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In the past decade, there has been a drastic decline in the number of Eastern Black rhinoceros (black rhinoceros) (Diceros bicornis michaeli), primarily because of poaching across their natural habitats, leaving few individuals in small, isolated populations that are vulnerable to demographic extinction, disease epidemics, genetic drift and inbreeding. However, genetic consequences of the demographic decline on the remaining populations have not been investigated. Using the mitochondrial control region, this study investigated how current levels of genetic diversity relate to historical patterns, quantified genetic differentiation between extant populations and assessed the impacts of previous translocations on genetic diversity across populations. A total of 74 individual eastern black rhinoceroses were sampled from five extant populations in Tanzania and one neighbouring cross-border population in the Maasai Mara in Kenya. Six maternal haplotypes were identified, with an overall haplotype diversity of h=0.7 but low overall nucleotide diversity within populations (π = 0.017) compared to historical populations from Tanzania (π = 0.021). There was extensive variation in haplotype distribution between populations, with more variation exists within (65.5 %) than among the populations (35.5%), which may indicate lack of migration between populations. Specifically, some geographically close populations with different histories of introductions didn’t share any haplotypes, suggesting that gene flow is currently restricted. The haplotypes were distributed among three east African haplogroups (CV, CE and EA) that have been described in previous studies, suggesting that multiple lineages have been preserved despite loss of haplotypes. One of the haplotypes was highly divergent and matched sequences previously classified as a subspecies that has not been recognised in recent years (D. b. ladoensis). We recommend that current levels of diversity be maintained by allowing natural movements of rhinoceroses between the populations, with the possibility of introducing additional variations by translocation of individuals between sites.

Reliable molecular identification of vertebrate species from morphologically unidentifiable tissue is critical for the prosecution of illegally-traded wildlife products, conservation-based biodiversity research, and identification of blood-meal hosts of hematophagous invertebrates. However, forensic identification of vertebrate tissue relies on sequencing of the mitochondrial cytochrome oxidase I (COI) ‘barcode’ gene, which remains costly for purposes of screening large numbers of unknown samples during routine surveillance. Here, we adapted a rapid, low-cost approach to differentiate 10 domestic and 24 wildlife species that are common in the East African illegal wildlife products trade based on their unique high-resolution melting profiles from COI, cytochrome b, and 16S ribosomal RNA gene PCR products. Using the approach, we identified (i) giraffe among covertly sampled meat from Kenyan butcheries, and (ii) forest elephant mitochondrial sequences among savannah elephant reference samples. This approach is being adopted for high-throughput pre-screening of potential bushmeat samples in East African forensic science pipelines.

BackgroundThe role of questing ticks in the epidemiology of tick-borne diseases in Kenya’s Maasai Mara National Reserve (MMNR), an ecosystem with intensified human-wildlife-livestock interactions, remains poorly understood. Therefore, we carried out a survey of the diversity of questing ticks, their blood-meal hosts, and tick-borne pathogens to understand potential effects to human and livestock health.MethodsQuesting ticks were collected by flagging and hand picks from vegetation in 25 localities and identified based on morphologic and molecular criteria. We used PCR with high-resolution melting (HRM) analysis, and sequencing to identify Anaplasma, Babesia, Coxiella, Ehrlichia, Rickettsia, and Theileria pathogen diversities and blood meals in 231 tick pools.ResultsA total of 1,465 host-seeking ticks were collected, including Rhipicephalus appendiculatus (n = 1,125), Rhipicephalus pulchellus (n = 6), Rhipicephalus evertsi (n = 5), Amblyomma cf. gemma (n = 178), Amblyomma gemma (n = 145), Amblyomma variegatum (n = 4), Amblyomma sp. (n = 1), and Haemaphysalis leachi (n = 1). Remnant blood-meals from humans, wildebeest, and African buffalo were detected in Rh. appendiculatus, goat in Rh. evertsi, sheep in Am. gemma, and cattle in Am. variegatum. Rickettsia africae was detected in Am. gemma (1/25 pools) that had blood-meal remnant from sheep and Am. variegatum (4/25 pools) that had fed on cattle. Rickettsia spp. were found in Am. gemma (4/25 pools) and Rh. evertsi (1/4 pools). Anaplasma ovis was detected in Rh. appendiculatus (1/172 pools) and Rh. evertsi (1/4 pools), while Anaplasma bovis was detected in Rh. appendiculatus (1/172 pools). Theileria parva was detected in Rh. appendiculatus (27/172 pools). Babesia, Ehrlichia and Coxiella pathogens were not found in any ticks. Unexpectedly, diverse Coxiella sp. endosymbionts were detected in all tick genera (174/231 pools).ConclusionsThe data shows that ticks from the rapidly-changing MMNR are infected with zoonotic R.africae and unclassified Rickettsia spp, demonstrating the persistent risk of African tick-bite fever and other and Spotted Fever Group rickettsioses to local dwellers and visitors to the Maasai Mara ecosystem. Protozoan pathogens that may pose risk to livestock production were also identified. We also highlight possible existence of morphotypic variants of Amblyomma species, based on the observation of Ambyomma cf. gemma, which may be potential human parasites or emergent disease vectors. Our findings also demonstrate that questing ticks in this ecosystem have dynamic vertebrate blood sources including humans, wildlife and domestic animals, which may amplify transmission of tickborne zoonoses and livestock diseases. Further studies are needed to determine the role of Coxiella endosymbionts in tick physiology and vector competence.

