Antimicrobial Usage by Small-Scale Commercial Poultry Farmers in Mid-Western District of Masindi Uganda: Patterns, Public Health Implications, and Antimicrobial Resistance of E. coli

Background Poultry production in Uganda is growing at a fast rate due to increasing demand, notwithstanding, poor husbandry practices, and diseases, prompting farmers to rear healthy productive flocks with antimicrobials. The study evaluated the knowledge and practices as regards the use of antibiotics among poultry farmers in Masindi district and determined the antibiotic susceptibility profiles of E. coli strains from chickens. Methods A cross-sectional study using a closed-end questionnaire was conducted in 140 selected small-scale commercial poultry farms in Masindi district between June and December, 2020. Analyzed qualitative data were presented as frequencies, percentages, and their 95% confidence intervals (95% CI). Fecal swabs from chickens were inoculated onto a MacConkey agar, and E. coli was confirmed by standard biochemical tests. Antimicrobial susceptibility was determined by the disk diffusion method for 7 antibiotics. Results Most farmers (74%) used antibiotics, mainly tetracycline (51.4%) and sulfonamides (28.6%), given to the chicks (45%), for both curative and prophylaxis purposes (80%), and via drinking water (67%). Farmers mainly used antibiotics recommended by the veterinarian (76.4%), more than relying on experience (10.7%), while 45% were involved in self-medicating the birds. On choosing the correct dosage, 45.7% read the instruction, and 42.9% consulted a veterinarian. Only 10.7% observed the drug withdrawal period, while 53.6% consumed eggs at home or sold eggs (35.7%) from birds under treatment. Of the 200 E. coli strains, 90 (45.0%) were resistant to one drug, 74 (37.0%) to two, and multidrug resistance to three classes of antibiotics was 36 (18.0%). Overall, E. coli resistance to tetracycline was (69.0%), ampicillin (37.0%), sulfonamides (36.0%), and to kanamycin (1.5%). Conclusions The small-scale poultry farmers frequently use antimicrobial drugs, mainly tetracycline and sulfonamides for curative and prophylaxis. Thus, enforcing measures against antibiotic use supported by a strong veterinary service sector and farmers' training on judicious use of antimicrobials are needed.


Background
Almost all rural and peri-urban homesteads in Uganda are engaged in agriculture, a sector that employs over 70% of the population [1]. Around 35 million birds are reared, mainly as free-ranging indigenous chickens (80%), with the rest under the intensive management of exotic breeds [1]. In comparison to other livestock, poultry production is growing at a faster rate due to the high national and regional demands. Consumption of poultry and poultry products is acceptable in most cultures, supporting food security, nutrition, and livelihoods for the rural poor [2]. Regardless, poultry production faces severe challenges such as poor husbandry practices, low-quality feeds, sanitary and housing defciencies [3,4] associated with the emergence of pathogens, and high disease burden. Inevitably, farmers opt to use antimicrobials for treatment and/or prevention of diseases and promotion of rapid growth [5,6].
Without doubt, the irrational use of antimicrobials in animals is a major contributor to increased antimicrobial resistance (AMR) [7,8] and the emergence of multidrugresistant (MDR) bacteria which is of global health concern [8,9]. AMR hampers the treatment of life-threatening infections in both humans and animals. Imprudent use of antimicrobials can selectively promote the emergence of drug-resistant commensal bacteria in the gut [10,11], which are excreted in feces, may contribute in food contamination and food-borne infections in humans [12][13][14].
Even though poor countries in sub-Saharan Africa (SSA) contribute a signifcant proportion to the global antibiotic use [15][16][17], the lack of reliable data on antimicrobial use in animals is an obstacle to the designing of mitigation strategies. Empirical data show the gravity of the problem; for instance, arbitrary determination of antimicrobial dosage before administration was reported by 93% of poultry keepers in Nigeria [18]. Similarly, the use of antibiotics without prescription and failure to observe antibiotic withdrawal periods were also reported [19,20]. Recent reports in Uganda indicate that 97% and 66% of farmers frequently used antibiotics in their poultry farms from Wakiso [21,22] and Nakaseke [23] districts, respectively, which are gotten from drug shops without prescriptions from veterinarians [19,20]. In general, the abuse of antimicrobial usage is exacerbated by weak enforcement of policies in Uganda [24,25].
Efective handling of AMR requires a multisectoral collaboration of human, animal, and environmental sector players, defned as one health [26,27]. At the national level, the Uganda government has established a multisectoral one health coordination ofce as a coordination frame and a National Action Plan to guide and counter AMR [28]. Tis is also in tandem with global eforts through the Quadripartite collaboration on AMR, consisting of the Food and Agriculture Organization (FAO), the World Organization for Animal Health (OIE), the World Health Organization (WHO), and the United Nations Environment Programme (UNEP) aims at antimicrobials stewardship [29][30][31]. Te WHO Global Action Plan on AMR [29] and the European Medicines Agency (EMA)/Antimicrobial Advice Ad Hoc Expert Group (AMEG) [32] explicitly aim to strengthen knowledge and evidence-based practices through surveillance and research to guide the development of appropriate strategies that ensure antimicrobial stewardship in human and animal health [33].
Clarifying the gaps in knowledge and practices of poultry farmers as regards antimicrobial use patterns is needed prior to designing meaningful mitigation strategies. Te study evaluated the knowledge and practices as regards the use of antibiotics among poultry farmers in Masindi district and determined the antibiotic susceptibility profles of E. coli. Te identifed knowledge and skill gaps of the farmers will be used to create awareness and infuence prudent behavioral change on antimicrobial use.

