Infection with endosymbiotic Spiroplasma disrupts tsetse (Glossina fuscipes fuscipes) metabolic and reproductive homeostasis.

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.

Genetic and phenotypic parameter estimates for selection within Ugandan indigenous chickens.

The high genetic variation within indigenous chickens (IC) which provides an opportunity to select superior stock for sustainable production and conservation is under-exploited. This study is aimed at estimating heritability and genetic and phenotypic correlation coefficients of productive and reproductive traits of Ugandan IC as a basis for selection. Data on traits were collected across two consecutive generations, weight (W) and shank length (SL) of chicks at hatching (HW) as well as at 2 (W2; SL2), 4 (W4; SL4), 6 (W6; SL6), 8 (W8; SL8), and 12 (W12; SL12) weeks of growth. Body weights at onset of lay (WFE) were also measured. In addition, egg number (EN-60), egg weight (EW), clutch number (CLN-60), and clutch size (CLS-60) over a period of 60 days were recorded. Genetic parameters were estimated using the univariate animal model analysis with restricted maximum likelihood procedure using the variability package of R, version 4.1.1. Heritability of traits ranged from 0.30 and 0.72 except SL4 (0.02), SL12 (0.14), and EN-60 (0.17). The traits EN-60 and W4 were negatively phenotypically correlated (- 0.49). Body weight at first egg was highly genetically correlated (0.99) with SL8. Egg number was significantly, negatively, and genetically correlated (- 0.96) with SL12. In conclusion, shank length is a potential phenotypic marker when selecting for live weight at onset of lay and egg yield. The shank length could, therefore, permit selection of superior chickens at an early age.

Apparent density, trypanosome infection rates and host preference of tsetse flies in the sleeping sickness endemic focus of northwestern Uganda.

BACKGROUND: African trypanosomiasis, caused by protozoa of the genus Trypanosoma and transmitted by the tsetse fly, is a serious parasitic disease of humans and animals. Reliable data on the vector distribution, feeding preference and the trypanosome species they carry is pertinent to planning sustainable control strategies. METHODOLOGY: We deployed 109 biconical traps in 10 villages in two districts of northwestern Uganda to obtain information on the apparent density, trypanosome infection status and blood meal sources of tsetse flies. A subset (272) of the collected samples was analyzed for detection of trypanosomes species and sub-species using a nested PCR protocol based on primers amplifying the Internal Transcribed Spacer (ITS) region of ribosomal DNA. 34 blood-engorged adult tsetse midguts were analyzed for blood meal sources by sequencing of the mitochondrial cytochrome c oxidase 1 (COI) and cytochrome b (cytb) genes. RESULTS: We captured a total of 622 Glossina fuscipes fuscipes tsetse flies (269 males and 353 females) in the two districts with apparent density (AD) ranging from 0.6 to 3.7 flies/trap/day (FTD). 10.7% (29/272) of the flies were infected with one or more trypanosome species. Infection rate was not significantly associated with district of origin (Generalized linear model (GLM), χ2 = 0.018, P = 0.895, df = 1, n = 272) and sex of the fly (χ2 = 1.723, P = 0.189, df = 1, n = 272). However, trypanosome infection was highly significantly associated with the fly's age based on wing fray category (χ2 = 22.374, P < 0.001, df = 1, n = 272), being higher among the very old than the young tsetse. Nested PCR revealed several species of trypanosomes: T. vivax (6.62%), T. congolense (2.57%), T. brucei and T. simiae each at 0.73%. Blood meal analyses revealed five principal vertebrate hosts, namely, cattle (Bos taurus), humans (Homo sapiens), Nile monitor lizard (Varanus niloticus), African mud turtle (Pelusios chapini) and the African Savanna elephant (Loxodonta africana). CONCLUSION: We found an infection rate of 10.8% in the tsetse sampled, with all infections attributed to trypanosome species that are causative agents for AAT. However, more verification of this finding using large-scale passive and active screening of human and tsetse samples should be done. Cattle and humans appear to be the most important tsetse hosts in the region and should be considered in the design of control interventions.

Genomic analysis of Sweet potato feathery mottle virus from East Africa.

