Prevalence of bovine trypanosomosis and tsetse fly density in Loka Abaya and Derara districts in Sidama Regional State, Ethiopia.

Animal trypanosomosis is a significant livestock disease with economic and social repercussions, reducing the supply of animal products and restricting the utilization of animals for traction and transportation. In Ethiopia, it is prevalent and poses a major hindrance to the advancement of animal production. This repeated cross-sectional study was aimed at assessing seasonal variation in bovine trypanosomosis prevalence and tsetse fly density and identifying the potential risk factors in the Loka Abaya and Derara districts of the Sidama National Regional State. Blood samples were collected from 964 cattle, 484 samples during the dry season, and 480 during the wet season. The buffy coat method was employed to analyze these samples. Furthermore, 78 standard NGU traps were set up at various locations in the two districts during both seasons for entomological investigation. The overall apparent prevalence of trypanosomosis was 9% (95% CI 7.3-11.0), without a significant difference (p > 0.05) between the dry season (7.4%) and wet season (10.6%). The apparent prevalence was significantly higher in Loka Abaya (11.8%) than in Derara (6.3%) district (OR = 2.04; p = 0.003) and in cattle with black coat color (29%) than in mixed color (6.8%) (OR = 5.3; p < 0.001). The majority of infections were caused by Trypanosoma congolense (70%), followed by T. vivax (29%), and mixed infections (1%) with the two species. The average packed cell volume (PCV) was significantly (p < 0.0001) lower in infected animals (20.7 ± 4%) compared to uninfected ones (25.5 ± 5.4%), in cattle examined during the dry season (24.1 ± 6%) versus the wet season (26.1 ± 4.7%), in cattle sampled from the Loka Abaya district (24.2 ± 5.5%) versus Derara district (26 ± 5.3%), and in cattle with poor body condition (23.6 ± 5.7%) compared to those with good body condition (26.5 ± 5.3%). A total of 5282 flies were captured during the study, with 4437 (84%) being tsetse flies (Glossina pallidipes), 439 (8.3%) Tabanids, 190 (3.6%) Stomoxys spp., and 216 (4.1%) Musca spp. The apparent density (AD) of G. pallidipes was 28.4 flies/trap/day, showing no statistically significant difference between wet (32.1) and dry (24.6) seasons (p > 0.05). The AD of G. pallidipes was significantly (p < 0.001) higher in the Loka Abaya district (57.3) than in the Derara district (0.9). The study highlights a moderate trypanosomosis apparent prevalence and high AD of G. pallidipes, showing significant variation between the study districts but no seasonal difference. The observed apparent prevalence of trypanosomosis and tsetse fly density notably affects animal health and productivity. As a result, strategies for vector control like insecticide-treated targets, trypanocidal medications for infected animals, and community-based initiatives such as education and participation in control programs are recommended.

Establishment and partial characterisation of a new cell line derived from adult tissues of the tsetse fly Glossina morsitans morsitans.

BACKGROUND: Insect cell lines play a vital role in many aspects of research on disease vectors and agricultural pests. The tsetse fly Glossina morsitans morsitans is an important vector of salivarian trypanosomes in sub-Saharan Africa and, as such, is a major constraint on human health and agricultural development in the region. METHODS: Here, we report establishment and partial characterisation of a cell line, GMA/LULS61, derived from tissues of adult female G. m. morsitans. GMA/LULS61 cells, grown at 28 °C in L-15 (Leibovitz) medium supplemented with foetal bovine serum and tryptose phosphate broth, have been taken through 23 passages to date and can be split 1:1 at 2-week intervals. Karyotyping at passage 17 revealed a predominantly haploid chromosome complement. Species origin and absence of contaminating bacteria were confirmed by PCR amplification and sequencing of fragments of the COI gene and pan-bacterial 16S rRNA gene respectively. However, PCR screening of RNA extracted from GMA/LULS61 cells confirmed presence of the recently described Glossina morsitans morsitans iflavirus and Glossina morsitans morsitans negevirus, but absence of Glossina pallipides salivary gland hypertrophy virus. GMA/LULS61 cells supported infection and growth of 6/7 different insect-derived strains of the intracellular bacterial symbiont Wolbachia. CONCLUSIONS: The GMA/LULS61 cell line has potential for application in a variety of studies investigating the biology of G. m. morsitans and its associated pathogenic and symbiotic microorganisms.

Entomological study on vectorial density, temporal variation of tsetse fly and other biting flies in intervention and non-intervention areas of South Omo Zone, Ethiopia.

