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Visceral and cutaneous leishmaniasis are endemic to specific regions due to the ecological preferences of phlebotomine sand flies and Leishmania spp. transmission. Sand fly entomological data in northern Kenya are scarce due to limited studies and neglect of leishmaniasis. The aim of this study was to investigate: (i) sand fly diversity and distribution; (ii) occurrence of Leishmania DNA within sand flies; and (iii) blood-meal sources of sand flies in Laisamis, northern Kenya. We conducted an entomological survey during February and March of 2021 in five areas of Laisamis sub-county using standard CDC light traps. A total of 1009 sand flies (394 male and 615 female) were morphologically identified, and representative samples verified by PCR amplification and sequencing of the cytochrome c oxidase subunit 1 ( cox 1) gene. Similarly, we identified blood-meal sources and Leishmania DNA in female sand flies by PCR amplicon sequencing of the vertebrate cytochrome b ( cyt b ) gene and internal transcribed spacer 1 (ITS1) of the 28S rRNA gene, respectively. Sergentomyia clydei (59.8%) was the most abundant sand fly species. Though collected mainly from one locality (Tirgamo), 14.8% of samples belonged to Phlebotomus ( Artemievus ) alexandri Sinton, 1928. We detected DNA of Leishmania major in 5.19% of Ph. alexandri , whereas Leishmania adleri DNA was detected in S. clydei (7.51%), Sergentomyia squamipleuris (8.00%), and Sergentomyia africanus (8.33%). Nine of 13 blood-fed sand flies had obtained blood from humans, of which 33.3% had L. major DNA. Both Ph. alexandri and S. clydei primarily fed on humans and could potentially be involved in the transmission of cutaneous leishmaniasis. The findings of this study contribute to the understanding of sand fly vector populations and their potential to transmit leishmaniasis in the area., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)

Akagera National Park and its surroundings are home to tsetse flies and a number of their mammalian hosts in Rwanda. A One-health approach is being used in the control and surveillance of both animal and human trypanosomosis in Rwanda. Determination of the infection level in tsetse flies, species of trypanosomes circulating in vectors, the source of tsetse blood meal and endosymbionts is crucial in understanding the epidemiology of the disease in animals and humans in the region. Tsetse flies ( n  = 1101), comprising Glossina pallidipes ( n  = 771) and Glossina morsitans centralis ( n  = 330) were collected from Akagera park and surrounding areas between May 2018 and June 2019. The flies were screened for trypanosomes, vertebrate host DNA to identify sources of blood meal, and endosymbionts by PCR - High Resolution Melting analysis and amplicon sequencing. The feeding frequency and the feeding indices (selection index - W) were calculated to identify the preferred hosts. An overall trypanosome infection rate of 13.9% in the fly's Head and Proboscis (HP) and 24.3% in the Thorax and Abdomen (TA) were found. Eight trypanosome species were identified in the tsetse fly HP and TA, namely: Trypanosoma (T.) brucei brucei , T. congolense Kilifi, T. congolense savannah, T. vivax , T. simiae , T. evansi , T. godfreyi , T. grayi and T. theileri . We found no evidence of human-infective T. brucei rhodesiense. We also identified eighteen species of vertebrate hosts that tsetse flies fed on, and the most frequent one was the buffalo ( Syncerus caffer ) (36.5%). The frequently detected host by selection index was the rhinoceros ( Diceros bicornis ) ( W  = 16.2). Most trypanosome infections in tsetse flies were associated with the buffalo blood meal. The prevalence of tsetse endosymbionts Sodalis and Wolbachia was 2.8% and 4.8%, respectively. No Spiroplasma and Salivary Gland Hypertrophy Virus were detected. These findings implicate the buffaloes as the important reservoirs of tsetse-transmitted trypanosomes in the area. This contributes to predicting the main cryptic reservoirs and therefore guiding the effective control of the disease. The study findings provide the key scientific information that supports the current One Health collaboration in the control and surveillance of tsetse-transmitted trypanosomosis in Rwanda., Competing Interests: None., (© 2023 Published by Elsevier B.V.)

Background: In sub-Saharan Africa, malaria is the common diagnosis for febrile illness and related clinical features, resulting in the under-diagnosis of other aetiologies, such as arboviruses and Rickettsia. While these may not be significant causes of mortality in malaria-endemic areas, they affect the daily life and performance of affected individuals. It is, therefore, important to have a clear picture of these other aetiologies to institute correct diagnoses at hospitals and improve patient outcomes., Methods: Blood samples were collected from patients with fever and other clinical features associated with febrile illness at selected hospitals in the malaria-endemic counties of Busia, Bungoma, and Kakamega, and screened for Crimean-Congo haemorrhagic fever, Sindbis, dengue and chikungunya viruses, Rickettsia africae, and Plasmodium spp. using high-throughput real-time PCR techniques. A logistic regression was performed on the results to explore the effect of demographic and socio-economic independent variables on malaria infection., Results: A total of 336 blood samples collected from hospital patients between January 2018 and February 2019 were screened, of which 17.6% (59/336) were positive for Plasmodium falciparum and 1.5% (5/336) for Plasmodium malariae. Two patients had dual P. falciparum/P. malariae infections. The most common clinical features reported by the patients who tested positive for malaria were fever and headache. None of the patients were positive for the arboviruses of interest or R. africae. Patients living in Busia (OR 5.2; 95% CI 2.46-11.79; p < 0.001) and Bungoma counties (OR 2.7; 95% CI 1.27-6.16; p = 0.013) had higher odds of being infected with malaria, compared to those living in Kakamega County., Conclusions: The reported malaria prevalence is in line with previous studies. The absence of arboviral and R. africae cases in this study may have been due to the limited number of samples screened, low-level circulation of arboviruses during inter-epidemic periods, and/or the use of PCR alone as a detection method. Other sero-surveys confirming their circulation in the area indicate that further investigations are warranted., (© 2022. The Author(s).)

Shimba Hills is a wildlife area in Kenya and a major focus of tsetse-borne trypanosomes in East Africa. In Shimba Hills, tsetse-borne trypanosomes constrain animal health and smallholder livelihoods. However, epidemiological data to guide hotspot-targeted control of infections are limited. This study assessed the dynamics of tsetse-borne trypanosome risk in Shimba Hills with the objective to describe infection hotspots for targeted control. Tsetse flies ( n = 696) collected in field surveys between November 2018 and September 2019 in Shimba Hills were characterized for chronological age and phenotypic sizes and screened for trypanosome and cattle DNA. Entomological inoculation rates for trypanosome risk assessment were derived from the product of fly abundance and molecular rates of vector infection and confirmed cattle bloodmeals in tsetse flies. In addition, cattle health indicators including anemia scores were assessed in contemporaneous parasitological surveys that screened livestock blood samples ( n = 1,417) for trypanosome using the buffy-coat technique. Compared with Glossina brevipalpis and G. austeni, G. pallidipes was the most abundant tsetse fly species in Shimba Hills and had a wider spatial distribution and greater likelihood for infectious bites on cattle. The risk of cattle infection was similar along the Shimba Hills human-wildlife-livestock interface and high within one thousand meters of the wildlife reserve boundary. Trypanosomes in tsetse flies were highly diverse and included parasites of wild-suids probably acquired from warthogs in Shimba Hills. Age and phenotypic sizes were similar between tsetse fly populations and did not affect the probability of infection or cattle bloodmeals in the vectors. Anemia was more likely in trypanosome-positive cattle whilst parasitological infection rates in cattle samples maintained a weak relationship with entomological inoculation rates probably because of the limited time scale of sample collection. Trypanosome risk in Shimba Hills is high in locations close to the wildlife reserve and driven by G. pallidipes infectious bites on cattle. Therefore, trypanosome vector control programmes in the area should be designed to reduce G. pallidipes abundance and tailored to target sites close to the wildlife reserve., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ebhodaghe, Bastos, Okal and Masiga.)

Background: Livestock are key sources of livelihood among pastoral communities. Livestock productivity is chiefly constrained by pests and diseases. Due to inadequate disease surveillance in northern Kenya, little is known about pathogens circulating within livestock and the role of livestock-associated biting keds (genus Hippobosca ) in disease transmission. We aimed to identify the prevalence of selected hemopathogens in livestock and their associated blood-feeding keds. Methods: We randomly collected 389 blood samples from goats (245), sheep (108), and donkeys (36), as well as 235 keds from both goats and sheep (116), donkeys (11), and dogs (108) in Laisamis, Marsabit County, northern Kenya. We screened all samples for selected hemopathogens by high-resolution melting (HRM) analysis and sequencing of PCR products amplified using primers specific to the genera: Anaplasma, Trypanosoma, Clostridium, Ehrlichia, Brucella, Theileria, and Babesia. Results: In goats, we detected Anaplasma ovis (84.5%), a novel Anaplasma sp. (11.8%), Trypanosoma vivax (7.3%), Ehrlichia canis (66.1%), and Theileria ovis (0.8%). We also detected A. ovis (93.5%), E. canis (22.2%), and T. ovis (38.9%) in sheep. In donkeys, we detected ' Candidatus Anaplasma camelii' (11.1%), T. vivax (22.2%), E. canis (25%), and Theileria equi (13.9%). In addition, keds carried the following pathogens; goat/sheep keds - T. vivax (29.3%) , Trypanosoma evansi (0.86%), Trypanosoma godfreyi (0.86%), and E. canis (51.7%); donkey keds - T. vivax (18.2%) and E. canis (63.6%); and dog keds - T. vivax (15.7%), T. evansi (0.9%), Trypanosoma simiae (0.9%) , E. canis (76%), Clostridium perfringens (46.3%), Bartonella schoenbuchensis (76%), and Brucella abortus (5.6%). Conclusions: We found that livestock and their associated ectoparasitic biting keds carry a number of infectious hemopathogens, including the zoonotic B. abortus . Dog keds harbored the most pathogens, suggesting dogs, which closely interact with livestock and humans, as key reservoirs of diseases in Laisamis. These findings can guide policy makers in disease control., Competing Interests: No competing interests were disclosed., (Copyright: © 2022 Mwaki DM et al.)