Sweet potato feathery mottle virus is a potyvirus that infect sweet potato. The genome of the virus was analysed to understand genetic diversity, evolution and gene flow. Motifs, nucleotide identity and a phylogenetic tree were used to determine phylogroup of the isolates. Gene flow and genetic diversity were tested using DnaSP v.5. Codons evolution were tested using three methods embedded in Datamonkey. The results indicate occurrence of an isolate of phylogroup B within East Africa. Low genetic differentiation was observed between isolates from Kenya and Uganda indicating evidence of gene flow between the two countries. Four genes were found to have positively selected codons bordering or occurring within functional motifs. A motif within P1 gene evolved differently between phylogroup A and B. The evidence of gene flow indicates frequent exchange of the virus between the two countries and P1 gene motif provide a possible marker that can be used for mapping the distribution of the phylogroups.

The population genomics of multiple tsetse fly (Glossina fuscipes fuscipes) admixture zones in Uganda.

Understanding the mechanisms that enforce, maintain or reverse the process of speciation is an important challenge in evolutionary biology. This study investigates the patterns of divergence and discusses the processes that form and maintain divergent lineages of the tsetse fly Glossina fuscipes fuscipes in Uganda. We sampled 251 flies from 18 sites spanning known genetic lineages and the four admixture zones between them. We apply population genomics, hybrid zone and approximate Bayesian computation to the analysis of three types of genetic markers: 55,267 double-digest restriction site-associated DNA (ddRAD) SNPs to assess genome-wide admixture, 16 microsatellites to provide continuity with published data and accurate biogeographic modelling, and a 491-bp fragment of mitochondrial cytochrome oxidase I and II to infer maternal inheritance patterns. Admixture zones correspond with regions impacted by the reorganization of Uganda's river networks that occurred during the formation of the West African Rift system over the last several hundred thousand years. Because tsetse fly population distributions are defined by rivers, admixture zones likely represent both old and new regions of secondary contact. Our results indicate that older hybrid zones contain mostly parental types, while younger zones contain variable hybrid types resulting from multiple generations of interbreeding. These findings suggest that reproductive barriers are nearly complete in the older admixture zones, while nearly absent in the younger admixture zones. Findings are consistent with predictions of hybrid zone theory: Populations in zones of secondary contact transition rapidly from early to late stages of speciation or collapse all together.

Farmers' practices and their knowledge of biotic constraints to sweetpotato production in East Africa.

Sweetpotato (Ipomoea batatas) is a vital crop for overcoming food insecurity in sub-Saharan Africa and its production is highest in East Africa where yields are high and the growing seasons are short. This cross-country study assessed farmers' local practices and their knowledge of the biotic constraints to sweetpotato production in Uganda, Rwanda, Kenya and Tanzania with the aim of providing empirical data that can ultimately be used to enhance sweetpotato production in these four countries. We collected data from 675 households using a standardized questionnaire integrated with a web-based mobile app. Survey results provided strong evidence that sweetpotato is valued as an important subsistence crop among smallholder farmers on pieces of land of less than 0.4 ha, and we observed that females were more involved than males in sweetpotato production. Sweetpotato was ranked as the second most important staple crop after cassava. Farmers noted an increase in sweetpotato production over the past five years in Uganda and Kenya but a decrease in Rwanda and Tanzania; the proportion of farmers who reported a decrease (33%) and an increase (36%) did not significantly differ. The main constraints to production were reported to be pests (32.6%), drought (21.6%), diseases (11.9%) and lack of disease-free planting materials (6.8%). Farmers recognized the signs and symptoms associated with sweetpotato diseases on leaves, root tubers, and whole plants, but most were unable to assign the disease type (bacterial, fungal or viral) correctly. We suggest that regional governments improve education, increase the provision of clean planting materials and strengthen breeding programs to improve disease resistance.

Analysis of the gut-specific microbiome from field-captured tsetse flies, and its potential relevance to host trypanosome vector competence.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Heterogeneity in the prevalence and intensity of bovine trypanosomiasis in the districts of Amuru and Nwoya, Northern Uganda.