A repeated cross-sectional entomological survey was conducted to estimate Glossina (tsetse) and other biting flies density, their seasonal variation and associated risk factors in intervention and non- intervention areas of South Omo Zone, Southwest Ethiopia from January 2019-November 2019. In both dry and wet seasons, a total of 96 NGU traps (64 traps in tsetse intervention districts and 32 traps in tsetse non- intervention districts) were deployed at an interval of about 100-200 m in purposively selected and suspected tsetse habitats. Thus, Glossina pallidipes was found to be the only cyclical vector along with mechanical vectors of Tabanus, Stomoxys and Haematopota. In tsetse intervention areas, G. pallidipes apparent density of 2.64 F/T/D and 0.42 F/T/D was recorded in dry and wet season respectively. Mechanical vectors (dry; wet) of Tabanus (205; 155), Stomoxys (34; 54) and Haematopota (50; 33) were also recorded in tsetse intervened areas. Whereas, in non- intervention areas, apparent density of G. pallidipes was 2.03 F/T/D and 0.56 F/T/D, respectively in dry and wet season. Similarly, Tabanus (22; 56), Stomoxys (10; 8) and Haematopota (5; 7) respectively in dry and wet (dry; wet) season were recorded in tsetse non- intervention areas. According to Negative Binomial Regression (NBR), season was the only variable significantly affecting (P < 0.05) the Glossina count in the current study area. Accordingly, the incidence G. pallidipes during wet season was decreased by the factor of 0.21 (CI; 0.097-0.47) when compared to its incidence in dry season by holding other variables constant. In conclusion, cyclical vectors were playing vital role in transmission of trypanosomosis in South Omo Zone along with numerous mechanical vectors even though there have been vector intervention activities in the areas. Therefore, strong, sustainable, environmentally friend and community participating vector control strategies should be followed to tackle the vector distribution in the area.

Trypanosoma brucei gambiense group 2 experimental in vivo life cycle: from procyclic to bloodstream form.

Trypanosoma brucei gambiense (Tbg) group 2 is a subgroup of trypanosomes able to infect humans and is found in West and Central Africa. Unlike other agents causing sleeping sickness, such as Tbg group 1 and Trypanosoma brucei rhodesiense, Tbg2 lacks the typical molecular markers associated with resistance to human serum. Only 36 strains of Tbg2 have been documented, and therefore, very limited research has been conducted despite their zoonotic nature. Some of these strains are only available in their procyclic form, which hinders human serum resistance assays and mechanistic studies. Furthermore, the understanding of Tbg2's potential to infect tsetse flies and mammalian hosts is limited. In this study, 165 Glossina palpalis gambiensis flies were experimentally infected with procyclic Tbg2 parasites. It was found that 35 days post-infection, 43 flies out of the 80 still alive were found to be Tbg2 PCR-positive in the saliva. These flies were able to infect 3 out of the 4 mice used for blood-feeding. Dissection revealed that only six flies in fact carried mature infections in their midguts and salivary glands. Importantly, a single fly with a mature infection was sufficient to infect a mammalian host. This Tbg2 transmission success confirms that Tbg2 strains can establish in tsetse flies and infect mammalian hosts. This study describes an effective in vivo protocol for transforming Tbg2 from procyclic to bloodstream form, reproducing the complete Tbg2 cycle from G. p. gambiensis to mice. These findings provide valuable insights into Tbg2's host infectivity, and will facilitate further research on mechanisms of human serum resistance.

Title: Cycle de vie expérimental in vivo de Trypanosoma brucei gambiense groupe 2 : de la forme procyclique à la forme sanguicole. Abstract: Trypanosoma brucei gambiense (Tbg) groupe 2 est un sous-groupe de trypanosomes capables d'infecter l'Homme, présent en Afrique de l'Ouest et en Afrique centrale. Contrairement aux autres agents responsables de la maladie du sommeil, tels que Tbg groupe 1 et Trypanosoma brucei rhodesiense, Tbg2 ne présente pas les marqueurs moléculaires habituellement associés à la résistance au sérum humain. Seules trente-six souches de Tbg2 ont été répertoriées, limitant considérablement les recherches sur ce sous-groupe malgré sa nature zoonotique. Certaines de ces souches ne sont disponibles que sous leur forme procyclique, ce qui freine la réalisation des tests de résistance au sérum humain et les études mécanistiques. De plus, la compréhension du potentiel de Tbg2 à infecter les glossines et les hôtes mammifères est limitée. Dans cette étude, 165 glossines Glossina palpalis gambiensis ont été infectées expérimentalement par des parasites Tbg2 sous leur forme procyclique. Trente-cinq jours après l'infection, 43 des 80 glossines encore en vie se sont révélées positives à Tbg2 en PCR sur leur salive. Ces glossines ont réussi à infecter trois des quatre souris utilisées pour leur repas de sang. La dissection des glossines a révélé que seules six d'entre elles étaient réellement porteuses d'infections matures dans leur intestin et leurs glandes salivaires. Il est important de noter qu'une seule glossine porteuse d'une infection mature a suffi pour infecter un hôte mammifère. Ce succès de transmission de Tbg2 confirme que les souches de Tbg2 peuvent s'établir dans les glossines et infecter des hôtes mammifères. Cette étude décrit un protocole in vivo pour transformer la forme procyclique de Tbg2 en forme sanguicole, en reproduisant le cycle complet de Tbg2 de G. p. gambiensis à la souris. Ces résultats fournissent des informations précieuses sur le potentiel infectieux de Tbg2 et faciliteront la recherche sur les mécanismes de résistance au sérum humain des souches.