BACKGROUND Simian immunodeficiency virus (SIV) naturally infects African non-human primates (NHPs) and poses a threat of transmission to humans through hunting and consumption of monkeys as bushmeat. This study investigated the as of yet unknown molecular diversity of SIV in free-ranging Chlorocebus species (African green monkeys-AGMs) and Papio anubis (olive baboons) within Mombasa, Kisumu and Naivasha urban centres in Kenya. METHODS We collected blood samples from 124 AGMs and 65 olive baboons in situ, and detected SIV by high-resolution melting analysis and sequencing of PCR products. RESULTS Simian immunodeficiency virus prevalence was 32% in AGMs and 3% in baboons. High-resolution melting (HRM) analysis demonstrated distinct melt profiles illustrating virus diversity confirmed by phylogenetic analysis. CONCLUSIONS There is persistent evolutionary diversification of SIVagm strains in its natural host, AGMs and cross-species infection to olive baboons is occurring. Further study is required to establish pathogenesis of the diverse SIVagm variants and baboon immunological responses.

Reliable molecular identification of vertebrate species from morphologically unidentifiable tissue is critical for the prosecution of illegally-traded wildlife products, conservation-based biodiversity research, and identification of blood-meal hosts of hematophagous invertebrates. However, forensic identification of vertebrate tissue relies on the sequencing of mitochondrial cytochrome oxidase I (COI) ‘barcode’ genes, which remains costly for purposes of screening large numbers of unknown samples during routine surveillance. Here, we adopted a rapid, low-cost approach to differentiate 10 domestic and 24 wildlife species that are common in the East African illegal wildlife products trade based on their unique high-resolution melting profiles from COI, cytochrome b, and 16S ribosomal RNA gene PCR products. Using the approach, we identified (i) giraffe among covertly sampled meat from Kenyan butcheries, and (ii) forest elephant mitochondrial sequences among savannah elephant reference samples. This approach is being adopted for high-throughput pre-screening of potential bushmeat samples in East African forensic science pipelines.

Rapid growth in world trade has enabled transnational criminal networks to conceal their contraband among the 1 billion containers shipped worldwide annually. Forensic methods are needed to identify the major cartels moving the contraband into transit. We combine DNA-based sample matching and geographic assignment of tusks to show that the two tusks from the same elephant are often shipped by the same trafficker in separate large consignments of ivory. The paired shipments occur close in time from the same initial place of export and have high overlap in the geographic origins of their tusks. Collectively, these paired shipments form a linked chain that reflects the sizes, interconnectedness, and places of operation of Africa’s largest ivory smuggling cartels.

Pilobolus are abundant in herbivore dung. Although they are non-pathogenic to herbivores, they are vectors to lungworms present in the dung that cause bronchitis if ingested. Thus, determining the presence of Pilobolus in the field might prove useful to assess a link between Pilobolus and lungworm infections, as well as the areas where they occur. Species identification within the genus has mostly relied on morphological data, which lacks accuracy due to the overlap of morphological characters. In this study, we applied genetic identification to assess the presence of Pilobolus species in Nairobi National Park, Kenya. This method is more reliable than the more commonly used morphometric analyses, and the generated sequences are useful for future reference. In this study, we collected dung samples from different herbivores in Nairobi National Park, and sporangium isolates were obtained for pure cultures. DNA was extracted from the pure cultures and fungal barcode primers were employed for PCR amplification and sequencing. Two species of Pilobolus were identified, Pilobolus pullus, with a high genetic affinity, and a second cryptic species, morphologically identified as Pilobolus crystallinus, but did not have a close genetic match to any species in Genbank. Lack of identification of other Pilobolus species, known as key species in lungworm transmission, may suggest the absence of such in the sampled area.