BACKGROUND: Livestock trypanosomiasis, transmitted mainly by tsetse flies of the genus Glossina is a major constraint to livestock health and productivity in the sub-Saharan Africa. Knowledge of the prevalence and intensity of trypanosomiasis is important in understanding the epidemiology of the disease. The objectives of this study were to (a) assess the prevalence and intensity of trypanosome infections in cattle, and (b) to investigate the reasons for the heterogeneity of the disease in the tsetse infested districts of Amuru and Nwoya, northern Uganda. METHODS: A cross-sectional study was conducted from September, 2011 to January, 2012. Blood samples were collected from 816 cattle following jugular vein puncture, and screened for trypanosomes by HCT and ITS-PCR. A Pearson chi-squared test and logistic regression analyses were performed to determine the association between location, age, sex, and prevalence of trypanosome infections. RESULTS: Out of the 816 blood samples examined, 178 (22 %) and 338 (41 %) tested positive for trypanosomiasis by HCT and ITS-PCR, respectively. Trypanosoma vivax infection accounted for 77 % of infections detected by ITS-PCR, T. congolense (16 %), T. brucei s.l (4 %) and mixed (T. vivax/ T. congolense/T.brucei) infections (3 %). The risk of trypanosome infection was significantly associated with cattle age (χ (2) = 220.4, df = 3, P < 0.001). The highest proportions of infected animals were adult males (26.7 %) and the least infected were the less than one year old calves (2.0 %). In addition, the risk of trypanosome infection was significantly associated with sex (χ (2) = 16.64, df = 1, P < 0.001), and males had a significantly higher prevalence of infections (26.8 %) than females (14.6 %). CONCLUSION: Our results indicate that the prevalence and intensity of trypanosome infections are highly heterogeneous being associated with cattle age, location and sex.

Analysis of multiple tsetse fly populations in Uganda reveals limited diversity and species-specific gut microbiota.

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.

Wolbachia association with the tsetse fly, Glossina fuscipes fuscipes, reveals high levels of genetic diversity and complex evolutionary dynamics.

BACKGROUND: Wolbachia pipientis, a diverse group of α-proteobacteria, can alter arthropod host reproduction and confer a reproductive advantage to Wolbachia-infected females (cytoplasmic incompatibility (CI)). This advantage can alter host population genetics because Wolbachia-infected females produce more offspring with their own mitochondrial DNA (mtDNA) haplotypes than uninfected females. Thus, these host haplotypes become common or fixed (selective sweep). Although simulations suggest that for a CI-mediated sweep to occur, there must be a transient phase with repeated initial infections of multiple individual hosts by different Wolbachia strains, this has not been observed empirically. Wolbachia has been found in the tsetse fly, Glossina fuscipes fuscipes, but it is not limited to a single host haplotype, suggesting that CI did not impact its population structure. However, host population genetic differentiation could have been generated if multiple Wolbachia strains interacted in some populations. Here, we investigated Wolbachia genetic variation in G. f. fuscipes populations of known host genetic composition in Uganda. We tested for the presence of multiple Wolbachia strains using Multi-Locus Sequence Typing (MLST) and for an association between geographic region and host mtDNA haplotype using Wolbachia DNA sequence from a variable locus, groEL (heat shock protein 60). RESULTS: MLST demonstrated that some G. f. fuscipes carry Wolbachia strains from two lineages. GroEL revealed high levels of sequence diversity within and between individuals (Haplotype diversity = 0.945). We found Wolbachia associated with 26 host mtDNA haplotypes, an unprecedented result. We observed a geographical association of one Wolbachia lineage with southern host mtDNA haplotypes, but it was non-significant (p = 0.16). Though most Wolbachia-infected host haplotypes were those found in the contact region between host mtDNA groups, this association was non-significant (p = 0.17). CONCLUSIONS: High Wolbachia sequence diversity and the association of Wolbachia with multiple host haplotypes suggest that different Wolbachia strains infected G. f. fuscipes multiple times independently. We suggest that these observations reflect a transient phase in Wolbachia evolution that is influenced by the long gestation and low reproductive output of tsetse. Although G. f. fuscipes is superinfected with Wolbachia, our data does not support that bidirectional CI has influenced host genetic diversity in Uganda.

Prevalence of aflatoxin along processing points of locally made complementary food formulae in northern Uganda: Safety and children's exposure across seasons.

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.

Status of food safety knowledge, attitude, and practices of caregivers of children in northern 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.

