The invertebrate microbiome contributes to multiple aspects of host physiology, including nutrient supplementation and immune maturation processes. We identified and compared gut microbial abundance ...and diversity in natural tsetse flies from Uganda using five genetically distinct populations of Glossina fuscipes fuscipes and multiple tsetse species (Glossina morsitans morsitans, G. f. fuscipes, and Glossina pallidipes) that occur in sympatry in one location. We used multiple approaches, including deep sequencing of the V4 hypervariable region of the 16S rRNA gene, 16S rRNA gene clone libraries, and bacterium-specific quantitative PCR (qPCR), to investigate the levels and patterns of gut microbial diversity from a total of 151 individuals. Our results show extremely limited diversity in field flies of different tsetse species. The obligate endosymbiont Wigglesworthia dominated all samples (>99%), but we also observed wide prevalence of low-density Sodalis (tsetse's commensal endosymbiont) infections (<0.05%). There were also several individuals (22%) with high Sodalis density, which also carried coinfections with Serratia. Albeit in low density, we noted differences in microbiota composition among the genetically distinct G. f. fuscipes flies and between different sympatric species. Interestingly, Wigglesworthia density varied in different species (10(4) to 10(6) normalized genomes), with G. f. fuscipes having the highest levels. We describe the factors that may be responsible for the reduced diversity of tsetse's gut microbiota compared to those of other insects. Additionally, we discuss the implications of Wigglesworthia and Sodalis density variations as they relate to trypanosome transmission dynamics and vector competence variations associated with different tsetse species.
Tsetse flies (Glossina spp.) house a population-dependent assortment of microorganisms that can include pathogenic African trypanosomes and maternally transmitted endosymbiotic bacteria, the latter ...of which mediate numerous aspects of their host's metabolic, reproductive, and immune physiologies. One of these endosymbionts, Spiroplasma, was recently discovered to reside within multiple tissues of field captured and laboratory colonized tsetse flies grouped in the Palpalis subgenera. In various arthropods, Spiroplasma induces reproductive abnormalities and pathogen protective phenotypes. In tsetse, Spiroplasma infections also induce a protective phenotype by enhancing the fly's resistance to infection with trypanosomes. However, the potential impact of Spiroplasma on tsetse's viviparous reproductive physiology remains unknown. Herein we employed high-throughput RNA sequencing and laboratory-based functional assays to better characterize the association between Spiroplasma and the metabolic and reproductive physiologies of G. fuscipes fuscipes (Gff), a prominent vector of human disease. Using field-captured Gff, we discovered that Spiroplasma infection induces changes of sex-biased gene expression in reproductive tissues that may be critical for tsetse's reproductive fitness. Using a Gff lab line composed of individuals heterogeneously infected with Spiroplasma, we observed that the bacterium and tsetse host compete for finite nutrients, which negatively impact female fecundity by increasing the length of intrauterine larval development. Additionally, we found that when males are infected with Spiroplasma, the motility of their sperm is compromised following transfer to the female spermatheca. As such, Spiroplasma infections appear to adversely impact male reproductive fitness by decreasing the competitiveness of their sperm. Finally, we determined that the bacterium is maternally transmitted to intrauterine larva at a high frequency, while paternal transmission was also noted in a small number of matings. Taken together, our findings indicate that Spiroplasma exerts a negative impact on tsetse fecundity, an outcome that could be exploited for reducing tsetse population size and thus disease transmission.
The tsetse fly (Glossina sp.) midgut is colonized by maternally transmitted and environmentally acquired bacteria. Additionally, the midgut serves as a niche in which pathogenic African trypanosomes ...reside within infected flies. Tsetse's bacterial microbiota impacts many aspects of the fly's physiology. However, little is known about the structure of tsetse's midgut-associated bacterial communities as they relate to geographically distinct fly habitats in east Africa and their contributions to parasite infection outcomes. We utilized culture dependent and independent methods to characterize the taxonomic structure and density of bacterial communities that reside within the midgut of tsetse flies collected at geographically distinct locations in Kenya and Uganda.
Using culture dependent methods, we isolated 34 strains of bacteria from four different tsetse species (G. pallidipes, G. brevipalpis, G. fuscipes and G. fuscipleuris) captured at three distinct locations in Kenya. To increase the depth of this study, we deep sequenced midguts from individual uninfected and trypanosome infected G. pallidipes captured at two distinct locations in Kenya and one in Uganda. We found that tsetse's obligate endosymbiont, Wigglesworthia, was the most abundant bacterium present in the midgut of G. pallidipes, and the density of this bacterium remained largely consistent regardless of whether or not its tsetse host was infected with trypanosomes. These fly populations also housed the commensal symbiont Sodalis, which was found at significantly higher densities in trypanosome infected compared to uninfected flies. Finally, midguts of field-captured G. pallidipes were colonized with distinct, low density communities of environmentally acquired microbes that differed in taxonomic structure depending on parasite infection status and the geographic location from which the flies were collected.
The results of this study will enhance our understanding of the tripartite relationship between tsetse, its microbiota and trypanosome vector competence. This information may be useful for developing novel disease control strategies or enhancing the efficacy of those already in use.
