The internal transcribed spacer 1 sequence polymorphism brings updates to tsetse species distribution in the northern Cameroon: Importance in planning efficient vector control.

Vector control remains one of the best strategies to prevent the transmission of trypanosome infections in humans and livestock and, thus, a good way to achieve the elimination of human African trypanosomiasis and animal African trypanosomiasis. A key prerequisite for the success of any vector control strategy is the accurate identification and correct mapping of tsetse species. In this work, we updated the tsetse fly species identification and distribution in many geographical areas in Cameroon. Tsetse flies were captured from six localities in Cameroon, and their species were morphologically identified. Thereafter, DNA was extracted from legs of each tsetse fly and the length polymorphism of internal transcribed spacer-1 (ITS1) region of each fly was investigated using PCR. ITS1 DNA fragments of each tsetse species were sequenced. The sequences obtained were analysed and compared to those available in GenBank. This enabled to confirm/infirm results of the morphologic identification and then, to establish the phylogenetic relationships between tsetse species. Morphologic features allowed to clearly distinguish all the tsetse species captured in the South Region of Cameroon, that is, Glossina palpalis palpalis, G. pallicera, G. caliginea and G. nigrofusca. In the northern area, G. morsitans submorsitans could also be distinguished from G. palpalis palpalis, G. tachinoides and G. fuscipes, but these three later could not be distinguished with routine morphological characters. The ITS1 length polymorphism was high among most of the studied species and allowed to identify the following similar species with a single PCR, that is, G. palpalis palpalis with 241 or 242 bp and G. tachinoides with 221 or 222 bp, G. fuscipes with 236 or 237 bp. We also updated the old distribution of tsetse species in the areas assessed, highlighting the presence of G. palpalis palpalis instead of G. fuscipes in Mbakaou, or in sympatry with G. morsitans submorsitans in Dodeo (northern Cameroon). This study confirms the presence of G. palpalis palpalis in the Adamawa Region of Cameroon. It highlights the limits of using morphological criteria to differentiate some tsetse species. Molecular tools based on the polymorphism of ITS1 of tsetse flies can differentiate tsetse species through a simple PCR before downstream analyses or vector control planning.

Prevalence of symbionts and trypanosome infections in tsetse flies of two villages of the "Faro and Déo" division of the Adamawa region of Cameroon.

BACKGROUND: Tsetse flies are vectors of human and animal African trypanosomiasis. In spite of many decades of chemotherapy and vector control, the disease has not been eradicated. Other methods like the transformation of tsetse fly symbionts to render the fly refractory to trypanosome infection are being evaluated. The aim of the present study was to evaluate the association between trypanosome infections and the presence of symbionts in these tsetse species. Tsetse flies were trapped in two villages of the "Faro and Déo" Division of the Adamawa region of Cameroon. In the field, tsetse fly species were identified and their infection by trypanosomes was checked by microscopy. In the laboratory, DNA was extracted from their midguts and the presence of symbionts (Sodalis glossinidius and Wolbachia sp.) and trypanosomes was checked by PCR. Symbionts/trypanosomes association tests were performed. RESULTS: Three tsetse fly species including Glossina tachinoides (90.1%), Glossina morsitans submorsitans (9.4%) and Glossina fuscipes fuscipes (0.5%) were caught. In all the population we obtained an occurrence rate of 37.2% for Sodalis glossinidius and 67.6% for Wolbachia irrespective to tsetse flies species. S. glossinidius and Wolbachia sp. occurrence rates were respectively 37 and 68% for G. tachinoides and 28.6 and 59.5% for G. m. submorsitans. Between Golde Bourle and Mayo Dagoum significant differences were observed in the prevalence of symbionts. Prevalence of trypanosomes were 34.8% for Glossina tachinoides and 40.5% for Glossina morsitans submorsitans. In G. tachinoides, the trypanosome infection rates were 11, 2.6 and 13.7%, respectively, for T. brucei s.l., T. congolense forest type and T. congolense savannah type. In G. m. submorsitans, these infection rates were 16.7, 9.5 and, 2.4% respectively, for T. brucei s.l., T. congolense forest type and T. congolense savannah type. CONCLUSIONS: The rate of tsetse fly infection by trypanosomes was low compared to those obtained in HAT foci of south Cameroon, and this rate was not statistically linked to the rate of symbiont occurrence. This study allowed to show for the first time the presence of Wolbachia sp. in the tsetse fly sub-species Glossina morsitans submorsitans and Glossina tachinoides.

Trypanosome Infections and Anemia in Cattle Returning from Transhumance in Tsetse-Infested Areas of Cameroon.

