Thierry Lefrançois

West Nile virus, Guadeloupe.

To determine whether West Nile virus (WNV) had reached the archipelago of Guadeloupe, a serologic study in horses and birds was conducted in 2002. Immunoglobulin (Ig) G, IgM, enzyme-linked immunosorbent assay, and seroneutralization tests identified WNV infection in horses and chickens. Six months later, a high rate of seroconversion was observed in horses.

Polymerase chain reaction as a diagnosis tool for detecting trypanosomes in naturally infected cattle in Burkina Faso

African animal trypanosomoses constitute the most important vector-borne cattle diseases in sub-Saharan Africa. Generally it is considered that there is a great lack of accurate tools for the diagnosis of the disease. During a trypanosomosis survey in the agro-pastoral zone of Sideradougou, Burkina Faso, 1036 cattle were examined for trypanosomes using microscopy. The PCR was applied on a subset of 260 buffy-coat samples using primers specific for Trypanosoma congolense savannah and riverine-forest groups, T. vivax, and T. brucei. Parasitological examination and the molecular technique were compared, showing a better efficiency of the latter. In the near future, the PCR is likely to become an efficient tool to estimate the prevalence of African trypanosomoses in affected areas.

Using species distribution models to optimize vector control in the framework of the tsetse eradication campaign in Senegal.

Significance Tsetse flies transmit human and animal trypanosomoses in sub-Saharan Africa, respectively a major neglected disease and the most important constraint to cattle production in infested countries. They are the target of the Pan African Tsetse and Trypanosomiasis Eradication Campaign (PATTEC). Here we show how distribution models can be used to optimize a tsetse eradication campaign in Senegal. Our results allow a better understanding of the relationships between tsetse presence and various environmental parameters measured by remote sensing. Furthermore, we argue that the methodology developed should be integrated into future tsetse control efforts that are planned under the umbrella of the PATTEC initiative. The results have a generic value for vector and pest control campaigns, especially when eradication is contemplated. Tsetse flies are vectors of human and animal trypanosomoses in sub-Saharan Africa and are the target of the Pan African Tsetse and Trypanosomiasis Eradication Campaign (PATTEC). Glossina palpalis gambiensis (Diptera: Glossinidae) is a riverine species that is still present as an isolated metapopulation in the Niayes area of Senegal. It is targeted by a national eradication campaign combining a population reduction phase based on insecticide-treated targets (ITTs) and cattle and an eradication phase based on the sterile insect technique. In this study, we used species distribution models to optimize control operations. We compared the probability of the presence of G. p. gambiensis and habitat suitability using a regularized logistic regression and Maxent, respectively. Both models performed well, with an area under the curve of 0.89 and 0.92, respectively. Only the Maxent model predicted an expert-based classification of landscapes correctly. Maxent predictions were therefore used throughout the eradication campaign in the Niayes to make control operations more efficient in terms of deployment of ITTs, release density of sterile males, and location of monitoring traps used to assess program progress. We discuss how the models’ results informed about the particular ecology of tsetse in the target area. Maxent predictions allowed optimizing efficiency and cost within our project, and might be useful for other tsetse control campaigns in the framework of the PATTEC and, more generally, other vector or insect pest control programs.

West Nile virus surveillance, Guadeloupe, 2003-2004.

We conducted extensive surveillance for West Nile virus infection in equines and chickens in Guadeloupe in 2003–2004. We showed a high seroprevalence in equines in 2003 related to biome, followed by a major decrease in virus circulation in 2004. No human or equine cases were reported during the study.

Amblyomma variegatum in cattle in Marie Galante, French Antilles: Prevalence, control measures, and infection by Ehrlichia ruminantium

We report Marie Galante as one of the Caribbean islands most heavily infested by the tropical bont tick (TBT) Amblyomma variegatum which is associated with two major diseases of ruminants: heartwater and dermatophilosis. In 2005, a survey was undertaken to assess the prevalence of TBT infestation in cattle, the prevalence of Ehrlichia ruminantium infection in TBTs, and the tick control measures implemented by livestock owners. A random sample of 195 cattle herds out of 1885 recorded on the island was investigated by thoroughly counting adult ticks on each animal and filling a questionnaire. A randomly collected sample of 136 TBTs was tested for infection by E. ruminantium by pCS20 nested PCR. Cattle herd prevalence (hp) was 73.8% for infestation by at least one TBT, 17.9% for infestation by at least one engorged female TBT, and 8.2% for clinical dermatophilosis. Cattle individual prevalence was 42.3% for infestation by at least one TBT, 6.6% for infestation by at least one engorged female TBT, and 2.2% for clinical dermatophilosis. The minimum, maximum and average numbers of TBTs per infested animal were, respectively 1, 108 and 11.5. Prevalence of TBT infection by E. ruminantium was 19.1%. No significant difference in herd prevalence was found among parishes or among ecological zones. For cattle owners treating against ticks (97.9% of all owners), all used aspersion of amitraz and herd prevalence was significantly different among those treating every 1-2-week (hp=69.6%, n=148), and less often than every 2-week (hp=88.6%, n=35) (P=0.031). Of the 42 herd subunits treated less than 4 days before the survey, 27 (64%) were infested with at least one TBT, and 6 (14%) with at least one engorged female TBT. These results indicate a high level of TBT infestation in Marie Galante, the inefficacy of tick treatments currently performed, and the need for an improved tick control strategy. Persisting high levels of infestation in Marie Galante threaten the success of on-going TBT eradication programs in the Caribbean because TBT can spread through migrating birds and trade of animals or of animal hides to other islands and potentially the American continent.

