{'fraction_non_alphanumeric': 0.03532576446853119, 'fraction_numerical': 0.022667035879583845, 'mean_word_length': 4.9217615366596394, 'pattern_counts': {'":': 0, '<': 2, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
The role of Escherichia coli in the etiology of piglet diarrhea in selected pig producing districts of central Uganda

Background: Pig production in Uganda is highly constrained by rampant piglet mortalities with diarrhea being a key feature. The present study was conducted to determine possible involvement of Escherichia coli (E. coli) as agents of diarrhea in piglets and elucidate the factors for their spread and virulence, towards development of mitigation strategies in the smallholder pig value chains in Uganda. Methodology: This was a cross-sectional study carried out from January to August 2020 on preand post-weaned piglets from households in Kayunga and Mityana districts of Central Uganda, selected by snowballing method to redundancy. Data about herd management and risk factors for colibacillosis were collected from selected farmers in the two districts. A total of 179 faecal samples were collected from randomly selected neonatal and pre-weaning piglets for bacteriological isolation of Escherichia coli. Virulence (enterotoxin and fimbrial) genes from the isolates were detected by multiplex polymerase chain reaction (PCR) assay. Results: From the 179 faecal samples, a total of 158 (88.3%) E. coli isolates were obtained. Virulence gene markers were detected in 18.4% (29/158) of the isolates. Among the investigated genes encoding for enterotoxin production, STb was the most prevalent (16/158, 10.13%), followed by STa (12/158, 7.59%), while gene for LT was not detected. The gene coding for F4 adhesin was the only one detected while F18 adhesin was not detected from the isolates. On multiple logistic regression analysis, only tertiary educational level (OR=0.141; 95% CI=0.30-0.666; p=0.013) and infrequent use of antibiotics (OR=0.231, 95% CI=0.062-0.859; p=0.029) among the farmers, were the two factors significantly protective of the piglets from diarrhoea. Conclusion: This study reports a high prevalence of enterotoxin gene markers among E. coli isolates in piglets and revealed the potential role of these bacteria in the aetiology of piglet diarrhoea and mortalities in Uganda. Additionally, this study identified risk factors that can be useful in formulating treatment and control strategies of infection caused by these bacteria. Further studies are needed to identify more adhesins these E. coli isolates employ for intestinal colonization, a step that will help inform vaccine development.


Introduction:
Pork is one of the most consumed meat products worldwide (1) and the demand for pig meat is expected to increase with the growth in global population and economy. After China, the European Union (EU) is the second biggest producer of pig meat with a yearly production of 22.3 million tons (2). Therefore, maintenance of good health of pigs is very important. Uganda is among sub-Saharan Africa countries where pig production is increasing rapidly. The pig prolificacy is helping provide food security, however, it is constrained by low post-natal piglet survival (3). A study to quantify and classify pre-weaning causes of piglet mortality and how mortality relates to piggery management was conducted in peri-urban areas of Kampala, and out of 681 piglets born, 222 piglets died pre-weaning, representing 32.6% mortality (4). This is very high mortality considering that efficiency of piggery depends on the surviving litter size. In that study, infectious diseases were among the major causal factors reported. Moreover, during the smallholder pig value chain assessment in Uganda conducted by International Livestock Research Institute (ILRI) (3), among the major complaints' farmers put forward was rampant diarrhea. Similarly, in another study conducted in Gulu and Soroti, 38% of the pig keeping households reported diarrhea as a problem (5).
Globally, poor piglet survival is mainly due to Escherichia coli-induced diarrhea (6) or due to other bacterial pathogens (7). However, the causes of diarrhea/mortalities in the piglets have not been determined in Central Uganda, the major pig-producing region of the country. Escherichia coli infections seriously affect pig productivity through diarrhea, growth retardation, high morbidity and mortality among diseased pigs (6). Two distinct diarrhea syndromes are majorly recognized; neonatal diarrhea in piglets from 1-7 days of life, and post-weaning diarrhea that manifests about a week after weaning (8). The objectives of this study are to establish the involvement of E. coli pathogens in diarrhea among Ugandan piglets and to characterize their virulence genes, which is aimed towards adoption/development of appropriate vaccines for application to prevent piglet diarrhea and related mortalities in the small holder pig value chains in Uganda.

Materials and method: Study area and design
This was a cross-sectional study carried out from January to August 2020 on pre-and post-weaned piglets from selected households in Kayunga and Mityana districts of Central Uganda. The location of Mityana district is between 00 27N and 32 03E while the coordinates for Kayunga district are 01 00N and 32 52E.

