Molecular characterization of Escherichia coli virulence markers in neonatal and postweaning piglets from major pig-producing districts of Uganda.

BACKGROUND: Piggery production is highly constrained by diseases, with diarrhoea in piglets being a major cause of economic losses to smallholder farmers in Uganda. Enterotoxigenic Escherichia coli (ETEC) is thought to be one of the major etiologies of this diarrhoea. A cross-sectional study was carried out in two high pig-producing districts of Uganda with the aim of determining the significance of piglet diarrhoea and the pathogenic determinants of causative E. coli. METHODOLOGY: A total of 40 households with piglets were visited in each district for a questionnaire survey and faecal sample collection. The questionnaire-based data collected included; demographic data and pig management practices. E. coli were isolated from diarrheic (43) and non-diarrheic (172) piglets and were subjected to antimicrobial susceptibility testing against nine commonly used antimicrobial agents. The E. coli isolates were further screened for the presence of 11 enterotoxin and fimbrial virulence gene markers using multiplex polymerase chain reaction. Data entry, cleaning, verification and descriptive statistics were performed using Microsoft Excel. Statistical analysis to determine any association between the presence of virulence markers and diarrhea in piglets was done using SPSS software (Version 23), with a p value of less than 0.05 taken as a statistically significant association. RESULTS: Escherichia coli were recovered from 81.4% (175/215) of the faecal samples. All the isolates were resistant to erythromycin, and most showed high resistance to tetracycline (71%), ampicillin (49%), and trimethoprim sulfamethoxazole (45%). More than half of the isolates (58.3%) carried at least one of the 11 virulence gene markers tested. EAST1 was the most prevalent virulence marker detected (35.4%), followed by STb (14.8%). Expression of more than one virulence gene marker was observed in 6.2% of the isolates, with the EAST1/STa combination being the most prevalent. Three adhesins; F17 (0.6%), F18 (6.3%) and AIDA-I (0.6%) were detected, with F18 being the most encountered. There was a statistically significant association between the occurrence of piglet diarrhoea and the presence of the AIDA-1 (p value = 0.037) or EAST1 (p value = 0.011) gene marker among the isolates. CONCLUSION AND RECOMMENDATION: The level of antimicrobial resistance among E. coli isolates expressing virulence markers were high in the sampled districts. The study established a significant association between presence of EAST1 and AIDA-I virulence markers and piglet diarrhea. Further studies should be carried out to elucidate the main adhesins borne by these organisms in Uganda and the actual role played by EAST1 in the pathogenesis of the infection since most isolates expressed this gene.

Phytochemical screening, antimycobacterial activity and acute toxicity of crude extracts of selected medicinal plant species used locally in the treatment of tuberculosis in Uganda.

BACKGROUND: Tuberculosis (TB) is one of the leading causes of death globally, and the rise in drug-resistant forms of TB has become a significant threat. Subsequently, it is crucial to explore new, effective and safe anti-TB agents. This study aimed at conducting phytochemical screening, antimycobacterial activity, and acute toxicity of the selected plant species' crude extracts to assess their toxicological potentials and efficacies against TB. METHODS: The aqueous and methanol/dichloromethane (DCM) (1:1) extracts of each selected plant species were subjected to phytochemical screening and antimycobacterial activity using microplate alamar blue assay. For acute toxicity, a single dose (2000 mg/kg) of the aqueous extracts was orally administered to each animal following the Organization for Economic Cooperation and Development (OECD) guidelines No. 425 and then observed for 14 days. The animals were closely observed on the general behavior and clinical signs of toxicity, and body weights were recorded. After the termination of the experiment, hematological, biochemical, and histopathological analyses were performed. RESULTS: The extracts contained alkaloids, flavonoids, tannins, saponins, steroids, terpenoids, resins, cardiac glycosides, phenolic compounds, and coumarins. Aqueous extracts showed moderate to weak activity against the susceptible (H37Rv) M. tuberculosis strain and weak activity against the MDR-TB strain with Minimum Inhibitory Concentrations (MIC µg/mL) ranging from 293.0-2344.0 and 1172.0-4688.0, respectively. Methanol/DCM extracts showed significant to moderate activity against the susceptible TB strain and moderate to weak activity against the MDR-TB strain with MIC (µg/mL) ranging from 98.0-586.0 and 293.0-781.0, respectively. One mortality was recorded from the A. coriaria treated group following the acute toxicity tests, but the LD50 of all the extracts was estimated to be above 2000 mg/kg. Histopathological analyses did not show any significant lesions in the examined organs except those from the A. coriaria treated group. CONCLUSION: Phytochemical screening of the extracts revealed the presence of alkaloids, tannins, saponins, flavonoids, steroids, terpenoids, resins, cardiac glycosides, phenolic compounds, and coumarins. All the methanol/DCM extracts of the plant species studied have promising antimycobacterial activity. The selected plant extracts studied exhibited low acute toxicity levels except for A. coriaria and could be safe for formulations into herbal products.

