Value chain hygiene practices and microbial contamination of street and market vended ready-to-eat grasshopper, Ruspolia differens in Uganda: Implications for food safety and public health.

Food safety is a major public health issue particularly in developing countries. Ready-to-eat street-vended foods contribute significantly to dietary intake in urban and peri-urban areas, but with elevated public health risk. In this study, hygiene and food safety practices as well as the microbial contamination in Uganda's edible grasshopper value chain were evaluated."A total of 29 grasshopper-processing households participated, and grasshopper samples collected. Indicator pathogens were analyzed using standard microbiological methods. In Kampala 50% and in Masaka 12% households had earth floors. All households in Kampala were one or two-roomed dwellings with no separate room as a kitchen, and shared a toilet. In contrast, 59% of households in Masaka had three or more rooms, 35% had a separate room for a kitchen and 47% did not share a toilet. 83% households in Kampala and 56% in Masaka obtained drinking water from public taps. Handwashing was inadequate and none of the actors was observed to wash their hands after taking a break or handling waste. For vendors, wearing protective clothing was not common, with only 28.5% in Kampala and 30.8% in Masaka wearing an apron. Containers for vending grasshoppers were largely uncovered and the utensils for measuring the grasshoppers were left mainly uncovered. Indicator organisms, Escherichia coli and Salmonella typhimurium, were detected. E. coli was the most common contaminant, but with lower levels in Masaka compared to Kampala. S. typhimurium was mainly a burden in Kampala. Our findings demonstrate that there are enormous contributors to poor hygiene and sanitation along the edible grasshopper value chain. The existence of pathogenic bacteria such as E. coli in ready-to-eat foods imply that their consumption poses a health risk.

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.

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.

Growth and Reproductive Performance of Edible Grasshopper (Ruspolia differens) on Different Artificial Diets.

Ruspolia differens (Serville) (Orthoptera: Tettigoniidae), also known as the 'edible grasshopper', 'African edible bush-cricket', and 'nsenene', is regarded as one of the most promising edible insect species that can be used for food, particularly in Sub-Saharan Africa. However, there is insufficient information on suitable diets and their effects on survival, adult weight, fecundity, and developmental time of this species, which are preconditions for large-scale production. In this study, we experimentally evaluated the effects of 12 diets (wheat bran, rice seed head, finger millet seed head, soya bran, maize bran, fresh maize comb, millet flour, chicken feed egg booster, simsim cake, sorghum seed head, powdered groundnut, and germinated finger millet), that are known to be accepted by R. differens, on their growth and reproductive parameters. The survival rate, developmental time, and adult weight varied considerably on the various diets. The highest nymphal survival rates, shortest development times, and highest adult weights were recorded for both sexes when fed fresh maize comb and germinated finger millet diet. Lifetime fecundity of females fed on germinated finger millet also was, on average, more than twice higher compared to other diets. The present study demonstrated that relatively inexpensive and locally available germinated finger millet, fresh maize seed (at the silking stage on the comb), sorghum seedhead, and finger millet seedhead could be successfully used to rear and sustain populations of R. differens. Our findings contribute to the future design of an effective mass-rearing system for this economically important edible insect.

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.

Prevalence of diarrhoea and associated risk factors among children under five years old in Pader District, northern Uganda.

BACKGROUND: Diarrhoea remains a major cause of morbidity and mortality in children under 5 years in sub-Saharan Africa. Of the three East African countries, Uganda has the worst mortality rate in children < 5 years, with 22% of these deaths attributed to diarrhoea. For proper planning and implementation of control, an understanding of the prevalence and determinants of the disease is crucial. This study assessed the prevalence of diarrhoea and related risk factors among children < 5 years in Pajule Subcounty, Pader District in northern Uganda. METHODS: A cross-sectional survey was conducted in April 2018, covering 244 randomly selected households having children < 5 years old in the study area. A semi-structured questionnaire was used to interview the households about diarrhoeal history in their children in the last 2 weeks preceding the survey, and on the risk factors predisposing children to diarrhoeal infections. Bivariate and multivariate logistic regression analyses with a 95% confidence interval and p < 0.05 was used to identify the risk factors associated with childhood diarrhoeal disease. RESULTS: We found a prevalence of diarrhoea of 29.1% [95% CI (23.7-35.0)] among children < 5 years in Pajule Subcounty during the 2 weeks preceding the survey. Use of unprotected water sources, age of child caretaker, child weaning time and family size had significant associations with diarrhoeal morbidity. CONCLUSION: The prevalence of childhood diarrhoea among children < 5 years of age in rural settings of Pajule Subcounty was higher than the Ugandan national average. Use of unprotected water sources, age of child caretaker, child weaning time and family size were identified as predictors of diarrhoeal occurrence. These findings underscore the need for improving access to clean water and providing community health education as the best methods for fighting childhood diarrhoea in the study area.

Developing Mass Egg-Laying Medium for the Edible Ruspolia differens (Orthoptera: Tettigonidae).