Sibling Species Composition and Susceptibility Status of Anopheles gambiae s.l. to Insecticides Used for Indoor Residual Spraying in Eastern Uganda.

Background: Malaria remains one of the most critical disease causing morbidity and mortality in Uganda. Indoor residual spraying (IRS) and the use of insecticide-treated bed nets are currently the predominant malaria vector control interventions. However, the emergence and spread of insecticide resistance among malaria vectors threaten the continued effectiveness of these interventions to control the disease, particularly in high transmission areas. To inform decisions on vector control, the current study evaluated the Anopheles malaria vector species and their susceptibility levels to 0.1% bendiocarb and 0.25% pirimiphos-methyl insecticides used in IRS intervention program in Namutumba district, Eastern Uganda. Methods: Anopheles larvae were collected between March and May 2017 from different breeding sites in the parishes of Nsinze and Nawaikona in Nsinze sub-county and reared to adults to assess the susceptibility status of populations in the study area. Mosquitoes were identified using morphological keys and species-specific polymerase chain reaction (PCR) assays. Susceptibility tests were conducted on 2- to 5-day-old non-blood-fed adult female Anopheles that emerged using insecticide-impregnated papers with 0.1% bendiocarb and 0.25% pirimiphos-methyl following standard World Health Organization (WHO) insecticide susceptibility bioassays. A Log-probit regression model was used to derive the knock-down rates for 50% and 95% of exposed mosquitoes. Results: A total of 700 mosquito larvae were collected from different breeding sites. Morphological identification showed that 500 individuals that emerged belonged to Anopheles gambiae sensu lato (s.l.), the main malaria vector. The PCR results showed that the dominant sibling species under the A. gambiae complex was Anopheles arabiensis 99.5% (395/397). WHO bioassay tests revealed that the population of mosquitoes exhibited high levels of susceptibility (24-hour post-exposure mortality 98-100%) to both insecticides tested. The median knock-down time, KDT50, ranged from 6.6 to 81.4 minutes, while the KDT95 ranged from 21.6 to 118.9 minutes for 0.25% pirimiphos-methyl. The KDT50 for 0.1% bendiocarb ranged from 2.8 to 62.9 minutes, whereas the KDT95 ranged from 36.0 to 88.5 minutes. Conclusions: These findings indicate that bendiocarb and pirimiphos-methyl are still effective against the major malaria vector, A. arabiensis in Nsinze sub-county, Namutumba district, Uganda and can be effectively used for IRS. The study has provided baseline information on the insecticide susceptibility status on malaria vectors in the study area. However, routine continuous monitoring program of insecticide susceptibility and malaria vector composition is required so as to guide future decisions on insecticide use for IRS intervention toward malaria elimination and to track future changes in vector population.

Genomic evidence of sex chromosome aneuploidy and infection-associated genotypes in the tsetse fly Glossina fuscipes, the major vector of African trypanosomiasis in Uganda.

The primary vector of the trypanosome parasite causing human and animal African trypanosomiasis in Uganda is the riverine tsetse fly Glossina fuscipes fuscipes (Gff). Our study improved the Gff genome assembly with whole genome 10× Chromium sequencing of a lab reared pupae, identified autosomal versus sex-chromosomal regions of the genome with ddRAD-seq data from 627 field caught Gff, and identified SNPs associated with trypanosome infection with genome-wide association (GWA) analysis in a subset of 351 flies. Results from 10× Chromium sequencing greatly improved Gff genome assembly metrics and assigned a full third of the genome to the sex chromosome. Results from ddRAD-seq suggested possible sex-chromosome aneuploidy in Gff and identified a single autosomal SNP to be highly associated with trypanosome infection. The top associated SNP was ∼1100 bp upstream of the gene lecithin cholesterol acyltransferase (LCAT), an important component of the molecular pathway that initiates trypanosome lysis and protection in mammals. Results suggest that there may be naturally occurring genetic variation in Gff in genomic regions in linkage disequilibrium with LCAT that can protect against trypanosome infection, thereby paving the way for targeted research into novel vector control strategies that can promote parasite resistance in natural populations.

Implications of microfauna-host interactions for trypanosome transmission dynamics in Glossina fuscipes fuscipes in Uganda.

