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.

Evolutionary changes in symbiont community structure in ticks.

Ecological specialization to restricted diet niches is driven by obligate, and often maternally inherited, symbionts in many arthropod lineages. These heritable symbionts typically form evolutionarily stable associations with arthropods that can last for millions of years. Ticks were recently found to harbour such an obligate symbiont, Coxiella-LE, that synthesizes B vitamins and cofactors not obtained in sufficient quantities from blood diet. In this study, the examination of 81 tick species shows that some Coxiella-LE symbioses are evolutionarily stable with an ancient acquisition followed by codiversification as observed in ticks belonging to the Rhipicephalus genus. However, many other Coxiella-LE symbioses are characterized by low evolutionary stability with frequent host shifts and extinction events. Further examination revealed the presence of nine other genera of maternally inherited bacteria in ticks. Although these nine symbionts were primarily thought to be facultative, their distribution among tick species rather suggests that at least four may have independently replaced Coxiella-LE and likely represent alternative obligate symbionts. Phylogenetic evidence otherwise indicates that cocladogenesis is globally rare in these symbioses as most originate via horizontal transfer of an existing symbiont between unrelated tick species. As a result, the structure of these symbiont communities is not fixed and stable across the tick phylogeny. Most importantly, the symbiont communities commonly reach high levels of diversity with up to six unrelated maternally inherited bacteria coexisting within host species. We further conjecture that interactions among coexisting symbionts are pivotal drivers of community structure both among and within tick species.

Multiple independent introductions of Plasmodium falciparum in South America.

The origin of Plasmodium falciparum in South America is controversial. Some studies suggest a recent introduction during the European colonizations and the transatlantic slave trade. Other evidence--archeological and genetic--suggests a much older origin. We collected and analyzed P. falciparum isolates from different regions of the world, encompassing the distribution range of the parasite, including populations from sub-Saharan Africa, the Middle East, Southeast Asia, and South America. Analyses of microsatellite and SNP polymorphisms show that the populations of P. falciparum in South America are subdivided in two main genetic clusters (northern and southern). Phylogenetic analyses, as well as Approximate Bayesian Computation methods suggest independent introductions of the two clusters from African sources. Our estimates of divergence time between the South American populations and their likely sources favor a likely introduction from Africa during the transatlantic slave trade.

Patterns of selection on Plasmodium falciparum erythrocyte-binding antigens after the colonization of the New World.

Pathogens, which have recently colonized a new host species or new populations of the same host, are interesting models for understanding how populations may evolve in response to novel environments. During its colonization of South America from Africa, Plasmodium falciparum, the main agent of malaria, has been exposed to new conditions in distinctive new human populations (Amerindian and populations of mixed origins) that likely exerted new selective pressures on the parasite's genome. Among the genes that might have experienced strong selective pressures in response to these environmental changes, the eba genes (erythrocyte-binding antigens genes), which are involved in the invasion of the human red blood cells, constitute good candidates. In this study, we analysed, in South America, the polymorphism of three eba genes (eba-140, eba-175, eba-181) and compared it to the polymorphism observed in African populations. The aim was to determine whether these genes faced selective pressures in South America distinct from what they experienced in Africa. Patterns of genetic variability of these genes were compared to the patterns observed at two housekeeping genes (adsl and serca) and 272 SNPs to separate adaptive effects from demographic effects. We show that, conversely to Africa, eba-140 seemed to be under stronger diversifying selection in South America than eba-175. In contrast, eba-181 did not show any sign of departure from neutrality. These changes in the patterns of selection on the eba genes could be the consequence of changes in the host immune response, the host receptor polymorphisms and/or the ability of the parasite to silence or express differentially its invasion proteins.

Detection of Anaplasma and Ehrlichia bacteria in humans, wildlife, and ticks in the Amazon rainforest.

Tick-borne bacteria of the genera Ehrlichia and Anaplasma cause several emerging human infectious diseases worldwide. In this study, we conduct an extensive survey for Ehrlichia and Anaplasma infections in the rainforests of the Amazon biome of French Guiana. Through molecular genetics and metagenomics reconstruction, we observe a high indigenous biodiversity of infections circulating among humans, wildlife, and ticks inhabiting these ecosystems. Molecular typing identifies these infections as highly endemic, with a majority of new strains and putative species specific to French Guiana. They are detected in unusual rainforest wild animals, suggesting they have distinctive sylvatic transmission cycles. They also present potential health hazards, as revealed by the detection of Candidatus Anaplasma sparouinense in human red blood cells and that of a new close relative of the human pathogen Ehrlichia ewingii, Candidatus Ehrlichia cajennense, in the tick species that most frequently bite humans in South America. The genome assembly of three new putative species obtained from human, sloth, and tick metagenomes further reveals the presence of major homologs of Ehrlichia and Anaplasma virulence factors. These observations converge to classify health hazards associated with Ehrlichia and Anaplasma infections in the Amazon biome as distinct from those in the Northern Hemisphere.