The objective of this work was to assess the anemic status and the use of an immunological test and PCR-based methods to determine the infection rates of trypanosomes species. Transhumance aims to provide cattle with greener pastures and greater water resources than in the Djerem region during the dry season. Two criteria were used to assess the health status of the animals, the prevalence of trypanosomiasis and the level of anemia. In addition, we have evaluated the effectiveness, in trypanosomiasis detection, of the Very Diag Kit (CEVA Santé animale), a Rapid diagnosis test (RDT) based on immunological identification of T. congolense s.l. and T. vivax, responsible for AAT. Four trypanosome species (Trypanosoma congolense savannah type (Tcs), T. congolense forest type (Tcf), T. brucei s.l. (Tbr) and T. vivax (Tvx)) were identified in cattle sampled in four villages. The overall infection rate determined by PCR (68.6%) was much higher than those generally reported in cattle from the Adamawa region (35 to 50%). Infections (including mixed infections) by Tc s.l. (Tcs + Tcf) were predominant (45.7%). The infection rates were also determined using the Very Diag Kit allowing us to identify Tc s.l. and Tvx in the field in less than 20 min. This method provided, for the global infection, a higher rate (76.5%) than that determined by PCR (68.6%), although it is supposed to be less sensitive than PCR. Tc s.l. infection rate (37.8%) was similar to that (38.8%) determined by PCR (Tcs + Tcf single infections). In contrast, the prevalence of Tvx single infections measured by RDT (18%) was nearly two-fold higher than that (9.4%) measured by PCR. Thus, further comparative analyses seem to be needed in order to more accurately assess the sensitivity and specificity of the Very Diag test under our conditions of use on blood samples. The mean PCVs in trypanosome-infected as well as in uninfected cattle were below 25%, the threshold below which an animal is considered anemic. Our study shows that cattle return from transhumance in poor health. It raises questions about its real benefit, especially since the herds are themselves likely to become vectors of trypanosomiasis and possibly of other diseases. At least, effective measures have to be undertaken to treat all cattle coming back from transhumance.

Trypanosome infections in animals from tsetse infected areas of Cameroon and their sensitivity and resistance molecular profiles for diminazene aceturate and isometamidium chloride.

Monitoring and assessment of control strategies for African trypanosomoses' elimination require not only updating data on trypanosome infections, but also to have an overview on the molecular profiles of trypanocides resistance in different epidemiological settings. This study was designed to determine, in animals from six tsetse-infested areas of Cameroon, the prevalence of trypanosome infections as well as the diminazene aceturate (DA) and isometamidium chloride (ISM) sensitivity/resistance molecular profiles of these trypanosomes. From 2016 to 2019, blood was collected in pigs, dogs, sheep, goats and cattle from six tsetse infested areas of Cameroon. DNA was extracted from blood and trypanosome species were identified by PCR. The sensitivity/resistance molecular profiles of trypanosomes to DA and ISM were investigated using PCR-RFLP. From 1343 blood samples collected, Trypanosoma vivax, Trypanosoma congolense forest and savannah, Trypanosoma theileri and trypanosomes of the sub-genus Trypanozoon were identified. The overall prevalence of trypanosome infections was 18.7%. These prevalence vary between trypanosome species, animal taxa, within and between sampling sites. Trypanosoma theileri was the predominant species with an infection rate of 12.1%. Trypanosomes showing resistant molecular profiles for ISM and DA were identified in animals from Tibati (2.7% for ISM and 65.6% for DA) and Kontcha (0.3% for ISM and 6.2% for DA). No trypanosome carrying resistant molecular profile for any of the two trypanocides was detected in animals from Fontem, Campo, Bipindi and Touboro. Mixed molecular profiles of sensitive/resistant trypanosomes were detected in animals from Tibati and Kontcha. Results of this study highlighted the presence of various trypanosome species as well as parasites carrying sensitive/resistant molecular profiles for DA and ISM in animals of tsetse infested areas of Cameroon. They indicate that the control strategies must be adapted according to epidemiological settings. The diversity of trypanosomes indicates that AAT remains a serious threat for animal breeding and animal health in these tsetse infested areas.

Detection of Brucella antibodies in domestic animals of southern Cameroon: Implications for the control of brucellosis.

