Impact of MiraNet® long-lasting insecticidal net against Anopheles arabiensis wild population of Northern Tanzania.

Despite high levels of pyrethroid resistance reported in malaria vectors, long-lasting insecticidal nets (LNs) still play a key role in controlling malaria transmission. This study tested the efficacy of MiraNet®, a pyrethroid-based LN against a wild population of Anopheles arabiensis in northern Tanzania. DuraNet® was used as a positive control in this evaluation. Standard WHO laboratory bioefficacy evaluations of MiraNet and DuraNet that were unwashed or had been washed 20 times indicated optimal knockdown and mortality for both net types against a susceptible strain of Anopheles gambiae s.s. Standard experimental hut evaluations were conducted to evaluate the efficacy of both nets against a wild population of An. arabiensis. The killing effect of MiraNet was 54.5% for unwashed and 50% for 20 times washed while DuraNet achieved 44.4% mortality for unwashed and 47.4% for 20 times washed against wild An. arabiensis. Both DuraNet and MiraNet exhibited significantly higher killing effects (> 44.4%). There was no significant difference in deterrence or induced exophily detected between the treatment arms for either net. Additionally, there were no adverse effects reported among hut sleepers. The results of this study indicate that the pyrethroid net MiraNet can be used effectively against wild populations of An. gambiae s.l. of low to moderate resistant levels from Northern Tanzania.

Population Structure and Migration Patterns of the Tsetse Fly Glossina fuscipes in Congo-Brazzaville.

Tsetse flies of the palpalis group, particularly Glossina fuscipes, are the main vectors of human African trypanosomiasis or sleeping sickness in Congo-Brazzaville. They transmit the deadly human parasite, Trypanosoma brucei gambiense and other trypanosomes that cause animal trypanosomiasis. Knowledge on diversity, population structure, population size, and gene flow is a prerequisite for designing effective tsetse control strategies. There is limited published information on these parameters including migration patterns of G. fuscipes in Congo-Brazzaville. We genotyped 288 samples of G. fuscipes from Bomassa (BMSA), Bouemba (BEMB), and Talangai (TLG) locations at 10 microsatellite loci and determined levels of genetic diversity, differentiation, structuring, and gene flow among populations. We observed high genetic diversity in all three localities. Mean expected heterozygosity was 0.77 ± 0.04, and mean allelic richness was 11.2 ± 1.35. Deficiency of heterozygosity was observed in all populations with positive and significant F IS values (0.077-0.149). Structure analysis revealed three clusters with genetic admixtures, evidence of closely related but potentially different taxa within G. fuscipes. Genetic differentiation indices were low but significant (F ST = 0.049, P < 0.05), indicating ongoing gene flow countered with a stronger force of drift. We recorded significant migration from all the three populations, suggesting exchange of genetic information between and among locations. Ne estimates revealed high and infinite population sizes in BEMB and TLG. These critical factors should be considered when planning area-wide tsetse control interventions in the country to prevent resurgence of tsetse from relict populations and/or reinvasion of cleared habitats.

Genetic diversity and phylogenetic relationships of tsetse flies of the palpalis group in Congo Brazzaville based on mitochondrial cox1 gene sequences.

