The composition and abundance of bacterial communities residing in the gut of Glossina palpalis palpalis captured in two sites of southern Cameroon.

BACKGROUND: A number of reports have demonstrated the role of insect bacterial flora on their host's physiology and metabolism. The tsetse host and vector of trypanosomes responsible for human sleeping sickness (human African trypanosomiasis, HAT) and nagana in animals (African animal trypanosomiasis, AAT) carry bacteria that influence its diet and immune processes. However, the mechanisms involved in these processes remain poorly documented. This underscores the need for increased research into the bacterial flora composition and structure of tsetse flies. The aim of this study was to identify the diversity and relative abundance of bacterial genera in Glossina palpalis palpalis flies collected in two trypanosomiasis foci in Cameroon. METHODS: Samples of G. p. palpalis which were either negative or naturally trypanosome-positive were collected in two foci located in southern Cameroon (Campo and Bipindi). Using the V3V4 and V4 variable regions of the small subunit of the 16S ribosomal RNA gene, we analyzed the respective bacteriome of the flies' midguts. RESULTS: We identified ten bacterial genera. In addition, we observed that the relative abundance of the obligate endosymbiont Wigglesworthia was highly prominent (around 99%), regardless of the analyzed region. The remaining genera represented approximately 1% of the bacterial flora, and were composed of Salmonella, Spiroplasma, Sphingomonas, Methylobacterium, Acidibacter, Tsukamurella, Serratia, Kluyvera and an unidentified bacterium. The genus Sodalis was present but with a very low abundance. Globally, no statistically significant difference was found between the bacterial compositions of flies from the two foci, and between positive and trypanosome-negative flies. However, Salmonella and Serratia were only described in trypanosome-negative flies, suggesting a potential role for these two bacteria in fly refractoriness to trypanosome infection. In addition, our study showed the V4 region of the small subunit of the 16S ribosomal RNA gene was more efficient than the V3V4 region at describing the totality of the bacterial diversity. CONCLUSIONS: A very large diversity of bacteria was identified with the discovering of species reported to secrete anti-parasitic compounds or to modulate vector competence in other insects. For future studies, the analyses should be enlarged with larger sampling including foci from several countries.

Detection of Wolbachia and different trypanosome species in Glossina palpalis palpalis populations from three sleeping sickness foci of southern Cameroon.

BACKGROUND: African trypanosomiases are caused by trypanosomes that are cyclically transmitted by tsetse. Investigations aiming to generate knowledge on the bacterial fauna of tsetse have revealed distinct symbiotic microorganisms. Furthermore, studies addressing the tripartite association between trypanosomes-tsetse-symbionts relationship have so far been contradictory. Most studies included Sodalis glossinudius and, consequently, the association involving Wolbachia is poorly understood. Understanding the vectorial competence of tsetse requires decrypting these tripartite associations. In this study, we identified Wolbachia and trypanosomes in Glossina palpalis palpalis from three human African trypanosomiasis (HAT) foci in southern Cameroon. METHODS: Tsetse flies were captured with pyramidal traps in the Bipindi, Campo and Fontem HAT foci. After morphological identification, DNA was extracted from whole tsetse flies and Wolbachia and trypanosomes were identified by PCR using different trypanosome-specific primers and two Wolbachia-specific primers (Wolbachia surface protein and 16S rRNA genes). Statistical analyses were performed to compare the trypanosome and Wolbachia infection rates between villages and different foci and to look for an association between these microorganisms. RESULTS: From a total of 2122 tsetse flies, 790 G. p. palpalis were analyzed. About 25.32% of flies hosted Wolbachia and 31.84% of non-teneral flies were infected by at least one trypanosome species. There was no significant difference between the global Wolbachia prevalence revealed by the two markers while some differences were observed between HAT foci. From 248 G. p. palpalis with trypanosome infections, 62.90% were with T. vivax, 34.68% with T. congolense forest, 16.13% with T. brucei (s.l.) and 2.42% with T. congolense savannah. Of all trypanosome-infected flies, 29.84% hosted Wolbachia and no association was observed between Wolbachia and trypanosome co-infections. CONCLUSIONS: This study revealed differences in the prevalence of Wolbachia and trypanosomes in G. p. palpalis according to HAT foci. The use of only one marker has underestimated the prevalence of Wolbachia, thus more markers in subsequent studies may improve its detection. The presence of Wolbachia seems to have no impact on the establishment of trypanosomes in G. p. palpalis. The tripartite association between tsetse, Wolbachia and trypanosomes varies according to studied areas. Studies aiming to evaluate the genetic polymorphism of Wolbachia and its density in tsetse flies could help to better understand this association.