Design and Setting of the Study.
Te study was conducted in Masindi district, located between coordinates 1°41" 0″ N and 31°44" 0″ E in Mid-Western Uganda, 214 kilometers from Kampala, the capital city of Uganda. Te district covers an area of 7,443 sq. km, with 6332 sq. km of arable land. It has a favorable climate with a bimodal rainfall pattern and an annual long-term average rainfall of 1,304 mm. Te district is divided into 5 subcounties of Budongo, Bwijanga, Kimengo, Miirya, and Pakanyi and two urban town councils of Bulima and Kabago, in Bwijanga and Budongo subcounties, respectively. In addition, the urbanised municipality comprises 4 divisions: central (urban) and three periurban divisions of Karujubu, Kigulya, and Nyangahya ( Figure 1). A total population of 292,951 people is majorly rural (68%) and engages in agriculture as the main economic activity (https://www.masindi.go.ug/district/population). Most households keep local chickens on a free-range system, with an intensive deep liter system for exotic birds, especially the layers confned to the urban and peri-urban areas. As one of the districts in Uganda's Albertine oil region, commercial poultry keeping is a target enterprise to meet the growing demand for eggs and meat. Small-scale commercial poultry farmers keep between 100 and 1000 exotic birds in an intensive management system, which also provides additional income for the urban and peri-urban dwellers in the district.
A cross-sectional study using a closed-end questionnaire survey was conducted in selected small-scale commercial poultry farms in Masindi between June and December, 2020. Prior to the survey, the study team met with local veterinary ofcials and leaders of poultry farmers to discuss the survey   Veterinary Medicine International plans. Te study population was small-scale commercial poultry farmers raising layer birds under a deep litter intensive husbandry system. A total of four divisions/subcounties were selected from Masindi district (central division, Kigulya, Karujubu, and Nyangahya) within the urban and peri-urban areas based on ease of access and the large number of farmers that reared exotic layer poultry. Te sample size was calculated at a 95% confdence level with the assumed prevalence of antibiotic use of 10% and margin of error at 5.0% of all farms, which gave 139 farms. From the selected divisions/subcounty, 140 households'/ poultry farms were selected from a list of farms (n � 400) obtained from Masindi district veterinary ofce (this was estimated based on their experience as there are no ofcial records on the number of farms). Te frst farm was randomly selected, while the second and subsequent farms were selected by consecutive sampling until the required number was obtained. A farm was included if the owner or an adult responsible for taking care of the layer birds was present and agreed to provide verbal informed consent. Farms were excluded if owners or a responsible adult could not be located at the time of the visit; however, the next available farm was considered for the interview if it met the selection criteria.
Prior to administering the questionnaire, the purpose of the study was outlined. Te team procured sachets and bottles of antibiotics available from the local veterinary drug shops in the area as samples which were displayed before the farmers to guide in the identifcation of familiar drugs used with their farms. Oral consent to participate in the study was obtained, and all consenting farmers present on the day of the interview participated. A closed-ended questionnaire was administered by a research assistant conversant in Runyoro, the local language used in the area. Te focus of the questionnaire was on poultry farmers' knowledge and practices believed to be associated with the imprudent use of antimicrobials. Te questionnaire aimed to explore participants' socio-demographic characteristics, common diseases experienced at the farm, the person(s) responsible for treating sick birds, and drugs commonly used among others. Te questionnaire was pretested on 10 individuals in Miirya subcounty with a similar setting in the district. Direct observations and farm records were also used to gather additional information related to antimicrobial usage practices.