BACKGROUND: Despite the morphological characterization established in the 1950s and 1960s, the identity of extant taxa that make up Glossina fuscipes (s.l.) in the Congo remains questionable. Previous claims of overlap between G. fuscipes (believed to be G. f. quanzensis) and G. palpalis palpalis around Brazzaville city further complicate the taxonomic status and population dynamics of the two taxa. This study aimed to determine the phylogenetic relationships between G. fuscipes (s.l.) and G. p. palpalis and to assess genetic variation among G. fuscipes (s.l.) populations in Congo Brazzaville. METHODS: We collected 263 G. fuscipes (s.l.) from northern and central regions, and 65 G. p. palpalis from southern part of the country. The mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was amplified using taxa-specific primer pairs. Sequence data were analyzed in DnaSP and Arlequin to assess the genetic diversity, differentiation and demographic history of G. fuscipes (s.l.) populations. RESULTS: The general BLAST analysis yielded a similarity of 99% for G. fuscipes (s.l.) and G. p. palpalis. BLASTn analysis for G. fuscipes (s.l.) showed > 98% identity with GenBank sequences for G. fuscipes (s.l.), with BEMB population showing 100% similarity with G. f. fuscipes. Glossina fuscipes (s.l.) populations showed high haplotype diversity (H = 46, Hd = 0.884), moderate nucleotide diversity ( = 0.012) and moderate (FST = 0.072) to high (FST = 0.152) genetic differentiation. Most of the genetic variation (89.73%) was maintained within populations. The mismatch analysis and neutrality tests indicated recent tsetse population expansions. CONCLUSIONS: Phylogenetic analysis revealed minor differences between G. fuscipes (s.l.) and G. p. palpalis. Genetic diversity of G. fuscipes (s.l.) was high in the populations sampled except one. Genetic differentiation ranged from moderate to high among subpopulations. There was a restricted gene flow between G. fuscipes (s.l.) populations in the north and central part of the country. Genetic signatures based on cox1 showed recent expansion and recovery of G. fuscipes (s.l.) populations from previous bottlenecks. To fully understand the species distribution limits, we recommend further studies involving a wider sampling scheme including the swampy Mossaka focus for G. fuscipes (s.l.) and the entire range of G. p. palpalis in South Congo.

Temporal genetic differentiation in Glossina pallidipes tsetse fly populations in Kenya.

BACKGROUND: Glossina pallidipes is a major vector of both Human and Animal African Trypanosomiasis (HAT and AAT) in Kenya. The disease imposes economic burden on endemic regions in Kenya, including south-western Kenya, which has undergone intense but unsuccessful tsetse fly control measures. We genotyped 387 G. pallidipes flies at 13 microsatellite markers to evaluate levels of temporal genetic variation in two regions that have been subjected to intensive eradication campaigns from the 1960s to the 1980s. One of the regions, Nguruman Escarpment, has been subject to habitat alteration due to human activities, while the other, Ruma National Park, has not. In addition, Nguruman Escarpment is impacted by the movement of grazing animals into the area from neighboring regions during the drought season. We collected our samples from three geographically close sampling sites for each of the two regions. Samples were collected between the years 2003 and 2015, spanning ~96 tsetse fly generations. RESULTS: We established that allelic richness averaged 3.49 and 3.63, and temporal Ne estimates averaged 594 in Nguruman Escarpment and 1120 in Ruma National Park. This suggests that genetic diversity is similar to what was found in previous studies of G. pallidipes in Uganda and Kenya, implying that we could not detect a reduction in genetic diversity following the extensive control efforts during the 1960s to the 1980s. However, we did find differences in temporal patterns of genetic variation between the two regions, indicated by clustering analysis, pairwise FST, and Fisher's exact tests for changes in allele and genotype frequencies. In Nguruman Escarpment, findings indicated differentiation among samples collected in different years, and evidence of local genetic bottlenecks in two locations previous to 2003, and between 2009 and 2015. In contrast, there was no consistent evidence of differentiation among samples collected in different years, and no evidence of local genetic bottlenecks in Ruma National Park. CONCLUSION: Our findings suggest that, despite extensive control measures especially between the 1960s and the 1980s, tsetse flies in these regions persist with levels of genetic diversity similar to that found in populations that did not experience extensive control measures. Our findings also indicate temporal genetic differentiation in Nguruman Escarpment detected at a scale of > 80 generations, and no similar temporal differentiation in Ruma National Park. The different level of temporal differentiation between the two regions indicates that genetic drift is stronger in Nugruman Escarpment, for as-yet unknown reasons, which may include differences in land management. This suggests land management may have an impact on G. pallidipes population genetics, and reinforces the importance of long term monitoring of vector populations in estimates of parameters needed to model and plan effective species-specific control measures.