A scoping review on tsetse fly blood meal sources and its assay methods since 1956 to 2022.

BACKGROUND: Tsetse flies (Glossina spp.) are the definitive biological vectors of African trypanosomes in humans and animals. Controlling this vector is the most promising method of preventing trypanosome transmission. This requires a comprehensive understanding of tsetse biology and host preference to inform targeted design and management strategies, such as the use of olfaction and visual cues in tsetse traps. No current review exists on host preference and blood meal analyses of tsetse flies. METHODS: This review presents a meta-analysis of tsetse fly blood meal sources and the methodologies used to identify animal hosts from 1956 to August 2022. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRIMA-ScR) was applied. This focused on tsetse-endemic countries, blood meal analysis methodologies and the blood meal hosts identified. The articles were retrieved and screened from databases using predetermined eligibility criteria. RESULTS: Only 49/393 of the articles retrieved matched the inclusion criteria. Glossina's main hosts in the wild included the bushbuck, buffalo, elephant, warthog, bushpig and hippopotamus. Pigs, livestock and humans were key hosts at the domestic interface. The least studied species included Glossina fuscipleuris, G. fusca, G. medicorum, G. tabaniformis and G. austeni. In the absence of preferred hosts, Glossina fed opportunistically on a variety of hosts. Precipitin, haemagglutination, disc diffusion, complement fixation, ELISA and PCR-based assays were used to evaluate blood meals. Cytochrome b (Cyt b) was the main target gene in PCR to identify the vertebrate hosts. CONCLUSIONS: Tsetse blood meal sources have likely expanded because of ecological changes that could have rendered preferred hosts unavailable. The major approaches for analysing tsetse fly blood meal hosts targeted Cyt b gene for species identification by Sanger sequencing. However, small-fragment DNAs, such as the mammalian 12S and 16S rRNA genes, along with second- and third-generation sequencing techniques, could increase sensitivity for host identification in multiple host feeders that Sanger sequencing may misidentify as "noise". This review of tsetse fly blood meal sources and approaches to host identification could inform strategies for tsetse control.

Tsetse fly ecology and risk of transmission of African trypanosomes related to a protected forest area at a military base in the city of Abidjan, Côte d'Ivoire.

African trypanosomoses, whose pathogens are transmitted by tsetse flies, are a threat to animal and human health. Tsetse flies observed at the military base of the French Forces in Côte d'Ivoire (FFCI base) were probably involved in the infection and death of military working dogs. Entomological and parasitological surveys were carried out during the rainy and dry seasons using "Vavoua" traps to identify tsetse fly species, their distribution, favorable biotopes and food sources, as well as the trypanosomes they harbor. A total of 1185 Glossina palpalis palpalis tsetse flies were caught, corresponding to a high average apparent density of 2.26 tsetse/trap/day. The results showed a heterogeneous distribution of tsetse at the FFCI base, linked to more or less favorable biotopes. No significant variation in tsetse densities was observed according to the season. The overall trypanosomes infection rate according to microscopic observation was 13.5%. Polymerase chain reaction (PCR) analyses confirmed the presence of Trypanosoma vivax and T. congolense forest type, responsible for African animal trypanosomosis. Our findings suggest that there is a risk of introduction and transmission of T. brucei gambiense, responsible for human African trypanosomiasis, on the study site. This risk of transmission of African trypanosomes concerns not only the FFCI base, but also inhabited peripheral areas. Our study confirmed the need for vector control adapted to the eco-epidemiological context of the FFCI base.

Title: Écologie des mouches tsé-tsé et risque de transmission des trypanosomes africains lié à une zone forestière protégée dans une base militaire de la ville d'Abidjan, Côte d'Ivoire. Abstract: Les trypanosomoses africaines, dont les agents pathogènes sont transmis par les mouches tsé-tsé, constituent une contrainte pour la santé animale et humaine. Des mouches tsé-tsé observées dans la base militaire des Forces françaises en Côte d'Ivoire (base FFCI) ont probablement été impliquées dans l'infection et la mort de chiens militaires. Des enquêtes entomologiques et parasitologiques ont été menées pendant la saison pluvieuse et la saison sèche à l'aide de pièges "Vavoua" afin d'identifier les espèces de mouches tsé-tsé, leur distribution, les biotopes favorables et leur source de nourriture ainsi que les trypanosomes qu'elles hébergent. Au total 1185 mouches tsé-tsé de l'espèce Glossina palpalis palpalis ont été capturées, ce qui correspond à une densité apparente moyenne élevée de 2,26 tsé-tsé/piège/jour. Les résultats ont montré une distribution hétérogène des tsé-tsé dans la base FFCI en lien avec des biotopes plus ou moins favorables. Aucune variation significative des densités de tsé-tsé n'a été observée en fonction de la saison. Le taux d'infection global par les trypanosomes était de 13,5 % selon l'observation microscopique. Les analyses PCR ont confirmé la présence de Trypanosoma vivax et T. congolense type forêt, responsable de la trypanosomose animale africaine. Nos résultats suggèrent qu'il existe un risque potentiel d'introduction et de transmission de T. brucei gambiense responsable de la trypanosomiase humaine africaine dans la zone d'étude. Ce risque de transmission des trypanosomes africains concerne non seulement l'intérieur de la base FFCI, mais aussi les espaces périphériques habités. Notre étude a confirmé la nécessité de mener une lutte antivectorielle adaptée au contexte éco-épidémiologique de la base FFCI.