A retrospective analysis of antimicrobial resistance in pathogenic Escherichia coli and Salmonella spp. isolates from poultry in Uganda.

There are increasing reports of antimicrobial treatment failures for bacterial diseases of poultry in Uganda. The paucity of data on antimicrobial resistance (AMR) of pathogenic bacteria in Uganda is a major setback to AMR control. This study investigated the occurrence of fowl typhoid, colibacillosis, and AMR in associated pathogens from 2012 to 2018. Laboratory records from the Central Diagnostic Laboratory (CDL), a National Veterinary Diagnostic Facility located at Makerere University, were reviewed. Archived isolates of the causative bacteria for the two diseases were also evaluated for AMR. The frequencies of the two disease conditions, their clinical and necropsy presentations and the demographic data of the diagnostic samples were summarized from the records. Archived bacterial isolates were revived before antimicrobial susceptibility testing. This was done on Mueller Hinton agar using the disk diffusion method, against 16 antimicrobials of medical and veterinary importance according to the Clinical Laboratory Standards Institute guidelines. A total of 697 poultry cases were presented for bacteriological investigations in the review period. Colibacillosis and salmonellosis had prevalence rates of 39.7% (277/697) and 16.2% (113/697), respectively. A total of 63 and 92 isolates of Escherichia coli and Salmonella spp., respectively, were archived but 43 (68.3%) E. coli and 47 (51.1%) Salmonella spp. isolates were recovered and evaluated for AMR. Multidrug resistance was more frequent in E. coli (38; 88.4%) than salmonellae (25; 53.2%), (p < 0.001). The high prevalence of colibacillosis, salmonellosis and the AMR of associated pathogens warrants immediate institution of appropriate disease control measures.

Survey of Candidate Single-Nucleotide Polymorphisms in SLC11A1, TLR4, NOD2, PGLYRP1, and IFNγ in Ankole Longhorn Cattle in Central Region of Uganda to Determine Their Role in Mycobacterium avium Subspecies paratuberculosis Infection Outcome.

Mycobacterium avium ssp. paratuberculosis (MAP) is the cause of Johne's disease (JD) in a wide range of domestic and wild ruminants. Single-nucleotide polymorphisms (SNPs) in several genes including solute-like carrier 11A1 (SLC11A1), interferon gamma (IFNγ), Toll-like receptor 4 (TLR4), nucleotide-binding oligomerization domain 2 gene (NOD2), and bovine peptidoglycan recognition protein 1 (PGLYRP1) have been implicated in influencing the infection outcome of MAP in cattle. We have carried out a survey in a population of Ankole cattle from three districts in the central region of Uganda including Isingiro, Lyantonde, and Rakai to determine the role played by several SNPs on the above genes in the infection outcome of local cattle in Uganda. Nine hundred fifty-five heads of cattle obtained from 93 herds were tested using ELISA. Thirty-five ELISA-positive cattle and 35 negative herd mates from a total of 955 cattle tested for MAP were genotyped using iPLEX MassARRAY genotyping systems to detect the presence of a total of 13 SNPS in five different genes (SLC11A1, IFNγ, TLR4, NOD2, and PGLYRP1). The cow-level prevalence of MAP infection in Ankole Longhorn cattle in the three districts was 3.98% (35/955), while the herd-level prevalence was 27.9% and within-herd prevalence was 12 ± 1.5% (95% CI = 9.1-14.8%). The genotypes and allele frequencies of the MAP-positive cattle were compared with those of their ELISA-negative herd mates to determine the significance of the polymorphisms. The results showed that SNPs rs109915208, rs110514940, and rs110905610 on SLC11A1, c.480G>A and c.625C>A on PGLYRP1, and c.2021C>T on TLR4 were monomorphic in both seropositive and seronegative cattle and therefore had no influence on the infection outcome. The remaining SNPs studied in the five genes [SLC11A1: rs109614179; TLR4: rs29017188 (c.226G>C), c.2021C>T; NOD2: rs110536091, rs111009394; PGLYRP1: c.102G>C, c.480G>A, c.625C>A; IFNγ: rs110853455] were polymorphic, but their allele and genotype frequencies did not show any significant difference between the seropositive and seronegative cattle. No significant difference was observed for any haplotype at the gene level.

