A Large Plasmodium vivax Reservoir and Little Population Structure in the South Pacific

Introduction

The importance of Plasmodium vivax in malaria elimination is increasingly being recognized, yet little is known about its population size and population genetic structure in the South Pacific, an area that is the focus of intensified malaria control.

Methods

We have genotyped 13 microsatellite markers in 295 P. vivax isolates from four geographically distinct sites in Papua New Guinea (PNG) and one site from Solomon Islands, representing different transmission intensities.

Results

Diversity was very high with expected heterozygosity values ranging from 0.62 to 0.98 for the different markers. Effective population size was high (12′872 to 19′533 per site). In PNG population structuring was limited with moderate levels of genetic differentiation. F _ST values (adjusted for high diversity of markers) were 0.14–0.15. Slightly higher levels were observed between PNG populations and Solomon Islands (F _ST = 0.16).

Conclusions

Low levels of population structure despite geographical barriers to transmission are in sharp contrast to results from regions of low P. vivax endemicity. Prior to intensification of malaria control programs in the study area, parasite diversity and effective population size remained high.     

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.     

Endothelial Protein C Receptor Gene Variants Not Associated with Severe Malaria in Ghanaian Children

Background

Two recent reports have identified the Endothelial Protein C Receptor (EPCR) as a key molecule implicated in severe malaria pathology. First, it was shown that EPCR in the human microvasculature mediates sequestration of Plasmodium falciparum-infected erythrocytes. Second, microvascular thrombosis, one of the major processes causing cerebral malaria, was linked to a reduction in EPCR expression in cerebral endothelial layers. It was speculated that genetic variation affecting EPCR functionality could influence susceptibility to severe malaria phenotypes, rendering PROCR, the gene encoding EPCR, a promising candidate for an association study.

Methods

Here, we performed an association study including high-resolution variant discovery of rare and frequent genetic variants in the PROCR gene. The study group, which previously has proven to be a valuable tool for studying the genetics of malaria, comprised 1,905 severe malaria cases aged 1–156 months and 1,866 apparently healthy children aged 2–161 months from the Ashanti Region in Ghana, West Africa, where malaria is highly endemic. Association of genetic variation with severe malaria phenotypes was examined on the basis of single variants, reconstructed haplotypes, and rare variant analyses.

Results

A total of 41 genetic variants were detected in regulatory and coding regions of PROCR, 17 of which were previously unknown genetic variants. In association tests, none of the single variants, haplotypes or rare variants showed evidence for an association with severe malaria, cerebral malaria, or severe malaria anemia.

Conclusion

Here we present the first analysis of genetic variation in the PROCR gene in the context of severe malaria in African subjects and show that genetic variation in the PROCR gene in our study population does not influence susceptibility to major severe malaria phenotypes.     

Chimpanzee population structure in Cameroon and Nigeria is associated with habitat variation that may be lost under climate change

Background

The Nigeria-Cameroon chimpanzee (Pan troglodytes ellioti) is found in the Gulf of Guinea biodiversity hotspot located in western equatorial Africa. This subspecies is threatened by habitat fragmentation due to logging and agricultural development, hunting for the bushmeat trade, and possibly climate change. Although P. t. ellioti appears to be geographically separated from the neighboring central chimpanzee (P. t. troglodytes) by the Sanaga River, recent population genetics studies of chimpanzees from across this region suggest that additional factors may also be important in their separation. The main aims of this study were: 1) to model the distribution of suitable habitat for P. t. ellioti across Cameroon and Nigeria, and P. t. troglodytes in southern Cameroon, 2) to determine which environmental factors best predict their optimal habitats, and 3) to compare modeled niches and test for their levels of divergence from one another. A final aim of this study was to examine the ways that climate change might impact suitable chimpanzee habitat across the region under various scenarios.