Specificity of resistance to dengue virus isolates is associated with genotypes of the mosquito antiviral gene Dicer-2.

In contrast to the prevailing view that invertebrate immunity relies on broad-spectrum recognition and effector mechanisms, intrinsic genetic compatibility between invertebrate hosts and their pathogens is often highly specific in nature. Solving this puzzle requires a better understanding of the molecular basis underlying observed patterns of invertebrate host-pathogen genetic specificity, broadly referred to as genotype-by-genotype interactions. Here, we identify an invertebrate immune gene in which natural polymorphism is associated with isolate-specific resistance to an RNA virus. Dicer-2 (dcr2) encodes a key protein upstream of the RNA interference (RNAi) pathway, a major antiviral component of innate immunity in invertebrates. We surveyed allelic polymorphism at the dcr2 locus in a wild-type outbred population and in three derived isofemale families of the mosquito Aedes aegypti that were experimentally exposed to several, genetically distinct isolates of dengue virus. We found that dcr2 genotype was associated with resistance to dengue virus in a virus isolate-specific manner. By contrast, no such association was found for genotypes at two control loci flanking dcr2, making it likely that dcr2 contains the yet-unidentified causal polymorphism(s). This result supports the idea that host-pathogen compatibility in this system depends, in part, on a genotype-by-genotype interaction between dcr2 and the viral genome, and points to the RNAi pathway as a potentially important determinant of intrinsic insect-virus genetic specificity.

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.

Changing distributions of ticks: causes and consequences.

Today, we are witnessing changes in the spatial distribution and abundance of many species, including ticks and their associated pathogens. Evidence that these changes are primarily due to climate change, habitat modifications, and the globalisation of human activities are accumulating. Changes in the distribution of ticks and their invasion into new regions can have numerous consequences including modifications in their ecological characteristics and those of endemic species, impacts on the dynamics of local host populations and the emergence of human and livestock disease. Here, we review the principal causes for distributional shifts in tick populations and their consequences in terms of the ecological attributes of the species in question (i.e. phenotypic and genetic responses), pathogen transmission and disease epidemiology. We also describe different methodological approaches currently used to assess and predict such changes and their consequences. We finish with a discussion of new research avenues to develop in order to improve our understanding of these host-vector-pathogen interactions in the context of a changing world.

Understanding the genetic, demographical and/or ecological processes at play in invasions: lessons from the southern cattle tick Rhipicephalus microplus (Acari: Ixodidae).

The southern cattle tick, Rhipicephalus microplus, is the ixodid species causing the largest economic losses in tropical agrosystems because of its recurrent invasive success, explosive demography on bovine herds, vector competence for diverse pathogens and frequent development of acaricide resistance. Its ecology and the physiological bases of the acaricide resistances it developed, as well as alternative tick control measures, have been intensively studied for decades. By contrast, the tick population genetic structure and its remarkable ability to quickly adapt to new environments have not yet received much attention. We investigated such issues using population genetics analyses in the recently invaded island New Caledonia. In this paper we aim to describe some guidelines for acarologists willing to investigate the processes at play in Acari invasions. Particular emphasis is given to the accuracy of sampling designs and sampling scales for population genetics to be actually informative on the demographical processes of the species (i.e., its mating rules, the determinants of population limits, population sizes, the relationships between genetic exchanges and geographical distances and relevant ecological factors).

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.

Emerging infectious diseases and new pandemics: dancing with a ghost! Lessons in inter- and transdisciplinary research in French Guiana, South America.

In light of current international public health challenges, calls for inter- and transdisciplinary research are increasing, particularly in response to complex and intersecting issues. Although widely used under the One Health flag, it is still unclear how inter- and transdisciplinary science should be applied to infectious disease research, public health, and the different stakeholders. Here, we present and discuss our common scientific and biomedical experience in French Guiana, South America to conduct and enrich research in vector-borne and zoonotic infectious diseases, with the aim to translate findings to public health and political stakeholders. We highlight the successful progressive dissolution of disciplinary boundaries that go beyond One Health positive-driven assumptions and argue that specific local conditions, as well as strong support from research and medical institutions, have facilitated an emulsion toward inter- and transdisciplinary science. This argument is intended to improve responses to public health concerns in French Guiana and other countries and regions of the world.

A need for null models in understanding disease transmission: the example of Mycobacterium ulcerans (Buruli ulcer disease).