Summary Black and white rhinoceros ( Diceros bicornis and Ceratotherium simum ) are iconic African species that are classified by the International Union for the Conservation of Nature (IUCN) as Critically Endangered and Near Threatened (http://www.iucnredlist.org/), respectively [1]. At the end of the 19 th century, Southern white rhinoceros ( Ceratotherium simum simum ) numbers had declined to fewer than 50 animals in the Hluhluwe-iMfolozi region of the KwaZulu-Natal (KZN) province of South Africa, mainly due to uncontrolled hunting [2,3]. Efforts by the Natal Parks Board facilitated an increase in population to over 20,000 in 2015 through aggressive conservation management [2]. Black rhinoceros ( Diceros bicornis ) populations declined from several hundred thousand in the early 19 th century to ∼65,000 in 1970 and to ∼2,400 by 1995 [1] with subsequent genetic reduction, also due to hunting, land clearances and later poaching [4]. In South Africa, rhinoceros poaching incidents have increased from 13 in 2007 to 1,215 in 2014 [1]. This has occurred despite strict trade bans on rhinoceros products and strict enforcement in recent years.

Coxiella burnetii is the causative agent of Q fever, a zoonotic disease of public health importance. The role of wildlife and their ticks in the epidemiology of C. burnetii in Kenya is unknown. This study analysed the occurrence and prevalence of the pathogen in wildlife and their ticks at two unique wildlife-livestock interfaces of Laikipia and Maasai Mara National Reserve (MMNR) with the aim to determine the potential risk of transmission to livestock and humans. Blood from 79 and 73 animals in Laikipia and MMNR, respectively, and 756 and 95 ixodid ticks in each of the areas, respectively, was analysed. Ticks were pooled before analyses into 137 and 29 samples in Laikipia and MMNR, respectively, of one to eight non-engorged ticks according to species and animal host. Real-time PCR amplifying the repetitive insertion element IS1111a of the transposase gene was used to detect C. burnetii DNA. Although none of the animals and ticks from MMNR tested positive, ticks from Laikipia had an overall pooled prevalence of 2.92% resulting in a maximum-likelihood estimate of prevalence of 0.54%, 95% CI 0.17-1.24. Ticks positive for C. burnetii DNA belonged to the genus Rhipicephalus at a pooled prevalence of 2.96% (maximum-likelihood estimate of prevalence of 0.54%, 95% CI 0.17-1.26). These ticks were Rhipicephalus appendiculatus, R. pulchellus and R. evertsi at pooled prevalence of 3.77, 3.03 and 2.04%, respectively. The presence of C. burnetii in ticks suggests circulation of the pathogen in Laikipia and demonstrates they may play a potential role in the epidemiology of Q fever in this ecosystem. The findings warrant further studies to understand the presence of C. burnetii in domestic animals and their ticks within both study areas.

Very little is known about the influence of massive and long distance migration on parasite epidemiology. Migration can simultaneously minimize exposure to common parasites in their habitats and increase exposure to novel pathogens from new environments and habitats encountered during migration, while physiological stress during long distance movement can lead to immune suppression, which makes migrants vulnerable to parasites. In this paper, we investigated the diversity, prevalence, parasite load, co-infection patterns and predilection sites of adult gastrointestinal helminths in 130 migrating wildebeests and tested for their relation with animal age, sex and migration time (which also could indicate different migration routes), and compared them with the non-migratory wildebeest. Surprisingly, only four parasite species were found, Oesophagostomum columbianum, Haemonchus placei, Calicophoron raja and Moniezia expansa, which were lower than in non-migratory wildebeest reported in the literature. These parasites were generalists, infecting livestock, and suggests that wildebeest and livestock, because of their interaction during migration, have a cross-infection risk. There was a negative relation between parasites diversity, prevalence and intensity of infection, and host age, which suggests that wildebeests acquire protective immunity against these parasites as they get older. Prevalence and intensity of infection were higher among wildebeest crossing the Mara Bridge (early migrants) compared to those crossing the Serena (late migrants), which suggests that early migrants (or migrants originating from different areas) have varying infection intensities. The prevalence and intensity of infection were higher in males compared to females and may be due to ecological, behavioural, or physiological differences between males and females. Our findings compared to those of previous studies suggest that migration may provide a mechanism to minimize exposure of hosts to common parasites through migratory escape, but this result awaits examination of helminths epidemiology of non-migratory wildebeests from areas of migrant origins. The potential parasitic cross-infection between wildebeests and livestock is a real risk to be taken into account in the management of wildebeest migration corridors., The authors also thank KWS for funding the research, and Sophia Masila for editing the manuscript.