Retrospective study on cattle and poultry diseases in Uganda.

Cattle and poultry enterprises are among the major contributors to food security and socioeconomic empowerment of households in Uganda. However, various diseases constrain their productivity. A two-year retrospective study between April 2012 and March 2014 was conducted using records for cattle and poultry diseases diagnosed at the Central Diagnostic Laboratory (CDL) to determine prevalent diseases in Uganda. The laboratory received 836 samples from poultry (36.3%) and cattle (63.7%). Of the 836 samples, 47.5% had a definitive diagnosis of disease causation. Most of the cattle and poultry diseases diagnosed were protozoan diseases (39.3%) followed by bacterial (21.4%), viral (17.1%), helminthiasis (11.1%), nutritional diseases (4%) and others (7.1%). For poultry, viral diseases (29.5%) and protozoan diseases (27.1%) especially newcastle disease (44.3%) and coccidiosis (100%) respectively, were the most diagnosed. While for cattle, hemo-protozoan parasites (52.1%) were the most prevalent, of which 92.9% were east coast fever infection. Bacterial infection (20.5%) in cattle were the second most diagnosed diseases and mastitis was the most diagnosed (46.2%). In summary, coccidioisis, collibacillosis, newcastle disease, gumboro disease, and avian helminthiasis were the most prevalent poultry diseases while in cattle, east coast fever, helminthiasis, mastitis, brucellosis and rabies were the most frequently diagnosed diseases. This study has identified the major diseases that hinder poultry and cattle production in Uganda. The data generated by CDL could be used for surveillance, monitoring and designing strategic interventions for control of poultry and cattle diseases in Uganda.

Prevalence of African swine fever virus in apparently healthy domestic pigs in Uganda.

BACKGROUND: African swine fever (ASF) is a contagious viral disease which can cause up to 100% mortality among domestic pigs leading to serious socio-economic impact on people's livelihoods. ASF is endemic in Uganda and there is paucity of information on the epidemiology of the disease. The major aim of this study was to determine the seroprevalence and prevalence of African swine fever virus (ASFV) in apparently healthy slaughter pigs at Wambizi slaughterhouse in Kampala city, Uganda. We also estimated the presence of ASFV antibodies and circulating viral antigens in pigs from selected districts of Uganda during targeted surveillance. We analysed 540 and 181 blood samples collected from slaughter pigs and pigs from targeted surveillance districts respectively. RESULTS: The prevalence of ASFV in slaughter pigs was 52.96% (95% CI, 48.75-57.14) and 11.5% (95% CI, 9.06-14.45) by ELISA and PCR respectively. In surveillance districts, the proportion of ASFV positive pigs was 53.59% (95% CI, 46.33-60.71) and 0.55% (95% CI, 0.1-3.06) by ELISA and PCR respectively. CONCLUSION: The study has found out a high seroprevalence of ASFV antibodies in apparently healthy slaughter pigs and also a high proportion of ASFV antibody seropositive pigs in surveyed districts in Uganda indicating exposure to ASFV. However, there was a lower prevalence of ASFV infection implying that there could be low virulent strains of ASFV circulating in domestic pigs in Uganda which requires further investigation.

Molecular characterization and phylogenetic study of African swine fever virus isolates from recent outbreaks in Uganda (2010-2013).