Understanding the interactions of ecosystems, humans and pathogens is important for disease risk estimation. This is particularly true for neglected and newly emerging diseases where modes and efficiencies of transmission leading to epidemics are not well understood. Using a model for other emerging diseases, the neglected tropical skin disease Buruli ulcer (BU), we systematically review the literature on transmission of the etiologic agent, Mycobacterium ulcerans (MU), within a One Health/EcoHealth framework and against Hill's nine criteria and Koch's postulates for making strong inference in disease systems. Using this strong inference approach, we advocate a null hypothesis for MU transmission and other understudied disease systems. The null should be tested against alternative vector or host roles in pathogen transmission to better inform disease management. We propose a re-evaluation of what is necessary to identify and confirm hosts, reservoirs and vectors associated with environmental pathogen replication, dispersal and transmission; critically review alternative environmental sources of MU that may be important for transmission, including invertebrate and vertebrate species, plants and biofilms on aquatic substrates; and conclude with placing BU within the context of other neglected and emerging infectious diseases with intricate ecological relationships that lead to disease in humans, wildlife and domestic animals.

The Chikungunya threat: an ecological and evolutionary perspective.

Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus. Although primarily African and zoonotic, it is known chiefly for its non-African large urban outbreaks during which it is transmitted by the same vectors as those of Dengue viruses. Unlike Dengue viruses, CHIKV displays a re-emergence pattern that closely depends on long-distance migrations including recent re-immigrations from African (putatively zoonotic) sources. Genus-based differences also emerged when comparing the evolution of Dengue-related (Flaviviruses) and of CHIKV-related (Alphaviruses) arboviruses. In this review, we discuss current information on CHIKV genetics, ecology and human infection. Further investigations on African CHIKV ecology and the differences between Flavivirus and Alphavirus members in adaptive changes and evolutionary constraints are likely to help delineate the potential of further CHIKV (re-)emergence.

Genetic specificity and potential for local adaptation between dengue viruses and mosquito vectors.

BACKGROUND: Several observations support the hypothesis that vector-driven selection plays an important role in shaping dengue virus (DENV) genetic diversity. Clustering of DENV genetic diversity at a particular location may reflect underlying genetic structure of vector populations, which combined with specific vector genotype x virus genotype (G x G) interactions may promote adaptation of viral lineages to local mosquito vector genotypes. Although spatial structure of vector polymorphism at neutral genetic loci is well-documented, existence of G x G interactions between mosquito and virus genotypes has not been formally demonstrated in natural populations. Here we measure G x G interactions in a system representative of a natural situation in Thailand by challenging three isofemale families from field-derived Aedes aegypti with three contemporaneous low-passage isolates of DENV-1. RESULTS: Among indices of vector competence examined, the proportion of mosquitoes with a midgut infection, viral RNA concentration in the body, and quantity of virus disseminated to the head/legs (but not the proportion of infected mosquitoes with a disseminated infection) strongly depended on the specific combinations of isofemale families and viral isolates, demonstrating significant G x G interactions. CONCLUSION: Evidence for genetic specificity of interactions in our simple experimental design indicates that vector competence of Ae. aegypti for DENV is likely governed to a large extent by G x G interactions in genetically diverse, natural populations. This result challenges the general relevance of conclusions from laboratory systems that consist of a single combination of mosquito and DENV genotypes. Combined with earlier evidence for fine-scale genetic structure of natural Ae. aegypti populations, our finding indicates that the necessary conditions for local DENV adaptation to mosquito vectors are met.

Environmental Variations in Mycobacterium ulcerans Transcriptome: Absence of Mycolactone Expression in Suboptimal Environments.

Buruli ulcer is a neglected tropical infectious disease, produced by the environmentally persistent pathogen Mycobacterium ulcerans (MU). Neither the ecological niche nor the exact mode of transmission of MU are completely elucidated. However, some environmental factors, such as the concentration in chitin and pH values, were reported to promote MU growth in vitro. We pursued this research using next generation sequencing (NGS) and mRNA sequencing to investigate potential changes in MU genomic expression profiles across in vitro environmental conditions known to be suitable for MU growth. Supplementing the growth culture medium in either chitin alone, calcium alone, or in both chitin and calcium significantly impacted the MU transcriptome and thus several metabolic pathways, such as, for instance, those involved in DNA synthesis or cell wall production. By contrast, some genes carried by the virulence plasmid and necessary for the production of the mycolactone toxin were expressed neither in control nor in any modified environments. We hypothesized that these genes are only expressed in stressful conditions. Our results describe important environmental determinants playing a role in the pathogenicity of MU, helping the understanding of its complex natural life cycle and encouraging further research using genomic approaches.

Survey of ticks in French Guiana.

