Identification of trypanosomes in wild animals from southern Cameroon using the polymerase chain reaction (PCR)

One possible explanation of the maintenance of many historical foci of sleeping sickness in Central Africa could be the existence of a wild animal reservoir. In this study, PCR was used to detect the different trypanosome species present in wild animal captured by hunters in the southern forest belt of Cameroon (Bipindi). Trypanosomes were also detected by a parasitological method (Quantitative buffy coat: QBC). Parasite could not be isolated in culture medium (Kit for in vitro isolation: KIVI). Specific primers of T. brucei s.l., T. congolense forest type, T. congolense savannah type, T. vivax, T. simiae and T. b. gambiense group 1 were used to identify parasites in the blood of 164 animals belonging to 24 different species including ungulates, rodents, pangolins, carnivores, reptiles and primates. Of the 24 studied species, eight were carrying T. b. gambiense group 1. Those parasites pathogenic to man were found in monkeys (Cercocebus torquatus and Cercopithecus nictitans), in ungulates (Cephalophus dorsalis and C. monticola), in carnivores (Nandinia binotata and Genetta servalina) and in rodents (Cricetomys gambianus and Atherurus africanus). 13 species (54%) were carrying T. brucei s.l. identified as non-gambiense group 1.     

Bushmeat genetics: setting up a reference framework for the DNA typing of African forest bushmeat

The bushmeat trade in tropical Africa represents illegal, unsustainable off‐takes of millions of tons of wild game – mostly mammals – per year. We sequenced four mitochondrial gene fragments (cyt b, COI, 12S, 16S) in >300 bushmeat items representing nine mammalian orders and 59 morphological species from five western and central African countries (Guinea, Ghana, Nigeria, Cameroon and Equatorial Guinea). Our objectives were to assess the efficiency of cross‐species PCR amplification and to evaluate the usefulness of our multilocus approach for reliable bushmeat species identification. We provide a straightforward amplification protocol using a single ‘universal’ primer pair per gene that generally yielded >90% PCR success rates across orders and was robust to different types of meat preprocessing and DNA extraction protocols. For taxonomic identification, we set up a decision pipeline combining similarity‐ and tree‐based approaches with an assessment of taxonomic expertise and coverage of the GENBANK database. Our multilocus approach permitted us to: (i) adjust for existing taxonomic gaps in GENBANK databases, (ii) assign to the species level 67% of the morphological species hypotheses and (iii) successfully identify samples with uncertain taxonomic attribution (preprocessed carcasses and cryptic lineages). High levels of genetic polymorphism across genes and taxa, together with the excellent resolution observed among species‐level clusters (neighbour‐joining trees and Klee diagrams) advocate the usefulness of our markers for bushmeat DNA typing. We formalize our DNA typing decision pipeline through an expert‐curated query database – DNAbushmeat – that shall permit the automated identification of African forest bushmeat items.     

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.     

Field assessment in Cameroon of a reader of POC-CCA lateral flow strips for the quantification of Schistosoma mansoni circulating cathodic antigen in urine

BackgroundDetermining Schistosoma mansoni infection rate and intensity is challenging due to the low sensitivity of the Kato-Katz (KK) test that underestimates the true disease prevalence. Circulating cathodic antigen (CCA) excreted in urine is constantly produced by adult worms and has been used as the basis of a simple, non-invasive point of care test (POC-CCA) for Schistosoma mansoni infections. Although the abundance of CCA in urine is proportional to worm burden, the POC-CCA test is marketed as a qualitative test, making it difficult to investigate the wide range of infection intensities. This study was designed to compare the prevalence and intensity of S. mansoni by KK and POC-CCA and quantify, on fresh and frozen (<-20°C) urine samples, CCA using the visual scores and the ESEquant LR3 reader.MethodologyStool and urine samples were collected from 759 school-aged children. The prevalence and intensity of S. mansoni were determined using KK and POC-CCA. The degree of the positivity of POC-CCA was estimated by quantifying CCA on fresh and frozen urine samples using visual scores and strip reader. The prevalence, the infection intensity as well the relative amounts of CCA were compared.ResultsThe S. mansoni infection rates inferred from POC-CCA and KK were 40.7% and 9.4% respectively. Good correlations were observed between infection intensities recorded by; i) the reader and visual scoring system on fresh (Rho = 0.89) and frozen samples (Rho = 0.97), ii) the reader on fresh urine samples and KK (epg) (Rho = 0.44). Nevertheless, 238 POC-CCA positive children were negative for KK, and sixteen of them had high levels of CCA. The correlation between results from the reader on fresh and frozen samples was good (Rho = 0.85). On frozen samples, CCA was not detected in 55 samples that were positive in fresh urine samples.ConclusionThis study confirmed the low sensitivity of KK and the high capacity of POC-CCA to provide reliable data on the prevalence and intensity of S. mansoni infections. The lateral flow reader enabled accurate quantification of CCA under field conditions on fresh and frozen urine samples with less time and effort than KK.     