Brucellosis is one of the world's most widespread bacterial zoonoses caused by Brucella. It leads to considerable economic losses as a result of low productivity of infected animals and the long debilitating illness in humans. Despite its impact on human and animal health, little attention has been paid on Brucella infections in domestic animals. It is in this light that the prevalence of Brucella antibodies was determined in domestic animals with the overarching goal of improving our knowledge on brucellosis in southern Cameroon. During cross-sectional studies conducted from December 2016 to August 2018 in five sites of southern Cameroon, blood samples were collected in cattle, sheep, goat, pig and dog. Plasma was obtained from each blood sample and Brucella antibodies were detected using the Rose Bengal test and the enzyme-linked immunosorbent assay (ELISA). From 1873 animals that were sampled, the overall prevalence of Brucella antibodies using Indirect enzyme-linked immunosorbent assay (i-ELISA) was 6.35% (118/1873): 9.12% (78/855) in cattle; 8.04% (30/373) in sheep; 6.06% (2/33) in dog, 1.87% (3/160) in pig and 1.1% (5/452) in goat. Between animal species (p-value < .0001, x2  = 33.63) as well as sampling sites (p-value = .0001, x2  = 18.97), significant differences were observed in the prevalence of Brucella antibodies. Yoko and Noun localities have shown the highest prevalence of 8.6% (30/348) and 7.2% (78/1070), respectively. This prevalence was significantly higher (p = .03, x2  = 1.25) in female than male cattle. Between adult (16.923%) and young cattle (7.8%), significant difference (p = .04, x2  = 6.42) was observed in the prevalence of Brucella antibodies. This study shows that the prevalence of Brucella antibodies varies between animal species and localities. It also shows several domestic animals of southern Cameroon that have been in contact with Brucella. It enabled to identify villages where investigations on the transmission dynamic must be focused for the final goal of developing control measures for this neglected zoonotic disease.

Molecular identification of diminazene aceturate resistant trypanosomes in tsetse flies from Yoko in the Centre region of Cameroon and its epidemiological implications.

African animal trypanosomiases are caused by trypanosomes cyclically or mechanically transmitted by tsetse and other biting flies. Although molecular tools have been developed to identify drug-resistant trypanosomes in mammals, little or no investigation on drug-resistance has been undertaken on trypanosomes harbored by tsetse flies. Moreover, no data on mechanical vectors of African trypanosomes is available in most endemic areas of Cameroon. This study was designed to update our knowledge on the cyclical and mechanical vectors of African trypanosomes, and using molecular tools to identify different trypanosome species as well as diminazene aceturate resistant trypanosomes in tsetse flies trapped at Yoko in the Centre region of Cameroon. For this study, traps were used to catch tsetse and mechanical vectors of African trypanosomes. The flies trapped were counted and identified by sex and species. DNA was extracted from tsetse and species-specific primers were used to identify different trypanosome species. PCR-RFLP was used to detect diminazene aceturate resistant strains of Trypanosoma congolense. In all, 454 flies comprising 168 (37%) Tabanus spp., 71 (15.6%) Stomoxys spp. and 215 (47.4%) tsetse fly (i.e. 107 (49.8%) Glossina fusca congolensis, 71 (33%) Glossina fusca fusca and 37 (17.2%) Glossina palpalis palpalis) were trapped. Trypanosome infections were identified in 12.6% (27/215) of tsetse flies: 13 in G. f. congolensis, 6 in G. p. palpalis and 5 in G. f. fusca. From 24 T. congolense positive samples, PCR-RFLP was successful on 37.5% of the samples. Four samples (16.2%) harbored T. congolense strains that were resistant to diminazene aceturate while the remaining samples had drug-sensitive strains. These results show for the first time the applicability of molecular tools for the identification of drug-resistant trypanosomes in tsetse. They revealed the existence of diminazene aceturate resistant strains of T. congolense in the tsetse-infested area of Yoko in the Centre region of Cameroon. Detection of drug-resistant trypanosomes in tsetse may enable scientists to map with accuracy specific areas where these parasites are transmitted. With such mapping, control strategies against African trypanosomiases could be improved by adapting control measures according to drug resistance distribution.

Molecular identification of diminazene aceturate-resistant strains of Trypanosoma congolense in naturally infected domestic animals of Yoko in the centre region of Cameroon.