Intestinal Bacterial Communities of Trypanosome-Infected and Uninfected Glossina palpalis palpalis from Three Human African Trypanomiasis Foci in Cameroon.

Glossina sp. the tsetse fly that transmits trypanosomes causing the Human or the Animal African Trypanosomiasis (HAT or AAT) can harbor symbiotic bacteria that are known to play a crucial role in the fly's vector competence. We hypothesized that other bacteria could be present, and that some of them could also influence the fly's vector competence. In this context the objectives of our work were: (a) to characterize the bacteria that compose the G. palpalis palpalis midgut bacteriome, (b) to evidence possible bacterial community differences between trypanosome-infected and non-infected fly individuals from a given AAT and HAT focus or from different foci using barcoded Illumina sequencing of the hypervariable V3-V4 region of the 16S rRNA gene. Forty G. p. palpalis flies, either infected by Trypanosoma congolense or uninfected were sampled from three trypanosomiasis foci in Cameroon. A total of 143 OTUs were detected in the midgut samples. Most taxa were identified at the genus level, nearly 50% at the species level; they belonged to 83 genera principally within the phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. Prominent representatives included Wigglesworthia (the fly's obligate symbiont), Serratia, and Enterobacter hormaechei. Wolbachia was identified for the first time in G. p. palpalis. The average number of bacterial species per tsetse sample was not significantly different regarding the fly infection status, and the hierarchical analysis based on the differences in bacterial community structure did not provide a clear clustering between infected and non-infected flies. Finally, the most important result was the evidence of the overall very large diversity of intestinal bacteria which, except for Wigglesworthia, were unevenly distributed over the sampled flies regardless of their geographic origin and their trypanosome infection status.

Xenomonitoring of sleeping sickness transmission in Campo (Cameroon).

BACKGROUND: The sleeping sickness focus of Campo in South Cameroon is still active, at a low endemic level, for more than a century, despite a regular medical surveillance. The present study focuses on the spatial distribution of xenomonitoring information obtained from an entomological survey performed in the dry season 2012. It appears that humans constitute a third of the blood meals and that the flies' densities were coherent with those classically observed in the different biotopes. Paradoxically, the epicenter of the focus is the place where the risk indicators are the lowest ones. METHODS: Particular attention was paid to the entomological device so that it covered the main part of human activities in the study area. One hundred and sixty-two pyramidal traps were used to catch tsetse flies twice a day that were identified, counted, dissected. Molecular analysis using classical and specific molecular markers was conducted to determine the importance of trypanosome infections and the nature of the feeding hosts. This information was used to calculate a Transmission Risk Index and to define a gradient of risk that was projected into a Geographical Information System. RESULTS: Conventional entomological indicators such as species identification of tsetse flies or the Apparent Density per Trap per day, show that Glossina palpalis palpalis is the main species in the campo area which is classically distributed into the different biotopes of the study area. Molecular analysis reveals that humans constitute a third of the blood feeding hosts and that 20 % of the dissected flies were infected with trypanosomes, principally with Nannomonas. Nevertheless, one fly was carrying Trypanosoma brucei gambiense, the pathogen agent of sleeping sickness, showing that the reservoir is still active in the epicenter of the focus. Paradoxically, the Transmission Risk Index is not important in the epicenter, demonstrating that endemic events are not only depending on the man/vector contact. CONCLUSION: Xenomonitoring provides a valuable guide/tool to determine places at higher risk for vector/human contact and to identify trypanosomes species circulating in the focus. This information from xenomonitoring demonstrates that decision makers should include a veterinary device in a control strategy.

Population genetics of forest type of Trypanosoma congolense circulating in Glossina palpalis palpalis of Fontem in the South-West region of Cameroon.

