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.

Impact of a small-scale tsetse fly control operation with deltamethrin impregnated "Tiny Targets" on tsetse density and trypanosomes' circulation in the Campo sleeping sickness focus of South Cameroon.

BACKGROUND: Significant progress has been made towards African sleeping sickness elimination in the last decade. Indeed, the World Health Organization (WHO) global goal of eliminating the chronic form of the disease as a public health problem was achieved in 2020 (i.e., < 2,000 new cases per year). Vector control has played an important role in achieving this goal. In this study, we evaluated the impact of the insecticide impregnated Tiny Targets on tsetse fly densities and their infection rates with Trypanosoma spp in the Campo sleeping sickness focus of South Cameroon. METHODS: The study site was divided into two areas: (i) the south-west experimental area, which included vector control, and (ii) the eastern part as the non-intervention area. After compiling the baseline entomological data (tsetse densities and trypanosome infection rates), around 2000 Tiny Targets were deployed in the South-West area and replaced every six months for two years. Post-intervention surveys were conducted every six months to determine tsetse densities and levels of trypanosome infections with PCR-based methods. RESULTS: Following the intervention, tsetse mean catches decreased by 61% after six months, and up to 73% after twelve months (pre-intervention: 2.48 flies/trap/day, 95%CI [1.92-3.14]; 12-months post-intervention: 0.66 tsetse/trap/day, 95%CI [0.42-0.94]). This decrease was not sustained after 18 months, and the mean catch doubled compared to that after 12 months. After 24 months, the mean catches still increased by 17% (18 months: 1.45 tsetse/trap/day, 95%CI [1.07-1.90] and 24 months: 1.71 tsetse/trap/day, 95%CI [1.27-2.24]). In the non-intervention area, a variation in tsetse catches was observed during the two years, with a general increase from 2.43 [0.73-5.77] to 3.64 [1.47-7.70] tsetse/trap/day. In addition, trypanosome infection rates dropped by 75% in both areas (P-value < 0.001) from 21.20% to 5.06% and from 13.14% to 3.45% in intervention and control areas respectively. CONCLUSION: Tiny targets have proven useful in reducing tsetse population densities and trypanosome infection rates, providing evidence for the integration of this tool in current strategies towards trypanosomiasis elimination in Campo. The non-sustained decrease of tsetse densities after one year may indicate reinvasions from neighbouring breeding sites or that the intervention area was not large enough. Our results show the need to scale up by accessing difficult breeding sites and extend the tiny targets to the whole transborder focus.

Environmental mutations in the Campo focus challenge elimination of sleeping sickness transmission in Cameroon.

Sleeping sickness is still prevalent in Campo, southern Cameroon, despite the efforts of World Health Organization and the National Control Programme in screening and treating cases. Reducing disease incidence still further may need the control of tsetse vectors. We update entomological and parasitological parameters necessary to guide tsetse control in Campo. Tsetse flies were trapped, their apparent densities were evaluated as the number of flies captured per trap per day and mapped using GIS tools. Polymerase chain reaction based methods were used to identify their trypanosome infection rates. Glossina palpalis palpalis was the dominant vector species representing 93.42% and 92.85% of flies captured respectively during the heavy and light dry seasons. This species presented high densities, that is, 3.87, 95% CI [3.84-3.91], and 2.51, 95% CI [2.49-2.53] flies/trap/day in the two seasons. Moreover, 16.79% (of 1054) and 20.23% (of 1132 flies) were found infected with at least 1 trypanosome species for the 2 seasons respectively, Trypanosoma congolense being the most prevalent species, and Trypanosoma. brucei gambiense identified in 4 samples. Tsetse flies are abundant in Campo and present high trypanosome infection rates. The detection of tsetse infected with human trypanosomes near the newly created palm grove show workers' exposition. Tsetse densities maps built will guide vector control with 'Tiny Targets'.

Single-strand conformation polymorphism (SSCP) of mitochondrial genes helps to estimate genetic differentiation, demographic parameters and phylogeny of Glossina palpalis palpalis populations from West and Central Africa.