Sample size determination
The sample size of households included in the study was arrived at using the Cochran formula; n = Zα 2 (P*Q)/E 2 (9), where, "n" is the sample size and "P" is the proportion of households keeping pigs. In this study, "P" was taken as 6%, which is the percentage of households rearing pigs in Kayunga (10) and "Q" is the proportion of households not keeping pigs (1-P).
The "E" is the level of acceptable error and was taken to be 5% while the "Zα" is the 95% confidence interval taken to be 1.96. Therefore, total number of households keeping pigs' targeted for the study was 87.

Ethical consideration
Ethical approval was obtained from the Ethical Review Committee of the School of Biosecurity, Biotechnical and Laboratory Sciences, College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University. Pig farmers were informed about the research and their consent to participate in the study was obtained before start of data collection.

Sampling, administration of questionnaire and collection of faecal samples
There was no information available regarding the households' keeping pigs in Uganda, therefore identification of the households was done using the Snowballing method to redundancy (11,12). Pig keeping households were visited to administer questionnaire and collect stool samples from diarrheic and non-diarrheic piglets. The first farmer was identified by the help of the extension agents and subsequent farmers were identified by the help of fellow farmers. A questionnaire was administered to selected farmers after obtaining their consent verbally in order to ascertain the factors that may underlie the spread of E. coli diarrhea in their piggeries. The roles of various household members in relation to pig care and management were also assessed particularly in relation to their gender.
Subsequently faecal samples were collected randomly from 2 piglets in each litter and before any antibiotic treatment was given in case of diarrhea, to ensure optimum recovery of E. coli. During the sample collection, each piglet was scored as being diarrheic or not. The faecal samples from each piglet were collected per rectum using sterile swabs (BD, Loveton, Maryland, USA) and were immediately placed into 5 ml of sterile chilled peptone water medium. The samples were then transported on ice in a cool box to the Microbiology laboratory at the College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, Uganda within 48 hrs of sampling for bacteriological culture and isolation.

Bacteriological isolation and identification
Each rectal swab was streaked out on sterile MacConkey agar (Condalab, Madrid, Spain) for the isolation of E. coli. Inoculated plates were incubated aerobically at 37°C for 18-24 hr. Preliminary identification of isolates was based on colonial morphology on all the media used. Lactose-fermenting colonies on MacConkey agar were biochemically confirmed as E. coli using tryptophan broth for indole test, methyl red for MR/VP test and citrate agar for citrate utilization test. Biochemically confirmed E. coli were stored in Brain heart infusion broth (Oxoid Ltd, Hampshire, United Kingdom) with 20% glycerol in duplicate at -20°C pending DNA extraction.

Antibiotic susceptibility tests
Antimicrobial susceptibility testing of E. coli isolates was performed by the standard Kirby-Bauer disk diffusion method against selected antibiotics (Oxoid Ltd, Hampshire, United Kingdom) which includes; kanamycin (K 30 µg), nalidixic acid (NA 30µg), tetracycline (TE 30 µg), ampicillin (AMP 10µg), chloramphenicol (C 30 µg), erythromycin (E 15µg), gentamicin (CN 10 µg) and ciprofloxacin (CIP 5µg) on Mueller-Hinton agar (Oxoid Ltd, Hampshire, United Kingdom). The 2018 Clinical and Laboratory Standards Institute (CLSI) guidelines (13) were used for the interpretation and categorization of the test strains as sensitive (S), intermediate (I) or resistant (R). Control E. coli strain ATCC 25922 was also set along with positive isolates during susceptibility testing for each antibiotic used.

Extraction of DNA from E. coli isolates
DNA was extracted from each isolate using the boiling method (5). Briefly, the stored isolates were thawed thoroughly before vortexing to ensure uniformity of the bacterial cell suspension. One ml of the sample was transferred into a correspondingly labelled 1.5 ml Eppendorf tubes, and centrifuged at 15,000 × g for 15 min in a microcentrifuge. The supernatant was discarded and remaining pellets were washed twice (by resuspending in 1 ml nuclease free water) followed by centrifugation at 15,000 × g for 10 min in a microcentrifuge. The pellets were then re-suspended in 100 μl of nuclease free water and boiled at 100°C for 10 min in a water bath. The tubes were thereafter frozen at -20°C for 10 min in a freezer and after thorough thawing, were centrifuged at 15,000 × g for 10 min in a microcentrifuge. Finally, 50 μl of the supernatant was transferred into correspondingly labelled Eppendorf tubes and stored at -20°C pending analysis by PCR.