Genetic diversity of Glossina fuscipes fuscipes along the shores of Lake Victoria in Tanzania and Kenya: implications for management.

BACKGROUND: Tsetse flies (Diptera: Glossinidae) are sole vectors for trypanosomiasis, which affect human health and livestock productivity in Africa. Little is known about the genetic diversity of Glossina fuscipes fuscipes, which is an important species in Tanzania and Kenya. The main objective of the study was to provide baseline data to determine the genetic variability and divergence of G. f. fuscipes in the Lake Victoria basin of Tanzania and Kenya in order to guide future vector control efforts in the region. FINDINGS: Two hundred and seventy five G. f. fuscipes from 8 sites along the shores of Lake Victoria were screened for genetic polymorphisms at 19 microsatellite loci. Samples were collected from two sites in Kenya and six sites in Tanzania. Four of the Tanzanian sites were located in the Rorya district, on the eastern shores of Lake Victoria, while the other two sites were from Ukerewe and Bukoba districts from the southern and western Lake Victoria shores, respectively. Four genetically distinct allopatric clusters were revealed by microsatellite analysis, which sorted the sampling sites according to geography, with sites separated by as little as ~65 km belonging to distinct genetic clusters, while samples located within ~35 km from each other group in the same cluster. CONCLUSION: Our results suggest that there is ongoing genetic admixture within sampling sites located ~35 km from each other, while sites located ~65 km apart are genetically isolated from each other. Similar patterns emerged from a parallel study on G. f. fuscipes analyzed from the Lake Victoria Uganda shores. From a control perspective these results suggest that for sites within the same genetic cluster, control efforts should be carried out in a coordinated fashion in order to avoid re-invasions. Future work should focus on better quantifying the extent and spatial patterns of the observed genetic discontinuities of the G. f. fuscipes populations along the Tanzanian shores. This will aid in their control by providing guidelines on the geographical extent of the area to be treated at the same time.

Comparative performance of traps in catching tsetse flies (Diptera: Glossinidae) in Tanzania.

This study was conducted to determine the efficiency of different tsetse traps in 28 sites across Tanzania. The traps used were biconical, H, NGU, NZI, pyramidal, S3, mobile, and sticky panels. Stationary traps were deployed at a distance of 200 m apart and examined 72 h after deployment. The results showed that 117 (52.2%) out of the 224 traps deployed captured at least one Glossina species. A total of five Glossina species were captured, namely Glossina brevipalpis, Glossina pallidipes, Glossina swynnertoni, Glossina morsitans, and Glossina fuscipes martinii. Biconical traps caught tsetse flies in 27 sites, pyramidal in 26, sticky panel in 20, mobile in 19, S3 in 15, NGU in 7, H in 2 and NZI in 1. A total of 21 107 tsetse flies were trapped, with the most abundant species being G. swynnertoni (55.9%), followed by G. pallidipes (31.1%), G. fuscipes martinii (6.9%) and G. morsitans (6.0%). The least caught was G. brevipalpis (0.2%). The highest number of flies were caught by NGU traps (32.5%), followed by sticky panel (16%), mobile (15.4%), pyramidal (13.0%), biconical (11.3%) and S3 (10.2%). NZI traps managed to catch 0.9% of the total flies and H traps 0.7%. From this study, it can be concluded that the most efficient trap was NGU, followed by sticky panel and mobile, in that order. Therefore, for tsetse fly control programmes, NGU traps could be the better choice. Conversely, of the stationary traps, pyramidal and biconical traps captured tsetse flies in the majority of sites, covering all three ecosystems better than any other traps; therefore, they would be suitable for scouting for tsetse infestation in any given area, thus sparing the costs of making traps for each specific Glossina species.

Mitochondrial DNA sequence divergence and diversity of Glossina fuscipes fuscipes in the Lake Victoria basin of Uganda: implications for control.

