Detection of antibodies against Ornithodoros moubata salivary antigens and their association with detection of African swine fever virus in pigs slaughtered in central Uganda.

Introduction: African swine fever (ASF) is an important disease of pigs in sub-Saharan Africa and Uganda and is threatening the pig population and agricultural economy of other continents. ASF virus (ASFV) can be transmitted from wild suids to domestic pigs through soft ticks of the Ornithodoros species. The aim of this study was to understand the relationship between domestic pigs' O. moubata tick exposure and ASFV status. Methods: Pigs were sampled from six abattoirs in the Kampala metropolitan area of Uganda from May 2021 through June 2022. Blood, serum, and tissue samples were collected. Serum was tested for antibodies against the rtTSGP1 salivary antigens of O. moubata ticks using an indirect ELISA assay. Blood and tissue samples from pigs were tested to detect ASFV using qPCR. Probability of tick exposure was categorized based on sample-to-positive ratio cut-off points. Results: Out of 1,328 serum samples tested, there were 828 (62.3%) samples with a negligible probability; 369 (27.8%) with a medium probability; 90 (6.8%) with a high probability, and 41 (3.1%) with a very high probability of exposure to the O. moubata salivary antigen. There was a statistically significant association between the pigs' O. moubata exposure and ASFV status with a higher proportion of pigs having a very high probability of infection if they were ASFV positive by blood, tonsil, and lymph nodes. Discussion: These results suggested that tick exposure was associated with ASFV transmission in Uganda. There were ASFV qPCR positive pigs that had no O. moubata exposure as well, which highlights that pig-to-pig and indirect contact transmission still play a significant role. This work highlights the need for further work in Uganda to investigate these transmission factors related to the O. moubata tick and ASFV transmission.

Spatiotemporal description of African swine fever virus nucleic acid and antibodies detected in pigs sampled at abattoirs in the greater Kampala metropolitan area, Uganda from May 2021 through June 2022.

BACKGROUND: African swine fever virus (ASFV) infections in Africa cause hemorrhagic disease in domestic pigs and is maintained by a sylvatic cycle in warthogs. It is endemic in Uganda, leading to significant economic losses. Previous studies performed in rural areas and in Kampala had differing diagnostic results. The purpose of this study was to provide a robust spatial, temporal, and diagnostic summary of pigs slaughtered in the greater Kampala metropolitan area over the course of one year. This study characterized 1208 to 1323 serum, blood, and tissue samples collected from pigs at six abattoirs in the greater Kampala metropolitan area of Uganda monthly from May 2021 through June 2022. Validated and standardized serologic and molecular diagnostics were used. RESULTS: Only 0.15% of pigs had detectable antibodies against ASFV, suggesting low survival rates or pre-clinical diagnosis. Yet, 59.5% of pigs were positive for ASFV DNA. Blood had the lowest detection rate (15.3%) while tonsil and lymph nodes had the highest (38% and 37.5%, respectively), spleen samples (31.5%) were in between. Agreement between sample types was fair to moderate overall. A significant seasonality of ASFV infections emerged with infections found predominately in the dry seasons. Spatial assessments revealed that the greater Kampala metropolitan area abattoirs have a catchment area that overlaps with Uganda's most pig dense regions. CONCLUSIONS: Pigs at greater Kampala metropolitan area abattoirs can be sentinels for acute disease throughout the pig dense region of Uganda, particularly in the dry seasons. The high prevalence detected suggests that pigs are sold in response to local reports of ASFV infections (panic sales). Serological surveillance is not useful, as very few pigs seroconverted in this study prior to slaughter. In contrast, tissue samples of pigs can be used to detect disease using qPCR methods.

A survey of biosecurity practices of pig farmers in selected districts affected by African swine fever in Uganda.