Summary: MicroRNAs (miRNAs) are single stranded gene regulators of 18-25 bp in length. They play a crucial role in regulating several biological processes in insects. However, the functions of miRNA in Glossina pallidipes , one of the biological vectors of African animal trypanosomosis in sub-Saharan Africa, remain poorly characterized. We used a combination of both molecular biology and bioinformatics techniques to identify miRNA genes at different developmental stages (larvae, pupae, teneral and reproductive unmated adults, gravid females) and sexes of G. pallidipes . We identified 157 mature miRNA genes, including 12 novel miRNAs unique to G. pallidipes . Moreover, we identified 93 miRNA genes that were differentially expressed by sex and/or in specific developmental stages. By combining both miRanda and RNAhybrid algorithms, we identified 5550 of their target genes. Further analyses with the Gene Ontology term and KEGG pathways for these predicted target genes suggested that the miRNAs may be involved in key developmental biological processes. Our results provide the first repository of G. pallidipes miRNAs across developmental stages, some of which appear to play crucial roles in tsetse fly development. Hence, our findings provide a better understanding of tsetse biology and a baseline for exploring miRNA genes in tsetse flies., Availability and Implementation: Raw sequence data are available from NCBI Sequence Read Archives (SRA) under Bioproject accession number PRJNA590626., Supplementary Information: Supplementary data are available at Bioinformatics Advances online., (© The Author(s) 2021. Published by Oxford University Press.)

Background: African Trypanosomiases threaten the life of both humans and animals. Trypanosomes are transmitted by tsetse and other biting flies. In Rwanda, the African Animal Trypanosomiasis (AAT) endemic area is mainly around the tsetse-infested Akagera National Park (NP). The study aimed to identify Trypanosoma species circulating in cattle, their genetic diversity and distribution around the Akagera NP., Methodology: A cross-sectional study was carried out in four districts, where 1,037 cattle blood samples were collected. The presence of trypanosomes was determined by microscopy, immunological rapid test VerY Diag and PCR coupled with High-Resolution Melt (HRM) analysis. A parametric test (ANOVA) was used to compare the mean Packed cell Volume (PCV) and trypanosomes occurrence. The Cohen Kappa test was used to compare the level of agreement between the diagnostic methods., Findings: The overall prevalence of trypanosome infections was 5.6%, 7.1% and 18.7% by thin smear, Buffy coat technique and PCR/HRM respectively. Microscopy showed a low sensitivity while a low specificity was shown by the rapid test (VerY Diag). Trypanosoma (T.) congolense was found at a prevalence of 10.7%, T. vivax 5.2%, T. brucei brucei 2% and T. evansi 0.7% by PCR/HRM. This is the first report of T.evansi in cattle in Rwanda. The non-pathogenic T. theileri was also detected. Lower trypanosome infections were observed in Ankole x Friesian breeds than indigenous Ankole. No human-infective T. brucei rhodesiense was detected. There was no significant difference between the mean PCV of infected and non-infected animals (p>0.162)., Conclusions: Our study sheds light on the species of animal infective trypanosomes around the Akagera NP, including both pathogenic and non-pathogenic trypanosomes. The PCV estimation is not always an indication of trypanosome infection and the mechanical transmission should not be overlooked. The study confirms that the area around the Akagera NP is affected by AAT, and should, therefore, be targeted by the control activities. AAT impact assessment on cattle production and information on the use of trypanocides are needed to help policymakers prioritise target areas and optimize intervention strategies. Ultimately, these studies will allow Rwanda to advance in the Progressive Control Pathway (PCP) to reduce or eliminate the burden of AAT., Competing Interests: The authors have declared that no competing interests exist.

Phlebotomine sand flies are vectors of Leishmania parasites that cause leishmaniases. Both sexes of sand flies feed on plants primarily for sugars, although the chemical cues that mediate attraction to host plants remain largely unknown. Previously, using coupled gas chromatography-mass spectrometry, the authors identified several volatile organic compounds (VOCs) common to preferred host plants for selected Afrotropical sand flies from the Fabaceae family. Of the identified volatiles, the significance of the monoterpenes linalool oxide, ocimene and p-cymene and the benzenoid m-cresol, p-cresol in sand fly behaviour is unknown. In olfactometer assays, the authors tested these compounds singly and in blends for their attractiveness to Phlebotomus duboscqi, cutaneous leishmaniasis vector in Kenya. In dose-response assays, single compounds increased the responses of males and females over controls, but their optimum attractive doses varied between the sexes. Two five-component blends, referred to as Blend-f and Blend-m for females and males respectively, were formulated and tested in dose-response assays against 1-octen-3-ol (positive control). The results of the present study showed that males and females were significantly attracted to varying levels of the two blends. In pairwise assays, the authors evaluated the most attractive of these blends to each sex (i.e., Blend Am for male against Blend Bf for female), revealing that males were attracted to both blends at varying levels, whereas females were indifferent. The study's results demonstrate that plant-derived VOCs can be exploited for sand fly management., (© 2021 The Royal Entomological Society.)

Background: Tick-borne pathogens (TBPs) are of global importance, especially in sub-Saharan Africa where they represent a major constraint to livestock production. Their association with human disease is also increasingly recognized, signalling their zoonotic importance. It is therefore crucial to investigate TBPs prevalence in livestock populations and the factors associated with their presence. We set out to identify TBPs present in cattle and to determine associated risk factors in western Kenya, where smallholder livestock production is important for subsistence and market-driven income., Results: Tick-borne pathogen infections in blood samples collected from cattle at livestock markets and slaughterhouses between May 2017 and January 2019 were identified by high-resolution melting analysis and sequencing of PCR products of genus-specific primers. Of the 422 cattle sampled, 30.1% (127/422) were infected with at least one TBP, while 8.8% (37/422) had dual infections. Anaplasma spp. (19.7%) were the most prevalent, followed by Theileria (12.3%), Ehrlichia (6.6%), and Babesia (0.2%) spp. Sequence analysis of the TBPs revealed them to be Anaplasma platys-like organisms (13.5%), Theileria velifera (7.4%), Anaplasma marginale (4.9%), Theileria mutans (3.1%), Theileria parva (1.6%), and Babesia bigemina (0.2%). Ehrlichia ruminantium, Rickettsia spp., and arboviruses were not detected. Exotic breeds of cattle were more likely to be infected with A. marginale compared to local breeds (OR: 7.99, 95% CI: 3.04-22.02, p <  0.001). Presence of ticks was a significant predictor for Anaplasma spp. (OR: 2.18, 95% CI: 1.32-3.69, p = 0.003) and Ehrlichia spp. (OR: 2.79, 95% CI: 1.22-7.23, p = 0.022) infection. Cattle sampled at slaughterhouses were more likely to be positive for Anaplasma spp. (OR: 1.64, 95% CI: 1.01-2.70, p = 0.048) and A. marginale (OR: 3.84, 95% CI: 1.43-12.21, p = 0.012), compared to those sampled at livestock markets., Conclusion: This study reports TBP prevalence and associated risk factors in western Kenya, factors which are key to informing surveillance and control measures., (© 2021. The Author(s).)

Trypanosomes are endemic and retard cattle health in Shimba Hills, Kenya. Wildlife in the area act as reservoirs of the parasites. However, wild animal species that harbor and expose cattle to tsetse-borne trypanosomes are not well known in Shimba Hills. Using xeno-monitoring surveillance to investigate wild animal reservoirs and sources of trypanosomes in Shimba Hills, we screened 696 trypanosome-infected and uninfected tsetse flies for vertebrate DNA using multiple-gene PCR-High Resolution Melting analysis and amplicon sequencing. Results revealed that tsetse flies fed on 13 mammalian species, preferentially Phacochoerus africanus (warthogs) (17.39%, 95% CI: 14.56-20.21) and Bos taurus (cattle) (11.35%, 95% CI: 8.99-13.71). Some tsetse flies showed positive cases of bloodmeals from multiple hosts (3.45%, 95% CI: 2.09-4.81), including warthog and cattle (0.57%, 95% CI: 0.01-1.14). Importantly, tsetse flies that took bloodmeals from warthog had significant risk of infections with Trypanosoma vivax (5.79%, 95% CI: 1.57-10.00), T. congolense (7.44%, 95% CI: 2.70-12.18), and T. brucei sl (2.48%, 95% CI: -0.33-5.29). These findings implicate warthogs as important reservoirs of tsetse-borne trypanosomes affecting cattle in Shimba Hills and provide valuable epidemiological insights to underpin the parasites targeted management in Nagana vector control programs in the area.