BACKGROUND: Glossina fuscipes fuscipes is the main vector of African Trypanosomiasis affecting both humans and livestock in Uganda. The human disease (sleeping sickness) manifests itself in two forms: acute and chronic. The Lake Victoria basin in Uganda has the acute form and a history of tsetse re-emergence despite concerted efforts to control tsetse. The government of Uganda has targeted the basin for tsetse eradication. To provide empirical data for this initiative, we screened tsetse flies from the basin for genetic variation at the mitochondrial DNA cytochrome oxidase II (mtDNA COII) gene with the goal of investigating genetic diversity and gene flow among tsetse, tsetse demographic history; and compare these results with results from a previous study based on microsatellite loci data in the same area. METHODS: We collected 429 Gff tsetse fly samples from 14 localities in the entire Ugandan portion of the Lake Victoria coast, covering 40,000 km(2). We performed genetic analyses on them and added data collected for 56 Gff individuals from 4 additional sampling sites in the basin. The 529 pb partial mitochondrial DNA cytochrome oxidase II (mtDNA COII) sequences totaling 485 were analysed for genetic differentiation, structuring and demographic history. The results were compared with findings from a previous study based on microsatellite loci data from the basin. RESULTS: The differences within sampling sites explained a significant proportion of the genetic variation. We found three very closely related mtDNA population clusters, which co-occurred in multiple sites. Although Φ ST (0 - 0.592; P < 0.05) and Bayesian analyses suggest some level of weak genetic differentiation, there is no correlation between genetic divergence and geographic distance (r = 0.109, P = 0.185), and demographic tests provide evidence of locality-based demographic history. CONCLUSION: The mtDNA data analysed here complement inferences made in a previous study based on microsatellite data. Given the differences in mutation rates, mtDNA afforded a look further back in time than microsatellites and revealed that Gff populations were more connected in the past. Microsatellite data revealed more genetic structuring than mtDNA. The differences in connectedness and structuring over time could be related to vector control efforts. Tsetse re-emergence after control interventions may be due to re-invasions from outside the treated areas, which emphasizes the need for an integrated area-wide tsetse eradication strategy for sustainable removal of the tsetse and trypanosomiasis problem from this area.

Recent outbreaks of rift valley Fever in East Africa and the middle East.

Rift Valley fever (RVF) is an important neglected, emerging, mosquito-borne disease with severe negative impact on human and animal health. Mosquitoes in the Aedes genus have been considered as the reservoir, as well as vectors, since their transovarially infected eggs withstand desiccation and larvae hatch when in contact with water. However, different mosquito species serve as epizootic/epidemic vectors of RVF, creating a complex epidemiologic pattern in East Africa. The recent RVF outbreaks in Somalia (2006-2007), Kenya (2006-2007), Tanzania (2007), and Sudan (2007-2008) showed extension to districts, which were not involved before. These outbreaks also demonstrated the changing epidemiology of the disease from being originally associated with livestock, to a seemingly highly virulent form infecting humans and causing considerably high-fatality rates. The amount of rainfall is considered to be the main factor initiating RVF outbreaks. The interaction between rainfall and local environment, i.e., type of soil, livestock, and human determine the space-time clustering of RVF outbreaks. Contact with animals or their products was the most dominant risk factor to transfer the infection to humans. Uncontrolled movement of livestock during an outbreak is responsible for introducing RVF to new areas. For example, the virus that caused the Saudi Arabia outbreak in 2000 was found to be the same strain that caused the 1997-98 outbreaks in East Africa. A strategy that involves active surveillance with effective case management and diagnosis for humans and identifying target areas for animal vaccination, restriction on animal movements outside the affected areas, identifying breeding sites, and targeted intensive mosquito control programs has been shown to succeed in limiting the effect of RVF outbreak and curb the spread of the disease from the onset.

Wolbachia, Sodalis and trypanosome co-infections in natural populations of Glossina austeni and Glossina pallidipes.

