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Background: African trypanosomiases are vector-borne diseases that affect humans and livestock in sub-Saharan Africa. Although data have been collected on tsetse fauna as well as trypanosome infections in tsetse flies and mammals in foci of sleeping sickness in Chad, the situation of tsetse fly-transmitted trypanosomes remains unknown in several tsetse-infested areas of Chad. This study was designed to fill this epidemiological knowledge gap by determining the tsetse fauna as well as the trypanosomes infecting tsetse flies in the area of Lake Iro in southeastern Chad., Methods: Tsetse flies were trapped along the Salamat River using biconical traps. The proboscis and tsetse body were removed from each fly. DNA was extracted from the proboscis using proteinase K and phosphate buffer and from the tsetse body using Chelex 5%. Tsetse flies were identified by amplifying and sequencing the cytochrome c oxydase I gene of each tsetse fly. Trypanosome species were detected by amplifying and sequencing the internal transcribed spacer 1 of infecting trypanosomes., Results: A total of 617 tsetse flies were trapped; the apparent density of flies per trap per day was 2. 6. Of the trapped flies, 359 were randomly selected for the molecular identification and for the detection of infecting trypanosomes. Glossina morsitans submorsitans (96.1%) was the dominant tsetse fly species followed by G. fuscipes fuscipes (3.1%) and G. tachinoides (0.8%). Four trypanosome species, including Trypanosoma vivax, T. simiae, T. godfreyi and T. congolense savannah, were detected. Both single infection (56.7%) and mixed infections of trypanosomes (4.6%) were detected in G. m. submorsitans. The single infection included T. simiae (20.5%), T. congolense savannah (16.43%), T. vivax (11.7%) and T. godfreyi (9.8%). The trypanosome infection rate was 61.4% in G. m. submorsitans, 72.7% in G. f. fuscipes and 66.6% in G. tachinoides. Trypanosome infections were more prevalent in tsetse bodies (40.6%) than in the proboscis (16.3%)., Conclusion: This study revealed the presence of different tsetse species and a diversity of trypanosomes pathogenic to livestock in the area of Lake Iro. The results highlight the risks and constraints that animal African trypanosomiasis pose to livestock breeding and the importance of assessing trypanosome infections in livestock in this area.

Background: Equine trypanosomiases are complex infectious diseases with overlapping clinical signs defined by their mode of transmission. Despite their economic impacts, these diseases have been neglected by the scientific community, the veterinary authorities and regulatory organizations. To fill the observed knowledge gap, we undertook the identification of different trypanosome species and subspecies naturally infecting horses and donkeys within the Chadian sleeping sickness focus. The objective of the study was to investigate the potential role of these domestic animals as reservoirs of the human-infective Trypanosoma brucei gambiense., Method: Blood samples were collected from 155 donkeys and 131 horses in three human African trypanosomiasis (HAT) foci in Chad. Rapid diagnostic test (RDT) and capillary tube centrifugation (CTC) test were used to search for trypanosome infections. DNA was extracted from each blood sample and different trypanosome species and subspecies were identified with molecular tools., Results: From 286 blood samples collected, 54 (18.9%) and 36 (12.6%) were positive for RDT and CTC, respectively. PCR revealed 101 (35.3%) animals with trypanosome infections. The Cohen's kappa coefficient used to evaluate the concordance between the diagnostic methods were low; ranging from 0.09 ± 0.05 to 0.48 ± 0.07. Trypanosomes of the subgenus Trypanozoon were the most prevalent (29.4%), followed by T. congolense forest (11.5%), Trypanosoma congolense savannah (4.9%) and Trypanosoma vivax (4.5%). Two donkeys and one horse from the Maro HAT focus were found with T. b. gambiense infections. No significant differences were observed in the infection rates of different trypanosomes between animal species and HAT foci., Conclusions: This study revealed several trypanosome species and subspecies in donkeys and horses, highlighting the existence of AAT in HAT foci in Chad. The identification of T. b. gambiense in donkeys and horses suggests considering these animals as potential reservoir for HAT in Chad. The presence of both human-infective and human non-infective trypanosomes species highlights the need for developing joint control strategies for HAT and AAT.