Monitoring the effectiveness of tsetse fly control interventions that aim to reduce transmission of African trypanosomiasis requires highly efficient sampling tools that can catch flies at low densities. The sticky small target (StS-target) has previously been shown to be more effective in sampling Glossina fuscipes fuscipes compared to the biconical trap. However, its efficiency in terms of the proportion of flies it catches out of those that visit it has not been reported. Furthermore, there are no reports on whether tsetse samples caught using the StS-target can be used for subsequent processes such as molecular tests. In this study, we evaluated the efficiency of the biconical trap and targets for sampling G. f. fuscipes. All targets were tiny (0.25 × 0.50 m) but varied in their capture system. We used targets with sticky surface (StS-targets) and those with an electrified surface (ES-targets). We also assessed the suitability of flies caught on the StS-target for molecular tests by amplifying DNA of bacterial communities. Randomized block design experiments were undertaken in Mbita area and Manga Island on Lake Victoria of western Kenya. Fly catches of each sampling tool were compared to those of the sampling tool flanked by electric (E) nets and analyzed using a negative binomial regression. The total catch for each sampling tool alone was divided by the total catch of the sampling tool flanked by two E-nets to obtain its efficiency expressed as a percentage. A proportion of flies caught on the StS-target was preserved for molecular tests. Overall, the efficiencies of the biconical trap, ES-target and StS-target were 7.7%, 13.3% and 27.0%, respectively. A higher proportion of females (69 to 79%) than males approached all the sampling tools, but the trap efficiency was greater for male G. f. fuscipes than females. Furthermore, sequencing the 16S rRNA gene from fly samples caught on the StS-target revealed the presence of Spiroplasma. Our results indicate that the SS-target is the most efficient trap to monitor G. f. fuscipes population during interventions, with the biconical trap being the least efficient, and samples collected from StS-targets are suitable for molecular studies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Trypanosoma vivax is a vector-borne protozoan parasite of livestock endemic to Africa and South America. To date, fifteen genotypes of the parasite have been described in vertebrate and insect hosts in East Africa. However, information regarding T. vivax diversity remains limited in many endemic countries in the sub-region, including Kenya. Such information could deepen insight into the local epidemiology of animal trypanosomiasis in Shimba Hills, a wildlife area in southeast Kenya where T. vivax is endemic and infects livestock. We employed two-gene conventional-PCR-sequencing and phylogenetic analysis to characterize T. vivax genotypes in tsetse flies collected between November 2018 and September 2019 in the wildlife-livestock interface of the Shimba Hills National Reserve. Phylogenetic analysis of Internal Transcribed Spacer-1 (ITS-1) sequences of T. vivax isolates confirmed the presence of two T. vivax genotypes in Shimba Hills of which >80% of T. vivax isolates from tsetse flies clustered within the virulent Tvv4-genotype clade. Tsetse infections with the Tvv4 genotype were also confirmed based on 18S rRNA gene sequencing. Expanded gene characterization identified three closely related haplotypes within the Tvv4-clade. The Tvv4-isolates were detected in male and female Glossina pallidipes tsetse flies, most of which were collected from grasslands and within two kilometres of the Shimba Hills National Reserve boundary. Considering that T. vivax is the most common trypanosome in the Shimba Hills area and causes severe clinical conditions in livestock, the Tvv4 genotype reported here for the first time in Kenya contributes to our understanding of these pathologies. The effectiveness of trypanocidal drugs in the management of Tvv4 is presently not clearly understood. Therefore, the parasite management in Shimba Hills should focus on vector control to reduce the density of G. pallidipes, especially in grasslands near the wildlife protectorate., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Tsetse-transmitted Animal African Trypanosomosis (AAT) is one of the most important constraints to livestock development in Africa. Use of trypanocides has been the most widespread approach for the management of AAT, despite the associated drug resistance and health concerns associated with drug metabolites in animal products. Alternative control measures that target tsetse fly vectors of AAT, though effective, have been hard to sustain in part because these are public goods applied area-wide. The International Centre of Insect Physiology and Ecology (icipe) and partners have developed and implemented a novel tsetse repellent collar (TRC) applied on animals to limit contact of tsetse flies and livestock, thereby reducing AAT transmission. The TRC has now advanced to commercialization. A household-level survey involving 632 cattle keeping households, was conducted in Shimba Hills region of Kwale County, where field trials of the TRC have been previously conducted to assess farmers' knowledge, perception, and practices towards the management of tsetse flies, their willingness to pay (WTP) for the TRC, and factors affecting the WTP. Almost all the respondents (90%) reported that tsetse flies were the leading cattle infesting pests in the area. About 22% of these correctly identified at least four AAT clinical signs, and even though many (68%) used trypanocidal drugs to manage the disease, 50% did not perceive the drug as being effective in AAT management (50%). Few respondents (8%) were aware of the harmful effects of trypanocidal drugs. About 89% of the respondents were aware of icipe TRC, and 30% of them were using the field trial collars during the survey. Sixty-three (63%) of them were willing to pay for the TRC at the same cost they spend treating an animal for AAT. On average farmers were willing to pay KES 3,352 per animal per year. Male educated household heads are likely to pay more for the TRC. Moreover, perceived high AAT prevalence and severity further increases the WTP. Wider dissemination and commercialization of the herd-level tsetse control approach (TRC) should be encouraged to impede AAT transmission and thus enhance food security and farm incomes among the affected rural communities. Besides the uptake of TRC can be enhanced through training, especially among women farmers., Competing Interests: The authors have declared that no competing interests exist.

Anaplasmosis, caused by infection with bacteria of the genus Anaplasma, is an important veterinary and zoonotic disease. Transmission by ticks has been characterized but little is known about non-tick vectors of livestock anaplasmosis. This study investigated the presence of Anaplasma spp. in camels in northern Kenya and whether the hematophagous camel ked, Hippobosca camelina, acts as a vector. Camels (n = 976) and > 10,000 keds were sampled over a three-year study period and the presence of Anaplasma species was determined by PCR-based assays targeting the Anaplasmataceae 16S rRNA gene. Camels were infected by a single species of Anaplasma, 'Candidatus Anaplasma camelii', with infection rates ranging from 63-78% during the dry (September 2017), wet (June-July 2018), and late wet seasons (July-August 2019). 10-29% of camel keds harbored 'Ca. Anaplasma camelii' acquired from infected camels during blood feeding. We determined that Anaplasma-positive camel keds could transmit 'Ca. Anaplasma camelii' to mice and rabbits via blood-feeding. We show competence in pathogen transmission and subsequent infection in mice and rabbits by microscopic observation in blood smears and by PCR. Transmission of 'Ca. Anaplasma camelii' to mice (8-47%) and rabbits (25%) occurred readily after ked bites. Hence, we demonstrate, for the first time, the potential of H. camelina as a vector of anaplasmosis. This key finding provides the rationale for establishing ked control programmes for improvement of livestock and human health., Competing Interests: The authors have declared that no competing interests exist.

Vectors of emerging infectious diseases have expanded their distributional ranges in recent decades due to increased global travel, trade connectivity and climate change. Transboundary range shifts, arising from the continuous movement of humans and livestock across borders, are of particular disease control concern. Several tick-borne diseases are known to circulate between eastern Uganda and the western counties of Kenya, with one fatal case of Crimean-Congo haemorrhagic fever (CCHF) reported in 2000 in western Kenya. Recent reports of CCHF in Uganda have highlighted the risk of cross-border disease translocation and the importance of establishing inter-epidemic, early warning systems to detect possible outbreaks. We therefore carried out surveillance of tick-borne zoonotic pathogens at livestock markets and slaughterhouses in three counties of western Kenya that neighbour Uganda. Ticks and other ectoparasites were collected from livestock and identified using morphological keys. The two most frequently sampled tick species were Rhipicephalus decoloratus (35%) and Amblyomma variegatum (30%); Ctenocephalides felis fleas and Haematopinus suis lice were also present. In total, 486 ticks, lice and fleas were screened for pathogen presence using established molecular workflows incorporating high-resolution melting analysis and identified through sequencing of PCR products. We detected CCHF virus in Rh. decoloratus and Rhipicephalus sp. cattle ticks, and 82 of 96 pools of Am. variegatum were positive for Rickettsia africae. Apicomplexan protozoa and bacteria of veterinary importance, such as Theileria parva, Babesia bigemina and Anaplasma marginale, were primarily detected in rhipicephaline ticks. Our findings show the presence of several pathogens of public health and veterinary importance in ticks from livestock at livestock markets and slaughterhouses in western Kenya. Confirmation of CCHF virus, a Nairovirus that causes haemorrhagic fever with a high case fatality rate in humans, highlights the risk of under-diagnosed zoonotic diseases and calls for continuous surveillance and the development of preventative measures., (© 2020 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

Background: Glossina (tsetse flies) biologically transmit trypanosomes that infect both humans and animals. Knowledge of their distribution patterns is a key element to better understand the transmission dynamics of trypanosomosis. Tsetse distribution in Rwanda has not been well enough documented, and little is known on their current distribution. This study determined the current spatial distribution, abundance, diversity, and seasonal variations of tsetse flies in and around the Akagera National Park., Methods: A longitudinal stratified sampling following the seasons was used. Biconical traps were deployed in 55 sites for 6 consecutive days of each study month from May 2018 to June 2019 and emptied every 48 h. Flies were identified using FAO keys, and the number of flies per trap day (FTD) was used to determine the apparent density. Pearson chi-square (χ2) and parametrical tests (t-test and ANOVA) were used to determine the variations between the variables. The significance (p < 0.05) at 95% confidence interval was considered. Logistic regression was used to determine the association between tsetse occurrence and the associated predictors., Results: A total of 39,516 tsetse flies were collected, of which 73.4 and 26.6% were from inside Akagera NP and the interface area, respectively. Female flies accounted for 61.3 while 38.7% were males. Two species were identified, i.e. G. pallidipes [n = 29,121, 7.4 flies/trap/day (FTD)] and G. morsitans centralis (n = 10,395; 2.6 FTD). The statistical difference in numbers was significant between the two species (p = 0.000). The flies were more abundant during the wet season (15.8 FTD) than the dry season (4.2 FTD). Large numbers of flies were trapped around the swamp areas (69.1 FTD) inside the park and in Nyagatare District (11.2 FTD) at the interface. Glossina morsitans was 0.218 times less likely to occur outside the park. The chance of co-existing between the two species reduced outside the protected area (0.021 times)., Conclusions: The occurrence of Glossina seems to be limited to the protected Akagera NP and a narrow band of its surrounding areas. This finding will be crucial to design appropriate control strategies. Glossina pallidipes was found in higher numbers and therefore is conceivably the most important vector of trypanosomosis. Regional coordinated control and regular monitoring of Glossina distribution are recommended.

Aedes aegypti and Culex pipiens complex mosquitoes are prolific vectors of arboviruses that are a global threat to human and animal health. Increased globalization and ease of travel have facilitated the worldwide dissemination of these mosquitoes and the viruses they transmit. To assess disease risk, we determined the frequency of arboviruses in western Kenyan counties bordering an area of high arboviral activity. In addition to pathogenic viruses, insect-specific flaviviruses (ISFs), some of which are thought to impair the transmission of specific pathogenic arboviruses, were also evaluated. We trapped mosquitoes in the short and long rainy seasons in 2018 and 2019 at livestock markets and hospitals. Mosquitoes were screened for dengue, chikungunya and other human pathogenic arboviruses, ISFs, and their blood-meal sources as determined by high-resolution melting analysis of (RT-)PCR products. Of 6,848 mosquitoes collected, 89% were trapped during the long rainy season, with A. aegypti (59%) and Cx. pipiens sensu lato (40%) being the most abundant. Most blood-fed mosquitoes were Cx. pipiens s.l. with blood-meals from humans, chicken, and sparrow (Passer sp.). We did not detect dengue or chikungunya viruses. However, one Culex poicilipes female was positive for Sindbis virus, 30 pools of Ae. aegypti had cell fusing agent virus (CFAV; infection rate (IR) = 1.27%, 95% CI = 0.87%-1.78%); 11 pools of Ae. aegypti had Aedes flavivirus (AeFV; IR = 0.43%, 95% CI = 0.23%-0.74%); and seven pools of Cx. pipiens s.l. (IR = 0.23%, 95% CI = 0.1%-0.45%) and one pool of Culex annulioris had Culex flavivirus. Sindbis virus, which causes febrile illness in humans, can complicate the diagnosis and prognosis of patients with fever. The presence of Sindbis virus in a single mosquito from a population of mosquitoes with ISFs calls for further investigation into the role ISFs may play in blocking transmission of other arboviruses in this region., Competing Interests: The authors have declared that no competing interests exist.

