Retrospective in silico mutation profiling of SARS-CoV-2 structural proteins circulating in Uganda by July 2021: Towards refinement of COVID-19 disease vaccines, diagnostics, and therapeutics.

The SARS-CoV-2 virus, the agent of COVID-19, caused unprecedented loss of lives and economic decline worldwide. Although the introduction of public health measures, vaccines, diagnostics, and therapeutics disrupted the spread of the SARS-CoV-2, the emergence of variants poses substantial threat. This study traced SARS-CoV-2 variants circulating in Uganda by July 2021 to inform the necessity for refinement of the intervention medical products. A comprehensive in silico analysis of the SARS-CoV-2 genomes detected in clinical samples collected from COVID-19 patients in Uganda revealed occurrence of structural protein variants with potential of escaping detection, resisting antibody therapy, or increased infectivity. The genome sequence dataset was retrieved from the GISAID database and the open reading frame encoding the spike, envelope, membrane, or nucleocapsid proteins was translated. The obtained protein sequences were aligned and inspected for existence of variants. The variant positions on each of the four alignment sets were mapped on predicted epitopes as well as the 3D structures. Additionally, sequences within each of the sets were clustered by family. A phylogenetic tree was constructed to assess relationship between the encountered spike protein sequences and Wuhan-Hu-1 wild-type, or the Alpha, Beta, Delta and Gamma variants of concern. Strikingly, the frequency of each of the spike protein point mutations F157L/Del, D614G and P681H/R was over 50%. The furin and the transmembrane serine protease 2 cleavage sites were unaffected by mutation. Whereas the Delta dominated the spike sequences (16.5%, 91/550), Gamma was not detected. The envelope protein was the most conserved with 96.3% (525/545) sequences being wild-type followed by membrane at 68.4% (397/580). Although the nucleocapsid protein sequences varied, the variant residue positions were less concentrated at the RNA binding domains. The dominant nucleocapsid sequence variant was S202N (34.5%, 205/595). These findings offer baseline information required for refining the existing COVID-19 vaccines, diagnostics, and therapeutics.

Animal health beyond the single disease approach - A role for veterinary herd health management in low-income countries?

In order to identify and evaluate health related constraints faced by Ugandan pig farmers, a veterinary herd health management approach (VHHM) was applied in 20 randomly selected pig farms in the Lira district, Uganda. Regular herd visits were conducted between July 2018 and June 2019, using e.g. interviews, observations, clinical examinations and laboratory analyzes to gather qualitative and quantitative data on relevant aspects of the production. The pig farmers kept on average 18.6 pigs, including 2.6 sows/year. The production figures varied considerably but were generally poor. The sows produced 1.6 litters/year and 8.2 piglets born alive per litter, the average daily gain was 101 g/day, and the mortality in growers was 9.7%. Four major constraints were identified; poor nutrition, infectious diseases, inferior biosecurity, and poor reproductive management. The quantity and quality of feed was suboptimal. Endo- and ectoparasites were very common, causing diarrhea, bronchitis, pneumonia, skin lesions and pruritus. Post-weaning diarrhea associated with enterotoxigenic Escherichia coli was important in the two largest herds, and parvoviral antibodies were found in seven herds, two experiencing problems with mummified fetuses. Biosecurity practices were insufficient and inconsistent, with free-ranging pigs and the use of village boars being the major risks. Reproductive figures were affected by poor estrus detection and service management. Overall, farmers lacked important knowledge on good management practices. In conclusion, the VHHM identified several important constraints that should be addressed in order to increase the productivity of Ugandan pig herds.

RA92A recombinant protein as immunogen to protect cattle against tick challenge in Brazil and Uganda

In this study, the recombinant gut protein rRa92A produced in Pichia pastoris yeast cells was used to immunize cattle in two experiments, one in Brazil and the other in Uganda. In both experiments, the animals were intramuscularly (IM) injected with 200 µg of recombinant protein in Brazil on days 0, 30 and 51 and in Uganda on days 0, 30. Blood samples for sera separation were collected from different days in both experiments. These samples were analyzed by ELISAs. In Brazil, ticks collected from the animals during the experimental period were analyzed for biological parameters. At Uganda, blood was collected to assess blood parameters, clinical signs were recorded and adult tick (Rhipicephalus appendiculatus) counts were performed. All animals of the vaccinated groups were shown to produce antibodies, and it was not possible to detect an effect on Rhipicephalus microplus. All the clinical parameters were considered within the normal ranges for both the experimental and control groups in Uganda. Antibody absorbance was elevated after each immunization and remained high until the end of the experiments, remaining low in the control animals. The results of stall test carried out in Brazil using R. microplus tick showed efficacy of 21.95%. The rRa92A immunization trial experiments in Uganda showing a decrease of 55.2% in the number of engorged adult ticks, which was statistically significant (p<0.05). Assessment of the immunogenicity of Ra92A produced in the P. pastoris expression system in bovines is reported for the first time, and the protein acted as a concealed antigen.