Results

Ecological niche models (ENMs) were created using the software package Maxent for the three populations of chimpanzees that have been inferred to exist in Cameroon and eastern Nigeria: (i) P. t. troglodytes in southern Cameroon, (ii) P. t. ellioti in northwestern Cameroon, and (iii) P. t. ellioti in central Cameroon. ENMs for each population were compared using the niche comparison test in ENMtools, which revealed complete niche divergence with very little geographic overlap of suitable habitat between populations.

Conclusions

These findings suggest that a positive relationship may exist between environmental variation and the partitioning of genetic variation found in chimpanzees across this region. ENMs for each population were also projected under three different climate change scenarios for years 2020, 2050, and 2080. Suitable habitat of P. t. ellioti in northwest Cameroon / eastern Nigeria is expected to remain largely unchanged through 2080 in all considered scenarios. In contrast, P. t. ellioti in central Cameroon, which represents half of the population of this subspecies, is expected to experience drastic reductions in its ecotone habitat over the coming century.

Electronic supplementary material

The online version of this article (doi:10.1186/s12862-014-0275-z) contains supplementary material, which is available to authorized users.     

Genetic structure, nestmate recognition and behaviour of two cryptic species of the invasive big-headed ant Pheidole megacephala

Background

Biological invasions are recognized as a major cause of biodiversity decline and have considerable impact on the economy and human health. The African big-headed ant Pheidole megacephala is considered one of the world''s most harmful invasive species.

Methodology/Principal Findings

To better understand its ecological and demographic features, we combined behavioural (aggression tests), chemical (quantitative and qualitative analyses of cuticular lipids) and genetic (mitochondrial divergence and polymorphism of DNA microsatellite markers) data obtained for eight populations in Cameroon. Molecular data revealed two cryptic species of P. megacephala, one inhabiting urban areas and the other rainforests. Urban populations belong to the same phylogenetic group than those introduced in Australia and in other parts of the world. Behavioural analyses show that the eight populations sampled make up four mutually aggressive supercolonies. The maximum distance between nests from the same supercolony was 49 km and the closest distance between two nests belonging to two different supercolonies was 46 m. The genetic data and chemical analyses confirmed the behavioural tests as all of the nests were correctly assigned to their supercolony. Genetic diversity appears significantly greater in Africa than in introduced populations in Australia; by contrast, urban and Australian populations are characterized by a higher chemical diversity than rainforest ones.

Conclusions/Significance

Overall, our study shows that populations of P. megacephala in Cameroon adopt a unicolonial social structure, like invasive populations in Australia. However, the size of the supercolonies appears several orders of magnitude smaller in Africa. This implies competition between African supercolonies and explains why they persist over evolutionary time scales.     

Microsatellite DNA Analysis Revealed a Drastic Genetic Change of Plasmodium vivax Population in the Republic of Korea During 2002 and 2003

Background

Vivax malaria was successfully eliminated in the Republic of Korea (South Korea) in the late 1970s, but it was found to have re-emerged from 1993. In order to control malaria and evaluate the effectiveness of malaria controls, it is important to develop a spatiotemporal understanding of the genetic structure of the parasite population. Here, we estimated the population structure and temporal dynamics of the transmission of Plasmodium vivax in South Korea by analyzing microsatellite DNA markers of the parasite.

Methodology/Principal Findings

We analyzed 14 microsatellite DNA loci of the P. vivax genome from 163 South Korean isolates collected from 1994 to 2008. Allelic data were used to analyze linkage disequilibrium (LD), genetic differentiation and population structure, in order to make a detailed estimate of temporal change in the parasite population. The LD analysis showed a gradual decrease in LD levels, while the levels of genetic differentiation between successive years and analysis of the population structure based on the Bayesian approach suggested that a drastic genetic change occurred in the South Korean population during 2002 and 2003.

Conclusions/Significance

Although relapse and asymptomatic parasite carriage might influence the population structure to some extent, our results suggested the continual introduction of P. vivax into South Korea through other parasite population sources. One possible source, particularly during 2002 and 2003, is North Korea. Molecular epidemiology using microsatellite DNA of the P. vivax population is effective for assessing the population structure and temporal dynamics of parasite transmission; information that can assist in the elimination of vivax malaria in endemic areas.     