BACKGROUND: Tsetse flies harbor at least three bacterial symbionts: Wigglesworthia glossinidia, Wolbachia pipientis and Sodalis glossinidius. Wigglesworthia and Sodalis reside in the gut in close association with trypanosomes and may influence establishment and development of midgut parasite infections. Wolbachia has been shown to induce reproductive effects in infected tsetse. This study was conducted to determine the prevalence of these endosymbionts in natural populations of G. austeni and G. pallidipes and to assess the degree of concurrent infections with trypanosomes. METHODS: Fly samples analyzed originated from Kenyan coastal forests (trapped in 2009-2011) and South African G. austeni collected in 2008. The age structure was estimated by standard methods. G. austeni (n=298) and G. pallidipes (n= 302) were analyzed for infection with Wolbachia and Sodalis using PCR. Trypanosome infection was determined either by microscopic examination of dissected organs or by PCR amplification. RESULTS: Overall we observed that G. pallidipes females had a longer lifespan (70 d) than G. austeni (54 d) in natural populations. Wolbachia infections were present in all G. austeni flies analysed, while in contrast, this symbiont was absent from G. pallidipes. The density of Wolbachia infections in the Kenyan G. austeni population was higher than that observed in South African flies. The infection prevalence of Sodalis ranged from 3.7% in G. austeni to about 16% in G. pallidipes. Microscopic examination of midguts revealed an overall trypanosome infection prevalence of 6% (n = 235) and 5% (n = 552), while evaluation with ITS1 primers indicated a prevalence of about 13% (n = 296) and 10% (n = 302) in G. austeni and G. pallidipes, respectively. The majority of infections (46%) were with T. congolense. Co-infection with all three organisms was observed at 1% and 3.3% in G. austeni and G. pallidipes, respectively. Eleven out of the thirteen (85%) co-infected flies harboured T. congolense and T. simiae parasites. While the association between trypanosomes and Sodalis infection was statistically significant in G. pallidipes (P = 0.0127), the number of co-infected flies was too few for a definite conclusion. CONCLUSIONS: The tsetse populations analyzed differed in the prevalence of symbionts, despite being sympatric and therefore exposed to identical environmental factors. The density of infections with Wolbachia also differed between G. austeni populations. There were too few natural co-infections detected with the Sodalis and trypanosomes to suggest extensive inter-relations between these infections in natural populations. We discuss these findings in the context of potential symbiont-mediated control interventions to reduce parasite infections and/or fly populations.

Comparative diagnostic and analytical performance of PCR and LAMP-based trypanosome detection methods estimated using pooled whole tsetse flies and midguts.

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.

Prevalence of SGHV among tsetse species of economic importance in Tanzania and their implication for SIT application.

Sterile Insect technique is an important component in area-wide integrated tsetse control. The presence of the salivary glands hypertrophy virus (SGHV) in the wild tsetse, which are the seeds for colony adaptations in the laboratory has become a stumbling block in establishing and maintaining colonies in the laboratory. The virus is transmitted both vertically (in the wild) and horizontally (in the laboratory). However, its prevalence is magnified in the laboratory as a result of the use of in vitro membrane feeding regimen. Fly species of Glossina fuscipes fuscipes, G. pallidipes, G. morsitans and G. swynnertoni were collected from the coastal and inland areas of Tanzania and virus infection rates were assessed microscopically and by PCR. The data showed that in a period of 4years, the virus was present in all species tested irrespective of their ages, sex, and season of the year. However, infection levels differed among species and from one location to another. Symptomatic infection determined by dissection was 1.2% (25/2164) from the coast as compared to 0.4% (6/1725) for inland collected flies. PCR analysis indicated a higher infection rate of 19.81% (104/525) of asymptomatic flies. From these observations, we conclude that care should be taken when planning to initiate tsetse laboratory colonies for use in SIT eradication program. All efforts should be made to select non-infected flies when initiating laboratory colonies and to try to minimize the infection with SGHV. Also management of SGHV infection in the established colony should be applied.

The population structure of Glossina fuscipes fuscipes in the Lake Victoria basin in Uganda: implications for vector control.