Background: Visceral leishmaniasis (VL) and zoonotic cutaneous leishmaniasis (ZCL) are of public health concern in Merti sub-County, Kenya, but epidemiological data on transmission, vector abundance, distribution, and reservoir hosts remain limited. To better understand the disease and inform control measures to reduce transmission, we investigated the abundance and distribution of sand fly species responsible for Leishmania transmission in the sub-County and their blood-meal hosts., Methods: We conducted an entomological survey in five villages with reported cases of VL in Merti sub-County, Kenya, using CDC miniature light traps and castor oil sticky papers. Sand flies were dissected and identified to the species level using standard taxonomic keys and PCR analysis of the cytochrome c oxidase subunit 1 (cox1) gene. Leishmania parasites were detected and identified by PCR and sequencing of internal transcribed spacer 1 (ITS1) genes. Blood-meal sources of engorged females were identified by high-resolution melting analysis of vertebrate cytochrome b (cyt-b) gene PCR products., Results: We sampled 526 sand flies consisting of 8 species, Phlebotomus orientalis (1.52%; n = 8), and 7 Sergentomyia spp. Sergentomyia squamipleuris was the most abundant sand fly species (78.71%; n = 414) followed by Sergentomyia clydei (10.46%; n = 55). Leishmania major, Leishmania donovani, and Trypanosoma DNA were detected in S. squamipleuris specimens. Humans were the main sources of sand fly blood meals. However, we also detected mixed blood meals; one S. squamipleuris specimen had fed on both human and mouse (Mus musculus) blood, while two Ph. orientalis specimens fed on human, hyrax (Procavia capensis), and mouse (Mus musculus) blood., Conclusions: Our findings implicate the potential involvement of S. squamipleuris in the transmission of Leishmania and question the dogma that human leishmaniases in the Old World are exclusively transmitted by sand flies of the Phlebotomus genus. The presence of Trypanosoma spp. may indicate mechanical transmission, whose efficiency should be investigated. Host preference analysis revealed the possibility of zoonotic transmission of leishmaniasis and other pathogens in the sub-County. Leishmania major and L. donovani are known to cause ZCL and VL, respectively. However, the reservoir status of the parasites is not uniform. Further studies are needed to determine the reservoir hosts of Leishmania spp. in the area.

The riverine tsetse fly Glossina fuscipes fuscipes is a major vector of trypanosome pathogens causing African trypanosomiasis. This fly species uses a combination of olfactory and visual cues to locate its hosts. Previously, traps and targets baited with visual cues have been used in vector control, but the development of olfactory-based tools has been challenging. Recently, repellents have shown promise as olfactory-based tools in tsetse vector control. Here, we evaluated a three-component blend comprising 6-methyl-5-hepten-2-one, acetophenone and geranyl acetone (blend K), previously identified as a repellent for savannah tsetse flies in zebra skin odor, on G. f. fuscipes populations. Using a series of 6 × 6 randomized Latin square-designed experiments, G. f. fuscipes catches in biconical traps were monitored on four islands of Lake Victoria in western Kenya between July and September 2019, after the long rainy season. Traps were baited with blend K and individual components of this blend. The known tsetse repellent blend WRC (waterbuck repellent compounds) and trap alone were included as controls. Daily catch data in thirty-six replicate trials were analyzed using generalized linear model with negative binomial error structure using the package "MASS" in R. Treatment, day and site were set as predictor variables. Our results showed that, blend K significantly reduced G. f. fuscipes catches by 25.6% (P < 0.01) compared to the control trap alone but was not significantly different from WRC which reduced catches by 20.7% (P < 0.05). Of the individual compounds, geranyl acetone solely significantly reduced catches by 29.1% (P < 0.01) which did not differ from blend K or WRC. We conclude that geranyl acetone accounts for the repellent effect of blend K on the riverine tsetse fly, G. f. fuscipes, demonstrating the ecological importance of animal skin odors in the host-seeking behavior of medically-important tsetse fly vectors., (Copyright © 2020. Published by Elsevier B.V.)

Background: Major constraints to camel production include pests and diseases. In northern Kenya, little information is available about blood-borne pathogens circulating in one-humped camels ( Camelus dromedarius ) or their possible transmission by the camel haematophagous ectoparasite, Hippobosca camelina , commonly known as camel ked or camel fly. This study aimed to: (i) identify the presence of potentially insect-vectored pathogens in camels and camel keds, and (ii) assess the potential utility of keds for xenodiagnosis of camel pathogens that they may not vector. Methods: In Laisamis, northern Kenya, camel blood samples (n = 249) and camel keds (n = 117) were randomly collected from camels. All samples were screened for trypanosomal and camelpox DNA by PCR, and for Anaplasma, Ehrlichia, Brucella, Coxiella, Theileria , and Babesia by PCR coupled with high-resolution melting (PCR-HRM) analysis. Results: In camels, we detected Trypanosoma vivax (41%), Trypanosoma evansi (1.2%), and " Candidatus Anaplasma camelii" (68.67%). In camel keds, we also detected T. vivax (45.3%), T. evansi (2.56%), Trypanosoma melophagium (1/117) (0.4%), and " Candidatus Anaplasma camelii" (16.24 %). Piroplasms ( Theileria spp. and Babesia spp.), Coxiella burnetii , Brucella spp., Ehrlichia spp., and camel pox were not detected in any samples. Conclusions: This study reveals the presence of epizootic pathogens in camels from northern Kenya. Furthermore, the presence of the same pathogens in camels and in keds collected from sampled camels suggests the potential use of these flies in xenodiagnosis of haemopathogens circulating in camels., Competing Interests: No competing interests were disclosed., (Copyright: © 2020 Kidambasi KO et al.)

Background: Major constraints to camel production include pests and diseases. In northern Kenya, little information is available about disease pathogens circulating in one-humped camels ( Camelus dromedarius ) or their possible transmission by the camel haematophagous ectoparasite, Hippobosca camelina , commonly known as camel ked or camel fly. This study aimed to: (i) identify the presence of potentially insect-vectored pathogens in camels and camel keds, and (ii) assess the potential utility of keds for xenodiagnosis of camel disease pathogens that they may not vector. Methods: In Laisamis, northern Kenya, camel blood samples (n = 249) and camel keds (n = 117) were randomly collected from camels. All samples were screened for trypanosomal and camelpox DNA by PCR, and for Anaplasma, Ehrlichia, Brucella, Coxiella, Theileria , and Babesia by PCR coupled with high-resolution melting (PCR-HRM) analysis. Results: In camels, we detected Trypanosoma vivax (102/249) (41%), Trypanosoma evansi (3/249) (1.2%), and " Candidatus Anaplasma camelii" (137/200) (68.5%). In camel keds, we also detected T. vivax (53/117) (45.3%), T. evansi (3/117) (2.56%), Trypanosoma melophagium (1/117) (0.4%), and " Candidatus Anaplasma camelii" (19/117) (16.24 %). Piroplasms ( Theileria spp. and Babesia spp.), Coxiella burnetii , Brucella spp., Ehrlichia spp., and camel pox were not detected in any samples. Conclusions: This study reveals the presence of epizootic pathogens in camels from northern Kenya. Furthermore, the presence of the same pathogens in camels and in keds collected from sampled camels suggests the potential use of these flies in xenodiagnosis of haemopathogens circulating in camels., Competing Interests: No competing interests were disclosed., (Copyright: © 2019 Kidambasi KO et al.)

Background: Phlebotomus (Larroussius) guggisbergi is among the confirmed vectors for cutaneous leishmaniasis (CL) transmission in Kenya. This scarring and stigmatizing form of leishmaniasis accounts for over one million annual cases worldwide. Most recent CL epidemics in Kenya have been reported in Gilgil, Nakuru County, where the disease has become a public health issue. However, little is known about the factors that drive its transmission. Here, we sought to determine the occurrence, distribution and host blood feeding preference of the vectors, and to identify Leishmania species and infection rates in sandflies using molecular techniques. This information could lead to a better understanding of the disease transmission and improvement of control strategies in the area., Methodology/ Principal Findings: An entomological survey of sandflies using CDC light traps was conducted for one week per month in April 2016, and in June and July 2017 from five villages of Gilgil, Nakuru county; Jaica, Sogonoi, Utut, Gitare and Njeru. Sandflies were identified to species level using morphological keys and further verified by PCR analysis of cytochrome c oxidase subunit I (COI) gene. Midguts of female sandflies found to harbour Leishmania were ruptured and the isolated parasites cultured in Novy-MacNeal-Nicolle (NNN) media overlaid with Schneider's insect media to identify the species. Leishmania parasite screening and identification in 198 randomly selected Phlebotomus females and parasite cultures was done by PCR-RFLP analysis of ITS1 gene, nested kDNA-PCR and real-time PCR-HRM followed by sequencing. Bloodmeal source identification was done by real-time PCR-HRM of the vertebrate cytochrome-b gene. A total of 729 sandflies (males: n = 310; females: n = 419) were collected from Utut (36.6%), Jaica (24.3%), Sogonoi (34.4%), Njeru (4.5%), and Gitare (0.1%). These were found to consist of nine species: three Phlebotomus spp. and six Sergentomyia spp. Ph. guggisbergi was the most abundant species (75.4%, n = 550) followed by Ph. saevus sensu lato (11.3%, n = 82). Sandfly species distribution across the villages was found to be significantly different (p<0.001) with Jaica recording the highest diversity. The overall Leishmania infection rate in sandflies was estimated at 7.07% (14/198). Infection rates in Ph. guggisbergi and Ph. saevus s.l. were 9.09% (12/132) and 3.57% (2/56) respectively. L. tropica was found to be the predominant parasite in Gilgil with an overall infection rate of 6.91% (13/188) in Ph. guggisbergi (n = 11) and Ph. saevus s.l. (n = 2) sandflies. However, PCR analysis also revealed L. major infection in one Ph. guggisbergi specimen. Bloodmeal analysis in the 74 blood-fed sandflies disclosed a diverse range of vertebrate hosts in Ph. guggisbergi bloodmeals, while Ph. saevus s.l. fed mainly on humans., Conclusions/ Significance: The high infection rates of L. tropica and abundance of Ph. guggisbergi in this study confirms this sandfly as a vector of L. tropica in Kenya. Furthermore, isolation of live L. tropica parasites from Ph. saevus s.l. suggest that there are at least three potential vectors of this parasite species in Gilgil; Ph. guggisbergi, Ph. aculeatus and Ph. saevus s.l. Molecular identification of L. major infections in Ph. guggisbergi suggested this sandfly species as a potential permissive vector of L. major, which needs to be investigated further. Sandfly host preference analysis revealed the possibility of zoonotic transmissions of L. tropica in Gilgil since the main vector (Ph. guggisbergi) does not feed exclusively on humans but also other vertebrate species. Further investigations are needed to determine the potential role of these vertebrate species in L. tropica and L. major transmission in the area., Competing Interests: The authors have declared that no competing interests exist.