African animal trypanosomiases (AAT) remain the major constraint for livestock production, agriculture and food security in Africa. Although several control measures have been developed to fight AAT, the use of trypanocides remains the main strategy in most affected poor and rural communities. However, several studies have highlighted drug-resistant-trypanosome infections in many African countries, though this phenomenon is still not well described. This study aims to detect trypanosome species and the molecular profiles of drug-resistant-trypanosomes in naturally infected domestic animals of Yoko in the centre region of southern Cameroon. Therefore, in October 2017, 348 animals were blood sampled. The level of packed cell volume (PCV) was evaluated in each animal and trypanosome infections were investigated with the capillary tube centrifugation technique (CTC). Thereafter, DNA was extracted from blood samples and different trypanosome species were identified by PCR. The resistant/sensitive molecular profiles of trypanosomes for diminazene aceturate (DA) and isometamidium chloride (ISM) were investigated by PCR-RFLP. About 18.4% (64/348) of animals analyzed by PCR were found with trypanosome infections including Trypanosoma vivax, Trypanosoma brucei s.l. and Trypanosoma congolense forest and savannah. Trypanosoma congolense savannah was the predominant species with an infection rate of 15.2%. Between villages, significant (p˂0.0001) differences were found in the overall trypanosome infection rates. No molecular profile for ISM resistant-trypanosomes was identified. Conversely, about 88.9% (40/45) of T. congolense positive samples have shown molecular profiles of DA-resistant strains while the remaining 11.1% (5/45) showed mixed molecular profiles of resistant/sensitive strains. Results showed that the molecular profiles of DA-resistant strains of T. congolense in domestic animals of Yoko were widespread. This data needs to be confirmed by testing in vivo the drug susceptibilities of the trypanosome strains herein detected. In conclusion, appropriate future control measures are required. In addition to the intensification of vector control, ISM is advised for the treatment of animals infected by trypanosomes.

Molecular identification of Wolbachia and Sodalis glossinidius in the midgut of Glossina fuscipes quanzensis from the Democratic Republic of Congo.

During the last 30 years, investigations on the microbiome of different tsetse species have generated substantial data on the bacterial flora of these cyclical vectors of African trypanosomes, with the overarching goal of improving the control of trypanosomiases. It is in this context that the presence of Wolbachia and Sodalis glossinidius was studied in wild populations of Glossina fuscipes quanzensis from the Democratic Republic of Congo. Tsetse flies were captured with pyramidal traps. Of the 700 Glossina f. quanzensis captured, 360 were dissected and their midguts collected and analyzed. Sodalis glossinidius and Wolbachia were identified by PCR. The Wolbachia-positive samples were genetically characterized with five molecular markers. PCR revealed 84.78% and 15.55% midguts infected by Wolbachia and S. glossinidius, respectively. The infection rates varied according to capture sites. Of the five molecular markers used to characterize Wolbachia, only the fructose bis-phosphate aldolase gene was amplified for about 60% of midguts previously found with Wolbachia infections. The sequencing results confirmed the presence of Wolbachia and revealed the presence of S. glossinidius in the midgut of Glossina f. quanzensis. A low level of midguts were naturally co-infected by both bacteria. The data generated in this study open a framework for investigations aimed at understanding the contribution of these symbiotic microorganisms to the vectorial competence of Glossina fuscipes quanzensis.

Prevalence of Sodalis glossinidius and different trypanosome species in Glossina palpalis palpalis caught in the Fontem sleeping sickness focus of the southern Cameroon.

Tsetse flies are the cyclical vector of human and animal African trypanosomiasis. To improve vector control in order to achieve the elimination of human African trypanosomiasis (HAT) and boost the control of animal diseases, investigations have been undertaken on the tripartite association between tsetse, trypanosome, and symbionts. It is in this light that Sodalis glossinidius and different trypanosomes were identified in Glossina palpalis palpalis caught in Fontem in southern Cameroon. For this study, DNA was extracted from whole flies, and S. glossinidius and different trypanosome species were identified by polymerase chain reaction (PCR). Statistical analyses were performed to compare the trypanosome and S. glossinidius infection rates and to look for an association between these microorganisms. Of the 274 G. p. palpalis caught, 3.3% (9/274) were teneral. About 35% (96/274) of these flies harbored S. glossinidius. Of the 265 non-teneral flies, 37.7% were infected by trypanosomes. The infection rates of Trypanosoma congolense "forest type" and Trypanosoma vivax were 26.04% and 18.11%, respectively. About 6.41% of tsetse harbored mixed infections of T. congolense and T. vivax. Of the 69 tsetse with T. congolense infections, 33.33% (23/69) harbored S. glossinidius while 71.86% (69/96) of flies harboring S. glossinidius were not infected by trypanosomes. No association was observed between S. glossinidius and trypanosome infections. Some wild tsetse harbor S. glossinidius and trypanosomes, while others have no infection or are infected by only one of these microorganisms. We conclude that the presence of S. glossinidius does not favor trypanosome infections in G. p. palpalis of the Fontem focus.

Trypanosome infection rates in tsetse flies in the "silent" sleeping sickness focus of Bafia in the Centre Region in Cameroon.

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.

Microsatellite genotyping reveals diversity within populations of Sodalis glossinidius, the secondary symbiont of tsetse flies.