BACKGROUND: Glossina fuscipes fuscipes is the primary vector of trypanosomiasis in humans and livestock in Uganda. The Lake Victoria basin has been targeted for tsetse eradication using a rolling carpet initiative, from west to east, with four operational blocks (3 in Uganda and 1 in Kenya), under a Pan-African Tsetse and Trypanosomiasis Eradication Campaign (PATTEC). We screened tsetse flies from the three Ugandan PATTEC blocks for genetic diversity at 15 microsatellite loci from continental and offshore populations to provide empirical data to support this initiative. METHODS: We collected tsetse samples from 11 sites across the Lake Victoria basin in Uganda. We performed genetic analyses on 409 of the collected tsetse flies and added data collected for 278 individuals in a previous study. The flies were screened across 15 microsatellite loci and the resulting data were used to assess the temporal stability of populations, to analyze patterns of genetic exchange and structuring, to estimate dispersal rates and evaluate the sex bias in dispersal, as well as to estimate demographic parameters (NE and NC). RESULTS: We found that tsetse populations in this region were stable over 4-16 generations and belong to 4 genetic clusters. Two genetic clusters (1 and 2) corresponded approximately to PATTEC blocks 1 and 2, while the other two (3 and 4) fell within PATTEC block 3. Island populations grouped into the same genetic clusters as neighboring mainland sites, suggesting presence of gene flow between these sites. There was no evidence of the stretch of water separating islands from the mainland forming a significant barrier to dispersal. Dispersal rates ranged from 2.5 km per generation in cluster 1 to 14 km per generation in clusters 3 and 4. We found evidence of male-biased dispersal. Few breeders are successfully dispersing over large distances. Effective population size estimates were low (33-310 individuals), while census size estimates ranged from 1200 (cluster 1) to 4100 (clusters 3 and 4). We present here a novel technique that adapts an existing census size estimation method to sampling without replacement, the scheme used in sampling tsetse flies. CONCLUSION: Our study suggests that different control strategies should be implemented for the three PATTEC blocks and that, given the high potential for re-invasion from island sites, mainland and offshore sites in each block should be targeted at the same time.

Polyandry is a common event in wild populations of the Tsetse fly Glossina fuscipes fuscipes and may impact population reduction measures.

BACKGROUND: Glossina fuscipes fuscipes is the main vector of human and animal trypanosomiasis in Africa, particularly in Uganda. Attempts to control/eradicate this species using biological methods require knowledge of its reproductive biology. An important aspect is the number of times a female mates in the wild as this influences the effective population size and may constitute a critical factor in determining the success of control methods. To date, polyandry in G.f. fuscipes has not been investigated in the laboratory or in the wild. Interest in assessing the presence of remating in Ugandan populations is driven by the fact that eradication of this species is at the planning stage in this country. METHODOLOGY/PRINCIPAL FINDINGS: Two well established populations, Kabukanga in the West and Buvuma Island in Lake Victoria, were sampled to assess the presence and frequency of female remating. Six informative microsatellite loci were used to estimate the number of matings per female by genotyping sperm preserved in the female spermathecae. The direct count of the minimum number of males that transferred sperm to the spermathecae was compared to Maximum Likelihood and Bayesian probability estimates. The three estimates provided evidence that remating is common in the populations but the frequency is substantially different: 57% in Kabukanga and 33% in Buvuma. CONCLUSIONS/SIGNIFICANCE: The presence of remating, with females maintaining sperm from different mates, may constitute a critical factor in cases of re-infestation of cleared areas and/or of residual populations. Remating may enhance the reproductive potential of re-invading propagules in terms of their effective population size. We suggest that population age structure may influence remating frequency. Considering the seasonal demographic changes that this fly undergoes during the dry and wet seasons, control programmes based on SIT should release large numbers of sterile males, even in residual surviving target populations, in the dry season.

Genetic diversity and population structure of Glossina pallidipes in Uganda and western Kenya.

