Rhipicephalus appendiculatus, introduced from Tanzania in 2002, is now well established on Grande Comore but has not yet reached the other islands of the archipelago (Mohéli, Anjouan and Mayotte). Only one of the two clades identified in Africa has settled so far. Amblyomma variegatum, which was not supposed to be able to persist in the Antananarivo region (1300 m) nor in other Malagasy regions of high altitude without regular introductions of ticks by infested cattle, is now endemic as a general rule up to 1600 m although other regions of lower altitude (1400 m) are still free of the tick. This species remains confined in a small area of the west coast on La Reunion Island. On the contrary, Hyalomma dromedarii could not settle on Madagascar where it was introduced in 2008 and Rhipicephalus evertsi evertsi is not yet present in Grande Comore despite regular introductions by infested cattle from Tanzania. A phylogeographic approach has been carried out at an intra-specific level for A. variegatum. This study has led to the identification of two main lineages, one covering all species distribution and one restricted to East Africa and the Indian Ocean area. These two lineages are in sympatry in Madagascar where a high genetic diversity has been described, whereas a lower genetic diversity is observed on other islands. These results seem to agree with the historical data concerning the introduction of the tick in the Indian Ocean area.

Treating cattle to protect people? Impact of footbath insecticide treatment on tsetse density in Chad.

BACKGROUND: In Chad, several species of tsetse flies (Genus: Glossina) transmit African animal trypanosomoses (AAT), which represents a major obstacle to cattle rearing, and sleeping sickness, which impacts public health. After the failure of past interventions to eradicate tsetse, the government of Chad is now looking for other approaches that integrate cost-effective intervention techniques, which can be applied by the stake holders to control tsetse-transmitted trypanosomoses in a sustainable manner. The present study thus attempted to assess the efficacy of restricted application of insecticides to cattle leg extremities using footbaths for controlling Glossina m. submorsitans, G. tachinoides and G. f. fuscipes in southern Chad. METHODOLOGY/PRINCIPAL FINDINGS: Two sites were included, one close to the historical human African trypanosomiasis (HAT) focus of Moundou and the other to the active foci of Bodo and Moissala. At both sites, a treated and an untreated herd were compared. In the treatment sites, cattle were treated on a regular basis using a formulation of deltamethrin 0.005% (67 to 98 cattle were treated in one of the sites and 88 to 102 in the other one). For each herd, tsetse densities were monthly monitored using 7 biconical traps set along the river and beside the cattle pen from February to December 2009. The impact of footbath treatment on tsetse populations was strong (p < 10(-3)) with a reduction of 80% in total tsetse catches by the end of the 6-month footbath treatment. CONCLUSIONS/SIGNIFICANCE: The impact of footbath treatment as a vector control tool within an integrated strategy to manage AAT and HAT is discussed in the framework of the "One Health" concept. Like other techniques based on the treatment of cattle, this technology should be used under controlled conditions, in order to avoid the development of insecticide and acaricide resistance in tsetse and tick populations, respectively.

Tick control practices in Burkina Faso and acaricide resistance survey in Rhipicephalus (Boophilus) geigyi (Acari: Ixodidae).

Traditional systems account for 95 % of the livestock produced in Burkina Faso. Tick infestation hampers livestock productivity in this area. However, little information exists on tick-control practices used by livestock farmers. We interviewed 60 stockbreeders working in traditional farming systems to obtain the first data on tick-control practices. Sixteen farmers (27 %) did not use conventional practices: seven removed ticks by hand or plastered cattle with dung or engine oil; nine farmers treated cattle with crop pesticides. Forty-four farmers (73 %) used mainly synthetic pyrethroids (SP; either alphacypermethrin or deltamethrin in 20 and 18 farms, respectively) and occasionally amitraz (N = 6). Intervals between treatments varied significantly depending on the chemical used: most farmers using crop pesticides (100 %), amitraz (100 %) or alphacypermethrin (80 %) adjusted tick-control to tick-burden, whereas farmers using deltamethrin tended more to follow a tick-control schedule. Perception of tick-control effectiveness significantly varied among practices: tick-control failures were more frequently reported by farmers using alphacypermethrin (55 %) than by those using either other conventional acaricides (17 %) or crop pesticides (0 %). We investigated whether this could indicate actual development of SP-resistance in cattle ticks. First, using the larval packet test technique, we confirmed that the computation of LC50 and LC90 was repeatable and remained stable across generations of the Rhipicephalus (Boophilus) geigyi Houndé laboratory strain. We then collected from the field fully-engorged female R. geigyi to evaluate the SP-resistance relative to the Houndé reference strain. We did not detect any case of SP-resistance in the field-derived R. geigyi ticks.

Restricted application of insecticides: a promising tsetse control technique, but what do the farmers think of it?