Theileria is a genus of tick-borne protozoan that is globally widespread and infects nearly all ungulates in which they cause either latent infection or lethal disease. Wild animals are considered reservoir hosts of many species of Theileria and their diversity in wildlife species is increasingly becoming of interest. The molecular characterization and identification of Theileria infecting wildlife has been studied in a few species including buffalo, which are considered reservoir host for Theileria parva infecting cattle. In this study, we sequenced Theileria species infecting wildebeest (Connochaetes taurinus) and used molecular-genetic and phylogenetic analysis of the 18 Small Subunit of the Ribosomal RNA (18S rRNA) to identify their relationships with known species of Theileria. Our results revealed three new Theileria haplotypes infecting wildebeest. Phylogenetic analysis revealed that haplotype 1 and 2 clustered in the same clade as Theileria separata and with Theileria sp. isolated from other small to medium sized antelopes. Haplotype 3 clustered close to the Theileria ovis clade. This is the first molecular description and characterization of Theileria species infecting blue wildebeest in East Africa. This study demonstrates the potential for Theileria transmission between wildebeest and small domestic ungulates, such as sheep and goats.

[Background] A huge effort in rhinoceros conservation has focused on poaching and habitat loss as factors leading to the dramatic declines in the endangered eastern black rhinoceros (Diceros bicornis michaeli) and the southern white rhinoceros (Ceratotherium simum simum). Nevertheless, the role disease and parasite infections play in the mortality of protected populations has largely received limited attention. Infections with piroplasmosis caused by Babesia bicornis and Theileria bicornis has been shown to be fatal especially in small and isolated populations in Tanzania and South Africa. However, the occurrence and epidemiology of these parasites in Kenyan rhinoceros is not known., [Results] Utilizing 18S rRNA gene as genetic marker to detect rhinoceros infection with Babesia and Theileria, we examined blood samples collected from seven rhinoceros populations consisting of 114 individuals of black and white rhinoceros. The goal was to determine the prevalence in Kenyan populations, and to assess the association of Babesia and Theileria infection with host species, age, sex, location, season and population mix (only black rhinoceros comparing to black and white rhinoceros populations). We did not detect any infection with Babesia in the sequenced samples, while the prevalence of T. bicornis in the Kenyan rhinoceros population was 49.12% (56/114). White rhinoceros had significantly higher prevalence of infection (66%) compared to black rhinoceros (43%). The infection of rhinoceros with Theileria was not associated with animal age, sex or location. The risk of infection with Theileria was not higher in mixed species populations compared to populations of pure black rhinoceros., [Conclusion] In the rhinoceros studied, we did not detect the presence of Babesia bicornis, while Theileria bicornis was found to have a 49.12% prevalence with white rhinoceros showing a higher prevalence (66%) comparing with black rhinoceros (43%). Other factors such as age, sex, location, and population mix were not found to play a significant role., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

Hard ticks are blood feeding ectoparasites that infest humans and animals and are vectors of pathogenic microorganism that cause severe infectious diseases. Morphological identification has been the main approach of identifying ticks but the technique is considered inaccurate and difficult. Molecular techniques have recently been considered to be appropriate approaches for accurate and rapid identification and Internal Transcribed Spacer 2 (ITS 2) has been shown to differentiate genus of hard ticks. Currently, genetic identification of ticks using ITS 2 has not been carried out in Kenya. In this study, 80 tick samples were collected from Lake Nakuru and Tsavo National Parks and were identified morphologically using appropriate identification keys. DNA was extracted from the appendages using DNA extraction kit followed by partial amplification of ITS 2 gene. The PCR products were then analyzed by gel electrophoresis and positive PCR products were sequenced. Of the tick samples four genera were identified morphologically; Amblyomma, Hyalomma, Rhipicephalus and Dermacentor. Of the tick samples identified and compared with the sequences in the GenBank, six and seven samples showed 98-100% homology with A. variegatum and R. pulchellus respectively and they clustered in their respective monophyletic group in the phylogeny tree with a bootstrap of 99%. Two samples showed 92% homology with H. dromedarii and the study sequences clustered with the reference sequence with a bootstrap of 99% while six samples showed 95% homology with H. marginatum rufipes, however, only four of these samples clustered together with the reference sequence in the phylogeny with a bootstrap of 95%. One sample showed 91% homology with A. humerali and did not cluster together in the phylogeny tree. Congruency between both techniques was high with a correlation coefficient of 0.941. This is the first report of phenotypic and genotypic traits of tick species in Kenya and the findings will add value to the existing knowledge in identification of ticks.