Prevalence of adhesin and toxin genes in E. coli strains isolated from diarrheic and non-diarrheic pigs from smallholder herds in northern and eastern Uganda.

BACKGROUND: Enterotoxigenic E. coli (ETEC) significantly contribute to diarrhea in piglets and weaners. The smallholder pig producers in Uganda identified diarrhea as one of the major problems especially in piglets. The aim of this study was to; i) characterize the virulence factors of E. coli strains isolated from diarrheic and non-diarrheic suckling piglets and weaners from smallholder herds in northern and eastern Uganda and ii) identify and describe the post-mortem picture of ETEC infection in severely diarrheic piglets. Rectal swab samples were collected from 83 piglets and weaners in 20 herds and isolated E. coli were characterized by PCR, serotyping and hemolysis. RESULTS: The E. coli strains carried genes for the heat stable toxins STa, STb and EAST1 and adhesins F4 and AIDA-I. The genes for the heat labile toxin LT and adhesins F5, F6, F18 and F41 were not detected in any of the E. coli isolates. Where the serogroup could be identified, E. coli isolates from the same diarrheic pig belonged to the same serogroup. The prevalence of EAST1, STb, Stx2e, STa, AIDA-I, and F4 in the E. coli isolates from suckling piglets and weaners (diarrheic and non-diarrheic combined) was 29, 26.5, 2.4, 1.2, 16, and 8.4 %, respectively. However the prevalence of F4 and AIDA-I in E. coli from diarrheic suckling piglets alone was 22.2 and 20 %, respectively. There was no significant difference in the prevalence of the individual virulence factors in E. coli from the diarrheic and non-diarrheic pigs (p > 0.05). The main ETEC strains isolated from diarrheic and non-diarrheic pigs included F4/STb/EAST1 (7.2 %), F4/STb (1.2 %), AIDA/STb/EAST1 (8 %) and AIDA/STb (8 %). At post-mortem, two diarrheic suckling piglets carrying ETEC showed intact intestinal villi, enterocytes and brush border but with a layer of cells attached to the brush border, suggestive of ETEC infections. CONCLUSION: This study has shown that the F4 fimbriae is the most predominant in E. coli from diarrheic piglets in the study area and therefore an F4-based vaccine should be considered one of the preventive measures for controlling ETEC infections in the piglets in northern and eastern Uganda.

Prevalence of and factors associated with Brucella sero-positivity in cattle in urban and peri-urban Gulu and Soroti towns of Uganda.

Brucellosis is a key zoonosis of major public health, animal welfare and economic significance, and is endemic in livestock in Uganda. A cross-sectional epidemiological study was carried out to estimate the sero-prevalence of brucellosis and identify factors associated with sero-positivity in cattle in urban and peri-urban Gulu and Soroti towns of Northern and Eastern Uganda, respectively. A total of 1007 sera and data on biologically plausible risk factors from 166 herds and their spatial locations, were collected from cattle reared in urban and peri-urban Gulu and Soroti towns of Uganda. The sera were analyzed using indirect ELISA and sero-positive reactors confirmed by competitive ELISA. Multivariable models were used to investigate for risk factors. The overall animal-level and herd-level sero-prevalence was 7.5% (76/1007, 95% Confidence Interval (CI): 6.15-9.4%) and 27.1% (45/166, 95% CI: 20.9-34.3%), respectively. Herd-level sero-prevalence was significantly (P<0.001) higher in Soroti than Gulu. In Gulu town, sero-positivity increased with an increase in herd size (P=0.03) and age (P=0.002), and was higher in cattle brought in from western Uganda (P<0.0001). In Soroti town, introduction of new cattle into a herd was significantly (P=0.027) associated with herd sero-positivity. There was a geographically differential risk (clustering) of Brucella sero- positivity in herds in Soroti, while sero-positivity was homogeneously distributed in Gulu. The data highlight brucellosis occurrence and major risk factors for its transmission in cattle in urban and peri-urban areas.