Amplification of fimbrial and enterotoxin genes by multiplex PCR assay
Multiplex PCR assay was used to amplify the fragments of genes encoding enterotoxins (STa, STb and LT) and fimbriae (F4 and F18) of each E. coli isolate. The primers used are as shown in Table 1. Each reaction consisted of 1× PCR buffer II, 3 mM MgCl2, 200 μM each of dATP, dTTP, dCTP and dGTP and 1.5U of AmpliTaq Gold DNA polymerase (Applied Biosystems, ThermoFisher Scientific Corporation, Massachusetts, USA). The cycling conditions were set as follows; 95°C for 5 min, followed by 35 cycles of 94°C for 60 sec, 59°C for 60 sec and 72°C for 60 sec, followed by a final extension at 72°C for 5 min. DNA from E. coli strains 853/67; O149 (F4 + , F6 + , LT + , STa + , STb + and EAST1 + ) (Uppsala, Sweden) and a blank sample without DNA were used as positive and negative controls, respectively.

Agarose gel electrophoresis
Eight microliters of each of the PCR amplicons were mixed with 2 μL of the loading buffer and resolved on 2% agarose gel in 1×TBE buffer at 125 V for 45 min using mupid-exu submarine electrophoresis system. The gels were stained by ethidium bromide, visualized by UVtransillumination and gel pictures captured by Smartphone camera.

Statistical analysis
Data obtained from the questionnaires were entered into a Microsoft Excel spreadsheet and descriptive statistics was performed. A univariate analysis using Chi-square test or Fisher's exact test was used to analyze the laboratory data and also test the associations between the independent variables and the outcome variable. Variables with p ≤ 0.25 were then tested for confounding by cross tabulation of two variables. If the variables were confounding (p ≤ 0.05), only one was taken for multivariate logistic regression analysis based on biological plausibility. The model fitness was assessed by Hosmer and Lemeshow (HL) test.

Results:
Socio-demographics of the farmers involved in the study.
In total, samples were collected from 74 households; 35 and 39 in Mityana and Kayunga districts, respectively ( Table 2). Male gender dominated pig farming in the both districts, with 19 (52.3%) and 28 (71.8%) from Mityana and Kayunga, respectively. Majority of farmers in Mityana were in age group of 31-40 years (34. 3%), while in Kayunga, piggery was practiced mostly by farmers who were between 51-60 years of age (33.3%), which indicated that the youth were less frequently involved in piggery in the two districts (Table 3).    "n" denotes the number of selected households; * = statistically significant at p < 0.05 In Mityana, 14/35 (40%) farmers had primary level of education only, while in Kayunga, 18/39 (46.2%) farmers were educated up to secondary level. These statistics show that 13.5% of all those involved in keeping pigs did not have formal education. The difference in the education level of pig farmers in the two districts was however not statistically significant (p= 0.113). However, 48.6% of those engaged in piggery in both districts were involved in farming as their major occupation. Wife owned pigs in 32 of the 74 households (43.2%) while in Mityana alone, 48.6% (17/35) of pigs were owned by wife. There was significant difference (p=0.016) in the ownership of pigs in both districts, with wife owning more pigs than husband. In both Kayunga and Mityana, housewives were most frequently involved in caring for the pigs (32.4%). Hired labourers were more frequently involved in pig care in Kayunga (25.6%) compared to Mityana (8.6%). On the other hand, husbands were less frequently involved in pig care (14.9%) ( Table 3).
Fifty-nine percent of farmers in Kayunga reared exotic pig breeds, but Mityana farmers mostly reared local breeds (62.9%). In both districts, farmers were mostly involved in small scale pig rearing of about 1-10 per household. Additionally, 5.4% of farmers in this study were keeping more than 30 pigs per herd. However, the differences in herd size in both districts were not significant (p > 0.05).

E. coli strains isolated
From the total of 179 faecal samples collected, 158 (88.3%) E. coli isolates were obtained following bacteriological culture. Majority of the E. coli isolates (85, 53.8%) were from Kayunga while 73 (46.2%) were from Mityana district (Table 4). Multiplex PCR assay revealed that 18.4% (29/ 158) of the isolates harbored virulence genes. Among the investigated genes coding for toxin production, STb was the most prevalent (16/ 158, 10.13%), followed by STa (12/158, 7.59 %), while gene for LT was not detected. Gene for fimbriae was present in 0.63% (1/158) of the isolates. The F4 was the only fimbrial gene detected in this study while F18 was not detected in any of the isolates. The one E. coli strain carrying F4 gene also carried enterotoxin STa gene, and no fimbrial adhesin was detected in the rest of 157 (99.4%) E. coli isolates (Fig 2).