BACKGROUND: Genetic variation of microsatellite loci is a widely used method for the analysis of population genetic structure of several organisms. To improve our knowledge on the population genetics of trypanosomes, Trypanosoma congolense forest and savannah types were identified in the mid-guts of Glossina palpalis palpalis caught in five villages of Fontem in the South-West region of Cameroon. From the positive samples of Trypanosoma congolense forest, the genetic diversity and the population genetic structure of these parasites were evaluated. METHOD: For this study, pyramidal traps were set up during three entomological surveys and 3347 tsetse flies were collected, dissected and 1903 midguts collected. DNA was extracted from midguts and specific primers were used to identify Trypanosoma congolense forest and savannah. All Trypanosoma congolense forest positive samples were characterized with seven microsatellite markers. RESULTS: Microscopic examination revealed 25 (1.31%) mid-gut infections with trypanosomes while the PCR method identified 120 (6.3%) infections due to Trypanosoma congolense: 94 (78.33%) Trypanosoma congolense forest and 28 (21.77%) Trypanosoma congolense savannah. The trypanosome infection rates varied significantly between villages and years of capture. Menji recorded the highest infection rate (15.11%); and samples captured in 2009 were more infected (14.33%). The microsatellite markers revealed a genetic variability between Trypanosoma congolense forest populations of Fontem villages and 6.38% of mixed infections due to different genotypes of T. congolense "forest type". CONCLUSION: Our data on the population genetics play in favor of a clonal reproduction of this parasite. The microsatellite markers used here showed a low genetic differentiation and an absence of sub-structuration (FST ≤ 0.0003) between Trypanosoma congolense forest populations of Fontem villages. However, the high FST value (FST ≥ 0.3911) between samples of the Democratic Republic of Congo and those of Fontem villages indicates low migration rates between trypanosomes of these subpopulations.

Population genetics of Trypanosoma brucei circulating in Glossina palpalis palpalis and domestic animals of the Fontem sleeping sickness focus of Cameroon.

BACKGROUND: Human African Trypanosomiasis is still a public health threat in Cameroon. To assess Trypanosoma brucei strains circulating in the Fontem sleeping sickness focus, we conducted a genetic structure study using microsatellites to assess genotypes circulating in both tsetse flies and domestic animals. METHOD: For this study, pyramidal traps were set up and 2695 tsetse flies were collected and 1535 (57%) living flies were dissected and their mid-guts collected. Furthermore, blood samples were collected from 397 domestic animals (pigs, goats, sheep and dogs). DNA was extracted from midguts and blood samples, and specific primers were used to identify trypanosomes of the subgenus Trypanozoon. All positive samples were genetically characterized with seven microsatellite markers. RESULTS: Seventy five (4.7%) midguts of tsetse flies and 140 (35.2%) domestic animals were found infected by trypanosomes of the subgenus Trypanozoon. The genetic characterization of 215 Trypanozoon positive samples (75 from tsetse and 140 from animals) revealed a genetic diversity between Trypanosoma brucei circulating in tsetse and domestic animals. Of these positive samples, 87 (40.5%) single infections were used here to investigate the population genetics of Trypanosoma brucei circulating in tsetse and domestic animals. The dendrogram illustrating the genetic similarities between Trypanosoma brucei genotypes was subdivided into four clusters. The samples from tsetse belonged to the same cluster whereas the samples from domestic animals and espcially pigs were distributed in the four clusters. CONCLUSION: Pigs appeared as the animal species harboring the highest number of different Trypanosoma brucei strains. They may play an important role in the propagation of different genotypes. The FST values revealed a sub structuration of Trypanosoma brucei according to hosts and sometimes villages. The data obtained from this study may have considerable importance for the understanding of the transmission and the spread of specific genotypes of Trypanosoma brucei.

Population genetics of Glossina palpalis palpalis from central African sleeping sickness foci.

BACKGROUND: Glossina palpalis palpalis (Diptera: Glossinidae) is widespread in west Africa, and is the main vector of sleeping sickness in Cameroon as well as in the Bas Congo Province of the Democratic Republic of Congo. However, little is known on the structure of its populations. We investigated G. p. palpalis population genetic structure in five sleeping sickness foci (four in Cameroon, one in Democratic Republic of Congo) using eight microsatellite DNA markers. RESULTS: A strong isolation by distance explains most of the population structure observed in our sampling sites of Cameroon and DRC. The populations here are composed of panmictic subpopulations occupying fairly wide zones with a very strong isolation by distance. Effective population sizes are probably between 20 and 300 individuals and if we assume densities between 120 and 2000 individuals per km2, dispersal distance between reproducing adults and their parents extends between 60 and 300 meters. CONCLUSIONS: This first investigation of population genetic structure of G. p. palpalis in Central Africa has evidenced random mating subpopulations over fairly large areas and is thus at variance with that found in West African populations of G. p. palpalis. This study brings new information on the isolation by distance at a macrogeographic scale which in turn brings useful information on how to organise regional tsetse control. Future investigations should be directed at temporal sampling to have more accurate measures of demographic parameters in order to help vector control decision.