BACKGROUND: African swine fever (ASF) is a highly lethal and economically significant disease of domestic pigs in Eastern Africa particularly in Uganda where outbreaks regularly occur. Sequence analysis of variable genome regions have been extensively used for molecular epidemiological studies of African swine fever virus (ASFV) isolates. By combining p72, P54 and pB602L (CVR), a high level resolution approach is achieved for viral discrimination. The major aim of this study therefore, was to investigate the genetic relatedness of ASF outbreaks that occurred between 2010 and 2013 in Uganda to contribute to the clarification of the epidemiological situation over a four year period. METHODS: Tissue samples from infected domestic pigs associated with an ASF outbreak from 15 districts in Uganda were confirmed as being infected with ASFV using a p72 gene-based polymerase chain reaction amplification (PCR) assay recommended by OIE. The analysis was conducted by genotyping based on sequence data from three single copy ASFV genes. The E183L gene encoding the structural protein P54 and part of the gene encoding the p72 protein was used to delineate genotypes. Intra-genotypic resolution of viral relationships was achieved by analysis of tetramer amino acid repeats within the hypervariable CVR of the B602L gene. RESULTS: Twenty one (21) ASF outbreaks were confirmed by the p72 ASF diagnostic PCR, however; only 17 isolates were successfully aligned after sequencing. Our entire isolates cluster with previous ASF viruses in genotype IX isolated in Uganda and Kenya using p72 and P54 genes. Analysis of the CVR gene generated three sub-groups one with 23 tetrameric amino acid repeats (TRS) with an additional CAST sequence, the second with 22 TRS while one isolate Ug13. Kampala1 had 13 TRS. CONCLUSION: We identified two new CVR subgroups different from previous studies. This study constitutes the first detailed assessment of the molecular epidemiology of ASFV in domestic pigs in the different regions of Uganda.

Detection of Antibodies and Confirmation of Mycobacterium avium Subspecies paratuberculosis Using Nested PCR in Bulk Milk Samples from Nakasongola and Sembabule Districts, Uganda.

Mycobacterium avium subspecies paratuberculosis (MAP) is an emerging pathogen in many livestock and wildlife populations around the world. Concerns range from the serious economic impacts on livestock productivity to its suspected role in the human inflammatory bowel disease syndrome. Milk and faeces of infected animals are the main vehicles through which the organism spreads from infected to susceptible hosts. In this study, a survey was done in Nakasongola and Sembabule districts of Uganda involving a total of seven dairy collection centres to determine the prevalence of antibodies to MAP in bulk milk samples. The milk was tested with a commercial ELISA kit for MAP testing in milk. Positive and suspicious milk samples were further tested using nested PCR. Of the 257 milk samples tested, 11 (4.3%) were positive and five (1.9%) were suspicious. All the ELISA positive and suspicious milk samples were positive using nested PCR. The results show that MAP infection occurs in cattle from the two districts and highlight the need for a paratuberculosis control program in these and other districts where MAP infection has been reported.

Epidemiological Overview of African Swine Fever in Uganda (2001-2012).

African swine fever (ASF) is a contagious viral disease, which can cause up to 100% mortality among domestic pigs. In Uganda there is paucity of information on the epidemiology of the disease, hence a study was carried out to elucidate the patterns of ASF outbreaks. Spatial and temporal analyses were performed with data collected monthly by the district veterinary officers (DVOs) and sent to the central administration at MAAIF from 2001 to 2012. Additionally, risk factors and the associated characteristics related to the disease were assessed based on semistructured questionnaires sent to the DVOs. A total of 388 ASF outbreaks were reported in 59 districts. Of these outbreaks, 201 (51.8%) were reported in districts adjacent to the national parks while 80 (20.6%) were adjacent to international borders. The number of reported ASF outbreaks changed over time and by geographical regions; however, no outbreak was reported in the North-Eastern region. ASF was ranked as second most important disease of pigs, and it occurred mostly during the dry season (P = 0.01). Pig movements due to trade (OR 15.5, CI 4.9-49.1) and restocking (OR 6.6, CI 2.5-17.3) were the major risk factors. ASF control strategies should focus on limiting pig movements in Uganda.