BACKGROUND: Glossina pallidipes has been implicated in the spread of sleeping sickness from southeastern Uganda into Kenya. Recent studies indicated resurgence of G. pallidipes in Lambwe Valley and southeastern Uganda after what were deemed to be effective control efforts. It is unknown whether the G. pallidipes belt in southeastern Uganda extends into western Kenya. We investigated the genetic diversity and population structure of G. pallidipes in Uganda and western Kenya. RESULTS: AMOVA indicated that differences among sampling sites explained a significant proportion of the genetic variation. Principal component analysis and Bayesian assignment of microsatellite genotypes identified three distinct clusters: western Uganda, southeastern Uganda/Lambwe Valley, and Nguruman in central-southern Kenya. Analyses of mtDNA confirmed the results of microsatellite analysis, except in western Uganda, where Kabunkanga and Murchison Falls populations exhibited haplotypes that differed despite homogeneous microsatellite signatures. To better understand possible causes of the contrast between mitochondrial and nuclear markers we tested for sex-biased dispersal. Mean pairwise relatedness was significantly higher in females than in males within populations, while mean genetic distance was lower and relatedness higher in males than females in between-population comparisons. Two populations sampled on the Kenya/Uganda border, exhibited the lowest levels of genetic diversity. Microsatellite alleles and mtDNA haplotypes in these two populations were a subset of those found in neighboring Lambwe Valley, suggesting that Lambwe was the source population for flies in southeastern Uganda. The relatively high genetic diversity of G. pallidipes in Lambwe Valley suggest large relict populations remained even after repeated control efforts. CONCLUSION: Our research demonstrated that G. pallidipes populations in Kenya and Uganda do not form a contiguous tsetse belt. While Lambwe Valley appears to be a source population for flies colonizing southeastern Uganda, this dispersal does not extend to western Uganda. The complicated phylogeography of G. pallidipes warrants further efforts to distinguish the role of historical and modern gene flow and possible sex-biased dispersal in structuring populations.

Tracking the feeding patterns of tsetse flies (Glossina genus) by analysis of bloodmeals using mitochondrial cytochromes genes.

Tsetse flies are notoriously difficult to observe in nature, particularly when populations densities are low. It is therefore difficult to observe them on their hosts in nature; hence their vertebrate species can very often only be determined indirectly by analysis of their gut contents. This knowledge is a critical component of the information on which control tactics can be developed. The objective of this study was to determine the sources of tsetse bloodmeals, hence investigate their feeding preferences. We used mitochondrial cytochrome c oxidase 1 (COI) and cytochrome b (cytb) gene sequences for identification of tsetse fly blood meals, in order to provide a foundation for rational decisions to guide control of trypanosomiasis, and their vectors. Glossina swynnertoni were sampled from Serengeti (Tanzania) and G. pallidipes from Kenya (Nguruman and Busia), and Uganda. Sequences were used to query public databases, and the percentage identities obtained used to identify hosts. An initial assay showed that the feeds were from single sources. Hosts identified from blood fed flies collected in Serengeti ecosystem, included buffaloes (25/40), giraffes (8/40), warthogs (3/40), elephants (3/40) and one spotted hyena. In Nguruman, where G. pallidipes flies were analyzed, the feeds were from elephants (6/13) and warthogs (5/13), while buffaloes and baboons accounted for one bloodmeal each. Only cattle blood was detected in flies caught in Busia and Uganda. Out of four flies tested in Mbita Point, Suba District in western Kenya, one had fed on cattle, the other three on the Nile monitor lizard. These results demonstrate that cattle will form an integral part of a control strategy for trypanosomiasis in Busia and Uganda, while different approaches are required for Serengeti and Nguruman ecosystems, where wildlife abound and are the major component of the tsetse fly food source.

Landmarks in the evolution of technologies for identifying trypanosomes in tsetse flies.