The edible katydid, Ruspolia differens (Serville), is an economically and nutritionally important insect species that is common and widespread in Africa. Currently, there are aims to develop mass-rearing methods suitable for this species, but a critical first step would require a suitable oviposition medium to ensure economic and easy production of eggs with high hatchability. The objective of the present study was to determine the oviposition preference and egg hatchability in different oviposition media. Based on preliminary testing of a wide range of different materials, two oviposition media, namely, artificial (folded plastic cloth) and natural medium (leaf sheath of Pennisetum purpureum Schumach attached to a stem), were selected for a detailed experiment. A two-choice laboratory experiment was conducted in which R. differens females were able to select between the folded plastic cloth (one side covered with short fleece) and a leaf sheath of P. purpureum (wrapped in polyester fiber). The results show that R. differens laid almost three times more eggs on the plastic cloth (mean ± SE, 169.3 ± 18.0) than on the natural substrate, P. purpurem (69.0 ± 13.9). However, the hatchability (overall mean% ± SE, 56.5 ± 4.7) of eggs in the two media did not differ. Folded plastic cloth, therefore, provided a more effective medium for egg laying by R. differens, and is thus a promising oviposition medium for getting natural populations to start laying eggs in future mass rearing.

Host plants of the non-swarming edible bush cricket Ruspolia differens.

The edible Ruspolia differens (Orthoptera: Tettigoniidae) is a widely-consumed insect in East Africa but surprisingly little is known of its host plant use in the field. We studied host plants used by non-swarming R. differens for 15 months, in central Uganda. In particular, we assessed the use of host plant species with respect to host cover in the field and host parts used by R. differens, also recording their sex, developmental stages, and colour morph. Ruspolia differens were found on 19 grass and two sedge species and they were observed predominantly (99% of 20,915 observations) on seven grasses (namely, Panicum maximum, Brachiaria ruziziensis, Chloris gayana, Hyparrhenia rufa, Cynodon dactylon, Sporobolus pyramidalis, and Pennisetum purpureum). Ruspolia differens was most frequently observed on the most common grass of each study site but P. maximum, and S. pyramidalis were used more frequently than expected from their cover in the field. Furthermore, R. differens were observed predominantly on inflorescences (97% of feeding observations) and much less frequently on the leaves (3.0%), stems (0.1%), and inflorescence stalks (0.1%) of grasses and sedges. Host use was not independent of sex, developmental stage, or colour morph. Panicum maximum was the preferred host of the youngest nymphs of R. differens. Overall, our findings indicate that a continuous supply of diverse grass resources with inflorescences is necessary for the management and conservation of wild populations of R. differens.

High prevalence of Pfmdr-1 N86 and D1246 genotypes detected among febrile malaria outpatients attending Lira Regional Referral Hospital, Northern Uganda.

OBJECTIVE: 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. RESULTS: 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.

Fatty acid content and composition in edible Ruspolia differens feeding on mixtures of natural food plants.

OBJECTIVES: To develop successful mass-rearing programs of edible insects, knowledge of the feeds and their influence on nutritional content is critical. We assessed the influence of natural food plants (grass inflorescences) and their mixtures on fatty acid profiles of edible Ruspolia differens. We reared neonate nymphs to adult on six dietary treatments consisting of one, and mixtures of two, three, five, six and eight plants. RESULTS: The contents of saturated, monounsaturated and polyunsaturated fatty acids, omega-6/omega-3 ratio, and adult body weight did not differ among dietary treatments. However, the composition of fatty acids differed significantly among insects fed on six dietary treatments, but only for the rare fatty acids. Our results demonstrate that even if natural diets (grass inflorescences) do not strongly modify fatty acid contents or compositions of R. differens, when reared from neonate nymphs to adults, their n - 6/n - 3 fatty acid ratio is generally low and thus good for a healthy human diet.

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.

Bottom-up impact on the cecidomyiid leaf galler and its parasitism in a tropical rainforest.

The relative importance of host-plant resources, natural enemies or their interactions in controlling the population of galling insects and their parasitism is poorly known for tropical gallers. In this study, we assessed the impacts of plant quality and density of host trees in regulating the densities of a galler species, the cecidomyiid leaf galler (Cecidomyiini sp. 1EJV) and its parasitoids and inquilines on Neoboutonia macrocalyx trees in Uganda. We manipulated the nutritional quality (or vigour) and the resource concentration with four levels each of fertilization and the group size of host tree. We then recorded the effects of these treatments on the growth rate and total leaf area of host plants, the density of gallers and their mortality by parasitoids and inquilines. Higher levels of fertilization and host density resulted in significantly higher total leaf area than did ambient nutrient levels, and lowest tree densities, respectively. Fertilization also caused significant change in the growth rate of leaf area. Both higher fertilization and host density caused higher density of gallers. Total leaf area was positively associated with galler density, but within galled replicates, the galled leaves were larger than the ungalled leaves. Although highest levels of fertilization and density of host trees caused significant change in the densities of parasitoids, the rate of parasitism did not change. However, tree-density manipulations increased the rate of inquilinism, but on a very low level. Our results demonstrate a trophic cascade in the tropical galler and its parasitoids as a response to bottom-up effects.