A good understanding of tsetse fly population structure and migration is essential to optimize the control of sleeping sickness. This can be done by studying the genetics of tsetse fly populations. In this work, we estimated the genetic differentiation within and among geographically separated Glossina palpalis palpalis populations from Cameroon, the Democratic Republic of the Congo and Ivory Coast. We determined the demographic history of these populations and assessed phylogenetic relationships among individuals of this sub-species. A total of 418 tsetse flies were analysed: 258 were collected in four locations in Cameroon (Bipindi, Campo, Fontem and Bafia), 100 from Azaguié and Nagadoua in Ivory Coast and 60 from Malanga in the Democratic Republic of the Congo. We examined genetic variation at three mitochondrial loci: COI, COII-TLII, and 16S2. 34 haplotypes were found, of which 30 were rare, since each was present in <5% of the total number of individuals. No haplotype was shared among Cameroon, Ivory Coast and the Democratic Republic of the Congo populations. The fixation index FST of 0.88 showed a high genetic distance between Glossina palpalis palpalis populations from the three countries. That genetic distance was correlated to the geographic distance between populations. We also found that there is substantial gene flow between flies from locations separated by over 100 km in Cameroon and between flies from locations separated by over 200 km in Ivory Coast. Demographic parameters suggest that the tsetse flies from Fontem (Cameroon) had reduced in population size in the recent past. Phylogenetic analysis confirms that Glossina palpalis palpalis originating from the Democratic Republic of the Congo are genetically divergent from the two other countries as already published in previous studies.

PCR-based molecular identification of two intermediate snail hosts of Schistosoma mansoni in Cameroon.

BACKGROUND: Snails of the genus Biomphalaria are intermediate hosts of Schistosoma mansoni, the causative agent of the human intestinal schistosomiasis. Two Biomphalaria species (Biomphalaria pfeifferi and Biomphalaria camerunensis) are involved in the transmission in Cameroon, where the disease is present nationwide. However, difficulty in the identification of both vectors impedes proper assessment of the epidemiological burden caused by each species. To overcome this issue, we designed a PCR-based molecular diagnostic tool to improve the identification of these species. METHODS: We analyzed the internal transcribed spacer 2 (ITS2) region of Biomphalaria ribosomal DNA (rDNA) using polymerase chain reaction amplification (PCR) and restriction fragment length polymorphism (RFLP). RESULTS: The amplification of the ITS2 region of Biomphalaria snails resulted in a 490 bp fragment and produced two profiles for each species after digestion with the restriction enzyme Hpa II. The profile 1 (Bc-HpaII-1: 212-bp and 139-bp bands) for B. camerunensis, was common in all the sampling points; the profile 2 (Bc-HpaII-2: 212-bp and 189-bp bands), was only observed in the Lake Monoun Njindoun sampling site. Biomphalaria pfeifferi profile 1 (Bpf-HpaII-1: 211-bp and 128-bp bands) was common in most of B. pfeifferi sampling points; the profile 2 (Bpf-HpaII-2: 289-bp and 128-bp bands) was only observed in Mokolo (Far North Cameroon).The second restriction enzyme TaqαI, revealed three band profiles, Bc-TaqαI-1 (243-bp, 136-bp and 118-bp bands) and Bc-TaqαI-2 (244-bp, 136-bp and 99-bp) for B. camerunensis and Bpf-TaqαI-1 (242-bp, 135-bp and 107-bp bands) for B. pfeifferi. Sequencing analysis revealed the occurrence of six haplotypes for B. camerunensis and three haplotypes for B. pfeifferi. The level of gene flow was low and the Biomphalaria populations were not in demographic expansion according to neutrality tests (Tajima's D and Fu's Fs). CONCLUSIONS: The PCR-RFLP technique revealed genetic diversity in Biomphalaria snails, and the combination with the morphological method could improve the identification of B. pfeifferi and B. camerunensis in Cameroon. This could help focus on the infection to evaluate the transmission risk with respect of the different species and to develop efficient and cost-effective control measures.