Social determinants of long lasting insecticidal hammock use among the Ra-glai ethnic minority in Vietnam: implications for forest malaria control

Background

Long-lasting insecticidal hammocks (LLIHs) are being evaluated as an additional malaria prevention tool in settings where standard control strategies have a limited impact. This is the case among the Ra-glai ethnic minority communities of Ninh Thuan, one of the forested and mountainous provinces of Central Vietnam where malaria morbidity persist due to the sylvatic nature of the main malaria vector An. dirus and the dependence of the population on the forest for subsistence - as is the case for many impoverished ethnic minorities in Southeast Asia.

Methods

A social science study was carried out ancillary to a community-based cluster randomized trial on the effectiveness of LLIHs to control forest malaria. The social science research strategy consisted of a mixed methods study triangulating qualitative data from focused ethnography and quantitative data collected during a malariometric cross-sectional survey on a random sample of 2,045 study participants.

Results

To meet work requirements during the labor intensive malaria transmission and rainy season, Ra-glai slash and burn farmers combine living in government supported villages along the road with a second home at their fields located in the forest. LLIH use was evaluated in both locations. During daytime, LLIH use at village level was reported by 69.3% of all respondents, and in forest fields this was 73.2%. In the evening, 54.1% used the LLIHs in the villages, while at the fields this was 20.7%. At night, LLIH use was minimal, regardless of the location (village 4.4%; forest 6.4%).

Discussion

Despite the free distribution of insecticide-treated nets (ITNs) and LLIHs, around half the local population remains largely unprotected when sleeping in their forest plot huts. In order to tackle forest malaria more effectively, control policies should explicitly target forest fields where ethnic minority farmers are more vulnerable to malaria.     

Environmental variation and rivers govern the structure of chimpanzee genetic diversity in a biodiversity hotspot

Background

The mechanisms that underlie the diversification of tropical animals remain poorly understood, but new approaches that combine geo-spatial modeling with spatially explicit genetic data are providing fresh insights on this topic. Data about the diversification of tropical mammals remain particularly sparse, and vanishingly few opportunities exist to study endangered large mammals that increasingly exist only in isolated pockets. The chimpanzees of Cameroon represent a unique opportunity to examine the mechanisms that promote genetic differentiation in tropical mammals because the region is home to two chimpanzee subspecies: Pan troglodytes ellioti and P. t. trogolodytes. Their ranges converge in central Cameroon, which is a geographically, climatically and environmentally complex region that presents an unparalleled opportunity to examine the roles of rivers and/or environmental variation in influencing the evolution of chimpanzee populations.

Results

We analyzed microsatellite genotypes and mtDNA HVRI sequencing data from wild chimpanzees sampled at a fine geographic scale across Cameroon and eastern Nigeria using a spatially explicit approach based upon Generalized Dissimilarity Modeling. Both the Sanaga River and environmental variation were found to contribute to driving separation of the subspecies. The importance of environmental variation differed among subspecies. Gene-environment associations were weak in P. t. troglodytes, whereas environmental variation was found to play a much larger role in shaping patterns of genetic differentiation in P. t. ellioti.

Conclusions

We found that both the Sanaga River and environmental variation likely play a role in shaping patterns of chimpanzee genetic diversity. Future studies using single nucleotide polymorphism (SNP) data are necessary to further understand how rivers and environmental variation contribute to shaping patterns of genetic variation in chimpanzees.

Electronic supplementary material

The online version of this article (doi:10.1186/s12862-014-0274-0) contains supplementary material, which is available to authorized users.     

Rapid loss of genetic variation in a founding population of Primula elatior (Primulaceae) after colonization

Background and Aims

Land-use changes and associated extinction/colonization dynamics can have a large impact on population genetic diversity of plant species. The aim of this study was to investigate genetic diversity in a founding population of the self-incompatible forest herb Primula elatior and to elucidate the processes that affect genetic diversity shortly after colonization.