Understanding what the trypanosome pathogens are, their vectors and mode of transmission underpin efforts to control the disease they cause in both humans and livestock. The risk of transmission is estimated by determining what proportion of the vector population is carrying the infectious pathogens. This risk also depends on the infectivity of the trypanosomes to humans and livestock. Most livestock pathogens are not infective to humans, whereas the two sub-species that infect humans also infect livestock. As with other infectious diseases, we can therefore trace the foundation of many continuing disease control programs for trypanosomiasis to the discovery of the pathogens and their vectors more than a century ago. Over this period, methods for detecting and identifying trypanosomes have evolved through various landmark discoveries. This review describes the evolution of methods for identifying African trypanosomes in their tsetse fly vectors.

Phylogeography and population structure of Glossina fuscipes fuscipes in Uganda: implications for control of tsetse.

BACKGROUND: Glossina fuscipes fuscipes, a riverine species of tsetse, is the main vector of both human and animal trypanosomiasis in Uganda. Successful implementation of vector control will require establishing an appropriate geographical scale for these activities. Population genetics can help to resolve this issue by characterizing the extent of linkage among apparently isolated groups of tsetse. METHODOLOGY/PRINCIPAL FINDINGS: We conducted genetic analyses on mitochondrial and microsatellite data accumulated from approximately 1000 individual tsetse captured in Uganda and neighboring regions of Kenya and Sudan. Phylogeographic analyses suggested that the largest scale genetic structure in G. f. fuscipes arose from an historical event that divided two divergent mitochondrial lineages. These lineages are currently partitioned to northern and southern Uganda and co-occur only in a narrow zone of contact extending across central Uganda. Bayesian assignment tests, which provided evidence for admixture between northern and southern flies at the zone of contact and evidence for northerly gene flow across the zone of contact, indicated that this structure may be impermanent. On the other hand, microsatellite structure within the southern lineage indicated that gene flow is currently limited between populations in western and southeastern Uganda. Within regions, the average F(ST) between populations separated by less than 100 km was less than approximately 0.1. Significant tests of isolation by distance suggested that gene flow is ongoing between neighboring populations and that island populations are not uniformly more isolated than mainland populations. CONCLUSIONS/SIGNIFICANCE: Despite the presence of population structure arising from historical colonization events, our results have revealed strong signals of current gene flow within regions that should be accounted for when planning tsetse control in Uganda. Populations in southeastern Uganda appeared to receive little gene flow from populations in western or northern Uganda, supporting the feasibility of area wide control in the Lake Victoria region by the Pan African Tsetse and Trypanosomiasis Eradication Campaign.

Loop-mediated Isothermal Amplification (LAMP) test for detection of Trypanosoma evansi strain B.

Camel Trypanosomiasis (Surra) is mainly caused by Trypanosoma evansi strains that express variable surface glycoprotein (VSG) RoTat 1.2. However, in Kenya a second causative strain that does not express RoTat 1.2 VSG (T. evansi type B) has been identified. The prevalence of T. evansi type B largely remains unknown due to inadequate diagnostic assay. This work reports the development of a sensitive and specific diagnostic assay capable of detecting T. evansi type B based on the strategy of Loop-mediated Isothermal Amplification (LAMP) of DNA. The test is rapid and amplification is achieved within 20-25min at 63 degrees C using a real time PCR machine. Restriction enzyme AluI digestion of the amplicon gave the predicted 83bp and 89bp sized bands and the LAMP product melt curves showed consistent melting temperature (T(m)) of approximately 89 degrees C. The assay analytical sensitivity is approximately 0.1tryps/ml while that of classical PCR test targeting the same gene is approximately 10tryps/ml. There was a 100% agreement in detection of the LAMP amplification product in real time, gel electrophoresis, on addition of SYBR Green I, and when using chromatographic Lateral Flow Dipstick (LFD) format. The use of the LAMP test revealed nine more T. evansi type B DNA samples that were not initially detected through PCR. The robustness and higher sensitivity of the T. evansi type B LAMP assay coupled with the visual detection of the amplification product indicate that the technique has strong potential as a point-of-use test in surra endemic areas.