Introduction: In Uganda, pig production is an important source of livelihood for many people and contributes to food security. African swine fever (ASF) is a major constraint to pig production in Uganda, threatening the food supply and sustainable livelihoods. Prevention of ASF primarily relies on good biosecurity practices along the pig value chain. Previous studies showed that biosecurity along the pig value chain and on farms in Uganda is poor. However, the biosecurity practices of pig farmers in ASF affected areas of Uganda and their opinions on on-farm ASF morbidity and mortality were previously not comprehensively characterized. The objectives of this study were to document pig farmers' experiences with ASF in their farms and to describe the pig biosecurity practices in districts of Uganda that were highly affected by ASF. Methods: A total of 99 farmers were interviewed in five districts. Data were collected by way of triangulation through farmer interviews, field observations during the farmer interviews, and a survey of key informants. However, farmer interviews were considered the primary source of data for this study. Farmers' biosecurity practices were scored using a biosecurity scoring algorithm. Results: Forty-one out of 96 (42.7%) farmers reported having pigs with ASF in the past 12 months. The level of pig farming experience (p = 0.0083) and herd size (p < 0.0001) were significantly associated with the reported occurrence of ASF. Overall, the biosecurity scores for the respondents were considered poor with 99% (98/99) scoring <70% and just one farmer obtaining a fair score of 72.2%. District (p = 0.0481), type of husbandry system (p = 0.014), and type of pig breed raised (p = 0.004) were significantly associated with farmer's biosecurity score. Conclusion: Continued farmer education on ASF and the importance of good biosecurity practices is necessary. More in-depth scientific inquiry into the factors influencing the biosecurity practices among pig farmers in Uganda is necessary.

The live pig supply chain as reported by farmers in selected districts affected by African swine fever in Uganda, June and July 2022.

Introduction: Uganda is a pork-producing country in East Africa. The African swine fever virus (ASFV) has had a devastating impact on the country's pig industry. The movements of pigs and pork are a major pathway of spreading ASFV. This study was done to describe the live pig supply chain within and through districts that are impacted by African swine fever (ASF) in Uganda. Methods: A pig farmer survey in districts known to have ASFV was done using a semi-structured questionnaire available in English and two local languages. In total, 99 farmers were interviewed across five districts. Farmers were conveniently and purposively selected by local government veterinary officials. An online key informant survey was also used to validate farmer responses. Results: Most farmers interviewed in all districts reported to source and sell most of their pigs from within their district the farm was in, although there was variation by district and pig type. In relation to pig type, 89.7% of farmers sourced sows, 80.0% sourced boars, and 96.4% sourced weaned pigs from the district where the farm was located. As for sales, 91.3% of farmers sold sows, 92.7% sold boars, 91.9% sold weaned pigs, and 92.2% sold market pigs in the district where the farm was located. There was also variation to whom pigs were sold and sourced by pig type. Conclusion: This information is useful when planning the scale and focus of disease control programs based on animal movement. This study revealed that pig disease control programs can be targeted to smaller regions. Furthermore, there is a need for farmers and pig traders to be educated on and adhere to veterinary regulations of animal movement and good biosecurity practices to reduce disease spread when purchasing and selling pigs from known ASFV infected areas.

Whole Genome Sequencing Shows that African Swine Fever Virus Genotype IX Is Still Circulating in Domestic Pigs in All Regions of Uganda.

Blood samples were collected from pigs at six abattoirs in the Kampala, Uganda metropolitan area from May 2021 through June 2022, and tested for African swine fever virus. Thirty-one samples with cycle threshold values < 26 from pigs with different geographic origins, clinical and pathologic signs, and Ornithodoros moubata exposure underwent whole genome sequencing. The p72 gene was used to genotype the isolates, and all were found to be genotype IX; whole genome sequences to previous genotype IX isolates confirmed their similarity. Six of the isolates had enough coverage to evaluate single nucleotide polymorphisms (SNPs). Five of the isolates differed from historic regional isolates, but had similar SNPs to one another, and the sixth isolate also differed from historic regional isolates, but also differed from the other five isolates, even though they are all genotype IX. Whole genome sequencing data provide additional detail on viral evolution that can be useful for molecular epidemiology, and understanding the impact of changes in genes to disease phenotypes, and may be needed for vaccine targeting should a commercial vaccine become available. More sequencing of African swine fever virus isolates is needed in Uganda to understand how and when the virus is changing.