Bacterial Culture and Antibiotic Susceptibility Testing.
Two composite fecal swabs obtained from fve fresh chicken droppings in each poultry farm were placed in sterile tubes containing Stuart transport media and transported on ice in a cooler box to the Microbiology Laboratory in the College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, Kampala. Samples were inoculated onto a MacConkey agar (MAC) (Remel, Lenexa, KS, USA) and incubated aerobically at 37°C for 18−24 hrs. Presumptive E. coli was confrmed by standard biochemical testing using API20E (Analytical Profle Index; BioM6rieux, Hazelwood, MO).

Data Management and Analysis.
Te collected data were coded and entered into an Excel worksheet (Microsoft ® Excel for Windows, 2017). Data cleaning and analysis were carried out in Statistical Package for Social Sciences (SPSS version 17) to obtain descriptive statistics for each variable. Results were presented in the form of frequencies, percentages, and their 95% confdence intervals (95% CI) to determine the extent of antimicrobial use in poultry production in the study area. Antibiotics used for poultry production in Masindi district were grouped into four categories A to D based on EMA/AMEG classifcation [32] (i) category A: avoid includes antibiotics that are currently not authorized for veterinary use and may only be given to companion animals under exceptional circumstances; (ii) category B: restrict, antibiotics are critically important in human medicine, and their use in animals should be restricted to mitigate the risk to public health, and only used when there are no antibiotics in categories C or D; (iii) category C: caution includes antibiotics for which alternatives in human medicine generally exist, but only few alternatives are available in certain veterinary indications. Should only be used when there are no antimicrobial substances in category D that would be clinically efective; and (iv) category D: prudence includes antibiotics which Veterinary Medicine International should be used as frst line treatments, whenever possible, which should be used prudently and only when medically needed.

Antibiotic Use Practices and Associated Public Health
Risks. As regards the public health risks associated with antibiotic use practices, farmers mostly selected antibiotics, recommended by the veterinarians (76.4%). While 10.7% of the farmers used previous experience, 7.2% chose only available drugs and only 2.1% relied on drugs that were advertised (Table 4)

Health Challenges Reported by Poultry Farmers in Masindi
District. Te fock size of small holder poultry farmers ranged between 100 and 300 birds, with a median of 240 birds. Te three major health problems experienced on poultry farms were Gumboro (133; 34%), new castle disease (127; 32%), and respiratory diseases (16%). Coccidiosis and ectoparasites were also experienced by poultry farmers, among others (Figure 2).
Ninety (45%) of the 200 resistant isolates were only resistant to one drug; for TRI, TET, and AMP, the percentages were 18%, 31%, and 51%, respectively. While 62% were resistant to TET and SUL, 24% and 18% were resistant to TET and AMP or TRM, respectively. Overall, 36 (18%) of the bacterial isolates were resistant to at least one drug in three classes of antibiotics (multiple drug resistance). Tese include resistance to TET, SUL, and NEO (42%), TET, AMP, and TRM (19%) or KAN (8%). Te remaining 31% of the isolates were resistant to TET, SUL, NEO, and GEN ( Figure 3).