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.

Crateva adansonii DC, an African ethnomedicinal plant, exerts cytotoxicity in vitro and prevents experimental mammary tumorigenesis in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Crateva adansonii DC is a plant traditionally used in Cameroon to treat constipation, asthma, snakebites, postmenopausal complaints and cancers. AIM: The anticancer potential of the dichloromethane/methanol extract of C. adansonii stem barks was investigated using human breast cancer cell and 7,12 dimethylbenz(a)anththracene (DMBA)-induced mammary tumorigenesis model in rats. MATERIAL AND METHODS: The cytotoxicity of C. adansonii extract was assessed in vitro towards breast carcinoma (MCF-7 and MDA-MB-231) and non-tumoral cell lines (NIH/3T3 and HUVEC) by Alamar Blue assay. Furthermore, in vivo studies were performed on female Wistar rats treated either with C. adansonii extract at a dose of 75 or 300mg/kg body weight or with tamoxifen (3.3mg/kg body weight), starting 1 week prior DMBA treatment and lasted 12 weeks. The investigation focused on tumour burden, tumour DNA fingerprint, morphological, histological, hematological, and biochemical parameters. RESULTS: CC50 values for the in vitro assays were 289µg/mL against MCF-7 cells and >500µg/mL in others cells, leading to a selectivity index ≥1.73. C. adansonii extract significantly (p<0.001) revealed in vivo the reduction of the cumulative tumour yield (87.23%), total tumour burden (88.64%), average tumour weight (71.11%) and tumour volume (78.07%) at the dose of 75mg/kg as compared to DMBA control group. A weak effect was also observed at 300mg/kg. This extract showed a moderate hyperplasia at the dose of 75mg/kg while at 300mg/kg no significant change was noted as compared to DMBA group. It protected rats from the DNA alteration induced by DMBA and increased antioxydant enzymes activities in mammary gland tissue homogenates. In addition, Ultra-High Performance Liquid Chromatography/ESI-QTOF-Mass Spectrometry analysis of C. adansonii extract detected structure-related of many well-known anticancer agents such as flavane gallate, flavonol, phenylpropanoïds, sesquiterpene derivatives, gallotannins and lignans. The LD50 of C. adansonii was estimated to be greater than 5000mg/kg. CONCLUSIONS: These aforementioned results suggest that the C. adansonii extract may possess antitumor constituents, which could combat breast cancer and prevent chemically-induced breast cancer in rats.

Effect of sampling methods, effective population size and migration rate estimation in Glossina palpalis palpalis from Cameroon.

Human and animal trypanosomiases are two major constraints to development in Africa. These diseases are mainly transmitted by tsetse flies in particular by Glossina palpalis palpalis in Western and Central Africa. To set up an effective vector control campaign, prior population genetics studies have proved useful. Previous studies on population genetics of G. p. palpalis using microsatellite loci showed high heterozygote deficits, as compared to Hardy-Weinberg expectations, mainly explained by the presence of null alleles and/or the mixing of individuals belonging to several reproductive units (Wahlund effect). In this study we implemented a system of trapping, consisting of a central trap and two to four satellite traps around the central one to evaluate a possible role of the Wahlund effect in tsetse flies from three Cameroon human and animal African trypanosomiases foci (Campo, Bipindi and Fontem). We also estimated effective population sizes and dispersal. No difference was observed between the values of allelic richness, genetic diversity and Wright's FIS, in the samples from central and from satellite traps, suggesting an absence of Wahlund effect. Partitioning of the samples with Bayesian methods showed numerous clusters of 2-3 individuals as expected from a population at demographic equilibrium with two expected offspring per reproducing female. As previously shown, null alleles appeared as the most probable factor inducing these heterozygote deficits in these populations. Effective population sizes varied from 80 to 450 individuals while immigration rates were between 0.05 and 0.43, showing substantial genetic exchanges between different villages within a focus. These results suggest that the "suppression" with establishment of physical barriers may be the best strategy for a vector control campaign in this forest context.