Tsetse flies (Diptera: Glossinidae) are vectors for African trypanosomes (Euglenozoa: kinetoplastida), protozoan parasites that cause African trypanosomiasis in humans (HAT) and nagana in livestock. In addition to trypanosomes, two symbiotic bacteria (Wigglesworthia glossinidia and Sodalis glossinidius) and two parasitic microbes, Wolbachia and a salivary gland hypertrophy virus (SGHV), have been described in tsetse. Here we determined the prevalence of and coinfection dynamics between Wolbachia, trypanosomes, and SGHV in Glossina fuscipes fuscipes in Uganda over a large geographical scale spanning the range of host genetic and spatial diversity. Using a multivariate analysis approach, we uncovered complex coinfection dynamics between the pathogens and statistically significant associations between host genetic groups and pathogen prevalence. It is important to note that these coinfection dynamics and associations with the host were not apparent by univariate analysis. These associations between host genotype and pathogen are particularly evident for Wolbachia and SGHV where host groups are inversely correlated for Wolbachia and SGHV prevalence. On the other hand, trypanosome infection prevalence is more complex and covaries with the presence of the other two pathogens, highlighting the importance of examining multiple pathogens simultaneously before making generalizations about infection and spatial patterns. It is imperative to note that these novel findings would have been missed if we had employed the standard univariate analysis used in previous studies. Our results are discussed in the context of disease epidemiology and vector control.

Characterization of Ugandan Endemic Aspergillus Species and Identification of Non-Aflatoxigenic Isolates for Potential Biocontrol of Aflatoxins.

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.

Seasonal Monitoring of Glossina Species Occurrence, Infection Rates, and Trypanosoma Species Infections in Pigs in West Nile Region, Uganda.

Introduction: Trypanosomiasis is a parasitic infection caused by the protozoa Trypanosoma. It is exclusively associated with Glossina species habitats and, therefore, restricted to specific geographical settings. It affects a wide range of hosts, including humans. Animals may carry different Trypanosoma spp. while being asymptomatic. They are, therefore, potentially important in unpremeditated disease transmission. Aim: The aim of this study was to study the potential impact of the government tsetse fly control program, and to elucidate the role of pigs in the Trypanosoma epidemiology in the West Nile region in Uganda. Methods: A historically important human African trypanosomiasis (HAT) hotspot was selected, with sampling in sites with and without a government tsetse fly control program. Pigs were screened for infection with Trypanosoma and tsetse traps were deployed to monitor vector occurrence, followed by tsetse fly dissection and microscopy to establish infection rates with Trypanosoma. Pig blood samples were further analyzed to identify possible Trypanosoma infections using internal transcribed spacer (ITS)-PCR. Results: Using microscopy, Trypanosoma was detected in 0.56% (7/1262) of the sampled pigs. Using ITS-PCR, 114 of 341 (33.4%) pig samples were shown to be Trypanosoma vivax positive. Of the 360 dissected tsetse flies, 13 (3.8%) were positive for Trypanosoma under the microscope. The difference in captured tsetse flies in the government intervention sites in comparison with the control sites was significant (p < 0.05). Seasonality did not play a substantial role in the tsetse fly density (p > 0.05). Conclusion: This study illustrated the impact of a government control program with low vector abundance in a historical HAT hotspot in Uganda. The study could not verify that pigs in the area were carriers for the causative agent for HAT, but showed a high prevalence of the animal infectious agent T. vivax.

Population structure, molecular characterization, and phylogenetic analysis of Fasciola gigantica from two locations in Uganda.