Reproductive Status of Onchocerca volvulus after Ivermectin Treatment in an Ivermectin-Na?ve and a Frequently Treated Population from Cameroon

Background

For two decades, onchocerciasis control has been based on mass treatment with ivermectin (IVM), repeated annually or six-monthly. This drug kills Onchocerca volvulus microfilariae (mf) present in the skin and the eyes (microfilaricidal effect) and prevents for 3–4 months the release of new mf by adult female worms (embryostatic effect). In some Ghanaian communities, the long-term use of IVM was associated with a more rapid than expected skin repopulation by mf after treatment. Here, we assessed whether the embryostatic effect of IVM on O. volvulus has been altered following frequent treatment in Cameroonian patients.

Methodology

Onchocercal nodules were surgically removed just before (D0) and 80 days (D80) after a standard dose of IVM in two cohorts with different treatment histories: a group who had received repeated doses of IVM over 13 years, and a control group with no history of large-scale treatments. Excised nodules were digested with collagenase to isolate adult worms. Embryograms were prepared with females for the evaluation of their reproductive capacities.

Principal Findings

Oocyte production was not affected by IVM. The mean number of intermediate embryos (morulae and coiled mf) decreased similarly in the two groups between D0 and D80. In contrast, an accumulation of stretched mf, either viable or degenerating, was observed at D80. However, it was observed that the increase in number of degenerating mf between D0 and D80 was much lower in the frequently treated group than in the control one (Incidence Rate Ratio: 0.25; 95% CI: 0.10–0.63; p = 0.003), which may indicate a reduced sequestration of mf in the worms from the frequently treated group.

Conclusion/Significance

IVM still had an embryostatic effect on O. volvulus, but the effect was reduced in the frequently treated cohort compared with the control population.     

Trypanosoma brucei s.l.: characterisation of stocks from Central Africa by PCR analysis of mobile genetic elements

To better understand the epidemiology of sleeping sickness in the Central African sub-region, notably the heterogeneity of Human African Trypanosomiasis (HAT) foci, the mobile genetic element PCR (MGE-PCR) technique was used to genotype Trypanosoma brucei s.l. (T. brucei s.l.) isolates from this sub-region. Using a single primer REV B, which detects positional variation of the mobile genetic element RIME, via amplification of flanking regions, MGE-PCR revealed a micro genetic variability between Trypanosoma brucei gambiense (T. b. gambiense) isolates from Central Africa. The technique also revealed the presence of several T. b. gambiense genotypes and allowed the identification of minor and major ubiquitous genotypes in HAT foci. The presence of several T. b. gambiense genotypes in HAT foci may explain the persistence and the resurgence phenomena of the disease and also the epidemic and the endemic status of some Central African sleeping sickness foci. The MGE-PCR technique represents a simple, rapid, and specific method to differentiate Central African T. brucei s.l. isolates.     

Genetic structure of the tiger mosquito, Aedes albopictus, in Cameroon (Central Africa)

Background

Aedes albopictus (Skuse, 1884) (Diptera: Culicidae), a mosquito native to Asia, has recently invaded all five continents. In Central Africa it was first reported in the early 2000s, and has since been implicated in the emergence of arboviruses such as dengue and chikungunya in this region. Recent genetic studies of invasive species have shown that multiple introductions are a key factor for successful expansion in new areas. As a result, phenotypic characters such as vector competence and insecticide susceptibility may vary within invasive pest species, potentially affecting vector efficiency and pest management. Here we assessed the genetic variability and population genetics of Ae. albopictus isolates in Cameroon (Central Africa), thereby deducing their likely geographic origin.

Methods and Results

Mosquitoes were sampled in 2007 in 12 localities in southern Cameroon and analyzed for polymorphism at six microsatellite loci and in two mitochondrial DNA regions (ND5 and COI). All the microsatellite markers were successfully amplified and were polymorphic, showing moderate genetic structureamong geographic populations (F_ST = 0.068, P<0.0001). Analysis of mtDNA sequences revealed four haplotypes each for the COI and ND5 genes, with a dominant haplotype shared by all Cameroonian samples. The weak genetic variation estimated from the mtDNA genes is consistent with the recent arrival of Ae. albopictus in Cameroon. Phylogeographic analysis based on COI polymorphism indicated that Ae. albopictus populations from Cameroon are related to tropical rather than temperate or subtropical outgroups.