Comparison of diaphragm meat juice and muscle swab samples to spleen and spleen swab samples for the detection of African swine fever viral nucleic acid.

Use of meat juice and muscle swabs at slaughterhouses may provide an easy-to-collect sample for African swine fever (ASF) surveillance. Meat juice has been experimentally shown to be a reliable sample for the detection of ASF virus (ASFV). We compared the detection of ASFV nucleic acid from diaphragm meat juice, diaphragm muscle swab, spleen, and spleen swabs from pigs with signs of ASFV infection at slaughterhouses around Kampala, Uganda. Pigs with ≥2 clinical or pathology signs at the time of slaughter had a spleen sample, spleen swab, diaphragm muscle sample, and diaphragm muscle swab collected. Meat juice was collected from muscle samples through a freeze-thaw cycle. Each sample was tested individually, and 72 spleen, meat juice, and muscle swab sample pools of 4 negative and 1 positive sample were tested, as well. Standard operating procedures from the USDA-Foreign Animal Disease Diagnostic Laboratory for viral DNA extraction and real-time PCR (rtPCR) were used. Of the 493 pigs evaluated, we classified as positive 357 (72.4%) diaphragm meat juice samples, 218 (44.2%) diaphragm muscle swabs, 247 (50.1%) spleen samples, and 241 (48.9%) spleen swabs. All spleen sample pools were positive (72 of 72; 100%), as were 71 of 72 (98.6%) meat juice pools and 67 of 72 (93.1%) muscle swab pools. Meat juice samples provided a reliable sample type for the detection by rtPCR of ASFV in pigs with natural infections.

An Investigation of Burkholderia pseudomallei Seroprevalence in Market Pigs Slaughtered at Selected Pig Abattoirs in Uganda.

Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis, a disease of humans and animals. It is primarily transmitted through direct contact with contaminated soil and surface water. The epidemiology of this pathogen in Africa, including Uganda, is largely unknown. The objectives of this study were to estimate the seroprevalence of B. pseudomallei in pigs slaughtered in central Uganda and to identify potential hotspots for this pathogen in the country. A total of 1035 pig sera were analyzed for serological responses to B. pseudomallei with type A and type B LPS using OPS type A and OPS type B ELISAs. Of the 1035 samples, 75 (7.25%, 95% CI: 5.8-9%) were seropositive to the OPS-A ELISA using a two standard deviations (SD) cutoff and 19 (1.84%, 95% CI: 1.2-2.9%) at 3 SD. For the OPS-B ELISA, 93/1035 (8.99%, 95% CI: 7.4-10.9%) were seropositive at the 2 SD cutoff, and 28/1035 (2.71%, 95% CI: 1.9-3.9%) at the 3 SD cutoff. Pigs slaughtered in central Uganda were exposed to B. pseudomallei, and there is a higher seroprevalence in the rainy months. Public health awareness campaigns about melioidosis may be needed.

"How Are My Age and Cows Related?" Cognitive Interviewing as a Tool to Pretest Survey Questions in Two Limited Resource Settings.

Antimicrobial resistance is a complex topic requiring interdisciplinary solutions embedded in One Health thinking. Currently, many surveys are underway in low- and middle-income countries to study how antimicrobial use in the livestock sector is driving resistance. In a survey, the respondents must understand and answer the questions correctly to produce accurate and valuable results. Pretesting survey questions is therefore important but sometimes not performed due to limited time and resources. Cognitive interviewing is a pretesting method to give insights into the respondent's way of interpreting and mentally processing the survey questions to identify problems and finding ways to improve the questions. It has previously been suggested that cognitive interviews may be difficult to use in some cultural settings. This study aimed to use cognitive interviews in a respondent-adjusted way to study how survey questions related to antimicrobial use are understood and answered by 12 small-scale farmers in Kenya and Uganda. The results show that even a small number of interviews and using interviewers with limited knowledge of cognitive interviewing can identify many problems in survey questions and the survey tool. Cognitive interviews may provide a feasible and affordable way of pretesting questionnaires in situations where time and resources are limited, for example, during a disease outbreak.