Salmonella species in piglets and weaners from Uganda: prevalence, antimicrobial resistance and herd-level risk factors.

Non-typhoidal salmonellosis is of concern in humans in sub-Saharan Africa, and this is partly due to the high number of immunocompromised persons. Pork and pork products could be among the sources of these non-typhi Salmonella spp. The aim of this study was to identify Salmonella spp. in piglets and weaners in northern and eastern Uganda, characterize their antimicrobial resistance patterns and determine herd-level risk factors. Fecal samples were collected from 465 piglets and weaners from 93 herds (49 and 44 from northern and eastern Uganda, respectively). In addition, information about the herd management and potential risk factors were collected. The fecal samples were cultured for the identification of Salmonella spp. The Salmonella spp. confirmed by serotyping were further characterized by determination of minimum inhibitory concentration (MIC) to 12 antimicrobials by broth microdilution. At individual level, the total prevalence of Salmonella spp. was 12% (12.2% in northern and 11.9% in eastern Uganda). At herd level, the total prevalence was 39% (43% in northern and 34% in eastern Uganda). From 56 samples with Salmonella spp., 20 serovars were identified including two serovars identified only by their antigenic formulae. The predominant serovars were S. Zanzibar, S. Heidelberg, S. Infantis, S. Typhimurium, S. Stanleyville, S. Aberdeen and S. Kampala. In total, 57% of the 53 Salmonella spp. analyzed, originating from 27% of the herds, were resistant to at least one antimicrobial agent. The majority of drug-resistant isolates (60%) were from northern Uganda. Eight multidrug-resistant (MDR) isolates were from northern Uganda and three MDR isolates were from eastern Uganda. Increased prevalence of Salmonella spp. was associated with feeding the young and adults separately as compared to feeding the young and adults together (p=0.043, OR=4.3; 95% CI 1.1, 17.38). Protective factors were "intensive" method of keeping the pigs versus "tethering and roaming" (p=0.016, OR=0.11; 95% CI 0.02, 0.64), "intensive" method versus "semi-intensive" method (p=0.048, OR=0.12; 95% CI 0.01, 0.96) and cleaning feeders after every two days versus daily (p=0.017, OR=0.18; 95% CI 0.05, 0.72). This study has revealed a high prevalence of infection of piglets and weaners with diverse non-typhi Salmonella serovars and highlights the potential role of pork and pork products as sources of these organisms for humans. In addition, this study has identified protective factors that could be promoted to control Salmonella spp. and in antimicrobial resistance reduction programs in rural pigs from Uganda.

Ethnopharmacological practices by livestock farmers in Uganda: survey experiences from Mpigi and Gulu districts.

BACKGROUND: There is continued reliance on conventional veterinary drugs including anthelmintics, to some of which resistance has developed. Loss of indigenous technical knowledge (ITK) from societies affects the opportunities for utilization of ethnopharmacological practices unless properly documented. This study was conducted to identify common traditional practices using medicinal plants against helminthosis and other livestock diseases in Mpigi and Gulu districts of Uganda. METHODS: Seven focus group discussions with ten farmers per group plus 18 key informant interviews were held in each district from August to November 2011. Ranking was used to quantify disease burdens and to identify priority livestock and breeds. Samples of each plant were submitted to Makerere University herbarium for identification and documentation. The local name, relative availability and International Union for Conservation of Nature (IUCN) status were recorded. RESULTS: Seventy six farmers in Mpigi and 74 in Gulu were interviewed. Theileriosis and helminthosis were the most common disease conditions in cattle and goats, respectively. Forty plant species within 34 genera from 22 botanical families were identified, with 20 of these used against helminthosis. Other plants treated wounds and ecto-parasites, theileriosis, retained placenta and bovine ephemeral fever. Non-plant practices (7) and plants cited were used in combination depending on availability. Males older than 40 years had most ethnopharmacological knowledge. Most plants (75%, n = 40) were common, but 10 were rare. IUCN status was not evaluated for 95% of these plants. Conventional and traditional drug use in Gulu and Mpigi districts was different (χ2 = 24; p < 0.001). The scientific, English, Luganda and Acholi names of all plants and their availability within the communities are documented herein. CONCLUSION: This is the first detailed livestock-related ethnopharmacological study in Gulu district. Farmers in Uganda are still using a variety of practices to treat livestock ailments. Scientific validation and evaluation of conservation status are urgently needed to ensure future availability and knowledge about these plant resources.