The aim of this study was to develop a PCR-based microsatellite genotyping method for identifying genetic diversity in Sodalis glossinidius, a symbiont associated with tsetse fly infection by trypanosomes causing human and animal trypanosomiasis. Allelic polymorphism at three loci, investigated on 40 fly gut extracts, evidenced eight alleles and the existence of five genotypes. This novel approach was shown to be efficient and suitable for routine large-scale genotyping of S. glossinidius present in the biologically complex tsetse fly extracts; it could favor progress in the fields of diagnosis, epidemiology, population genetics, and fly/symbiont/trypanosome interactions.

Genetic diversity and population structure of the secondary symbiont of tsetse flies, Sodalis glossinidius, in sleeping sickness foci in Cameroon.

BACKGROUND: Previous studies have shown substantial differences in Sodalis glossinidius and trypanosome infection rates between Glossina palpalis palpalis populations from two Cameroonian foci of human African trypanosomiasis (HAT), Bipindi and Campo. We hypothesized that the geographical isolation of the two foci may have induced independent evolution in the two areas, resulting in the diversification of symbiont genotypes. METHODOLOGY/PRINCIPAL FINDINGS: To test this hypothesis, we investigated the symbiont genetic structure using the allelic size variation at four specific microsatellite loci. Classical analysis of molecular variance (AMOVA) and differentiation statistics revealed that most of the genetic diversity was observed among individuals within populations and frequent haplotypes were shared between populations. The structure of genetic diversity varied at different geographical scales, with almost no differentiation within the Campo HAT focus and a low but significant differentiation between the Campo and Bipindi HAT foci. CONCLUSIONS/SIGNIFICANCE: The data provided new information on the genetic diversity of the secondary symbiont population revealing mild structuring. Possible interactions between S. glossinidius subpopulations and Glossina species that could favor tsetse fly infections by a given trypanosome species should be further investigated.

Tsetse fly blood meal modification and trypanosome identification in two sleeping sickness foci in the forest of southern Cameroon.

The blood meal origins of 222 tsetse flies (213 Glossina palpalis palpalis, 7 Glossina pallicera pallicera, one Glossina nigrofusca and one Glossina caliginea) caught in 2008 in two Human African trypanosomiasis foci (Bipindi and Campo) of south Cameroon were investigated. 88.7% of tsetse flies blood meals were identified using the heteroduplex method and the origin of the remaining blood meals (11.3%) was identified by sequencing the cytochrome B gene. Most of the meals were from humans (45.9%) and pigs (37.4%), 16.7% from wild animals. Interestingly, new tsetse fly hosts including turtle (Trionyx and Kinixys) and snake (Python sebae) were identified. Significant differences were recorded between Bipindi where the blood meals from pigs were predominant (66.7% vs 23.5% from humans) and Campo where blood meals from humans were predominant (62.9% vs 22.7% from pigs). Comparison with the data recorded in 2004 in the same foci (and with the same molecular approach) demonstrated significant modifications of the feeding patterns: increase in blood meals from pigs in Bipindi (66.7% in 2008 vs 44.8% in 2004) and in Campo (20.5% in 2008 vs 6.8% in 2004), decrease in that from human (significant in Bipindi only). 12.6%, 8.1% and 2.7% of the flies were, respectively, Trypanosoma congolense forest type, Trypanosoma congolense savannah type and Trypanosoma brucei gambiense infected. These results demonstrate that tsetse fly feeding patterns can be specific of a given area and can evolve rapidly with time. They show an active circulation of a variety of trypanosomes in sleeping sickness foci of southern Cameroon.

Tripartite interactions between tsetse flies, Sodalis glossinidius and trypanosomes--an epidemiological approach in two historical human African trypanosomiasis foci in Cameroon.

Epidemiological surveys were conducted in two historical human African trypanosomiasis foci in South Cameroon, Bipindi and Campo. In each focus, three sampling areas were defined. In Bipindi, only Glossina palpalis was identified, whereas four species were identified in Campo, G. palpalis being highly predominant (93%). For further analyses, 75 flies were randomly chosen among the flies trapped in each of the six villages. Large and statistically significant differences were recorded between both (1) the prevalence of Sodalis glossinidius (tsetse symbiont) and the prevalence of trypanosome infection of the major fly species G. p. palpalis and (2) the respective prevalence of symbiont and infection between the two foci. Despite these differences, the rate of infected flies harbouring the symbiont was very similar (75%) in both foci, suggesting that symbionts favour fly infection by trypanosomes. This hypothesis was statistically tested and assessed, showing that S. glossinidius is potentially an efficient target for controlling tsetse fly vectorial competence and consequently sleeping sickness.