Development and characterization of microsatellite markers for the tsetse species Glossina brevipalpis and preliminary population genetics analyses.

Tsetse flies, the vectors of African trypanosomes are of key medical and economic importance and one of the constraints for the development of Africa. Tsetse fly control is one of the most effective and sustainable strategies used for controlling the disease. Knowledge about population structure and level of gene flow between neighbouring populations of the target vector is of high importance to develop appropriate strategies for implementing effective management programmes. Microsatellites are commonly used to identify population structure and assess dispersal of the target populations and have been developed for several tsetse species but were lacking for Glossina brevipalpis. In this study, we screened the genome of G. brevipalpis to search for suitable microsatellite markers and nine were found to be efficient enough to distinguish between different tsetse populations. The availability of these novel microsatellite loci will help to better understand the population biology of G. brevipalpis and to assess the level of gene flow between different populations. Such information will help with the development of appropriate strategies to implement the sterile insect technique (SIT) in the framework of an area-wide integrated pest management (AW-IPM) approach to manage tsetse populations and ultimately address the trypanosomoses problem in these targeted areas.

Title: Développement et caractérisation de marqueurs microsatellites pour l'espèce de mouche tsé-tsé Glossina brevipalpis et analyses génétiques préliminaires des populations. Abstract: Les mouches tsé-tsé, vecteurs des trypanosomes africains, sont d'une importance médicale et économique majeure et l'une des contraintes pour le développement de l'Afrique. La lutte contre la mouche tsé-tsé est l'une des stratégies les plus efficaces et durables utilisées pour contrôler la maladie. La connaissance de la structure de la population et du niveau de flux de gènes entre les populations voisines du vecteur cible est d'une grande importance pour développer des stratégies appropriées pour la mise en œuvre de programmes de gestion efficaces. Les microsatellites sont couramment utilisés pour identifier la structure de la population et évaluer la dispersion des populations cibles et ont été développés pour plusieurs espèces de glossines mais manquaient pour Glossina brevipalpis. Dans cette étude, nous avons criblé le génome de G. brevipalpis pour rechercher des marqueurs microsatellites appropriés et neuf ont été trouvés suffisamment efficaces pour faire la distinction entre différentes populations de glossines. La disponibilité de ces nouveaux locus microsatellites aidera à mieux comprendre la biologie des populations de G. brevipalpis et à évaluer le niveau de flux de gènes entre différentes populations. Ces informations aideront à l'élaboration de stratégies appropriées pour mettre en œuvre la technique de l'insecte stérile dans le cadre d'une approche de lutte antiparasitaire intégrée à l'échelle de la zone pour gérer les populations de glossines et, en fin de compte, résoudre le problème des trypanosomoses dans les zones concernées.

Trypanosomes infection, endosymbionts, and host preferences in tsetse flies (Glossina spp.) collected from Akagera park region, Rwanda: A correlational xenomonitoring study.

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.

Improved models for the relationship between age and the probability of trypanosome infection in female tsetse, Glossina pallidipes Austen.

Between 1990 and 1999, at Rekomitjie Research Station, Zambezi Valley, Zimbabwe, 29,360 female G. pallidipes were dissected to determine their ovarian category and trypanosome infection status. Overall prevalences were 3.45 and 2.66% for T. vivax and T. congolense, respectively, declining during each year as temperatures increased from July - December. Fits to age-prevalence data using Susceptible-Exposed-Infective (SEI) and SI compartmental models were statistically better than those obtained using a published catalytic model, which made the unrealistic assumption that no female tsetse survived more than seven ovulations. The improved models require knowledge of fly mortality, estimated separately from ovarian category distributions. Infection rates were not significantly higher for T. vivax than for T. congolense. For T. congolense in field-sampled female G. pallidipes, we found no statistical support for a model where the force of infection was higher at the first feed than subsequently. The long survival of adult female tsetse, combined with feeding at intervals ≤3 days, ensures that post-teneral feeds, rather than the first feed, play the dominant role in the epidemiology of T. congolense infections in G. pallidipes. This is supported by estimates that only about 3% of wild hosts at Rekomitjie were harbouring sufficient T. congolense to ensure that tsetse feeding off them take an infected meal, so that the probability of ingesting an infected meal is low at every meal.