Background: Strategies for combatting residual malaria by targeting vectors outdoors are gaining importance as the limitations of primary indoor interventions are reached. Strategies to target ovipositing females or her offspring are broadly applicable because all mosquitoes require aquatic habitats for immature development irrespective of their biting or resting preferences. Oviposition site selection by gravid females is frequently studied by counting early instar larvae in habitats; an approach which is valid only if the number of larvae correlates with the number of females laying eggs. This hypothesis was tested against the alternative, that a higher abundance of larvae results from improved survival of a similar or fewer number of families., Methods: In a controlled experiment, 20 outdoor artificial ponds were left uncovered for 4 days to allow oviposition by wild mosquitoes, then covered with netting and first and second instar larvae sampled daily. Natural Anopheles habitats of two different types were also identified, and all visible larvae sampled. All larvae were identified to species, and most samples of the predominant species, Anopheles arabiensis, were genotyped using microsatellites for sibling group reconstructions using two contrasting softwares, BAPS and COLONY., Results: In the ponds, the number of families reconstructed by each software significantly predicted larval abundance (BAPS R 2  = 0.318, p = 0.01; COLONY R 2  = 0.476, p = 0.001), and suggested that around 50% of females spread larvae across multiple ponds (skip oviposition). From natural habitats, the mean family size again predicted larval abundance using BAPS (R 2  = 0.829, p = 0.017) though not using COLONY (R 2  = 0.218, p = 0.68), but both softwares once more suggested high rates of skip oviposition (in excess of 50%)., Conclusion: This study shows that, whether in closely-located artificial habitats or natural breeding sites, higher early instar larval densities result from more females laying eggs in these sites. These results provide empirical support for use of early instar larval abundance as an index for oviposition site preference. Furthermore, the sharing of habitats by multiple females and the high skip-oviposition rate in An. arabiensis suggest that larviciding by auto-dissemination of insecticide may be successful.

Background: African trypanosomosis, primarily transmitted by tsetse flies, remains a serious public health and economic challenge in sub-Saharan Africa. Interventions employing natural repellents from non-preferred hosts of tsetse flies represent a promising management approach. Although zebras have been identified as non-preferred hosts of tsetse flies, the basis for this repellency is poorly understood. We hypothesized that zebra skin odors contribute to their avoidance by tsetse flies., Methodology/principal Findings: We evaluated the effect of crude zebra skin odors on catches of wild savannah tsetse flies (Glossina pallidipes Austen, 1903) using unbaited Ngu traps compared to the traps baited with two known tsetse fly management chemicals; a repellent blend derived from waterbuck odor, WRC (comprising geranylacetone, guaiacol, pentanoic acid and δ-octalactone), and an attractant comprising cow urine and acetone, in a series of Latin square-designed experiments. Coupled gas chromatography-electroantennographic detection (GC/EAD) and GC-mass spectrometry (GC/MS) analyses of zebra skin odors identified seven electrophysiologically-active components; 6-methyl-5-hepten-2-one, acetophenone, geranylacetone, heptanal, octanal, nonanal and decanal, which were tested in blends and singly for repellency to tsetse flies when combined with Ngu traps baited with cow urine and acetone in field trials. The crude zebra skin odors and a seven-component blend of the EAD-active components, formulated in their natural ratio of occurrence in zebra skin odor, significantly reduced catches of G. pallidipesby 66.7% and 48.9% respectively, and compared favorably with the repellency of WRC (58.1%- 59.2%). Repellency of the seven-component blend was attributed to the presence of the three ketones 6-methyl-5-hepten-2-one, acetophenone and geranylacetone, which when in a blend caused a 62.7% reduction in trap catch of G. pallidipes., Conclusions/significance: Our findings reveal fundamental insights into tsetse fly ecology and the allomonal effect of zebra skin odor, and potential integration of the three-component ketone blend into the management toolkit for tsetse and African trypanosomosis control., Competing Interests: The authors have declared that no competing interests exist.

Most malaria-endemic countries are heavily reliant upon rapid diagnostic tests (RDT) for malaria case identification and treatment. RDT previously used for malaria diagnosis can subsequently be used for molecular assays, including qualitative assessment of parasite species present or the carriage of resistance markers, because parasite DNA can be extracted from the blood inside the RDT which remains preserved on the internal components. However, the quantification of parasite density has not previously been possible from used RDT. In this study, blood samples were collected from school-age children in Western Kenya, in the form of both dried blood spots on Whatman filter paper, and the blood spot that is dropped into rapid diagnostic tests during use. Having first validated a robotic DNA extraction method, the parasite density was determined from both types of sample by duplex qPCR, and across a range of densities. The methods showed good agreement. The preservation of both parasite and human DNA on the nitrocellulose membrane inside the RDT was stable even after more than one year's storage. This presents a useful opportunity for researchers or clinicians wishing to gain greater information about the parasite populations that are being studied, without significant investment of resources.

Background: Anopheles funestus is among the major malaria vectors in Kenya and sub-Saharan Africa and has been recently implicated in persistent malaria transmission. However, its ecology and genetic diversity remain poorly understood in Kenya., Methods: Using 16 microsatellite loci, we examined the genetic structure of An. funestus sampled from 11 locations (n = 426 individuals) across a wide geographical range in Kenya spanning coastal, western and Rift Valley areas., Results: Kenyan An. funestus resolved as three genetically distinct clusters. The largest cluster (FUN1) broadly included samples from western and Rift Valley areas of Kenya with two clusters identified from coastal Kenya (FUN2 and FUN3), not previously reported. Geographical distance had no effect on population differentiation of An. funestus. We found a significant variation in the mean Plasmodium infectivity between the clusters (χ 2 = 12.1, df = 2, P = 0.002) and proportional to the malaria prevalence in the different risk zones of Kenya. Notably, there was variation in estimated effective population sizes between the clusters, suggesting possible differential impact of anti-vector interventions in represented areas., Conclusions: Heterogeneity among Kenyan populations of An. funestus will impact malaria vector control with practical implications for the development of gene-drive technologies. The difference in Plasmodium infectivity and effective population size between the clusters could suggest potential variation in phenotypic characteristics relating to competence or insecticide resistance. This is worth examining in future studies.

Background: Many arboviruses transmitted by mosquitoes have been implicated as causative agents of both human and animal illnesses in East Africa. Although epidemics of arboviral emerging infectious diseases have risen in frequency in recent years, the extent to which mosquitoes maintain pathogens in circulation during inter-epidemic periods is still poorly understood. This study aimed to investigate whether arboviruses may be maintained by vertical transmission via immature life stages of different mosquito vector species., Methodology: We collected immature mosquitoes (egg, larva, pupa) on the shores and islands of Lake Baringo and Lake Victoria in western Kenya and reared them to adults. Mosquito pools (≤25 specimens/pool) of each species were screened for mosquito-borne viruses by high-resolution melting analysis and sequencing of multiplex PCR products of genus-specific primers (alphaviruses, flaviviruses, phleboviruses and Bunyamwera-group orthobunyaviruses). We further confirmed positive samples by culturing in baby hamster kidney and Aedes mosquito cell lines and re-sequencing., Principal Findings: Culex univittatus (2/31pools) and Anopheles gambiae (1/77 pools) from the Lake Victoria region were positive for Bunyamwera virus, a pathogenic virus that is of public health concern. In addition, Aedes aegypti (3/50), Aedes luteocephalus (3/13), Aedes spp. (2/15), and Culex pipiens (1/140) pools were positive for Aedes flaviviruses at Lake Victoria, whereas at Lake Baringo, three pools of An. gambiae mosquitoes were positive for Anopheles flavivirus. These insect-specific flaviviruses (ISFVs), which are presumably non-pathogenic to vertebrates, were found in known medically important arbovirus and malaria vectors., Conclusions: Our results suggest that not only ISFVs, but also a pathogenic arbovirus, are naturally maintained within mosquito populations by vertical transmission, even in the absence of vertebrate hosts. Therefore, virus and vector surveillance, even during inter-epidemics, and the study of vector-arbovirus-ISFV interactions, may aid in identifying arbovirus transmission risks, with the potential to inform control strategies that lead to disease prevention., Competing Interests: The authors have declared that no competing interests exist.

Background: Most malaria vectors belong to species complexes. Sibling species often exhibit divergent behaviors dictating the measures that can be deployed effectively in their control. Despite the importance of the Anopheles funestus complex in malaria transmission in sub-Saharan Africa, sibling species have rarely been identified in the past and their vectoring potential remains understudied., Methods: We analyzed 1149 wild-caught An. funestus (senso lato) specimens from 21 sites in Kenya, covering the major malaria endemic areas including western, central and coastal areas. Indoor and outdoor collection tools were used targeting host-seeking and resting mosquitoes. The identity of sibling species, infection with malaria Plasmodium parasites, and the host blood meal sources of engorged specimens were analyzed using PCR-based and sequencing methods., Results: The most abundant sibling species collected in all study sites were Anopheles funestus (59.8%) and Anopheles rivulorum (32.4%) among the 1062 successfully amplified specimens of the An. funestus complex. Proportionally, An. funestus dominated in indoor collections whilst An. rivulorum dominated in outdoor collections. Other species identified were Anopheles leesoni (4.6%), Anopheles parensis (2.4%), Anopheles vaneedeni (0.1%) and for the first time in Kenya, Anopheles longipalpis C (0.7%). Anopheles funestus had an overall Plasmodium infection rate of 9.7% (62/636), predominantly Plasmodium falciparum (59), with two infected with Plasmodium ovale and one with Plasmodium malariae. There was no difference in the infection rate between indoor and outdoor collections. Out of 344 An. rivulorum, only one carried P. falciparum. We also detected P. falciparum infection in two non-blood-fed An. longipalpis C (2/7) which is the first record for this species in Kenya. The mean human blood indices for An. funestus and An. rivulorum were 68% (93/136) and 64% (45/70), respectively, with feeding tendencies on a broad host range including humans and domestic animals such as cow, goat, sheep, chicken and pig., Conclusions: Our findings underscore the importance of active surveillance through application of molecular approaches to unravel novel parasite-vector associations possibly contributed by cryptic species with important implications for effective malaria control and elimination.