Background: Ticks and tick-borne diseases are a major impediment to livestock production worldwide. Cattle trade and transnational transhumance create risks for the spread of ticks and tick-borne diseases and threaten cattle production in the absence of an effective tick control program. Few studies have been undertaken on cattle ticks in the Central African region; therefore, the need to assess the occurrence and the spatial distribution of tick vectors with the aim of establishing a baseline for monitoring future spread of tick borne-diseases in the region is urgent., Results: A total of 7091 ixodid ticks were collected during a countrywide cross-sectional field survey and identified using morphological criteria. Of these, 4210 (59.4%) ticks were Amblyomma variegatum, 1112 (15.6%) Rhipicephalus (Boophilus) microplus, 708 (10.0%) Rhipicephalus (Boophilus) decoloratus, 28 (0.4%) Rhipicephalus (Boophilus) annulatus, 210 (3.0%) Hyalomma rufipes, 768 (10.8%) Hyalomma truncatum, and 19 (0.3%) Rhipicephalus sanguineus. Three ticks of the genus Hyalomma spp. and 33 of the genus Rhipicephalus spp. were not identified to the species level. Cytochrome c oxidase subunit 1 (cox1) gene sequencing supported the data from morphological examination and led to identification of three additional species, namely Hyalomma dromedarii, Rhipicephalus sulcatus and Rhipicephalus pusillus. The finding of the invasive tick species R. microplus in such large numbers and the apparent displacement of the indigenous R. decoloratus is highly significant since R. microplus is a highly efficient vector of Babesia bovis., Conclusions: This study reports the occurrence and current geographical distribution of important tick vectors associated with cattle in Cameroon. It appears that R. microplus is now well established and may be displacing native Rhipicephalus (Boophilus) species, such as R. decoloratus. This calls for an urgent response to safeguard the livestock sector in western central Africa.

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.

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.

Background: The Bafia sleeping sickness focus of Cameroon is considered as "silent" with no case reported for about 20 years despite medical surveys performed during the last decades. In this focus, all epidemiological factors that can contribute to trypanosomes transmission are present. To update our knowledge on the current risks of Human and Animal African trypanosomiases, different trypanosome species were identified in midguts of tsetse flies captured in the Bafia focus., Methods: Tsetse flies were trapped using pyramidal traps. Each tsetse fly was identified and live flies were dissected and their midguts collected. DNA was extracted from each midgut and thereafter, blood meals and different trypanosome species were identified with molecular tools. The biological data were transported onto maps in order to have their distribution., Results: Of the 98 traps set up, 461 Glossina palpalis palpalis were captured; 322 (69.8 %) tsetse flies were dissected and 49 (15.2 %) teneral flies identified. The average apparent density of tsetse flies per day was 1.18. Of the 35 (10.9 %) blood meals collected, 82 % were taken on pigs and 17.6 % on humans. Eighty two (25.5 %) trypanosome infections were identified: 56 (17.4 %) T. congolense savannah, 17 (5.3 %) T. congolense forest, 5 (1.6 %) T. vivax and 4 (1.2 %) T. brucei s.l. No infection of T. simiae and T. b. gambiense was identified. Sixty seven (81.7 %) infections were single and 15 (18.3 %) mixed involving one triple infection (T. congolense forest, T. brucei and T. vivax) and 14 double infections: 11 T. congolense forest and T. congolense savannah, two T. congolense savannah and T. brucei, and one of T. brucei and T. vivax. The generated maps show the distribution of tsetse flies and trypanosome infections across the focus., Conclusion: This study has shown that animal trypanosomes remain an important problem in this region. Meanwhile, it is very likely that HAT does not seem anymore to be a public health problem in this focus. The generated maps enabled us to define high risk transmission areas for AAT, and where disease control must be focused in order to improve animal health as well as the quantity of animal proteins.

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.