Near-infrared imaging for automated tsetse pupae sex sorting in support of the sterile insect technique.

Tsetse flies are the cyclical vectors of African trypanosomes and one of several methods to manage this vector is the sterile insect technique (SIT). The ability to determine the sex of tsetse pupae with the objective to separate the sexes before adult emergence has been a major goal for decades for tsetse management programmes with an SIT component. Tsetse females develop faster and pharate females inside the pupae melanise 1-2 days before males. This earlier melanisation can be detected by infrared cameras through the pupal shell, and the newly developed Near InfraRed Pupae Sex Sorter (NIRPSS) takes advantage of this. The melanisation process is not homogeneous for all fly organs and the pupa needs to be examined ventrally, dorsally and laterally to ensure accurate classification by an image analysis algorithm. When the pupae are maturing at a constant temperature of 24 °C and sorted at the appropriate age, 24 days post-larviposition for Glossina palpalis gambiensis, the sorting machine can efficiently separate the sexes. The recovered male pupae can then be sterilised for field releases of males, while the rest of the pupae can be used to maintain the laboratory colony. The sorting process with the new NIRPSS had no negative impact on adult emergence and flight ability. A mean male recovery of 62.82 ± 3.61% was enough to provide sterile males to an operational SIT programme, while mean contamination with females (4.69 ± 3.02%) was low enough to have no impact on the maintenance of a laboratory colony.

Title: Imagerie dans l'infrarouge proche pour le tri automatisé du sexe des pupes de glossines comme aide à la technique de l'insecte stérile. Abstract: Les glossines sont les vecteurs cycliques des trypanosomes africains et la technique de l'insecte stérile (TIS) est l'une des méthodes de gestion de ce vecteur. La capacité à déterminer le sexe des pupes de glossines dans le but de séparer les sexes avant l'émergence des adultes a été un objectif majeur, pendant des décennies, pour les programmes de lutte contre les glossines avec une composante TIS. Les femelles tsé-tsé se développent plus rapidement et les pharates femelles à l'intérieur des pupes se mélanisent 1 à 2 jours avant les mâles. Cette mélanisation précoce peut être détectée par des caméras infrarouges à travers la coque de la pupe, ce que le nouveau trieur de sexe des pupes dans le proche infrarouge (TSPPIR) utilise. Le processus de mélanisation n'est pas homogène pour tous les organes de la mouche et la pupe doit être examinée ventralement, dorsalement et latéralement pour assurer une classification précise par un algorithme d'analyse d'image. Lorsque les pupes mûrissent à une température constante de 24 °C et sont triées à l'âge approprié, 24 jours après la larviposition pour Glossina palpalis gambiensis, la machine de tri peut séparer efficacement les sexes. Les pupes mâles récupérées peuvent ensuite être stérilisées pour les lâchers de mâles sur le terrain tandis que le reste des pupes peut être utilisé pour maintenir la colonie de laboratoire. Le processus de tri avec le nouveau TSPPIR n'a eu aucun impact négatif sur l'émergence et la capacité de vol des adultes. Une récupération moyenne des mâles de 62,82 ± 3,61% était suffisante pour fournir des mâles stériles à un programme TIS opérationnel, tandis que la contamination moyenne par les femelles (4,69 ± 3,02%) était suffisamment faible pour n'avoir aucun impact sur le maintien d'une colonie de laboratoire.

Tsetse flies (Glossina morsitans morsitans) choose birthing sites guided by substrate cues with no evidence for a role of pheromones.

Tsetse flies significantly impact public health and economic development in sub-Saharan African countries by transmitting the fatal disease African trypanosomiasis. Unusually, instead of laying eggs, tsetse birth a single larva that immediately burrows into the soil to pupate. Where the female chooses to larviposit is, therefore, crucial for offspring survival. Previous laboratory studies suggested that a putative larval pheromone, n-pentadecane, attracts gravid female Glossina morsitans morsitans to appropriate larviposition sites. However, this attraction could not be reproduced in field experiments. Here, we resolve this disparity by designing naturalistic laboratory experiments that closely mimic the physical characteristics found in the wild. We show that gravid G. m. morsitans were neither attracted to the putative pheromone nor, interestingly, to pupae placed in the soil. By contrast, females appear to choose larviposition sites based on environmental substrate cues. We conclude that, among the many cues that likely contribute to larviposition choice in nature, substrate features are a main determinant, while we failed to find evidence for a role of pheromones.

Improved estimates of abortion rates in tsetse (Glossina spp.).