To determine the prevalence of Plasmodium falciparum multi-drug resistant gene-1 (Pfmdr-1) N86Y and D1246Y genotypes among febrile malaria outpatients attending Lira Regional Referral Hospital, ...Uganda.
Overall, 92.3% (n = 48/52) and 90% (n = 45/50) of the parasites detected carried the wild type alleles 1246D and N86, respectively. Only 7.7% (n = 4/52) and 10% (n = 5/50) of these P. falciparum isolates carried the Pfmdr-1 mutant alleles 1246Y and 86Y, respectively. Our results show high prevalence of wild type alleles N86 and D1246 in P. falciparum isolates from Lira Regional Referral Hospital, which could translate to a decreased sensitivity to artemether-lumefantrine. Continued monitoring of prevalence of single nucleotide polymorphisms is warranted to timely inform malaria treatment policies and guidelines.
Acute stunting in children, liver cancer, and death often occur due to human exposure to aflatoxins in food. The severity of aflatoxin contamination depends on the type of Aspergillus fungus ...infecting the crops. In this study, Aspergillus species were isolated from households’ staple foods and were characterized for different aflatoxin chemotypes. The non-aflatoxigenic chemotypes were evaluated for their ability to reduce aflatoxin levels produced by aflatoxigenic A. flavus strains on maize grains. Aspergillus flavus (63%), A. tamarii (14%), and A. niger (23%) were the main species present. The A. flavus species included isolates that predominantly produced aflatoxins B1 and B2, with most isolates producing a high amount (>20 ug/µL) of aflatoxin B1 (AFB1), and a marginal proportion of them also producing G aflatoxins with a higher level of aflatoxin G1 (AFG1) than AFB1. Some non-aflatoxigenic A. tamarii demonstrated a strong ability to reduce the level of AFB1 by more than 95% when co-inoculated with aflatoxigenic A. flavus. Therefore, field evaluation of both non-aflatoxigenic A. flavus and A. tamarii would be an important step toward developing biocontrol agents for mitigating field contamination of crops with aflatoxins in Uganda.
The safety of homemade weaning foods in low‐ and middle‐income countries is of great concern as rural households have limited access to standardized commercial weaning foods. In the Acholi subregion ...of Uganda, complementary foods are locally produced. However, there is limited information on the Food safety knowledge (FSK), food safety attitude (FSA), and food hygiene practices (FHP) of the caregivers. This study examined food safety knowledge, attitude, and practices of the caregivers of children 6–23 months of age in Amuru and Nwoya districts, Northern Uganda, between March 2019 and June 2019. A cross‐sectional study was conducted involving 180 caregivers. Data were collected using semi‐structured questionnaires and focus group discussions and analyzed using descriptive statistics, multivariate binary logistic regression, and thematic content analysis. Caregivers had sufficient FSK (74.1%) and positive FSA (68.1%). However, only 17.6% of them adhered to FHP. Frequency of food safety training (p =.041) and households with children who suffered from foodborne illness (p =.001) significantly predicted FSK. Conversely, both FSK and FSA were significantly predicted by gender roles in decision‐making on household income (p =.006) and households with older children (p =.041). A significant positive correlation was observed between FSK and FSA (r =.406, p =.000). However, major barriers to adherence to FHP were inadequate sanitation facilities and caregiver's workload. The overall nontranslation of sufficient FSK and positive FSA into proper FHP calls for future intervention to harness the sociodemographic factors that influence FSK and FSA and address the barriers to FHP among caregivers.
Aflatoxin contamination along the processing points of locally made complementary food composite needs to be ascertained and minimized to reduce exposure to weaning children. The study established ...the concentrations of total aflatoxin (TAF) and aflatoxin B1 (AFB1) along the processing points of locally made malted millet sesame soybean composite (MMSSC) across season one (wet) and season two (dry) and determined children's exposure to them. A total of 363 samples were collected in 2019. TAF and AFB1 concentrations were determined quantitatively using an enzyme-linked immunosorbent assay (ELISA). Consequently, exposure of individual children was assessed as Estimated Daily Intake (EDI), (ng kg−1 bw day−1). All the samples along the processing points had detectable concentrations of TAF and AFB1 ranging from 0.578 μg kg−1 to 1.187 μg kg−1 and 0.221 μg kg−1 to 0.649 μg kg−1 respectively. Contamination was highest in raw materials; soybean (Glycine max) > sesame (Sesamum indicum), followed by stored composite, freshly prepared composite, and least in millet (Eleusine coracana). Contamination varied significantly across seasons with the wet season having higher contamination than the dry season at P = 0.05. All samples (100%) were within the European Commission (EC) acceptable maximum tolerable level for TAF and AFB1 (4 μg kg−1 and 2 μg kg−1) respectively for processed foods for general consumption. But were below the EU acceptable maximum tolerable level for TAF and AFB1 (0.4 μg kg−1 and 0.1 μg kg−1) respectively for processed baby foods cereals. However, all were within the United States- Food and Drug Authority (US-FDA) and East African Community (EAC) set maximum acceptable limit of 20 μg kg−1 for TAFs, 10 μg kg−1 and 5 μg kg−1 for TAF and AFB1 respectively. Conversely, exposure to these toxins was much higher than the Provisional Maximum Tolerable Dietary Intake (PMTDI) of 0.4 ng kg−1 bw day−1 to 1.0 ng kg−1 bw day−1. A significant difference in exposure to both toxins was observed with the weight. The age of 5 months was the most exposed. A concerted effort is needed to reduce children's exposure to MMSSC to TAF and AFB1, taking sesame and soybean as priority ingredients and proper storage based on season to control contamination.