West Nile virus surveillance, Brazil, 2008-2010

BACKGROUND West Nile virus (WNV) is an emergent pathogen that is widely distributed in North and Central America. The recent introduction in South America has focused attention on the spread of WNV across Southern American countries. The transmission network involves mosquitoes, birds, horses and humans. METHODS The serological evaluation of sera from 678 equids and 478 birds was performed using a WNV-specific blocking ELISA, and only the positive results were confirmed by plaque reduction neutralisation tests (PRNTs). Molecular analysis was performed on sera from 992 healthy equids and on 63 macerates of brains from equids that died of encephalitis and had previously tested negative for other pathogens. We also tested swabs from 928 birds. The samples analysed were collected in different biomes of Brazil. RESULTS We identified WNV antibodies by ELISA in thirteen equids and five birds, and PRNT90 confirmed WNV positivity in four equid samples collected in 2009 in an area between the Amazon and the Pantanal. None of the ELISA positive bird samples were confirmed by PRNT90, and all samples tested by RT-PCR were negative. CONCLUSION WNV circulation is confirmed by this large scale survey even in the absence of detection of clinical cases.

Predicting West Nile Virus Seroprevalence in Wild Birds in Senegal

West Nile fever epidemiology is complex, and the role of birds in the maintenance, amplification, and dissemination of the West Nile virus (WNV) remains partially unknown. In 2003, a serological study was performed in Senegal, where West Nile infection is considered endemic. The goal was to identify potential reservoirs of WNV among bird species present in the Ferlo area (northern Senegal) and the Senegal River Valley, and to screen the ecological factors possibly related to West Nile infection. Serological data were analyzed using a generalized linear model. Statistical association between ecological factors and the risk of infection were then modeled to derive a species-specific risk. A cross-validation was conducted. The overall observed prevalence rate was 5.5% (n = 422). Thirteen bird species were found positive, from which five were migrating: Lanius senator, Anthus trivialis, Hippolais opaca, Jynx torquilla, and Cercotrichas galactotes. The nesting type in resident birds was positively correlated with the risk of infection (odds ratio [OR] = 11, p = 0.0003); the gregariousness level of birds appeared as a protective factor (OR = 0.3, p = 0.01). The predicted prevalence varied between 1% and 39% for resident species and between 1% and 7% for migrating species. Results of model internal validation were satisfactory at the individual and species level. However, more field and experimental investigations are needed to confirm these preliminary results and help target the future research and surveillance in Senegal.

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.

Innovative approach for transcriptomic analysis of obligate intracellular pathogen: selective capture of transcribed sequences of Ehrlichia ruminantium

BackgroundWhole genome transcriptomic analysis is a powerful approach to elucidate the molecular mechanisms controlling the pathogenesis of obligate intracellular bacteria. However, the major hurdle resides in the low quantity of prokaryotic mRNAs extracted from host cells. Our model Ehrlichia ruminantium (ER), the causative agent of heartwater, is transmitted by tick Amblyomma variegatum. This bacterium affects wild and domestic ruminants and is present in Sub-Saharan Africa and the Caribbean islands. Because of its strictly intracellular location, which constitutes a limitation for its extensive study, the molecular mechanisms involved in its pathogenicity are still poorly understood.ResultsWe successfully adapted the SCOTS method (Selective Capture of Transcribed Sequences) on the model Rickettsiales ER to capture mRNAs. Southern Blots and RT-PCR revealed an enrichment of ERs cDNAs and a diminution of ribosomal contaminants after three rounds of capture. qRT-PCR and whole-genome ER microarrays hybridizations demonstrated that SCOTS method introduced only a limited bias on gene expression. Indeed, we confirmed the differential gene expression between poorly and highly expressed genes before and after SCOTS captures. The comparative gene expression obtained from ER microarrays data, on samples before and after SCOTS at 96 hpi was significantly correlated (R2 = 0.7). Moreover, SCOTS method is crucial for microarrays analysis of ER, especially for early time points post-infection. There was low detection of transcripts for untreated samples whereas 24% and 70.7% were revealed for SCOTS samples at 24 and 96 hpi respectively.ConclusionsWe conclude that this SCOTS method has a key importance for the transcriptomic analysis of ER and can be potentially used for other Rickettsiales. This study constitutes the first step for further gene expression analyses that will lead to a better understanding of both ER pathogenicity and the adaptation of obligate intracellular bacteria to their environment.