Fasciola gigantica is a major pathogen that causes fasciolosis in Africa. A recent study in Uganda demonstrated that Fasciola flukes were present in 65.7% of slaughtered cattle. However, molecular identification of Fasciola species has not yet been performed in the country. In the present study, 292 Fasciola flukes were collected from Kampala and Gulu, Uganda. The samples were identified as F. gigantica using a multiplex polymerase chain reaction (PCR) assay for phosphoenolpyruvate carboxykinase (pepck) and a PCR-restriction fragment length polymorphism (RFLP) assay for DNA polymerase delta (pold). A significant genetic difference between F. gigantica obtained from cattle slaughtered at Kampala and Gulu was observed by analyzing the mitochondrial markers NADH dehydrogenase subunit 1 (nad1) and cytochrome C oxidase subunit 1 (cox1). Fasciola collected from Gulu had a more diversified population than that collected from Kampala, probably because of differences in livestock management systems. One of the possible reasons for this observation is that cattle slaughtered in Gulu were reared under an extensive communal grazing system, which is suitable for maintaining parasite diversity, whereas cattle slaughtered in Kampala mainly originated from fenced/closed farms, which limits parasite diversity. However, the cause of the difference between these two locations was not clearly defined by the results of this study. The F. gigantica population from Uganda was related to that obtained from Zambia. A star-like phylogeny was detected in a median-joining network analysis, which indicated rapid population expansion and suggested that the F. gigantica populations from both countries are maintained by domestic ruminants in eastern Africa. Interestingly, the F. gigantica population from Uganda was not related to those from Egypt and Nigeria. The results of the present study suggest that F. gigantica populations in African countries are indigenous to each country or region.

Spatial Distribution of Tsetse Flies and Trypanosome Infection Status in a Vector Genetic Transition Zone in Northern Uganda.

Background: Tsetse flies are vectors of the genus Trypanosoma that cause African trypanosomiasis, a serious parasitic disease of people and animals. Reliable data on the vector distribution and the trypanosome species they carry is pertinent for planning sustainable control strategies. This study was carried out to estimate the spatial distribution, apparent density, and trypanosome infection rates of tsetse flies in two districts that fall within a vector genetic transition zone in northern Uganda. Materials and Methods: Capturing of tsetse flies was done using biconical traps deployed in eight villages in Oyam and Otuke, two districts that fall within the vector genetic transition zone in northern Uganda. Trapped tsetse flies were sexed and morphologically identified to species level and subsequently analyzed for detection of trypanosome DNA. Trypanosome DNA was detected using a nested PCR protocol based on primers amplifying the internal transcribed spacer (ITS) region of ribosomal DNA. Results: A total of 717 flies (406 females; 311 males) were caught, all belonging to the Glossina fuscipes fuscipes species. The overall average flies/trap/day (FTD) was 2.20 ± 0.3527 (mean ± SE). Out of the 477 (201 male; 276 females) flies analyzed, 7.13% (34/477) were positive for one or more trypanosome species. Three species of bovine trypanosomes were detected, namely, Trypanosoma vivax, 61.76% (21/34), T. congolense, 26.47% (9/34), and T. brucei brucei, 5.88% (2/34), and two cases of mixed infection of T. congolense and T. brucei brucei, 5.88% (2/34). The infection rate was not significantly associated with the sex of the fly (generalized linear model (GLM), χ 2 = 0.051, p = 0.821, df = 1, n = 477) and district of origin (χ 2 = 0.611, p = 0.434, df = 1, n = 477). However, trypanosome infection was highly significantly associated with the fly's age based on wing fray category (χ 2 = 7.56, p = 0.006, df = 1, n = 477), being higher among the very old than the young. Conclusion: The relatively high tsetse density and trypanosome infection rate indicate that the transition zone is a high-risk area for perpetuating animal trypanosomiasis. Therefore, appropriate mitigation measures should be instituted targeting tsetse and other biting flies that may play a role as disease vectors, given the predominance of T. vivax in the tsetse samples.

RETRACTED: Gradual emergence followed by exponential spread of the SARS-CoV-2 Omicron variant in Africa.

The geographic and evolutionary origins of the SARS-CoV-2 Omicron variant (BA.1), which was first detected mid-November 2021 in Southern Africa, remain unknown. We tested 13,097 COVID-19 patients sampled between mid-2021 to early 2022 from 22 African countries for BA.1 by real-time RT-PCR. By November-December 2021, BA.1 had replaced the Delta variant in all African sub-regions following a South-North gradient, with a peak Rt of 4.1. Polymerase chain reaction and near-full genome sequencing data revealed genetically diverse Omicron ancestors already existed across Africa by August 2021. Mutations, altering viral tropism, replication and immune escape, gradually accumulated in the spike gene. Omicron ancestors were therefore present in several African countries months before Omicron dominated transmission. These data also indicate that travel bans are ineffective in the face of undetected and widespread infection.