BACKGROUND: Restricted application of insecticides to cattle is a cheap and safe farmer-based method to control tsetse. In Western Africa, it is applied using a footbath, mainly to control nagana and the tick Amblyomma variegatum. In Eastern and Southern Africa, it might help controlling the human disease, i.e., Rhodesian sleeping sickness as well. The efficiency of this new control method against ticks, tsetse and trypanosomoses has been demonstrated earlier. The invention, co-built by researchers and farmers ten years ago, became an innovation in Burkina Faso through its diffusion by two development projects. METHODOLOGY/PRINCIPAL FINDINGS: In this research, we studied the process and level of adoption in 72 farmers inhabiting the peri-urban areas of Ouagadougou and Bobo-Dioulasso. Variables describing the livestock farming system, the implementation and perception of the method and the knowledge of the epidemiological system were used to discriminate three clusters of cattle farmers that were then compared using indicators of adoption. The first cluster corresponded to modern farmers who adopted the technique very well. The more traditional farmers were discriminated into two clusters, one of which showed a good adoption rate, whereas the second failed to adopt the method. The economic benefit and the farmers' knowledge of the epidemiological system appeared to have a low impact on the early adoption process whereas some modern practices, as well as social factors appeared critical. The quality of technical support provided to the farmers had also a great influence. Cattle farmers' innovation-risk appraisal was analyzed using Rogers' adoption criteria which highlighted individual variations in risk perceptions and benefits, as well as the prominent role of the socio-technical network of cattle farmers. CONCLUSIONS/SIGNIFICANCE: Results are discussed to highlight the factors that should be taken into consideration, to move discoveries from bench to field for an improved control of trypanosomoses vectors.

Colonization of Grande Comore Island by a lineage of Rhipicephalus appendiculatus ticks.

BACKGROUND: Union of the Comoros suffered a severe East Coast Fever epidemic in 2004. Rhipicephalus appendiculatus was probably involved in pathogen transmission as this competent tick species, although previously absent from Comoros, was sampled on 4 animals on one geographical site during the epidemic. We carried out an entomological survey on all three islands of Union of the Comoros to establish cattle tick species distribution with a special emphasis on R. appendiculatus. We investigated R. appendiculatus intraspecific diversity as this species has been previously shown to be split off into two main cytoplasmic lineages with different ecology, physiology and vectorial competence. This survey also included sampling of live cattle imported from Tanzania to investigate the possibility of tick introduction through animal trade. RESULTS: Our data show that Comoros cattle are infested with Amblyomma variegatum, Rhipicephalus microplus and R. appendiculatus. This latter species has established throughout Grande Comore but is absent from Anjouan and Moheli. Interestingly, 43 out of the 47 sequenced R. appendiculatus ticks belong to one single highly competent lineage while ticks from the other lineage where only found on imported cattle or on cattle parked at the vicinity of the harbor. At last, 2 ticks identified as R. evertsi, a species so far virtually absent on Comoros, were sampled on imported cattle. CONCLUSIONS: This survey shows that importation of live cattle is clearly a source of vector introduction in Comoros. The wide distribution of one highly competent R. appendiculatus lineage on Grande Comore, together with the absence of this species on the two neighbouring islands is in accordance with the rapid and disastrous spread of East Coast Fever epidemics on Grande Comore Island only. Whether the other R. appendiculatus lineage as well as R. evertsi species will succeed in establishing permanently on Grande Comore needs to be monitored.

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.

Mining the genetic diversity of Ehrlichia ruminantium using map genes family.

Understanding bacterial genetic diversity is crucial to comprehend pathogenesis. Ehrlichia ruminantium (E. ruminantium), a tick-transmitted intracellular bacterial pathogen, causes heartwater disease in ruminants. This model rickettsia, whose genome has been recently sequenced, is restricted to neutrophils and reticulo-endothelial cells of its mammalian host and to the midgut and salivary glands of its vector tick. E. ruminantium harbors a multigene family encoding for 16 outer membrane proteins including MAP1, a major antigenic protein. All the 16 map paralogs are expressed in bovine endothelial cells and some are specifically translated in the tick or in the mammalian host. In this study, we carried out phylogenetic analyses of E. ruminantium using sequences of 6 MAP proteins, MAP1, MAP1-2, MAP1-6, MAP1-5, MAP1+1 and MAP1-14, localized either in the center or at the borders of the map genes cluster. We show that (i) map1 gene is a good tool to characterize the genetic diversity among Africa, Caribbean islands and Madagascar strains including new emerging isolates of E. ruminantium; (ii) the different map paralogs define different genotypes showing divergent evolution; (iii) there is no correlation between all MAP genotypes and the geographic origins of the strains; (iv) The genetic diversity revealed by MAP proteins is conserved whatever is the scale of strains sampling (village, region, continent) and thus was not related to the different timing of strains introduction, i.e. continuous introduction of strains versus punctual introduction (Africa versus Caribbean islands). These results provide therefore a significant advance towards the management of E. ruminantium diversity. The differential evolution of these paralogs suggests specific roles of these proteins in host-vector-pathogen interactions that could be crucial for developing broad-spectrum vaccines.