Methods

AFLP markers were used to analyse genetic diversity across three age classes and spatial genetic structure within a founding population of P. elatior in a recently established stand in central Belgium. Parentage analyses were used to assess the amount of gene flow from outside the population and to investigate the contribution of mother plants to future generations.

Results

The genetic diversity of second and third generation plants was significantly reduced compared with that of first generation plants. Significant spatial genetic structure was observed. Parentage analyses showed that <20 % of the youngest individuals originated from parents outside the study population and that >50 % of first and second generation plants did not contribute to seedling recruitment.

Conclusions

These results suggest that a small effective population size and genetic drift can lead to rapid decline of genetic diversity of offspring in founding populations shortly after colonization. This multigenerational study also highlights that considerable amounts of gene flow seem to be required to counterbalance genetic drift and to sustain high levels of genetic diversity after colonization in recently established stands.Key words: AFLP, colonization, forest regeneration, genetic diversity, genetic drift, parentage analysis, spatial genetic structure     

High genetic differentiation between the M and S molecular forms of Anopheles gambiae in Africa

Background

Anopheles gambiae, a major vector of malaria, is widely distributed throughout sub-Saharan Africa. In an attempt to eliminate infective mosquitoes, researchers are trying to develop transgenic strains that are refractory to the Plasmodium parasite. Before any release of transgenic mosquitoes can be envisaged, we need an accurate picture of the differentiation between the two molecular forms of An. gambiae, termed M and S, which are of uncertain taxonomic status.

Methodology/Principal Findings

Insertion patterns of three transposable elements (TEs) were determined in populations from Benin, Burkina Faso, Cameroon, Ghana, Ivory Coast, Madagascar, Mali, Mozambique, Niger, and Tanzania, using Transposon Display, a TE-anchored strategy based on Amplified Fragment Length Polymorphism. The results reveal a clear differentiation between the M and S forms, whatever their geographical origin, suggesting an incipient speciation process.

Conclusions/Significance

Any attempt to control the transmission of malaria by An. gambiae using either conventional or novel technologies must take the M/S genetic differentiation into account. In addition, we localized three TE insertion sites that were present either in every individual or at a high frequency in the M molecular form. These sites were found to be located outside the chromosomal regions that are suspected of involvement in the speciation event between the two forms. This suggests that these chromosomal regions are either larger than previously thought, or there are additional differentiated genomic regions interspersed with undifferentiated regions.     

No evidence of association between HIV-1 and malaria in populations with low HIV-1 prevalence

Background

The geographic overlap between HIV-1 and malaria has generated much interest in their potential interactions. A variety of studies have evidenced a complex HIV-malaria interaction within individuals and populations that may have dramatic effects, but the causes and implications of this co-infection at the population level are still unclear. In a previous publication, we showed that the prevalence of malaria caused by the parasite Plasmodium falciparum is associated with HIV infection in eastern sub-Saharan Africa. To complement our knowledge of the HIV-malaria co-infection, the objective of this work was to assess the relationship between malaria and HIV prevalence in the western region of sub-Saharan Africa.

Methodology/Principal Findings

Population-based cross-sectional data were obtained from the HIV/AIDS Demographic and Health Surveys conducted in Burkina Faso, Ghana, Guinea, Mali, Liberia and Cameroon, and the malaria atlas project. Using generalized linear mixed models, we assessed the relationship between HIV-1 and Plasmodium falciparum parasite rate (PfPR) adjusting for important socio-economic and biological cofactors. We found no evidence that individuals living in areas with stable malaria transmission (PfPR>0.46) have higher odds of being HIV-positive than individuals who live in areas with PfPR≤0.46 in western sub-Saharan Africa (estimated odds ratio 1.14, 95% confidence interval 0.86–1.50). In contrast, the results suggested that PfPR was associated with being infected with HIV in Cameroon (estimated odds ratio 1.56, 95% confidence interval 1.23–2.00).