Risk factors associated with diarrhea in piglets
Sixteen independent variables; sex of household head, education level, age of household head, occupation, ownership of the farm, district, breed of pigs, breeding method, pig herd size, pigs keeping methods, house provided for neonate, pig care taker, source of water, weaning period, diarrhea common in the farm and common use of antibiotics, were analyzed as potential risk factors in the causation of diarrhea in piglets in the total of 74 farms in Mityana and Kayunga districts. Univariate analysis shows that occupation, education level, breed of pigs, district and frequent use of antibiotics were significantly associated (p<0.05) with diarrhea in the piglets (Table 5). At multivariate analysis, two factors (education level and frequent use of antibiotics) were signifi-cantly associated with diarrhea in the piglets (Table 6). Tertiary education (OR=0.141, 95% CI=0.30-0.666, p=0.013) and infrequent use of antibiotics (OR=0.231, 95% CI=0.062-0.859, p=0.029) among the farmers were the two factors significantly protective of the piglets from diarrhoea.

Results of antimicrobial susceptibility test
The antibiotic to which the E. coli isolates exhibited the most resistance in both districts was erythromycin (Fig 3), followed by tetracycline. In general, the E. coli isolates from Kayunga district showed higher resistance to the antibiotics compared to those from Mityana district ( Table 7). The most effective antibiotic drug was kanamycin, followed by ciprofloxacin and gentamicin.