Discussion
Te study provides crucial information on antibiotics use practices among small-scale commercial poultry farmers in Masindi district, which is useful in planning and implementation of strategies to mitigate the emerging AMR. As the demand of poultry products increases, farmers in Masindi district are motivated to produce more; however, this calls for practices that ensure rational use of antimicrobial drugs mindful of consumers' health concerns. A larger segment of the farmers (74%) use antibiotics and  Veterinary Medicine International 48.6% always use antibiotics on their poultry farms. Tis was higher than previously reported in Tororo (15%) but comparable to Wakiso (74.0 versus 71.3%) in eastern and central Uganda [22], respectively. In an earlier study in Uganda, the antimicrobial use reported among livestockkeeping households was at 35% [37]. Te contrasting fndings of antimicrobial usage in our current study with the previous studies in Uganda could be due to knowledge diferences among study populations. It also suggests an increasing trend in accessing and use of antimicrobials by farmers. Our fndings are in agreement with reports of frequent antibiotic use among poultry farmers in Tanzania where 100% used antibiotics [38] and 65% in Abia State, Nigeria [39]. Te shift in production practices from traditional [40] rearing of scavenging indigenous birds to commercial intensive exotic poultry production, which     Veterinary Medicine International presumes that optimal health and production can be attained only with antimicrobials use, is the root cause of the problem. Te need to fnd alternatives to antimicrobials or optimal practices that enhance health and productivity is what the future needs. Arguably, the high frequency of antimicrobial usage could be attributed to the use of antimicrobials for curative and prophylaxis treatment, rather than for the promotion of growth. No growth promoters are used by poultry farmers in Masindi probably because they are not available in the market. Nevertheless, vitamin formulations containing tetracycline or sulfonamides are readily available for use. Typically, poultry reared under stressful conditions in overcrowded houses with inadequate ventilation and awful hygienic environments are highly predisposed to infection [41]. Most farmers opt to use antibiotics as a cover up for such poor animal husbandry practices, a justifcation which could be avoided. Farms that implement adequate biosecurity systems to limit undue distress in birds [42][43][44] can efectively maintain healthy productive focks with minimal antimicrobial use. As well, our fnding of excessive use of antimicrobials in poultry production raised public health concerns, which could lead to the transmission of resistant bacteria to humans and the environment [8,9]. It is well documented that antibiotic-resistant pathogens shed from birds can be transmitted to humans and cause food-borne illness with severe sequelae [13].
Te antibiotics frequently used in poultry were tetracycline and sulfonamides which were similar to the fndings of earlier studies in Uganda [21,22] and elsewhere in Tanzania [38], Nigeria [39], and Ghana [6]. In contrast, another report from poultry farmers, in Nigeria, indicated the frequent use of cotrimoxazole and neomycin in chicken production [45]. Although the drugs used might be diferent, category C and D antibiotics are readily available as cheap generics, accessible to farmers over-the-counter without prescription from veterinarians. In contrast, gentamycin, an antibiotic commonly available in an injectable formulation, is less likely to be used by poultry farmers.
Farmers treated sick birds without the involvement of veterinarians in 44.6% of the respondents and in most cases using antibiotics. Such practices are frequent in many countries in Africa [6,21,39] and could lead to under-dosing or over-dosing of birds. Often, farmers with more experience undertake treatment of their focks and consult a veterinarian only after the treatment has failed, which would increase antibiotic misuse and the risk of drug resistance. Tis is in concurrence with a previous report from Ghana which reported that over 50% of poultry farmers relied on personal experience to administer antimicrobials to their animals [6,46]. Probably, experienced farmers are biased towards seeking veterinary services in preference to consulting fellow farmers for advice in a bid to minimize the cost of treatment. Tis is because small-scale commercial poultry farmers may not be able to aford the services of veterinarians [47,48] and opt for other alternatives. Overall, the enforcement of stringent measures and guidelines on antibiotic use under veterinary supervision is needed, notwithstanding the prevailing weak veterinary services in Uganda [24,25,49]. A section of farmers used herbal preparations to treat various poultry diseases, such as newcastle disease and coccidiosis. Herbal drugs are frequently used by poultry farmers to treat various illnesses in Uganda [50]. Despite the availability of vaccines against most of the prevalent viral diseases, a good portion of the farmers kept their birds on antibiotics most of the time. Tis irrational use of drugs is promoted by aggressive marketing of antibiotics, the ease of obtaining drugs without prescription, and administration without veterinary advice.
Most farmers read instructions or relied on the veterinarian's instructions to estimate the dosage of drugs. Since the majority had attained secondary education and were between 20 and 50 years, it can be surmised that they had a relatively good understanding of written instruction. Moreover, a majority of urban poultry farmers are working class with higher education background and can easily follow the instructions given by veterinarians. However, it was observed that farmers that frequently used veterinary advice were more likely to use higher frequencies of antibiotic-containing agents for treatment and prevention of infections [6]. Tis could be due to the fact that veterinarians are not readily available to supervise and oversee antibiotic use at the farm, which gives farmers the confdence to misuse the drugs left in their hands.
