Ayodeji Olayemi

New Hosts of The Lassa Virus.

Lassa virus (LASV) causes a deadly haemorrhagic fever in humans, killing several thousand people in West Africa annually. For 40 years, the Natal multimammate rat, Mastomys natalensis, has been assumed to be the sole host of LASV. We found evidence that LASV is also hosted by other rodent species: the African wood mouse Hylomyscus pamfi in Nigeria, and the Guinea multimammate mouse Mastomys erythroleucus in both Nigeria and Guinea. Virus strains from these animals were isolated in the BSL-4 laboratory and fully sequenced. Phylogenetic analyses of viral genes coding for glycoprotein, nucleoprotein, polymerase and matrix protein show that Lassa strains detected in M. erythroleucus belong to lineages III and IV. The strain from H. pamfi clusters close to lineage I (for S gene) and between II & III (for L gene). Discovery of new rodent hosts has implications for LASV evolution and its spread into new areas within West Africa.

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.

Contribution of DNA-typing to bushmeat surveys: assessment of a roadside market in south-western Nigeria

Context Following recent socioeconomic transformations in western and central Africa, the volume of bushmeat hunting, a traditional source of proteins and revenue for rural populations, has reached unsustainable levels. The morphological identification of species sold on bushmeat market stalls may be challenging because of the presence of cryptic taxa and smoked or processed carcasses. Aims To assess the contribution of DNA-typing to traditional bushmeat surveys. We conducted a case study at a roadside bushmeat market in Asejire, south-western Nigeria, to characterise the mammalian diversity and sketch out the dynamics of the bushmeat trade. Methods We generated a 402-bp Cytochrome b fragment using a ‘universal’ mitochondrial primer pair that successfully amplified across five mammalian orders, and used assignment procedures to assess the taxonomic identification of the traded species. We combined DNA-typing with morphological-based market surveys and questionnaires to half (n = 20) of the market stakeholders. Key results Our combined morphological–DNA-based survey revealed a total of 17 species, representing seven mammalian orders (Rodentia, Lagomorpha, Primates, Hyracoidea, Carnivora, Pholidota and Artiodactyla). DNA-typing allowed identifying the Walter’s duiker, a cryptic, newly described species from the Dahomey Gap, and diagnosing an unidentified primate as the white-throated monkey, Cercopithecus erythrogaster, a species of high conservation concern in Nigeria. K2P pairwise genetic distances among all species exceeded the 11% threshold, indicative of species-level distinction. The most hunted species were the Walter’s duiker and, to a lesser extent, the greater cane rat, Thryonomys swinderianus. Questionnaires to traders revealed that the Asejire roadside market was a straightforward trader–hunter system centralising off-takes from distant hunting sites. Conclusions We showed how mitochondrial DNA-typing combined with assignment procedures improved the characterisation of the mammalian diversity sold on bushmeat markets. The hunted mammalian community consisted of versatile, small- to medium-sized secondary forest species characteristic of the Dahomey Gap assemblage; their sustainable management is in doubt because of the lack of conservation and health awareness within the traders’ community. Implications Given the utility of mitochondrial DNA-typing in identifying species sold in bushmeat markets, we argue in favour of multi-entry investigations to reach a comprehensive characterisation of the bushmeat trade. The building of a web-accessible mtDNA database covering the spectrum of the species hunted for bushmeat would appear to be a valuable diagnostic tool that may help Nigeria and neighbouring countries to set up a rigorous monitoring of wildlife extirpation.

Diversity and distribution of murid rodent populations between forest and derived savanna sites within south western Nigeria

Southward encroachment of the derived savanna zone with its attendant denudation of the rainforest in Nigeria adversely affects the diversity and distribution of mammals, particularly murid rodents. This study was carried out to establish the identities of murid rodent populations and to compare their diversity between forest and derived savanna sites within south western Nigeria. Identification of captured specimens was carried out by multivariate statistics of body and skull measurements, and also by analysis of certain discrete characters. Six species were identified: Praomys tullbergi, Hylomyscus stella, Mastomys natalensis, Arvicanthis rufinus, Lemniscomys striatus and Lophuromys sikapusi. Forest sites possessed a higher Shannon’s diversity index (1.685) than the derived savanna sites (0.978), containing a higher number of taxa and larger abundance of murid rodents. This underscores the negative impact that encroachment of the derived savanna has upon diversity of animals in areas that were once forest.

Arenavirus Diversity and Phylogeography of Mastomys natalensis Rodents, Nigeria.

Mastomys natalensis rodents are natural hosts for Lassa virus (LASV). Detection of LASV in 2 mitochondrial phylogroups of the rodent near the Niger and Benue Rivers in Nigeria underlines the potential for LASV emergence in fresh phylogroups of this rodent. A Mobala-like sequence was also detected in eastern Nigeria.

Phylogeography of the heavily poached African common pangolin (Pholidota, Manis tricuspis) reveals six cryptic lineages as traceable signatures of Pleistocene diversification

Knowledge on faunal diversification in African rainforests remains scarce. We used phylogeography to assess (i) the role of Pleistocene climatic oscillations in the diversification of the African common pangolin (Manis tricuspis) and (ii) the utility of our multilocus approach for taxonomic delineation and trade tracing of this heavily poached species. We sequenced 101 individuals for two mitochondrial DNA (mtDNA), two nuclear DNA and one Y‐borne gene fragments (totalizing 2602 bp). We used a time‐calibrated, Bayesian inference phylogenetic framework and conducted character‐based, genetic and phylogenetic delineation of species hypotheses within African common pangolins. We identified six geographic lineages partitioned into western Africa, Ghana, the Dahomey Gap, western central Africa, Gabon and central Africa, all diverging during the Middle to Late Pleistocene. MtDNA (cytochrome b + control region) was the sole locus to provide diagnostic characters for each of the six lineages. Tree‐based Bayesian delimitation methods using single‐ and multilocus approaches gave high support for ‘species’ level recognition of the six African common pangolin lineages. Although the diversification of African common pangolins occurred during Pleistocene cyclical glaciations, causative correlation with traditional rainforest refugia and riverine barriers in Africa was not straightforward. We conclude on the existence of six cryptic lineages within African common pangolins, which might be of major relevance for future conservation strategies. The high discriminative power of the mtDNA markers used in this study should allow an efficient molecular tracing of the regional origin of African common pangolin seizures.