Conclusion/Significance

Contrary to our previous research on eastern sub-Saharan Africa, this study did not identify an association between PfPR and infection with HIV in western sub-Saharan Africa, which suggests that malaria might not play an important role in the spread of HIV in populations where the HIV prevalence is low. Our work highlights the importance of understanding the epidemiologic effect of co-infection and the relevant factors involved in this relationship for the implementation of effective control strategies.     

Conservation Efforts May Increase Malaria Burden in the Brazilian Amazon

Background

Large-scale forest conservation projects are underway in the Brazilian Amazon but little is known regarding their public health impact. Current literature emphasizes how land clearing increases malaria incidence, leading to the conclusion that forest conservation decreases malaria burden. Yet, there is also evidence that proximity to forest fringes increases malaria incidence, which implies the opposite relationship between forest conservation and malaria. We compare the effect of these environmental factors on malaria and explore its implications.

Methods and Findings

Using a large malaria dataset (∼1,300,000 positive malaria tests collected over ∼4.5 million km²), satellite imagery, permutation tests, and hierarchical Bayesian regressions, we show that greater forest cover (as a proxy for proximity to forest fringes) tends to be associated with higher malaria incidence, and that forest cover effect was 25 times greater than the land clearing effect, the often cited culprit of malaria in the region. These findings have important implications for land use/land cover (LULC) policies in the region. We find that cities close to protected areas (PA’s) tend to have higher malaria incidence than cities far from PA’s. Using future LULC scenarios, we show that avoiding 10% of deforestation through better governance might result in an average 2-fold increase in malaria incidence by 2050 in urban health posts.

Conclusions

Our results suggest that cost analysis of reduced carbon emissions from conservation efforts in the region should account for increased malaria morbidity, and that conservation initiatives should consider adopting malaria mitigation strategies. Coordinated actions from disparate science fields, government ministries, and global initiatives (e.g., Reduced Emissions from Deforestation and Degradation; Millenium Development Goals; Roll Back Malaria; and Global Fund to Fight AIDS, Tuberculosis and Malaria), will be required to decrease malaria toll in the region while preserving these important ecosystems.     

Neutrophil paralysis in Plasmodium vivax malaria

Background

The activation of innate immune responses by Plasmodium vivax results in activation of effector cells and an excessive production of pro-inflammatory cytokines that may culminate in deleterious effects. Here, we examined the activation and function of neutrophils during acute episodes of malaria.

Materials and Methods

Blood samples were collected from P. vivax-infected patients at admission (day 0) and 30–45 days after treatment with chloroquine and primaquine. Expression of activation markers and cytokine levels produced by highly purified monocytes and neutrophils were measured by the Cytometric Bead Assay. Phagocytic activity, superoxide production, chemotaxis and the presence of G protein-coupled receptor (GRK2) were also evaluated in neutrophils from malaria patients.

Principal Findings

Both monocytes and neutrophils from P. vivax-infected patients were highly activated. While monocytes were found to be the main source of cytokines in response to TLR ligands, neutrophils showed enhanced phagocytic activity and superoxide production. Interestingly, neutrophils from the malaria patients expressed high levels of GRK2, low levels of CXCR2, and displayed impaired chemotaxis towards IL-8 (CXCL8).

Conclusion

Activated neutrophils from malaria patients are a poor source of pro-inflammatory cytokines and display reduced chemotactic activity, suggesting a possible mechanism for an enhanced susceptibility to secondary bacterial infection during malaria.     

Malaria vectors and transmission dynamics in coastal south-western Cameroon

Background

Malaria is a major public health problem in Cameroon. Unlike in the southern forested areas where the epidemiology of malaria has been better studied prior to the implementation of control activities, little is known about the distribution and role of anophelines in malaria transmission in the coastal areas.