Abortion rates were assessed among 170, 846 tsetse (154,228 Glossina pallidipes and 19,618 Glossina morsitans morsitans) sampled in Zimbabwe in 1988-1999. The study produced improved estimates of abortion rates and how these varied with fly age and size and temperatures experienced during pregnancy. An abortion was diagnosed if the uterus was empty and the largest oocyte <0.82 of the expected mature length. Abortion rates for G. pallidipes and G. m. morsitans were 0.64% (95% ci: 0.59-0.69) and 0.83% (0.62-1.10) for trapped flies and 2.03% (1.77-2.31) and 1.55% (1.20-1.98) for flies from artificial refuges. Abortion rates increased with increasing temperature and decreased with increasing wing length and wing fray. Contrary to laboratory findings, abortion rates did not increase in the oldest flies. Percentages of tsetse with empty uteri, regardless of abortion status, were significantly higher than estimated abortion percentages. For tsetse from traps, 4.01% (95% ci: 3.90-4.13) of G. pallidipes and 2.52% (2.14-2.95) of G. m. morsitans had empty uteri; for flies from artificial refuges, the percentages were 12.69% (12.07-13.34) and 14.90% (13.82-16.02), respectively. Abortion losses are small relative to losses at all other stages of life.

Evaluating the Effect of Irradiation on the Densities of Two RNA Viruses in Glossina morsitans morsitans.

Tsetse flies are cyclic vectors of Trypanosoma parasites, which cause debilitating diseases in humans and animals. To decrease the disease burden, the number of flies is reduced using the sterile insect technique (SIT), where male flies are sterilized through irradiation and released into the field. This procedure requires the mass rearing of high-quality male flies able to compete with wild male flies for mating with wild females. Recently, two RNA viruses, an iflavirus and a negevirus, were discovered in mass-reared Glossina morsitans morsitans and named GmmIV and GmmNegeV, respectively. The aim of this study was to evaluate whether the densities of these viruses in tsetse flies are affected by the irradiation treatment. Therefore, we exposed tsetse pupae to various doses (0-150 Gy) of ionizing radiation, either in air (normoxia) or without air (hypoxia), for which oxygen was displaced by nitrogen. Pupae and/or emerging flies were collected immediately afterwards, and at three days post irradiation, virus densities were quantified through RT-qPCR. Generally, the results show that irradiation exposure had no significant impact on the densities of GmmIV and GmmNegeV, suggesting that the viruses are relatively radiation-resistant, even at higher doses. However, sampling over a longer period after irradiation would be needed to verify that densities of these insect viruses are not changed by the sterilisation treatment.

Suitability of Raycell MK2 Blood X-ray Irradiator for the Use in the Sterile Insect Technique: Dose Response in Fruit Flies, Tsetse Flies and Mosquitoes.

The sterile insect technique (SIT) is based on the inundatory field release of a target pest following their reproductive sterilization via exposure to radiation. Until recently, gamma irradiation from isotopic sources has been the most widely used in SIT programs. As isotopic sources are becoming increasingly expensive, especially for small programs, and regulations surrounding their procurement and shipment increasingly strict, irradiation capacity is one of the limiting factors in smaller or newly developing SIT projects. For this reason, the possibility of using X-ray irradiators has been evaluated in the recent decade. The availability of "off-the-shelf" blood X-ray irradiators that meet the technical requirements for insect irradiation can provide irradiation capacity for those SIT projects in which the acquisition of gamma ray irradiators is not feasible. Following the recent technical characterization of a Raycell MK2 X-ray blood irradiator, it was found in this study, that MK2 instruments were suitable for the sterilization of fruit flies, tsetse flies and mosquitoes, inducing comparable, even slightly higher, sterility levels compared to those achieved by gamma ray irradiation. This, together with its estimated processing efficiency, shows that MK2 irradiators are suitable for small- to mid-sized SIT programs.

Sodalis glossinidius and Wolbachia infections in wild population of Glossina morsitans submorsitans caught in the area of Lake Iro in the south of Chad.

Investigations on the bacterial fauna and their association with trypanosome infections in tsetse fly have revealed contrasting results. This study aimed to detect Wolbachia and S. glossinidius in wild populations of G. m. submorsistans and subsequently, understand the influence that these bacteria may have on the vectorial competence of this tsetse species. Tsetse flies were captured in the area of Lake Iro in the south of Chad using biconical traps. After DNA extraction from each tsetse fly, Sodalis glossinidius and Wolbachia were detected using specific primers. Sodalis glossinidius and Wolbachia infection rates were compared and association studies involving trypanosome infections and S. glossinidius or Wolbachia were performed. From 345 G. m. submorsitans analyzed, 9.0% and 14.5% were respectively infected with S. glossinidius and Wolbachia. Only 2.31% of all tsetse flies were co-infected by the 2 bacteria. Of all trypanosome-infected flies, 7.1% and 9.8% harbored, respectively, S. glossinidius and Wolbachia. No association was observed between Wolbachia and trypanosome infections while a significant association (r = 4.992; P = 0.025) was found between S. glossinidius and the presence of trypanosomes. A significant association (r = 3.147; P = 0.043) was also observed between S. glossinidius and T. simiae; and none with T. congolense or T. godfreyi. This study revealed S. glossinidius and Wolbachia in G. m. submorsitans of the area of lake Iro. It showed that co-infections between Wolbachia and S. glossinidius are rare in wild populations of G. m. submorsitans and that the tripartite associations vary according to trypanosome species as well as symbiotic mricroorganisms.