Discussion:
This was the first study to elucidate the role of E. coli in the aetiology of piglet diarrhea as well as the risk factors and antimicrobial resistance of these pathogens in Central Uganda. The finding of 18.4% of the E. coli with virulence factors (fimbriae and/or enterotoxin genes) is significant since it confirms the possible involvement of these bacteria in the etiology of diarrhea and piglet mortalities in Uganda. In the present study, the only adhesin detected in E. coli was F4, which is in agreement with the previous study conducted in Northern and Eastern Uganda by Ikwap et al., (5) but differ from developed countries (14) where F4 was most predominant. In our study, F18 adhesin was not detected, however, F18 adhesin was recently reported in diarrheic weaners from large commercial farms in Central Uganda (15). Since F18 adhesin is associated with post weaning diarrhoea (PWD), the prevalence of PWD could be very low in weaners from small holder herds, since this condition is mainly related to intensive rearing systems with high infectious load, stress caused by early weaning, moving and mixing of pigs (5,16) Secondly, the nondetection of F18 could be due to the low number of diarrheic piglets tested in our study. Interestingly, no adhesins were detected in 99.4% of the E. coli isolates tested in this study, although we investigated only F4 and F18 fimbriae. There is the need to therefore conduct more research on other adhesins in order to determine the key adhesins used by these bacteria to colonize piglet intestines, which can subsequently lead to diarrhea. This step will enable identification of appropriate vaccines for disease prevention.
In our study, the prevalence of piglet diarrhea was 16.8%. This is similar to the finding of a study conducted in India by Vinodh-Kumar et al., (17) which reported a prevalence of 14.29%, although slightly higher than 10% rate reported in a similar study conducted in Spain by Mesonero-Escuredo et al., (18). Worldwide, E. coli expressing enterotoxins (ETEC) are well known to cause severe diarrhea often with high mortality rates in both neonatal and postweaning piglets (19). The ETEC strains associated with piglet diarrhea normally produce LT, STa or STb toxins or their combinations (20). In this present study, STa and STb were the only toxin genes detected, and in agreement with previous studies that reported high prevalence of STb in E. coli isolates from neonatal and weaned piglets (21,22), the most prevalent enterotoxin detected in E. coli from piglets in our study was STb (10.13%). However, the gene for LT was not detected in our isolates, suggesting that E. coli harboring genes encoding for this toxin are not yet widespread. The absence of the gene for LT in all of the E. coli isolates in our study is in agreement with previous study in Northern and Eastern Uganda by Ikwap et al., (5) where none of the LT gene was detected. This suggests that ETEC diarrhea in Central Uganda could be largely contributed by STb and STa in neonatal and post-weaning piglets. In order to cause colibacillosis, an ETEC strain needs to simultaneously carry at least one fimbria and one toxin gene (23). Considering this criterion, less than 1% of the isolates in our present study could be classified as ETEC since there was only one STa/F4 combination detected in this study. The other isolated strains might have been non-pathogenic or belonged to other pathotypes such as EPEC and the fimbrial type F5, F6 or F41, which were not investigated in this study. Two factors (tertiary education level and infrequent use of antibiotics) were significantly protective of the pigs from diarrhea from our study analysis. This may probably indicate that highly educated farmers (with tertiary level education) consider hygiene and other disease control measures more importantly than less educated farmers (with secondary level education) and therefore adhere to the required processes to control and prevent disease. However, if a farmer with formal education lacks training and experience in animal husbandry, it can lead to flaw in pig production as knowledge uptake itself does not necessarily guarantee an improvement in the everyday practices (24). Pig farmers in the study area were also involved in other farming activities, self-employed or were civil servants. This picture gives the impression that pig farming alone does not make farmers to make a living within the study area.
Infrequent use of antibiotics was significantly associated with lower frequency of diarrhea in the piglet probably because of the adherence to disease control measures such as environment hygiene and proper feeding. Farmers who frequently use antibiotics tend to assume that these drugs are a panacea to proper rearing of pigs, yet not all antibiotics are effective against bacteria pathogens. For treatment to be effective against disease-causing bacteria, antibiotics must reach the site of infection at sufficient concentrations and for sufficient amount of time. Inappropriate use of these antibiotics may not only promote antimicrobial resistance but also aggravate the disease. Furthermore, commensal beneficial bacteria provide a natural barrier and defense system against pathogenic bacteria, treatment with antibiotics results in killing of these beneficial bacteria, thereby rem-oving the natural defense system of the animals and making them more vulnerable to disease.
Antimicrobial resistance profiles of E. coli isolates in the present study revealed high resistance to erythromycin and tetracycline. This is highly concerning considering that the tetracyclines are the most frequently used antibiotics for treatment of infections in animals and poultry in Uganda, compared to other antimicrobial agents (25). Therefore, the high resistance levels in our study might be the result of widespread and frequent use of tetracycline to treat colibacillosis in the field. Interestingly one of the risk factors detected associated with piglet diarrhea was frequent use of antimicrobial agents against infections in the pig herds. This is attributable to probable emergence of antimicrobial resistance, hence the inability to control infections using the commonly available antimicrobials in these households.
Resistance to ampicillin, a commonly prescribed antibiotic for the treatment of colibacillosis, was relatively high with overall resistance of 41.8% in this study. A study done in Korea by Kyung-Hyo et al., (26) showed higher resistance of 84.9%. Overuse of ampicillin in Uganda to treat piglets against diseases such as clostridiosis and colibacillosis has probably led to such high degree of resistance of E. coli to this drug. Withdrawing such antibiotic from use by making it unavailable in the market and/or restricting its use may allow it to recover its potency, and may then subsequently be re-introduced, through a process of antibiotic cycling.
Majority of E. coli isolates in this study were susceptible to kanamycin, gentamicin, ciprofloxacin, nalidixic acid, and chloramphenicol. These findings are similar to that of a previous study on Salmonella spp in piglets and weaners from Northern and Eastern Uganda (27). Apart from gentamicin and kanamycin, the other three antimicrobials (chloramphenicol, ciprofloxacin and nalidixic acid) are mainly used in humans in Uganda. Furthermore, some of these antibiotics are very expensive and therefore not commonly used. Possibly this could be the reason for the high susceptibility of the piglet derived isolates of E. coli to these drugs in the present study. However, high levels of resistance to gentamicin were reported among E. coli isolated from diseased pigs in Belgium, Poland and Spain with rates of 46%, 45%, and 20% respectively (28). There is therefore the need for continuous monitoring, restrictions and judicious use of antimicrobials to ensure the future availability of effective antimicrobial drugs for use in both humans and veterinary medicine in Uganda.

Conclusion:
This study reports a high prevalence of enterotoxin gene markers in E. coli in tested piglets therefore highlighting the potential role of these bacteria in the aetiology of piglet diarrhea and mortalities in Uganda. Additionally, this study identified risk factors that can be useful in formulating treatment and control strategies for infections caused by these bacteria. Since no adhesins were identified in most of the E. coli isolates, further studies are needed to identify the adhesins these E. coli isolates employ for intestinal colonization, a step that will help inform vaccine development in Uganda settings.