Methods

A 12-month longitudinal entomological survey was conducted in Tiko, Limbe and Idenau from August 2001 to July 2002. Mosquitoes captured indoors on human volunteers were identified morphologically. Species of the Anopheles gambiae complex were identified using the polymerase chain reaction (PCR). Mosquito infectivity was detected by the enzyme-linked immunosorbent assay and PCR. Malariometric indices (plasmodic index, gametocytic index, parasite species prevalence) were determined in three age groups (<5 yrs, 5–15 yrs, >15 yrs) and followed-up once every three months.

Results

In all, 2,773 malaria vectors comprising Anopheles gambiae (78.2%), Anopheles funestus (17.4%) and Anopheles nili (7.4%) were captured. Anopheles melas was not anthropophagic. Anopheles gambiae had the highest infection rates. There were 287, 160 and 149 infective bites/person/year in Tiko, Limbe and Idenau, respectively. Anopheles gambiae accounted for 72.7%, An. funestus for 23% and An. nili for 4.3% of the transmission. The prevalence of malaria parasitaemia was 41.5% in children <5 years of age, 31.5% in those 5–15 years and 10.5% in those >15 years, and Plasmodium falciparum was the predominant parasite species.

Conclusion

Malaria transmission is perennial, rainfall dependent and An. melas does not contribute to transmission. These findings are important in the planning and implementation of malaria control activities in coastal Cameroon and West Africa.

     

Influences of intermittent preventive treatment and persistent multiclonal Plasmodium falciparum infections on clinical malaria risk

Background

Intermittent preventive treatment (IPT) of malaria involves administration of curative doses of antimalarials at specified time points to vulnerable populations in endemic areas, regardless whether a subject is known to be infected. The effect of this new intervention on the development and maintenance of protective immunity needs further understanding. We have investigated how seasonal IPT affects the genetic diversity of Plasmodium falciparum infections and the risk of subsequent clinical malaria.

Material and Methods

The study included 2227 Ghanaian children (3–59 months) who were given sulphadoxine-pyrimethamine (SP) bimonthly, artesunate plus amodiaquine (AS+AQ) monthly or bimonthly, or placebo monthly for six months spanning the malaria transmission season. Blood samples collected at three post-interventional surveys were analysed by genotyping of the polymorphic merozoite surface protein 2 gene. Malaria morbidity and anaemia was monitored during 12 months follow-up.

Results

Monthly IPT with AS+AQ resulted in a marked reduction in number of concurrent clones and only children parasite negative just after the intervention period developed clinical malaria during follow-up. In the placebo group, children without parasites as well as those infected with ≥2 clones had a reduced risk of subsequent malaria. The bimonthly SP or AS+AQ groups had similar number of clones as placebo after intervention; however, diversity and parasite negativity did not predict the risk of malaria. An interaction effect showed that multiclonal infections were only associated with protection in children without intermittent treatment.

Conclusion

Molecular typing revealed effects of the intervention not detected by ordinary microscopy. Effective seasonal IPT temporarily reduced the prevalence and genetic diversity of P. falciparum infections. The reduced risk of malaria in children with multiclonal infections only seen in untreated children suggests that persistence of antigenically diverse P. falciparum infections is important for the maintenance of protective malaria immunity in high transmission settings.     

Mosquito Feeding Assays to Determine the Infectiousness of Naturally Infected Plasmodium falciparum Gametocyte Carriers

Introduction

In the era of malaria elimination and eradication, drug-based and vaccine-based approaches to reduce malaria transmission are receiving greater attention. Such interventions require assays that reliably measure the transmission of Plasmodium from humans to Anopheles mosquitoes.

Methods

We compared two commonly used mosquito feeding assay procedures: direct skin feeding assays and membrane feeding assays. Three conditions under which membrane feeding assays are performed were examined: assays with i) whole blood, ii) blood pellets resuspended with autologous plasma of the gametocyte carrier, and iii) blood pellets resuspended with heterologous control serum.

