Tsetse blood-meal sources, endosymbionts and trypanosome-associations in the Maasai Mara National Reserve, a wildlife-human-livestock interface.

African trypanosomiasis (AT) is a neglected disease of both humans and animals caused by Trypanosoma parasites, which are transmitted by obligate hematophagous tsetse flies (Glossina spp.). Knowledge on tsetse fly vertebrate hosts and the influence of tsetse endosymbionts on trypanosome presence, especially in wildlife-human-livestock interfaces, is limited. We identified tsetse species, their blood-meal sources, and correlations between endosymbionts and trypanosome presence in tsetse flies from the trypanosome-endemic Maasai Mara National Reserve (MMNR) in Kenya. Among 1167 tsetse flies (1136 Glossina pallidipes, 31 Glossina swynnertoni) collected from 10 sampling sites, 28 (2.4%) were positive by PCR for trypanosome DNA, most (17/28) being of Trypanosoma vivax species. Blood-meal analyses based on high-resolution melting analysis of vertebrate cytochrome c oxidase 1 and cytochrome b gene PCR products (n = 354) identified humans as the most common vertebrate host (37%), followed by hippopotamus (29.1%), African buffalo (26.3%), elephant (3.39%), and giraffe (0.84%). Flies positive for trypanosome DNA had fed on hippopotamus and buffalo. Tsetse flies were more likely to be positive for trypanosomes if they had the Sodalis glossinidius endosymbiont (P = 0.0002). These findings point to complex interactions of tsetse flies with trypanosomes, endosymbionts, and diverse vertebrate hosts in wildlife ecosystems such as in the MMNR, which should be considered in control programs. These interactions may contribute to the maintenance of tsetse populations and/or persistent circulation of African trypanosomes. Although the African buffalo is a key reservoir of AT, the higher proportion of hippopotamus blood-meals in flies with trypanosome DNA indicates that other wildlife species may be important in AT transmission. No trypanosomes associated with human disease were identified, but the high proportion of human blood-meals identified are indicative of human African trypanosomiasis risk. Our results add to existing data suggesting that Sodalis endosymbionts are associated with increased trypanosome presence in tsetse flies.

Arboviruses and Blood Meal Sources in Zoophilic Mosquitoes at Human-Wildlife Interfaces in Kenya.

Background: Zoophilic mosquitoes play an important role in the transmission of arboviruses of medical importance at human-wildlife interfaces, yet arbovirus surveillance efforts have been focused mostly on anthropophilic mosquitoes. Understanding the diversity of zoophilic mosquitoes and their associated feeding patterns and arboviruses can inform better vector control strategies. Materials and Methods: We morphologically identified mosquitoes collected from two game reserves in Kenya, the Maasai Mara National Reserve (MMNR) and locations near the Shimba Hills National Reserve (SHNR). Representative mosquitoes were also identified by cytochrome c oxidase subunit 1 (COI) barcode sequencing. In addition, we identified the vertebrate hosts of mosquito blood meals from the contents of each mosquito's abdomen by high-resolution melting (HRM) analysis and sequencing of COI, 16S ribosomal RNA, and cytochrome b gene PCR products. Similarly, mosquito arbovirus infections were identified by HRM analysis and sequencing of Alphavirus- and Flavivirus-specific RT-PCR products. Results: Of 2858 mosquitoes collected, 51 were engorged with blood meals from seven different vertebrate hosts, including humans, birds, domestic, and peridomestic animals and wildlife. Culex was the most abundant mosquito genus, with Culex pipiens being the most abundant species in both study regions. Among MMNR samples, we detected dengue serotype-2 virus (DENV-2) for the first time in Aedes tarsalis and Aedes tricholabis, as well as Sindbis virus in male Cx. pipiens. We also detected DENV-2 in Aedes aegypti sampled from locations near the SHNR. Human and diverse wildlife blood meals were identified, including bushbuck blood in the dengue-infected Ae. tarsalis and both human and hippopotamus blood in a single Eretmapodites chrysogaster mosquito. Conclusions: Our findings highlight the potential risk of sylvatic dengue and Sindbis transmission to humans by zoophilic mosquitoes at human-wildlife interfaces in Africa. Of specific importance, we provide evidence of sylvatic DENV-2 in Ae. tarsalis and Ae. tricholabis, representing potential new dengue vectors.

Three-gene PCR and high-resolution melting analysis for differentiating vertebrate species mitochondrial DNA for biodiversity research and complementing forensic surveillance.

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.

Sero-epizootiologic survey of Trypanosoma brucei in Kenyan nonhuman primates.

Blood samples were collected from 121 individuals of three species of wild-caught nonhuman primates from Kenya, including African green monkeys (Cercopithecus aethiops), Syke's monkeys (C. mitis), and olive baboons (Papio cynocephalus anubis), and were examined for circulating Trypanosoma brucei and for T. brucei antigen and anti-trypanosome antibody. Indirect antibody enzyme-linked immunosorbent assay detected titers of anti-T. brucei antibodies in 13 of the primates sampled, and field-oriented latex agglutination test detected invariant T. brucei antigens in 10 (8.3%) of the primates. However, no trypanosomes were visible in blood smears, on wet blood films, or by buffy coat technique, nor were they demonstrable in a subset of C. aethiops individuals that were studied using mouse subinoculation.