Malaria parasites ( Plasmodium ) can change the attractiveness of their vertebrate hosts to Anopheles vectors, leading to a greater number of vector-host contacts and increased transmission. Indeed, naturally Plasmodium -infected children have been shown to attract more mosquitoes than parasite-free children. Here, we demonstrate Plasmodium -induced increases in the attractiveness of skin odor in Kenyan children and reveal quantitative differences in the production of specific odor components in infected vs. parasite-free individuals. We found the aldehydes heptanal, octanal, and nonanal to be produced in greater amounts by infected individuals and detected by mosquito antennae. In behavioral experiments, we demonstrated that these, and other, Plasmodium -induced aldehydes enhanced the attractiveness of a synthetic odor blend mimicking "healthy" human odor. Heptanal alone increased the attractiveness of "parasite-free" natural human odor. Should the increased production of these aldehydes by Plasmodium -infected humans lead to increased mosquito biting in a natural setting, this would likely affect the transmission of malaria., Competing Interests: Conflict of interest statement: A.R., J.G.d.B., J.G.L., and W.T. are inventors on a patent application filed with the UK Intellectual Property Office (application no. 1805023.7).

Background: Animal trypanosomosis is a major cause of economic loss in livestock production in Africa. A suggested control measure is to use breeds with traits of trypanotolerance. The study examines the effect of natural Trypanosoma vivax challenge on haematological parameters in two trypanotolerant cattle [N'Dama and West African Shorthorn (WASH)] herds. Methods: Trypanosoma vivax -specific primers were used to diagnose T. vivax infection in an N'Dama herd at Cape Coast in southern Ghana and a WASH herd at Chegbani in northern Ghana from May to July 2011 in a cross-sectional study. Levels of haematological parameters comprising packed cell volume (PCV), haemoglobin (Hb) concentration and red blood cell (RBC) and total white blood cell (TWBC) counts; differential WBC counts (neutrophils, lymphocytes, eosinophils, monocytes and basophils); and RBC indices of mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were determined in blood samples and then compared between infected and uninfected cattle. Results: We found that haematological indices for infected and uninfected animals in both breeds were within the normal range. However, the mean PCV values for T. vivax -infected WASH and N'Dama were lower in infected compared to uninfected animals. The difference was significant ( p < 0.05) in N'Dama but not in WASH. Conclusion: Despite the presence of infection by T. vivax , N'Dama and WASH cattle maintained their haematological parameters within acceptable normal ranges,  which confirms their trypanotolerant trait. This highlights the need for low-input traditional African farmers in medium, high and severe tsetse challenge areas to be educated on the advantages of N'Dama and WASH breeds to increase their utilization in integrated tsetse and trypanosomosis control programmes., Competing Interests: No competing interests were disclosed.

Background: Animal trypanosomosis is a major cause of economic loss in livestock production in Africa. A suggested control measure is to use breeds with traits of trypanotolerance. The study examines the effect of natural Trypanosoma vivax challenge on haematological parameters in two trypanotolerant cattle [N'Dama and West African Short Horn (WASH)] herds. Methods: T. vivax -specific primers were used to diagnose T. vivax infection in an N'Dama herd at Cape Coast in southern Ghana and a WASH herd at Chegbani in northern Ghana from May to July 2011 in a cross-sectional study. Levels of haematological parameters comprising packed cell volume (PCV), haemoglobin (Hb) concentration and total red blood cell (RBC) and white blood cell (WBC) counts; differential WBC counts (neutrophils, lymphocytes, eosinophils, monocytes and basophils); and RBC indices of mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were determined in blood samples and then compared between infected and uninfected cattle. Results: We found that haematological indices for infected and uninfected animals in both breeds were within the normal range. However, the mean PCV values for T. vivax -infected WASH and N'Dama were lower in infected compared to uninfected animals. The difference was significant ( p < 0.05) in N'Dama but not in WASH. The RBC indices were higher in infected N'Dama compared to infected WASH with a significant difference in total RBC ( p < 0.05). Conclusion: We conclude from our findings that despite the presence of infection by T. vivax , N'Dama and WASH cattle maintained their haematological parameters within acceptable normal ranges, and this underscores the need for routine diagnosis and treatment so that such trypanotolerant cattle do not serve as potential reservoirs of trypanosome parasites., Competing Interests: No competing interests were disclosed.

Background: Small islands serve as potential malaria reservoirs through which new infections might come to the mainland and may be important targets in malaria elimination efforts. This study investigated malaria vector species diversity, blood-meal hosts, Plasmodium infection rates, and long-lasting insecticidal net (LLIN) coverage on Mageta, Magare and Ngodhe Islands of Lake Victoria in western Kenya, a region where extensive vector control is implemented on the mainland., Results: From trapping for six consecutive nights per month (November 2012 to March 2015) using CDC light traps, pyrethrum spray catches and backpack aspiration, 1868 Anopheles mosquitoes were collected. Based on their cytochrome oxidase I (COI) and intergenic spacer region PCR and sequencing, Anopheles gambiae s.l. (68.52%), Anopheles coustani (19.81%) and Anopheles funestus s.l. (11.67%) mosquitoes were differentiated. The mean abundance of Anopheles mosquitoes per building per trap was significantly higher (p < 0.001) in Mageta than in Magare and Ngodhe. Mageta was also the most populated island (n = 6487) with low LLIN coverage of 62.35% compared to Ngodhe (n = 484; 88.31%) and Magare (n = 250; 98.59%). Overall, 416 (22.27%) engorged Anopheles mosquitoes were analysed, of which 41 tested positive for Plasmodium falciparum infection by high-resolution melting (HRM) analysis of 18S rRNA and cytochrome b PCR products. Plasmodium falciparum infection rates were 10.00, 11.76, 0, and 18.75% among blood-fed An. gambiae s.s. (n = 320), Anopheles arabiensis (n = 51), An. funestus s.s. (n = 29), and An. coustani (n = 16), respectively. Based on HRM analysis of vertebrate cytochrome b, 16S rRNA and COI PCR products, humans (72.36%) were the prominent blood-meal hosts of malaria vectors, but 20.91% of blood-meals were from non-human vertebrate hosts., Conclusions: These findings demonstrate high Plasmodium infection rates among the primary malaria vectors An. gambiae s.s. and An. arabiensis, as well as in An. coustani for the first time in the region, and that non-human blood-meal sources play an important role in their ecology. Further, the higher Anopheles mosquito abundances on the only low LLIN coverage island of Mageta suggests that high LLIN coverage has been effective in reducing malaria vector populations on Magare and Ngodhe Islands.

Although diverse tick-borne pathogens (TBPs) are endemic to East Africa, with recognized impact on human and livestock health, their diversity and specific interactions with tick and vertebrate host species remain poorly understood in the region. In particular, the role of reptiles in TBP epidemiology remains unknown, despite having been implicated with TBPs of livestock among exported tortoises and lizards. Understanding TBP ecologies, and the potential role of common reptiles, is critical for the development of targeted transmission control strategies for these neglected tropical disease agents. During the wet months (April-May; October-December) of 2012-2013, we surveyed TBP diversity among 4,126 ticks parasitizing livestock and reptiles at homesteads along the shores and islands of Lake Baringo and Lake Victoria in Kenya, regions endemic to diverse neglected tick-borne diseases. After morphological identification of 13 distinct Rhipicephalus, Amblyomma , and Hyalomma tick species, ticks were pooled (≤8 individuals) by species, host, sampling site, and collection date into 585 tick pools. By supplementing previously established molecular assays for TBP detection with high-resolution melting analysis of PCR products before sequencing, we identified high frequencies of potential disease agents of ehrlichiosis (12.48% Ehrlichia ruminantium , 9.06% Ehrlichia canis ), anaplasmosis (6.32% Anaplasma ovis , 14.36% Anaplasma platys , and 3.08% Anaplasma bovis ,), and rickettsiosis (6.15% Rickettsia africae , 2.22% Rickettsia aeschlimannii , 4.27% Rickettsia rhipicephali , and 4.95% Rickettsia spp.), as well as Paracoccus sp. and apicomplexan hemoparasites (0.51% Theileria sp., 2.56% Hepatozoon fitzsimonsi , and 1.37% Babesia caballi ) among tick pools. Notably, we identified E. ruminantium in both Amblyomma and Rhipicephalus pools of ticks sampled from livestock in both study areas as well as in Amblyomma falsomarmoreum (66.7%) and Amblyomma nuttalli (100%) sampled from tortoises and Amblyomma sparsum (63.6%) sampled in both cattle and tortoises at Lake Baringo. Similarly, we identified E. canis in rhipicephaline ticks sampled from livestock and dogs in both regions and Amblyomma latum (75%) sampled from monitor lizards at Lake Victoria. These novel tick-host-pathogen interactions have implications on the risk of disease transmission to humans and domestic animals and highlight the complexity of TBP ecologies, which may include reptiles as reservoir species, in sub-Saharan Africa.

A broad diversity of arthropod-borne viruses (arboviruses) of global health concern are endemic to East Africa, yet most surveillance efforts are limited to just a few key viral pathogens. Additionally, estimates of arbovirus diversity in the tropics are likely to be underestimated as their discovery has lagged significantly over past decades due to limitations in fast and sensitive arbovirus identification methods. Here, we developed a nearly pan-arbovirus detection assay that uses high-resolution melting (HRM) analysis of RT-PCR products from highly multiplexed assays to differentiate broad diversities of arboviruses. We differentiated 15 viral culture controls and seven additional synthetic viral DNA sequence controls, within Flavivirus, Alphavirus, Nairovirus, Phlebovirus, Orthobunyavirus and Thogotovirus genera. Among Bunyamwera, sindbis, dengue and Thogoto virus serial dilutions, detection by multiplex RT-PCR-HRM was comparable to the gold standard Vero cell plaque assays. We applied our low-cost method for enhanced broad-range pathogen surveillance from mosquito samples collected in Kenya and identified diverse insect-specific viruses, including a new clade in anopheline mosquitoes, and Wesselsbron virus, an arbovirus that can cause viral haemorrhagic fever in humans and has not previously been isolated in Kenya, in Culex spp. and Anopheles coustani mosquitoes. Our findings demonstrate how multiplex RT-PCR-HRM can identify novel viral diversities and potential disease threats that may not be included in pathogen detection panels of routine surveillance efforts. This approach can be adapted to other pathogens to enhance disease surveillance and pathogen discovery efforts, as well as the study of pathogen diversity and viral evolutionary ecology., (© 2016 John Wiley & Sons Ltd.)