Epidemiological Dynamics of Extended-Spectrum ß-Lactamase- or AmpC ß-Lactamase-Producing Escherichia coli Screened in Apparently Healthy Chickens in Uganda.

The dynamics of extended-spectrum ß-lactamase- (ESBL-) and AmpC ß-lactamase-producing bacteria (which are deadly groups of antimicrobial-resistant bacteria) have not been well understood in developing countries. This raises major concerns to antimicrobial resistance (AMR) control. We investigated the prevalence and factors linked to the fecal carriage of ESBL- or AmpC-producing Escherichia coli (ESBL-/AmpC-EC) in commercial chickens. Cloacal swabs from 400 birds were sampled and submitted to the Central Diagnostic Laboratory for ESBL-/AmpC-EC screening by culture methods using MacConkey agar supplemented with cefotaxime. Epidemiological data were collected using a structured questionnaire and plausible risk factor analyses prepared by R software using X 2 test and logistic regression modeling. Results showed that the prevalence of ESBL-/AmpC-EC was 17.5%. Univariable screening hypothesized that carriage was probably influenced by a type of commercial chicken, geographical location, age group, flock size, and housing system (p < 0.05). Modeling exposed that broiler birds were at a higher risk of being ESBL-/AmpC-EC carriers (COR = 9.82, CI = 3.85-25.07). Birds from Wakiso Town Council (COR = 4.89, CI = 2.04-11.72) and flocks of 700-1200 birds were also at a higher risk of harboring ESBL-/AmpC-EC (COR = 2.41, CI = 1.11-5.23). Birds aged 4 months and below were more susceptible to ESBL-/AmpC-EC carriage compared with those aged 1 month and below being 6.33 times (CI = 1.65-24.35) likely to be carriers. The occurrence of ESBL-/AmpC-EC in flocks suggests possible treatment failures while managing colibacillosis. Consequently, injudicious antimicrobial use should be replaced with an accurate diagnosis by bacterial culture and sensitivity testing so as to circumvent AMR emergence, spread, and associated losses.

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.

Investigation of Ebolavirus exposure in pigs presented for slaughter in Uganda.

In 2008, an outbreak of Reston ebolavirus (RESTV) in pigs in the Philippines expanded our understanding of the host range of ebolaviruses. Subsequent experimental infections with the human-pathogenic species Zaire ebolavirus (EBOV) confirmed that pigs are susceptible to African species of ebolaviruses. Pig keeping has become an increasingly important livelihood strategy throughout parts of sub-Saharan Africa, driven by increasing demand for pork. The growth in pig keeping is particularly rapid in Uganda, which has the highest per capita pork consumption in East Africa and a history of sporadic human outbreaks of Ebola virus disease (EVD). Using a systematic sampling protocol, we collected sera from 658 pigs presented for slaughter in Uganda between December 2015 and October 2016. Forty-six pigs (7%) were seropositive based on ELISA tests at two different institutions. Seropositive pigs had antibodies that bound to Sudan NP (n = 27), Zaire NP (Kikwit; n = 8) or both NPs (n = 11). Sera from 4 of the ELISA-positive pigs reacted in Western blot (EBOV NP = 1; RESTV NP = 2; both NPs = 2), and one sample had full neutralizing antibody against Sudan ebolavirus (SUDV) in virus neutralization tests. Pigs sampled in June 2016 were significantly more likely to be seropositive than pigs sampled in October 2016 (p = .03). Seropositive pigs were sourced from all regions except Western region. These observed temporal and spatial variations are suggestive of multiple introductions of ebolaviruses into the pig population in Uganda. This is the first report of exposure of pigs in Uganda to ebolaviruses and the first to employ systematic abattoir sampling for ebolavirus surveillance during a non-outbreak period. Future studies will be necessary to further define the role pigs play (if any) in ebolavirus maintenance and transmission so that potential risks can be mitigated.