Results

930 transmission experiments from Cameroon, The Gambia, Mali and Senegal were included in the analyses. Direct skin feeding assays resulted in higher mosquito infection rates compared to membrane feeding assays (odds ratio 2.39, 95% confidence interval 1.94–2.95) with evident heterogeneity between studies. Mosquito infection rates in membrane feeding assays and direct skin feeding assays were strongly correlated (p<0.0001). Replacing the plasma of the gametocyte donor with malaria naïve control serum resulted in higher mosquito infection rates compared to own plasma (OR 1.92, 95% CI 1.68–2.19) while the infectiousness of gametocytes may be reduced during the replacement procedure (OR 0.60, 95% CI 0.52–0.70).

Conclusions

Despite a higher efficiency of direct skin feeding assays, membrane feeding assays appear suitable tools to compare the infectiousness between individuals and to evaluate transmission-reducing interventions. Several aspects of membrane feeding procedures currently lack standardization; this variability makes comparisons between laboratories challenging and should be addressed to facilitate future testing of transmission-reducing interventions.     

Molecular Evidence for the Presence of Rickettsia Felis in the Feces of Wild-living African Apes

Background

Rickettsia felis is a common emerging pathogen detected in mosquitoes in sub-Saharan Africa. We hypothesized that, as with malaria, great apes may be exposed to the infectious bite of infected mosquitoes and release R. felis DNA in their feces.

Methods

We conducted a study of 17 forest sites in Central Africa, testing 1,028 fecal samples from 313 chimpanzees, 430 gorillas and 285 bonobos. The presence of rickettsial DNA was investigated by specific quantitative real-time PCR. Positive results were confirmed by a second PCR using primers and a probe targeting a specific gene for R. felis. All positive samples were sequenced.

Results

Overall, 113 samples (11%) were positive for the Rickettsia-specific gltA gene, including 25 (22%) that were positive for R. felis. The citrate synthase (gltA) sequence and outer membrane protein A (ompA) sequence analysis indicated 99% identity at the nucleotide level to R. felis. The 88 other samples (78%) were negative using R. felis-specific qPCR and were compatible with R. felis-like organisms.

Conclusion

For the first time, we detected R. felis in wild-living ape feces. This non invasive detection of human pathogens in endangered species opens up new possibilities in the molecular epidemiology and evolutionary analysis of infectious diseases, beside HIV and malaria.     

Sequential fragmentation of Pleistocene forests in an East Africa biodiversity hotspot: chameleons as a model to track forest history

Background

The Eastern Arc Mountains (EAM) is an example of naturally fragmented tropical forests, which contain one of the highest known concentrations of endemic plants and vertebrates. Numerous paleo-climatic studies have not provided direct evidence for ancient presence of Pleistocene forests, particularly in the regions in which savannah presently occurs. Knowledge of the last period when forests connected EAM would provide a sound basis for hypothesis testing of vicariance and dispersal models of speciation. Dated phylogenies have revealed complex patterns throughout EAM, so we investigated divergence times of forest fauna on four montane isolates in close proximity to determine whether forest break-up was most likely to have been simultaneous or sequential, using population genetics of a forest restricted arboreal chameleon, Kinyongia boehmei.

Methodology/Principal Findings

We used mitochondrial and nuclear genetic sequence data and mutation rates from a fossil-calibrated phylogeny to estimate divergence times between montane isolates using a coalescent approach. We found that chameleons on all mountains are most likely to have diverged sequentially within the Pleistocene from 0.93–0.59 Ma (95% HPD 0.22–1.84 Ma). In addition, post-hoc tests on chameleons on the largest montane isolate suggest a population expansion ∼182 Ka.

Conclusions/Significance

Sequential divergence is most likely to have occurred after the last of three wet periods within the arid Plio-Pleistocene era, but was not correlated with inter-montane distance. We speculate that forest connection persisted due to riparian corridors regardless of proximity, highlighting their importance in the region''s historic dispersal events. The population expansion coincides with nearby volcanic activity, which may also explain the relative paucity of the Taita''s endemic fauna. Our study shows that forest chameleons are an apposite group to track forest fragmentation, with the inference that forest extended between some EAM during the Pleistocene 1.1–0.9 Ma.