Diversity of tsetse flies and trypanosome species circulating in the highly infested cattle rearing area of the Faro and Deo subdivision, Adamawa region, Cameroon.

Animal African Trypanosomiasis (AAT) remains an animal health problem in sub-Saharan Africa and in Cameroon in particular. Despite more than 40 years of fighting against AAT in some tsetse infested areas, the disease prevalence is still a concern. Improving the control strategies in different settings requires to understand the current epidemiological situation of AAT. The aim of the present study was to update our knowledge on the diversity of tsetse fauna and trypanosome species in the tsetse infested area of Faro and Deo division, Adamawa region, Cameroon. Tsetse flies were caught using Vavoua trap in two villages and the apparent density per trap (ADP) were estimated. After morphological identification of tsetse fly species, flies were dissected and their midguts recovered. The presence of blood meal residues was recorded. Trypanosomes species were checked in the flies' midguts by microscopy followed by PCR method. The vertebrate taxa on which tsetse flies have taken blood meal were determined using the heteroduplex-PCR method. A total of 338 tsetse flies including 11 teneral flies (10 Glossina palpalis palpalis and 01 G. morsitans submorsitans) and 327 non-teneral were trapped in Mayo Lainde and Tchabal Mbabo. Amongst the caught tsetse flies, of the 327 non-teneral flies, 315 (96.3%) were G. p. palpalis, 8 (2.4%) were G. morsitans submorsitans and 4 (1.2%) G. fuscipes fuscipes. Trypanosome infections including Trypanosoma congolense forest (19.88%) and savanah (2.53%) "types", T. brucei s.l. (7.30%) and T. vivax (2.85%) were identified in 45.08% of non-teneral flies (32.38% for single infection and 12.70% for mixed infection). Amongst the 54 blood meals identified in tsetse midguts, 41% were from humans, 33% from cattle and 26% from other vertebrate hosts. About 51.9% of blood meals were found with various trypanosome species including 42.6% with T. congolense and 24% with T. brucei s.l. This study revealed the presence of three tsetse taxa and the circulation of four trypanosome taxa in villages of the Faro and Deo division. About 45% of captured tsetse fly are infected with trypanosome species causing AAT. Tsetse flies feed on humans, cattle and many other vertebrates. Strategies to eliminate the vectors must be improved to reduce the pathological impacts of trypanosome infections in this area.

Entomological assessment of tsetse-borne trypanosome risk in the Shimba Hills human-wildlife-livestock interface, Kenya.

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.

Seasonal Patterns: Bovine Trypanosomosis, Glossina pallidipes Density, and Infection in Rift Valleys of Gamo Zone, Southern Ethiopia.

Bovine trypanosomosis is a parasitic disease causing serious economic losses in livestock productivity and agricultural development. The disease has been reported in different parts of Ethiopia. However, seasonal pattern of trypanosomosis, tsetse fly apparent density, and infection are very limited in the southern rift valley of the country, particularly in Gamo Zone. Therefore, the objective of this cross-sectional study design was to estimate seasonal prevalence of bovine trypanosomosis, assessing tsetse fly apparent density and its infection by trypanosomes. For the parasitological study, a total of 600 cattle (300 in each season) were sampled and assayed using the buffy coat technique. A total of 80 standard NGU traps were deployed around the watering and grazing areas for the entomological survey. An overall prevalence of trypanosomosis was 10.17% (61/600), of which 7.33% (22/300) and 13% (39/300) accounted for the dry and wet seasons, respectively. The prevalence of trypanosomosis was significantly higher during the wet season (OR = 2.47; p < 0.05), in black coat color (OR = 7.2, p < 0.05), and poor body-conditioned (OR = 3.15; p < 0.05) animals. Two species of trypanosomes, Trypanosoma congolense, 68.85% (42/61), and Trypanosoma vivax, 31.15% (19/61), were circulating in the area. The mean PCV value in infected animals (22.56 ± 4.61) was significantly lower than in non-infected animals (25.3 ± 4.75). Entomological result indicated that Glossina pallidipes (G. pallidipes) was the only species of tsetse found in the study area. Totally, 3,789 flies were caught of which 81.42% (3,085/3,789) belong to G. pallidipes and 18.58% (704/3,789) were other biting flies. The overall apparent density of G. pallidipes was 12.85 flies/trap/day (FTD). Relatively higher G. pallidipes/trap/day were caught in the wet season (13.64 F/T/D) than in the dry season (12.07F/T/D). Of the flies caught, 342 G. pallidipes were randomly selected and dissected. The overall proportion of G. pallidipes infection was 18.42% (63/342) of which 12.28% (21/171) and 24.56% (42/171) were accounted in the dry and wet seasons, respectively. Infection in G. pallidipes was significantly higher during the wet season (OR = 2.32; p < 0.05) and in park grazing areas (OR = 2.45; p < 0.05). In conclusion, trypanosomosis is the major challenge for cattle productivity in the district. So this study warrants the need for strengthening the vector and parasite control interventions in the area.