Conclusion

The moderate genetic diversity observed among Cameroonian Ae. albopictus isolates is in keeping with recent introduction and spread in this country. The genetic structure of natural populations points to multiple introductions from tropical regions.     

Bacterial Diversity Associated with Populations of <Emphasis Type="Italic">Glossina</Emphasis> spp. from Cameroon and Distribution within the Campo Sleeping Sickness Focus

Tsetse flies were sampled in three villages of the Campo sleeping sickness focus in South Cameroon. The aim of this study was to investigate the flies’ gut bacterial composition using culture-dependent techniques. Out of the 32 flies analyzed (27 Glossina palpalis palpalis, two Glossina pallicera, one Glossina nigrofusca, and two Glossina caliginea), 17 were shown to be inhabited by diverse bacteria belonging to the Proteobacteria, the Firmicutes, or the Bacteroidetes phyla. Phylogenetic analysis based on 16S rRNA gene sequences indicated the presence of 16 bacteria belonging to the genera Acinetobacter (4), Enterobacter (4), Enterococcus (2), Providencia (1), Sphingobacterium (1), Chryseobacterium (1), Lactococcus (1), Staphylococcus (1), and Pseudomonas (1). Using identical bacterial isolation and identification processes, the diversity of the inhabiting bacteria analyzed in tsetse flies sampled in Cameroon was much higher than the diversity found previously in flies collected in Angola. Furthermore, bacterial infection rates differed greatly between the flies from the three sampling areas (Akak, Campo Beach/Ipono, and Mabiogo). Last, the geographic distribution of the different bacteria was highly uneven; two of them identified as Sphingobacterium spp. and Chryseobacterium spp. were only found in Mabiogo. Among the bacteria identified, several are known for their capability to affect the survival of their insect hosts and/or insect vector competence. In some cases, bacteria belonging to a given genus were shown to cluster separately in phylogenetic trees; they could be novel species within their corresponding genus. Therefore, such investigations deserve to be pursued in expanded sampling areas within and outside Cameroon to provide greater insight into the diverse bacteria able to infect tsetse flies given the severe human and animal sickness they transmit.     

Evolutionary relationships and biogeography of Biomphalaria (Gastropoda: Planorbidae) with implications regarding its role as host of the human bloodfluke, Schistosoma mansoni.

The wide geographic distribution of Schistosoma mansoni, a digenetic trematode and parasite of humans, is determined by the occurrence of its intermediate hosts, freshwater snails of the genus Biomphalaria (Preston 1910). We present phylogenetic analyses of 23 species of Biomphalaria, 16 Neotropical and seven African, including the most important schistosome hosts, using partial mitochondrial ribosomal 16S and complete nuclear ribosomal ITS1 and ITS2 nucleotide sequences. A dramatically better resolution was obtained by combining the data sets as opposed to analyzing each separately, indicating that there is additive congruent signal in each data set. Neotropical species are basal, and all African species are derived, suggesting an American origin for the genus. We confirm that a proto-Biomphalaria glabrata gave rise to all African species through a trans-Atlantic colonization of Africa. In addition, genetic distances among African species are smaller compared with those among Neotropical species, indicating a more recent origin. There are two species-rich clades, one African with B. glabrata as its base, and the other Neotropical. Within the African clade, a wide-ranging tropical savannah species, B. pfeifferi, and a Nilotic species complex, have both colonized Rift Valley lakes and produced endemic lacustrine forms. Within the Neotropical clade, two newly acquired natural hosts for S. mansoni (B. straminea and B. tenagophila) are not the closest relatives of each other, suggesting two separate acquisition events. Basal to these two species-rich clades are several Neotropical lineages with large genetic distances between them, indicating multiple lineages within the genus. Interesting patterns occur regarding schistosome susceptibility: (1) the most susceptible hosts belong to a single clade, comprising B. glabrata and the African species, (2) several susceptible Neotropical species are sister groups to apparently refractory species, and (3) some basal lineages are susceptible. These patterns suggest the existence of both inherent susceptibility and resistance, but also underscore the ability of S. mansoni to adapt to and acquire previously unsusceptible species as hosts. Biomphalaria schrammi appears to be distantly related to other Biomphalaria as well as to Helisoma, and may represent a separate or intermediate lineage.