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•TAF and AFB1 concentrations were highest in raw materials followed by stored composite, then freshly prepared composite.•TAF and AFB1 concentrations were significantly higher in the wet than in the dry season except in stored products.•All the samples met the standard limits for general consumption but were below EU standards for processed cereal baby food.•Estimated Daily intake for both total and AFB1 exceeded the Codex PMTDI with no variation between wet and dry seasons.
Tsetse flies (Glossina spp.) are vectors of parasitic trypanosomes, which cause human (HAT) and animal African trypanosomiasis (AAT) in sub-Saharan Africa. In Uganda, Glossina fuscipes fuscipes (Gff) ...is the main vector of HAT, where it transmits Gambiense disease in the northwest and Rhodesiense disease in central, southeast and western regions. Endosymbionts can influence transmission efficiency of parasites through their insect vectors via conferring a protective effect against the parasite. It is known that the bacterium Spiroplasma is capable of protecting its Drosophila host from infection with a parasitic nematode. This endosymbiont can also impact its host's population structure via altering host reproductive traits. Here, we used field collections across 26 different Gff sampling sites in northern and western Uganda to investigate the association of Spiroplasma with geographic origin, seasonal conditions, Gff genetic background and sex, and trypanosome infection status. We also investigated the influence of Spiroplasma on Gff vector competence to trypanosome infections under laboratory conditions. Generalized linear models (GLM) showed that Spiroplasma probability was correlated with the geographic origin of Gff host and with the season of collection, with higher prevalence found in flies within the Albert Nile (0.42 vs 0.16) and Achwa River (0.36 vs 0.08) watersheds and with higher prevalence detected in flies collected in the intermediate than wet season. In contrast, there was no significant correlation of Spiroplasma prevalence with Gff host genetic background or sex once geographic origin was accounted for in generalized linear models. Additionally, we found a potential negative correlation of Spiroplasma with trypanosome infection, with only 2% of Spiroplasma infected flies harboring trypanosome co-infections. We also found that in a laboratory line of Gff, parasitic trypanosomes are less likely to colonize the midgut in individuals that harbor Spiroplasma infection. These results indicate that Spiroplasma infections in tsetse may be maintained by not only maternal but also via horizontal transmission routes, and Spiroplasma infections may also have important effects on trypanosome transmission efficiency of the host tsetse. Potential functional effects of Spiroplasma infection in Gff could have impacts on vector control approaches to reduce trypanosome infections.
Uganda is the only country where the chronic and acute forms of human African Trypanosomiasis (HAT) or sleeping sickness both occur and are separated by < 100 km in areas north of Lake Kyoga. In ...Uganda, Glossina fuscipes fuscipes is the main vector of the Trypanosoma parasites responsible for these diseases as well for the animal African Trypanosomiasis (AAT), or Nagana. We used highly polymorphic microsatellite loci and a mitochondrial DNA (mtDNA) marker to provide fine scale spatial resolution of genetic structure of G. f. fuscipes from 42 sampling sites from the northern region of Uganda where a merger of the two disease belts is feared. Based on microsatellite analyses, we found that G. f. fuscipes in northern Uganda are structured into three distinct genetic clusters with varying degrees of interconnectivity among them. Based on genetic assignment and spatial location, we grouped the sampling sites into four genetic units corresponding to northwestern Uganda in the Albert Nile drainage, northeastern Uganda in the Lake Kyoga drainage, western Uganda in the Victoria Nile drainage, and a transition zone between the two northern genetic clusters characterized by high level of genetic admixture. An analysis using HYBRIDLAB supported a hybrid swarm model as most consistent with tsetse genotypes in these admixed samples. Results of mtDNA analyses revealed the presence of 30 haplotypes representing three main haplogroups, whose location broadly overlaps with the microsatellite defined clusters. Migration analyses based on microsatellites point to moderate migration among the northern units located in the Albert Nile, Achwa River, Okole River, and Lake Kyoga drainages, but not between the northern units and the Victoria Nile drainage in the west. Effective population size estimates were variable with low to moderate sizes in most populations and with evidence of recent population bottlenecks, especially in the northeast unit of the Lake Kyoga drainage. Our microsatellite and mtDNA based analyses indicate that G. f. fuscipes movement along the Achwa and Okole rivers may facilitate northwest expansion of the Rhodesiense disease belt in Uganda. We identified tsetse migration corridors and recommend a rolling carpet approach from south of Lake Kyoga northward to minimize disease dispersal and prevent vector re-colonization. Additionally, our findings highlight the need for continuing tsetse monitoring efforts during and after control.
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