The Lake Baringo and Lake Victoria regions of Kenya are associated with high seroprevalence of mosquito-transmitted arboviruses. However, molecular identification of potential mosquito vector species, including morphologically identified ones, remains scarce. To estimate the diversity, abundance, and distribution of mosquito vectors on the mainland shores and adjacent inhabited islands in these regions, we collected and morphologically identified adult and immature mosquitoes and obtained the corresponding sequence variation at cytochrome c oxidase 1 (COI) and internal transcribed spacer region 2 (ITS2) gene regions. A total of 63 species (including five subspecies) were collected from both study areas, 47 of which have previously been implicated as disease vectors. Fourteen species were found only on island sites, which are rarely included in mosquito diversity surveys. We collected more mosquitoes, yet with lower species composition, at Lake Baringo (40,229 mosquitoes, 32 species) than at Lake Victoria (22,393 mosquitoes, 54 species). Phylogenetic analysis of COI gene sequences revealed Culex perexiguus and Cx tenagius that could not be distinguished morphologically. Most Culex species clustered into a heterogeneous clade with closely related sequences, while Culex pipiens clustered into two distinct COI and ITS2 clades. These data suggest limitations in current morphological identification keys. This is the first DNA barcode report of Kenyan mosquitoes. To improve mosquito species identification, morphological identifications should be supported by their molecular data, while diversity surveys should target both adults and immatures. The diversity of native mosquito disease vectors identified in this study impacts disease transmission risks to humans and livestock., (© The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America.)

Rift Valley fever (RVF) outbreaks have occurred across eastern Africa from 1912 to 2010 approximately every 4-15 years, most of which have not been accompanied by significant epidemics in human populations. However, human epidemics during RVF outbreaks in eastern Africa have involved 478 deaths in 1998, 1107 reported cases with 350 deaths from 2006 to 2007 and 1174 cases with 241 deaths in 2008. We review the history of RVF outbreaks in eastern Africa to identify the epidemiological factors that could have influenced its increasing severity in humans. Diverse ecological factors influence outbreak frequency, whereas virus evolution has a greater impact on its virulence in hosts. Several factors could have influenced the lack of information on RVF in humans during earlier outbreaks, but the explosive nature of human RVF epidemics in recent years mirrors the evolutionary trend of the virus. Comparisons between isolates from different outbreaks have revealed an accumulation of genetic mutations and genomic reassortments that have diversified RVF virus genomes over several decades. The threat to humans posed by the diversified RVF virus strains increases the potential public health and socioeconomic impacts of future outbreaks. Understanding the shifting RVF epidemiology as determined by its evolution is key to developing new strategies for outbreak mitigation and prevention of future human RVF casualties.

Dengue outbreaks have persistently occurred in eastern African countries for several decades. We assessed each outbreak to identify risk factors and propose a framework for prevention and impact mitigation. Seven out of ten countries in eastern Africa and three islands in the Indian Ocean have experienced dengue outbreaks between 1823 and 2014. Major risk factors associated with past dengue outbreaks include climate, virus and vector genetics and human practices. Appropriate use of dengue diagnostic tools and their interpretation are necessary for both outbreak investigations and sero-epidemiological studies. Serosurvey findings during inter-epidemic periods have not been adequately utilised to prevent re-occurrence of dengue outbreaks. Local weather variables may be used to predict dengue outbreaks, while entomological surveillance can complement other disease-mitigation efforts during outbreaks and identify risk-prone areas during inter-epidemic periods. The limitations of past dengue outbreak responses and the enormous socio-economic impacts of the disease on human health are highlighted. Its repeated occurrence in East Africa refutes previous observations that susceptibility may depend on race. Alternate hypotheses on heterotypic protection among flaviviruses may not be applied to all ecologies. Prevention and mitigation of severe dengue outbreaks should necessarily consider the diverse factors associated with their occurrence. Implementation of phased dengue mitigation activities can enforce timely and judicious use of scarce resources, promote environmental sanitation, and drive behavioural change, hygienic practices and community-based vector control. Understanding dengue epidemiology and clinical symptoms, as determined by its evolution, are significant to preventing future dengue epidemics., (Copyright © 2016 John Wiley & Sons, Ltd.)

African trypanosomes thrive in the bloodstream and tissue spaces of a wide range of mammalian hosts. Infections of cattle cause an enormous socio-economic burden in sub-Saharan Africa. A hallmark of the trypanosome lifestyle is the flagellate's incessant motion. This work details the cell motility behavior of the four livestock-parasites Trypanosoma vivax, T. brucei, T. evansi and T. congolense. The trypanosomes feature distinct swimming patterns, speeds and flagellar wave frequencies, although the basic mechanism of flagellar propulsion is conserved, as is shown by extended single flagellar beat analyses. Three-dimensional analyses of the trypanosomes expose a high degree of dynamic pleomorphism, typified by the 'cellular waveform'. This is a product of the flagellar oscillation, the chirality of the flagellum attachment and the stiffness of the trypanosome cell body. The waveforms are characteristic for each trypanosome species and are influenced by changes of the microenvironment, such as differences in viscosity and the presence of confining obstacles. The distinct cellular waveforms may be reflective of the actual anatomical niches the parasites populate within their mammalian host. T. vivax displays waveforms optimally aligned to the topology of the bloodstream, while the two subspecies T. brucei and T. evansi feature distinct cellular waveforms, both additionally adapted to motion in more confined environments such as tissue spaces. T. congolense reveals a small and stiff waveform, which makes these parasites weak swimmers and destined for cell adherence in low flow areas of the circulation. Thus, our experiments show that the differential dissemination and annidation of trypanosomes in their mammalian hosts may depend on the distinct swimming capabilities of the parasites.

The blood-feeding patterns of mosquitoes are directly linked to the spread of pathogens that they transmit. Efficient identification of arthropod vector bloodmeal hosts can identify the diversity of vertebrate species potentially involved in disease transmission cycles. While molecular bloodmeal analyses rely on sequencing of cytochrome b (cyt b) or cytochrome oxidase 1 gene PCR products, recently developed bloodmeal host identification based on high resolution melting (HRM) analyses of cyt b PCR products is more cost-effective. To resolve the diverse vertebrate hosts that mosquitoes may potentially feed on in sub-Saharan Africa, we utilized HRM profiles of both cyt b and 16S ribosomal RNA genes. Among 445 blood-fed Aedeomyia, Aedes, Anopheles, Culex, Mansonia, and Mimomyia mosquitoes from Kenya's Lake Victoria and Lake Baringo regions where many mosquito-transmitted pathogens are endemic, we identified 33 bloodmeal hosts including humans, eight domestic animal species, six peridomestic animal species and 18 wildlife species. This resolution of vertebrate host species was only possible by comparing profiles of both cyt b and 16S markers, as melting profiles of some pairs of species were similar for either marker but not both. We identified mixed bloodmeals in a Culex pipiens from Mbita that had fed on a goat and a human and in two Mansonia africana mosquitoes from Baringo that each had fed on a rodent (Arvicanthis niloticus) in addition to a human or baboon. We further detected Sindbis and Bunyamwera viruses in blood-fed mosquito homogenates by Vero cell culture and RT-PCR in Culex, Aedeomyia, Anopheles and Mansonia mosquitoes from Baringo that had fed on humans and livestock. The observed mosquito feeding on both arbovirus amplifying hosts (including sheep and goats) and possible arbovirus reservoirs (birds, porcupine, baboons, rodents) informs arbovirus disease epidemiology and vector control strategies.

Background: Microscopy and rapid diagnostic tests (RDTs) are common tools for diagnosing malaria, but are deficient in detecting low Plasmodium parasitaemia. A novel molecular diagnostic tool (nPCR-HRM) that combines the sensitivity and specificity of nested PCR (nPCR) and direct PCR-high resolution melting analysis (dPCR-HRM) was developed. To evaluate patterns of anti-malarial drug administration when no parasites are detected, nPCR-HRM was employed to screen blood samples for low parasitaemia from febrile patients without microscopically detectable Plasmodium infections in a rural malaria-endemic setting., Methods: Blood samples (n = 197) were collected in two islands of Lake Victoria, Kenya, from febrile patients without Plasmodium detectable by microscopy or RDTs. 18S rRNA gene sequences were amplified from extracted DNA by nPCR-HRM, nPCR, and dPCR-HRM to detect and differentiate Plasmodium parasites. The limits of detection (LoD) were compared using serial dilutions of the WHO International Standard for P. falciparum DNA. Data on administration of anti-malarials were collected to estimate prescription of anti-malarial drugs to patients with and without low parasitaemia Plasmodium infections., Results: The coupled nPCR-HRM assay detected Plasmodium parasites with greater sensitivity (LoD = 236 parasites/mL) than either nPCR (LoD = 4,700 parasites/mL) or dPCR-HRM (LoD = 1,490 parasites/mL). Moreover, nPCR-HRM detected and differentiated low-parasitaemia infections in significantly greater proportions of patients than did either nPCR or dPCR-HRM (p-value <0.001). Among these low-parasitaemia infections, 67.7% of patients were treated with anti-malarials, whereas 81.5% of patients not infected with Plasmodium parasites were treated with anti-malarials., Conclusions: The enhanced sensitivity of nPCR-HRM demonstrates limitations of differential febrile illness diagnostics in rural malaria endemic settings that confound epidemiological estimates of malaria, and lead to inadvertent misadministration of anti-malarial drugs. This is the first study that employs low-parasitaemia Plasmodium diagnostics to quantify the prescription of anti-malarial drugs to both non-malaria febrile patients and patients with low-parasitaemia Plasmodium infections. nPCR-HRM enhances low-parasitaemia malaria diagnosis and can potentially surmount the deficiencies of microscopy and RDT-based results in determining low-parasitaemia Plasmodium infection rates for evaluating malaria elimination efforts. The findings highlight the need for improved differential diagnostics of febrile illness in remote malaria endemic regions.