The sleeping sickness focus of Campo lies along the Atlantic coast and extends along the Ntem River, which constitutes the Cameroonian and Equatorial Guinean border. It is a hypo-endemic focus with the disease prevalence varying from 0.3 to 0.86% during the last few decades. Investigations on animal reservoirs revealed a prevalence of Trypanosoma brucei gambiense of 0.6% in wild animals and 4.83% in domestic animals of this focus. From 2001 to 2012, about 19 931 tsetse were collected in this focus and five tsetse species including Glossina palpalis palpalis, G. pallicera, G. nigrofusca, G. tabaniformis and G. caliginea were identified. The analysis of blood meals of these flies showed that they feed on human, pig, goat, sheep, and wild animals such as antelope, duiker, wild pig, turtle and snake. The percentage of blood meals taken on these hosts varies according to sampling periods. For instance, 6.8% of blood meals from pig were reported in 2004 and 22% in 2008. This variation is subjected to considerable evolutions because the Campo HAT focus is submitted to socio-economic mutations including the reopening of a new wood company, the construction of autonomous port at "Kribi" as well as the dam at "Memve ele". These activities will bring more that 3000 inhabitants around Campo and induce the deforestation for the implementation of farmlands as well as breeding of domestic animals. Such mutations have impacts on the transmission and the epidemiology of sleeping sickness due to the modification of the fauna composition, the nutritional behavior of tsetse, the zoophilic/anthropophilic index. To achieve the elimination goal in the sleeping sickness focus of Campo, we report in this paper the current epidemiological situation of the disease, the research findings of the last decades notably on the population genetics of trypanosomes, the modifications of nutritional behavior of tsetse, the prevalence of T. b. gambiense in humans, domestic and wild animals. An overview on the types of mutations occurring in the region has been raised and a discussion on the strategies that can be implemented to achieve the elimination of the disease has been made.

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.

Background: Human African Trypanosomiasis (HAT) remains a public health problem in many poor countries. Due to lack of financial resources in these countries, cost-effective strategies are needed for efficient control of this scourge, especially the tsetse vector. It was shown that perennial water sources maintain a favourable biotope for tsetse flies and thus the transmission dynamics of sleeping sickness. The present paper aimed at assessing the transmission dynamics of HAT in a forest environment where the hydrographic network is important., Methods: Two entomological surveys were carried out in July 2009 and March 2010 in the Bipindi sleeping sickness focus of the South Region of Cameroon. Entomological and parasitological data were collected during both trapping periods (including the climate variations throughout a year) and compared to each other. The level of risk for transmission of the disease during each trapping period was also evaluated at the trap level and materialised on the map of the Bipindi focus., Results: Glossina palpalis palpalis was the most prevalent tsetse fly species captured in this focus. The overall densities of tsetse flies as well as the risk for transmission of HAT in the Bipindi focus were significantly higher in July than in March. At the trap level, we observed that these parameters were almost constant, whatever the trapping period, when the biotope included perennial water sources., Conclusions: This study shows that the spatial distribution of traps, as well as the temporal climatic variations might influence entomological and parasitological parameters of HAT and that the presence of perennial water sources in biotopes would favour the development of tsetse flies and thus the transmission of sleeping sickness. These factors should, therefore, be taken into account in order to provide more efficient vector control.

Background: The Malanga sleeping sickness focus of the Democratic Republic of Congo has shown an epidemic evolution of disease during the last century. However, following case detection and treatment, the prevalence of the disease decreased considerably. No active survey has been undertaken in this focus for a couple of years. To understand the current epidemiological status of sleeping sickness as well as the animal African trypanosomiasis in the Malanga focus, we undertook the identification of tsetse blood meals as well as different trypanosome species in flies trapped in this focus., Methods: Pyramidal traps were use to trap tsetse flies. All flies caught were identified and live flies were dissected and their mid-guts collected. Fly mid-gut was used for the molecular identification of the blood meal source, as well as for the presence of different trypanosome species., Results: About 949 Glossina palpalis palpalis were trapped; 296 (31.2%) of which were dissected, 60 (20.3%) blood meals collected and 57 (19.3%) trypanosome infections identified. The infection rates were 13.4%, 5.1%, 3.5% and 0.4% for Trypanosoma congolense savannah type, Trypanosoma brucei s.l., Trypanosoma congolense forest type and Trypanosoma vivax, respectively. Three mixed infections including Trypanosoma brucei s.l. and Trypanosoma congolense savannah type, and one mixed infection of Trypanosoma vivax and Trypanosoma congolense savannah type were identified. Eleven Trypanosoma brucei gambiense infections were identified; indicating an active circulation of this trypanosome subspecies. Of all the identified blood meals, about 58.3% were identified as being taken on pigs, while 33.3% and 8.3% were from man and other mammals, respectively., Conclusion: The presence of Trypanosoma brucei in tsetse mid-guts associated with human blood meals is indicative of an active transmission of this parasite between tsetse and man. The considerable number of pig blood meals combined with the circulation of Trypanosoma brucei gambiense in this focus suggests a transmission cycle involving humans and domestic animals and could hamper eradication strategies. The various species of trypanosomes identified in the Malanga sleeping sickness focus indicates the coexistence of animal and human African Trypanosomiasis. The development of new strategies integrating control measures for human and animal trypanosomiasis may enable the reduction of the control costs in this locality.

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.