Background: Very few studies have ever focused on the elephants that are wounded or killed as local communities attempt to scare these animals away from their settlements and farms, or on the cases in which local people take revenge after elephants have killed or injured humans. On the other hand, local communities live in close proximity to elephants and hence can play a positive role in elephant conservation by informing the authorities of the presence of injured elephants. Methodology/Principal Findings: Between 2007 and 2011, 129 elephants were monitored in Masai Mara (Kenya), of which 54 had various types of active (intentionally caused) or passive (non-intentionally caused) injuries. Also studied were 75 random control samples of apparently unaffected animals. The observed active injuries were as expected biased by age, with adults suffering more harm; on the other hand, no such bias was observed in the case of passive injuries. Bias was also observed in elephant sex since more males than females were passively and actively injured. Cases of passive and active injuries in elephants were negatively related to the proximity to roads and farms; the distribution of injured elephants was not affected by the presence of either human settlements or water sources. Overall more elephants were actively injured during the dry season than the wet season as expected. Local communities play a positive role by informing KWS authorities of the presence of injured elephants and reported 43% of all cases of injured elephants. Conclusions: Our results suggest that the negative effect of local communities on elephants could be predicted by elephant proximity to farms and roads. In addition, local communities may be able to play a more positive role in elephant conservation given that they are key informants in the early detection of injured elephants.

Parasite presence in any ecosystem generates complex navigating webs (Parasite-NW) within the system, through which parasites move from one to another host. The appropriate assimilation of parasite navigating web is pivotal for a better understanding of pathogen flow in the ecosystem, with implications for disease control. Sarcoptes mite has been approached from medical, veterinary, entomological, physiological and, recently, molecular sides, to understand its epidemiological navigating web between isolates from different hosts and geographical regions. The obtained conclusions are still a matter of debate. Sarcoptes navigating web (Sarcoptes-NW) is intricate and uncertain, with unexplainable pathogenic flow. In this review we summarize by which routes, under what conditions and at what levels the Sarcoptes mite moves among its hosts.

Background Rapid diagnostic tests (RDTs) for malaria are increasingly being used for management of patients. Different studies have shown significant failure rate of RDTs, especially in children and in areas of low malaria transmission. In this study, we sort molecular answers to RDT failure by re-screening RDT negative samples for malaria at genus and species level as well as for diversity of Plasmodium falciparum histidine-rich protein 2 (PfHRP-2) and lactate dehydrogenase (pLDH). PfHRP-2 and pLDH are the malaria target antigens for the commonly used commercial RDTs.

Background Habitat and food supply loss and disruption, together with man’s pursuit of the animal’s unique horn pose significant threats to the charismatic rhinoceros. Filarial worms have been thought to cause cutaneous lesions in black rhinoceros (Diceros bicornis) in Kenya and South Africa, but never in white rhinoceros (Ceratotherium simum) in the wild, despite the fact that the two species live often in close proximity. Stephanofilaria dinniki has been implicated in the past as the causal agents for such lesions. Findings In this paper we report a putative filariosis outbreak in both black and white rhinos at Meru National Park in Kenya. Four black and five white rhinos were affected by various degrees of filarioid-like lesions, while apparently all sympatric wild and domestic animals were filarial worm-free. Affected rhinos were captured and successfully treated. Comparison between the epidemiological aspects of white and black rhinoceros filariosis, and the possible relations between this outbreak and annual seasons, the presence of oxpeckers and other host species are discussed. Conclusions Our study highlights (i) that filarial infection is not restricted to black rhinos, but it affects both rhinoceros species, and (ii) the importance of the earlier detection and immediate treatment (capture-treat and release) of filarioid infections, which is of pivotal interest for wildlife conservation, and especially the endangered and isolated white and black rhinoceros populations., We also acknowledge institutional support from the Unit of Information Resources for Research at the "Consejo Superior de Investigaciones Científicas" (CSIC) for the article-processing charges contribution