Perspectives on Odor-Based Control of Tsetse Flies in Africa.

Tsetse-transmitted trypanosomiases are among the most neglected tropical diseases in sub-Sahara Africa. Although all tsetse species are susceptible to trypanosome infections, their differential attraction/feeding preferences for different wildlife, domestic animals, and/or humans constitute critical determinants of trypanosomes species they predominantly transmit. Artificial bait technologies, based on long-range tsetse olfactory responses to natural cues emitted by preferred hosts and blends of synthetic versions that mimic these cues, have successfully been applied in attractant-odor-based ("pull" tactic) reduction of field populations of some tsetse species. Olfactory attribute associated with active avoidance of tsetse-refractory non-hosts has similarly been exploited in design of repellent-odor-based ("push" tactic) protection of livestock. These tactics have opened possibility of spatially strategic deployment of the two sets of odor baits in "push-pull" tactics. Possibility of developing blends with enhanced attraction and repellence compared with those associated with savannah tsetse fly hosts and non-hosts, respectively, have been explored, where structure activity and blends of different components generated two novel blends. The studies evaluated structure activity and blends of different components. One based on attractive constituents associated with buffalo (Syncerus caffer) comprised of ε-nonalactone, nonanoic acid, 2-nonanone (in 1:3:2 proportion) delivered together with acetone, which showed significantly better attractancy on savannah tsetse fly than the standard blend comprised of 3-propylphenol, octenol, p-cresol, and acetone (POCA). The other blend comprised of δ-nonalactone, heptanoic acid, 4-methylguaiacol and geranylacetone (in 6:4:2:1 proportion) was significantly more repellent than previously characterized blend based on tsetse fly refractory waterbuck (Kobus defassa) constituents (δ-octalactone, pentanoic acid, guaiacol and geranylacetone). So far, no effective attractants or repellents of riverine tsetse fly species have been characterized. Optimized attractant and repellent blends for savannah tsetse flies lay down useful groundwork for future development of the "push-pull" deployment tactic for area-wide control of tsetse flies. Better understanding of the physiological, cellular, and molecular basis of response in the tsetse fly to odors can potentially augment the current tsetse fly-control interventions.

An atlas to support the progressive control of tsetse-transmitted animal trypanosomosis in Burkina Faso.

BACKGROUND: African animal trypanosomosis (AAT), transmitted by tsetse flies, is arguably the main disease constraint to integrated crop-livestock agriculture in sub-Saharan Africa, and African heads of state and governments adopted a resolution to rid the continent of this scourge. In order to sustainably reduce or eliminate the burden of AAT, a progressive and evidence-based approach is needed, which must hinge on harmonized, spatially explicit information on the occurrence of AAT and its vectors. METHODS: A digital repository was assembled, containing tsetse and AAT data collected in Burkina Faso between 1990 and 2019. Data were collected either in the framework of control activities or for research purposes. Data were systematically verified, harmonized, georeferenced and integrated into a database (PostgreSQL). Entomological data on tsetse were mapped at the level of individual monitoring traps. When this was not possible, mapping was done at the level of site or location. Epidemiological data on AAT were mapped at the level of location or village. RESULTS: Entomological data showed the presence of four tsetse species in Burkina Faso. Glossina tachinoides, present from the eastern to the western part of the country, was the most widespread and abundant species (56.35% of the catches). Glossina palpalis gambiensis was the second most abundant species (35.56%), and it was mainly found in the west. Glossina morsitans submorsitans was found at lower densities (6.51%), with a patchy distribution in the southern parts of the country. A single cluster of G. medicorum was detected (less than 0.25%), located in the south-west. Unidentified tsetse flies accounted for 1.33%. For the AAT component, data for 54,948 animal blood samples were assembled from 218 geographic locations. The samples were tested with a variety of diagnostic methods. AAT was found in all surveyed departments, including the tsetse-free areas in the north. Trypanosoma vivax and T. congolense infections were the dominant ones, with a prevalence of 5.19 ± 18.97% and 6.11 ± 21.56%, respectively. Trypanosoma brucei infections were detected at a much lower rate (0.00 ± 0.10%). CONCLUSIONS: The atlas provides a synoptic view of the available information on tsetse and AAT distribution in Burkina Faso. Data are very scanty for most of the tsetse-free areas in the northern part of the country. Despite this limitation, this study generated a robust tool for targeting future surveillance and control activities. The development of the atlas also strengthened the collaboration between the different institutions involved in tsetse and AAT research and control in Burkina Faso, which will be crucial for future updates and the sustainability of the initiative.