Background: The African malaria vector, Anopheles gambiae s.s., is known to feed selectively on certain plants for sugar sources. However, the adaptive significance of this behaviour especially on how the extracts of such plants impact on the fitness of this vector has not been explored. This study determined the toxicity and larvicidal activity on this vector of extracts from six selected plants found in Kenya and two compounds identified from Ricinus communis: 3-carbonitrile-4-methoxy-N-methyl-2-pyridone (ricinine), and its carboxylic acid derivative 3-carboxy-4-methoxy-N-methyl-2-pyridone, the latter compound being reported for the first time from this plant., Methods: Feeding assays tested for toxic effects of extracts from the plants Artemisia afra Jacq. ex Willd, Bidens pilosa L., Parthenium hysterophorus L., Ricinus coummunis L., Senna didymobotrya Fresen. and Tithonia diversifolia Hemsl. on adult females and larvicidal activity was tested against third-instar larvae of Anopheles gambiae s.s. Mortality of larvae and adult females was monitored for three and eight days, respectively; Probit analysis was used to calculate LC50. Survival was analysed with Kaplan-Meier Model. LC-MS was used to identify the pure compounds., Results: Of the six plants screened, extracts from T. diversifolia and R. communis were the most toxic against adult female mosquitoes after 7 days of feeding, with LC50 of 1.52 and 2.56 mg/mL respectively. Larvicidal activity of all the extracts increased with the exposure time with the highest mortality recorded for the extract from R. communis after 72 h of exposure (LC50 0.18 mg/mL). Mosquitoes fed on solutions of the pure compounds, 3-carboxy-4-methoxy-N-methyl-2-pyridone and ricinine survived almost as long as those fed on the R. communis extract with mean survival of 4.93 ± 0.07, 4.85 ± 0.07 and 4.50 ± 0.05 days respectively., Conclusions: Overall, these findings demonstrate that extracts from the six plant species exhibit varying bioactivity against the larvae and adult females of An. gambiae s.s. T. diversifolia and R. communis showed highest bioactivity against adult females An. gambiae and larvae while longevity of female An. gambiae s.s. decreased with exposure time to the two pure compounds.

Tsetse flies use olfactory and gustatory responses, through odorant and gustatory receptors (ORs and GRs), to interact with their environment. Glossina morsitans morsitans genome ORs and GRs were annotated using homologs of these genes in Drosophila melanogaster and an ab initio approach based on OR and GR specific motifs in G. m. morsitans gene models coupled to gene ontology (GO). Phylogenetic relationships among the ORs or GRs and the homologs were determined using Maximum Likelihood estimates. Relative expression levels among the G. m. morsitans ORs or GRs were established using RNA-seq data derived from adult female fly. Overall, 46 and 14 putative G. m. morsitans ORs and GRs respectively were recovered. These were reduced by 12 and 59 ORs and GRs respectively compared to D. melanogaster. Six of the ORs were homologous to a single D. melanogaster OR (DmOr67d) associated with mating deterrence in females. Sweet taste GRs, present in all the other Diptera, were not recovered in G. m. morsitans. The GRs associated with detection of CO2 were conserved in G. m. morsitans relative to D. melanogaster. RNA-sequence data analysis revealed expression of GmmOR15 locus represented over 90% of expression profiles for the ORs. The G. m. morsitans ORs or GRs were phylogenetically closer to those in D. melanogaster than to other insects assessed. We found the chemoreceptor repertoire in G. m. morsitans smaller than other Diptera, and we postulate that this may be related to the restricted diet of blood-meal for both sexes of tsetse flies. However, the clade of some specific receptors has been expanded, indicative of their potential importance in chemoreception in the tsetse.

Tsetse flies undergo drastic fluctuations in their water content throughout their adult life history due to events such as blood feeding, dehydration and lactation, an essential feature of the viviparous reproductive biology of tsetse. Aquaporins (AQPs) are transmembrane proteins that allow water and other solutes to permeate through cellular membranes. Here we identify tsetse aquaporin (AQP) genes, examine their expression patterns under different physiological conditions (blood feeding, lactation and stress response) and perform functional analysis of three specific genes utilizing RNA interference (RNAi) gene silencing. Ten putative aquaporins were identified in the Glossina morsitans morsitans (Gmm) genome, two more than has been previously documented in any other insect. All organs, tissues, and body parts examined had distinct AQP expression patterns. Two AQP genes, gmmdripa and gmmdripb ( = gmmaqp1a and gmmaqp1b) are highly expressed in the milk gland/fat body tissues. The whole-body transcript levels of these two genes vary over the course of pregnancy. A set of three AQPs (gmmaqp5, gmmaqp2a, and gmmaqp4b) are expressed highly in the Malpighian tubules. Knockdown of gmmdripa and gmmdripb reduced the efficiency of water loss following a blood meal, increased dehydration tolerance and reduced heat tolerance of adult females. Knockdown of gmmdripa extended pregnancy length, and gmmdripb knockdown resulted in extended pregnancy duration and reduced progeny production. We found that knockdown of AQPs increased tsetse milk osmolality and reduced the water content in developing larva. Combined knockdown of gmmdripa, gmmdripb and gmmaqp5 extended pregnancy by 4-6 d, reduced pupal production by nearly 50%, increased milk osmolality by 20-25% and led to dehydration of feeding larvae. Based on these results, we conclude that gmmDripA and gmmDripB are critical for diuresis, stress tolerance and intrauterine lactation through the regulation of water and/or other uncharged solutes.

Trypanosoma brucei relies on Spliced leader trans splicing to generate functional messenger RNAs. Trans splicing joins the specialized SL exon from the SL RNA to pre-mRNAs and is mediated by the trans-spliceosome, which is made up of small nuclear ribonucleoprotein particles and non-snRNP factors. Although the trans spliceosome is essential for trypanosomatid gene expression, not all spliceosomal protein factors are known and of these, only a few are completely characterized. In this study, we have characterized the trypanosome Splicing Factor, SF3a60, the only currently annotated SF3a component. As expected, epitope-tagged SF3a60 localizes in the trypanosome nucleus. SF3a60 is essential for cell viability but its depletion seem to have no detectable effect on trans-splicing. In addition, we used SF3a60 as bait in a Yeast-2-hybrid system screen and identified its interacting protein factors. The interactions with SF3a120, SF3a66 and SAP130 were confirmed by tandem affinity purification and mass spectrometry.

Background: The IAEA colony is the only one available for mass rearing of Glossina pallidipes, a vector of human and animal African trypanosomiasis in eastern Africa. This colony is the source for Sterile Insect Technique (SIT) programs in East Africa. The source population of this colony is unclear and its genetic diversity has not previously been evaluated and compared to field populations., Methodology/principal Findings: We examined the genetic variation within and between the IAEA colony and its potential source populations in north Zimbabwe and the Kenya/Uganda border at 9 microsatellites loci to retrace the demographic history of the IAEA colony. We performed classical population genetics analyses and also combined historical and genetic data in a quantitative analysis using Approximate Bayesian Computation (ABC). There is no evidence of introgression from the north Zimbabwean population into the IAEA colony. Moreover, the ABC analyses revealed that the foundation and establishment of the colony was associated with a genetic bottleneck that has resulted in a loss of 35.7% of alleles and 54% of expected heterozygosity compared to its source population. Also, we show that tsetse control carried out in the 1990's is likely reduced the effective population size of the Kenya/Uganda border population., Conclusions/significance: All the analyses indicate that the area of origin of the IAEA colony is the Kenya/Uganda border and that a genetic bottleneck was associated with the foundation and establishment of the colony. Genetic diversity associated with traits that are important for SIT may potentially have been lost during this genetic bottleneck which could lead to a suboptimal competitiveness of the colony males in the field. The genetic diversity of the colony is lower than that of field populations and so, studies using colony flies should be interpreted with caution when drawing general conclusions about G. pallidipes biology.

Detection of trypanosomes that cause disease in human beings and livestock within their tsetse fly hosts is an essential component of vector and disease control programmes. Several molecular-based diagnostic tests have been developed for this purpose. Many of these tests, while sensitive, require analysis of trypanosome DNA extracted from single flies, or from pooled tsetse fly heads and amplified trypanosome DNA. In this study, we evaluated the relative analytical and diagnostic sensitivities of two PCR-based tests (ITS and TBR) and a Trypanozoon specific LAMP assay using pooled whole tsetse flies and midguts spiked with serially diluted procyclics of a laboratory strain of Trypanosoma brucei brucei (KETRI 3386). Test sensitivity was also evaluated using experimentally infected tsetse flies. The aim was to determine the most appropriate pooling strategy for whole tsetse and midguts. RIME-LAMP had the highest diagnostic sensitivity (100%) followed by TBR-PCR (95%) and ITS-PCR (50%) in detecting trypanosome DNA from pooled tsetse midguts. RIME-LAMP also had the best diagnostic specificity (75%) followed by ITS-PCR (68%) and TBR-PCR (50%). The relative detection limit determined by serial dilution of procyclics was below 10(-6) (equivalent to 1parasite/ml). Using TBR-PCR, ITS-PCR and RIME-LAMP, it was possible to detect trypanosome DNA in single flies or in pools of 2, 3, 4, 5, 10, or 15 flies/midguts. The proportion of positive pools declined by up to 60% when testing pools of 15 whole flies as opposed to testing pools of 5-10 flies. Additionally, it was possible to detect DNA in a single infected tsetse fly in the background of 4, 9, or 14 uninfected tsetse flies. Averaged across pool sizes and tsetse species, RIME-LAMP detected the highest proportion of positive pools in spiked whole tsetse and midguts (86.6% and 87.2%) followed by TBR-PCR (78. 6% and 79.2%) and ITS-PCR (34.3% and 40.2%). There were no significant differences between the proportions of positive pools detected in whole flies and midguts. We conclude that pooling of whole tsetse/midguts is an effective strategy to reduce hands-on-time and hence has potential application in large scale xenomonitoring to generate epidemiological data for decision making. RIME-LAMP offers the best diagnostic sensitivity and specificity on pooled tsetse midguts, thus demonstrating its superior diagnostic performance when compared with TBR-PCR and ITS-PCR. Using pools of whole tsetse or midguts as source of DNA does not have any significant effect on test results and is more representative of the field conditions where the proportion of flies with infected midguts tends to be higher than flies with infected salivary glands. Therefore to save time and minimize costs, pooling of whole tsetse flies is recommended., (Copyright © 2013 Elsevier B.V. All rights reserved.)