In this study, we examine the current pattern of tick diversity and host use in French Guiana, South America, from 97 sampling localities encompassing peri-urban, rural and natural habitats. We collected 3395 ticks, including 1485 specimens from 45 vertebrate species (humans, domestic and wild animals) and 1910 questing specimens from vegetation. Morphological examinations identified 22 species belonging to six genera: Amblyomma (16 species), Rhipicephalus (two species), Ixodes (one species), Dermacentor (one species), Haemaphysalis (one species), Ornithodoros (one species). To facilitate future identification, we produced a bank of pictures of different stages for all these species. Taxonomic identification was then confirmed by molecular characterization of two mitochondrial genes, cytochrome c oxidase CO1 and 16S rDNA. Eleven of the 22 reported species were collected on humans, six on domestic animals and 12 on wild animals. The most widespread tick species collected were A. cajennense sensu stricto and, to a lesser extent, A. oblongoguttatum; both of these species were frequently found on humans. We used these results to discuss the tick-associated risks for human and animal health in French Guiana.

Phylogenetics of the Spiroplasma ixodetis endosymbiont reveals past transfers between ticks and other arthropods.

The bacterium Spiroplasma ixodetis is a maternally inherited endosymbiont primarily described from ticks but also found widespread across other arthropods. While it has been identified as a male-killing agent in some insect species, the consequences of infection with S. ixodetis in ticks are entirely unknown, and it is unclear how this endosymbiont spreads across tick species. Here, we have investigated this aspect through the examination of the diversity and evolutionary history of S. ixodetis infections in 12 tick species and 12 other arthropod species. Using a multi-locus typing approach, we identified that ticks harbor a substantial diversity of divergent S. ixodetis strains. Phylogenetic investigations revealed that these S. ixodetis strains do not cluster within a tick-specific subclade but rather exhibit distinct evolutionary origins. In their past, these strains have undergone repeated horizontal transfers between ticks and other arthropods, including aphids and flies. This diversity pattern strongly suggests that maternal inheritance and horizontal transfers are key drivers of S. ixodetis spread, dictating global incidence of infections across tick communities. We do not, however, detect evidence of S. ixodetis-based male-killing since we observed that infections were widely present in both males and females across populations of the African blue tick Rhipicephalus decoloratus.

Chitin Increases Mycobacterium ulcerans Growth in Acidic Environments.

Species with a chitinous exoskeleton are overrepresented among the aquatic organisms carrying Mycobacterium ulcerans (MU) in nature and laboratory experiments have demonstrated the enhancing effects of chitin on the growth of MU. Field surveys identified pH as one of the key parameters delineating the distribution of MU in tropical regions. The present study investigated the relationship between chitin and pH in MU growth. By focusing on pH variations in the field, our results revealed that chitin enhanced MU growth in acidic environments. The present study provides new information on the ecological conditions favoring the development of this mycobacterium in nature.

Population genetics and molecular epidemiology or how to "débusquer la bête".

Parasites represent a great proportion of the world's living organisms and are of overwhelming significance because of their impact on hosts (evolutionarily, medically, agronomical and economically). The knowledge of the population biology of such organisms is thus of fundamental importance to population biologists. Most parasites cannot be studied by direct methods and their biology has to be assessed via indirect means, most notably using molecular markers. In this review, we present the molecular tools, the null models employed, the statistical tools available and the kinds of inferences one can make when using molecular markers to study the ecology/epidemiology of host-parasite systems (molecular ecology/molecular epidemiology). We conclude with relevant examples, most issued from our laboratory, to illustrate the pros and cons of such methods for the study of parasites, vectors, micropathogens and their hosts and briefly discuss future needs.

Direct and indirect inferences on parasite mating and gene transmission patterns. Pangamy in the cattle tick Rhipicephalus (Boophilus) microplus.

Mating system plays a determinant role in the maintenance and distribution of genetic variation. Difficulties in applying standard methods of indirect inferences onto parasitic life-cycles partly explain the current lack of knowledge on parasite mating systems. The present study develops a combination of direct and indirect inference methods circumventing such difficulties, and illustrates in particular how such a combination modifies our understanding of the biology of the southern cattle tick, Rhipicephalus microplus. To directly assess how females and males pair in natural populations, we sampled 225 mating pairs, genotyped them at six microsatellite loci, and analysed the correlation between mating status and genetic relatedness. This analysis revealed pangamy, i.e. a random association of male and female genotypes. In addition, indirect methods inferred that sib-groups of ticks exploit distinct individual-cows, and hence that ticks probably move among cows during their parasitic lifetime. Altogether, these results negate the expectation of a high frequency of sib-mating, and show the coexistence of genetically diverging stocks within tick cohorts and populations. These results have several consequences for our understanding of R. microplus. For instance, while existing models of pesticide resistance management look perfectly applicable to this species, the epidemiological models of the micropathogens it vectors needs revision. More largely, the methods developed here would help clarify the evolutionary patterns of any dioceous parasite.