Small mammal diversity and dynamics within Nigeria, with emphasis on reservoirs of the lassa virus

Nigeria has a rich small mammal community, with several species implicated as carriers of zoonotic microbes such as the Lassa virus (LASV). We sought to elucidate the diversity and distribution of these animals (including known LASV reservoirs) geographically, habitat-wise and seasonally. Our DNA-assisted survey detected at least 19 small mammal species amongst 790 specimens. Diversity indices were similar between ecological zones and also between endemic and non-endemic areas for Lassa fever. Mastomys natalensis, the most renowned LASV host, was present in eight out of nine localities sampled. We also described the spatial occurrence of other known LASV hosts such as M. erythroleucus and Hylomyscus pamfi, including carriers of LASV-like arenaviruses such as Mus (Nannomys) spp. The most numerous rodents (Mastomys natalensis, M. erythroleucus, and Praomys daltoni) were captured mainly inside human dwellings. Reproductive activity occurred throughout the year, but led to population peaks for M. natalensis in the dry season and for M. erythroleucus and P. daltoni in the rainy season. Extensive geographic distribution of LASV rodent reservoirs, with population peaks in different seasons, shows that the risk of rodent-to-human transmission of LASV is greater than currently realized.

Morphometric characterization of the Giant Pouched Rat (Cricetomys Waterhouse 1840) in the forest zone of South Western Nigeria

Abstract This study was carried out to identify distinguishing morphometric characters between taxa from the genus Cricetomys in the rainforest. Specimens were captured from localities in the rainforest zone of South Western Nigeria, and 22 classic morphometric distances were taken on the skulls. Statistical analysis of morphometric data was carried out using principal component analysis and canonical variate analysis. In the principal component analysis, zygomatic width, rostrum depth, mandible length, incisive foramen length, and interorbital width represent the heaviest loading characters involved in separating male specimens of C. emini from C. gambianus. Among males, morphometric separation of these two species of Cricetomys was found to concur with pelage characterizations. Female specimens of C. emini and C. gambianus, however, could not be distinctly separated morphometrically, but nasal length, upper diastema length, rostrum depth, mandible length, mandible depth, zygomatic width, and parietal width are among the heaviest loadings involved in their partial separation. Moreover, morphometric data and pelage patterns do not produce identical characterizations of female specimens. Findings from this study reinforce the view that pelage characterizations are not entirely reliable in distinguishing taxa within Cricetomys. The morphometric characters identified in this study will contribute to more a reliable identification of giant pouched rat species within forests of Africa.

Widespread arenavirus occurrence and seroprevalence in small mammals, Nigeria

BackgroundLassa fever, killing thousands of people annually, is the most reported viral zoonotic disease in Nigeria. Recently, different rodent species carrying diverse lineages of the Lassa virus (LASV) in addition to a novel Mobala-like genetic sequence were detected within the country. Here, screening 906 small mammal specimens from 11 localities for IgG antibodies and incorporating previous PCR detection data involving the same populations, we further describe arenavirus prevalence across Nigeria in relation to host species and geographical location.MethodsSmall mammals were trapped during the period 2011–2015 according to geographical location (endemic and non-endemic zones for Lassa fever), season (rainy and dry seasons between 2011 and 2012 for certain localities) and habitat (indoors, peridomestic settings and sylvatic vegetation). Identification of animal specimens from genera such as Mastomys and Mus (Nannomys) was assisted by DNA sequencing. Small mammals were tested for LASV IgG antibody using an indirect immunofluorescence assay (IFA).ResultsSmall mammals were infected in both the endemic and non-endemic zones for Lassa fever, with a wider range of species IgG-positive (n = 8) than those which had been previously detected to be PCR-positive (n = 3). IgG-positive species, according to number of infected individuals, were Mastomys natalensis (n = 40), Mastomys erythroleucus (n = 15), Praomys daltoni (n = 6), Mus baoulei (n = 5), Rattus rattus (n = 2), Crocidura spp. (n = 2), Mus minutoides (n = 1) and Praomys misonnei (n = 1). Multimammate mice (Mastomys natalensis and M. erythroleucus) were the most ubiquitously infected, with animals testing positive by either PCR or IgG in 7 out of the 11 localities sampled. IgG prevalence in M. natalensis ranged from 1% in Abagboro, 17–36 % in Eguare Egoro, Ekpoma and Ngel Nyaki, up to 52 % in Mayo Ranewo. Prevalence according to locality, season and age was not, however, statistically significant for M. natalensis in Eguare Egoro and Ekpoma, localities that were sampled longitudinally.ConclusionsOverall, our study demonstrates that arenavirus occurrence is probably more widely distributed geographically and in extent of host taxa than is currently realized. This expanded scope should be taken into consideration in Lassa fever control efforts. Further sampling should also be carried out to isolate and characterize potential arenaviruses present in small mammal populations we found to be seropositive.