Te observance of the antibiotic withdrawal period is required to ensure animal products are free from drug residues. Nonetheless, most poultry farmers in our study used antibiotics without considering the drug withdrawal period or the expiry dates, which was similar to another study in Ghana [6]. Tis in part shows a lack of adequate knowledge on the deleterious efects of consuming eggs with high concentrations of antibiotics but also suggested that farmers do not care about the public health implications of such a practice. Farmers deliberately ignore the withdrawal periods and sell prohibited eggs to limit fnancial losses [46]. A lack of monitoring from governmental agencies had been previously identifed as a reason why withholding periods were not adhered to by farmers [51]. Largely, the limited facilities in Uganda cannot efectively test and monitor the presence of drug residues in eggs [25,49].
Only a small proportion of the poultry farmers was aware of and followed the expiry date, suggesting the need for farmer sensitization. Furthermore, a majority of farmers were not acquiescent to observe the expiry dates of antibiotics. Use of expired drugs raises concerns of low efcacy and efectiveness which contributes to AMR. As earlier on, reported and expired oral pediatric antibiotics have been associated with high AMR in a study from Nigeria [47]. Tus, it is of necessity to have policies that ensure expired antibiotics are not administered in animals.
Tat 76.3% of E. coli, from the poultry in Masindi were resistant to at least one drug, was not surprising. Tis was comparable to an earlier study that reported 70% resistance in Uganda [52]. Other studies reported a higher prevalence of AMR of 88.4% [53] and 87% [54] of E. coli isolates in poultry in Uganda. Overall, 56% of the E. coli isolates were resistant to at least two antibiotics, suggesting that the exotic chicken from small commercial layers is beset by high level Veterinary Medicine International of AMR. Te high AMR reported in this study is similar to previous studies reported in Uganda [53]. Te emergence of drug-resistant bacteria in animal populations and their potential transfer to humans is of global public health concern [8,9]. Te likelihood of misuse is high in Uganda, a country with weak veterinary services and poor regulation of antibiotic usage [25,49].
Te commensal E. coli strains that colonize the gut of animals are used as indicator bacteria for changes within the microfora ecosystem [55]. Te use of antimicrobials in poultry production can create selective pressure for E. coli leading to the emergence of antibiotic resistant strains in the gut [10,11]. Tus, the observed AMR can be construed as a predictor of increased selective pressure as a result of excessive use of antimicrobial agents in poultry production. Tis can also be used as an early warning sign for the emergence of AMR in various species of microbes within the gut. Tis may require another study to investigate the susceptibility of various species of gut microfora to antibiotics.
Category C and D antibiotic drugs used in poultry production are simultaneously listed as drugs for treating various human illnesses in the country [56,57]. Tere should be a clear justifcation with a reasonable need for their use in poultry health, as opposed to routine administration. Notably, prudent antimicrobial usage and curbing of the emerging resistance in livestock at the community and national scale would require interdisciplinary and multisectoral collaboration, referred to as one health, of diferent sectors: animal, human, and environmental health [27,[29][30][31]. Tus, through the National Action Plan (NAP), the government of Uganda has designed comprehensive strategies to contain and control AMR threats in the human and animal health sectors at the national level [28]. Tese eforts will in the long run prevent, slow down, and control the spread of resistant pathogens [28].

Conclusion
Te small-scale poultry farmers in Masindi continue to use antimicrobial drugs, mainly tetracycline and sulfonamides for curative and prophylaxis treatments, without consulting veterinarians. Te experienced farmers tended to avoid seeking veterinary services in a bid to minimize the cost of treatment. Adherence to the recommended drug withdraws period and expiry dates for drugs was glaringly lacking putting the consumers health at risk. Majorly, E. coli strains were resistant to multiple antimicrobial drugs suggesting increasing selective pressure due to excessive use of drugs in poultry production. Te enforcement of stringent measures and guidelines on antibiotic use supported by a strong veterinary service sector in Uganda as well as farmers training on judicious use of antimicrobials are needed. Tetracycline.

Data Availability
Te data supporting the current study are available from the corresponding author upon request.

Ethical Approval
Te study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Makerere University College of Veterinary Medicine, Animal Resources, and Biosecurity. Permission was sought from Masindi District Local Government. We sought verbal informed consent in the local language from respondents. Tey were informed that their participation was voluntary and their refusal would not result in any negative consequences. During data analysis, we used unique identifers to ensure confdentiality.