425 Please refer to the forthcoming article as: Sadiq et al. 2022. Prevalence and molecular characterization of non-typhoidal Salmonel- la isolated from meat and environmental samples of retail shops of Lahore Punjab, Pakistan. Vet Ital. 10.12834/VetIt.2392.14037.1. 1Department of Epidemiology and Public Health, University of Veterinary and Animal Sciences, Lahore, Pakistan. 2Department of Environmental Sciences, Quaid-i-Azam University, Islamabad, Pakistan 3Italian National and OIE Reference Laboratory for Salmonella. Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), Padua, Italy. *Corresponding author at: Department of Epidemiology and Public Health, University of Veterinary and Animal Sciences, Lahore, Pakistan. E-mail: shakera.sadiq@uvas.edu.pk. Shakera Sadiq1*, Mamoona Chaudhry1, Muhammad Hassan Mushtaq1, Junaid Sadiq2, Saima Hasan1, Lettini Antonia Anna3 Salmonella is one of the leading causes of bacterial diarrhea worldwide, accounts for one of the four key global causes of diarrheal illnesses (WHO, 2018). Non-typhoidal Salmonellae are important foodborne bacterial pathogens that can cause bacteremia, gastroenteritis, and subsequent infection (Acheson and Hohmann, 2001). Every year, non-typhoidal Salmonella infections results in approximately 153 Introduction Foodborne diseases pose a significant impediment to socio-economic development globally and represent a constant threat to public health (Devleesschauwer, et al., 2018). Almost 1 in 10 people become infected every year due to food- borne diseases. Diarrheal diseases are the most common illnesses from contaminated unsafe food. Keywords Prevalence, Zoonotic, Salmonella, Retail shops. Veterinaria Italiana 2022, 58 (4), 425-434. doi: 10.12834/VetIt.2392.14037.1 Accepted: 28.05.2021 | Available on line: 31.12.2022 Summary Non-typhoidal Salmonellae are important foodborne bacterial pathogens that can cause bacteremia, gastroenteritis, and subsequent infection. The aim of the study was to determine the prevalence of Salmonella in the live bird market and retail shops of Lahore (Pakistan). A total of 720 samples of chicken meat, chopping board, cages, hands, and transportation vans were collected. Salmonella was recovered from 103 (14.36%) samples. The highest prevalence (33.33%) was found in transportation van samples followed by chicken meat samples (17.26%). In the towns of Lahore, the highest prevalence was found in Samanabad Town (19 %) followed by Data Ganj Bakhsh Town (17%) with the lowest in Gulberg Town (6.9%). Salmonella Typhimurium was most common (35.92%) followed by S. Enteritidis (25.24%), S. Dublin (14.56%), S. Gallinarum biovar Gallinarum (8.74%), and untyped Salmonella species (15.53%). This was the first baseline study of the prevalence of non-typhoidal Salmonella at the live bird market and retail shops of Lahore. Implementation of appropriate control measures is required at both the human side and poultry food production chain to reduce the burden and transmission of the zoonotic Salmonellae. Prevalence and molecular characterization of typhoidal and non-typhoidal Salmonella isolated from meat and environmental samples of retail shops of Lahore Punjab, Pakistan Non typhoidal Salmonellae in of Pakistan Sadiq et al. 426 Veterinaria Italiana 2022, 58 (4), 425-434. doi: 10.12834/VetIt.2392.14037.1 million cases of gastroenteritis and 57,000 deaths globally (Brunette, 2017). According to the data of the global burden of diseases, among the total deaths due to salmonellosis, 38,500 deaths per year occur in children less than five years of age (Delahoy, et al., 2018) Poultry is considered as the most important reservoir of Salmonella and also considered as one of the common sources of human salmonellosis globally (Antunes, et al., 2016; Yang, et al., 2011; Zwe, et al., 2018). Contaminated foods particularly broiler retail chicken meat is considered as the most commonly implicated foods for non-typhoidal salmonellosis in humans (Alali, et al., 2012; Donado-Godoy, et al., 2012; Lamas, et al., 2016; Li, et al., 2017; Shang, et al., 2019). Salmonella bacteria own the ability to reside in the healthy chicken without showing any symptoms so it may go unnoticed, therefore poses the risk of human infection through contaminated chicken at retail level (Zwe, et al., 2018). Salmonella is the leading cause of foodborne disease worldwide especially in the Middle East, Eastern Europe, and Southeast Asia (Ta, et al., 2012). It is also one of the most important zoonotic pathogens both in animals and humans in developing countries (Ejo, et al., 2016) Important stages for transmission of Salmonella to chicken and its products include the status of infection in the host population at the farm after rearing, fecal contamination during transport by vehicles to the slaughtering and processing facility, and cross-contamination between chicken carcasses and equipment during cutting and processing. It is also documented in studies that during transport or shipping, the prevalence of Salmonella in positive birds increases due to fecal contamination of feathers and skin by companion birds (McCrea, et al., 2006). In Pakistan when the broiler flock is ready after rearing in broiler farms, transport vehicles are used to transport live birds either to processing plants, live bird markets or directly to retail shops of the county. Live bird markets or wet markets are common in Asian countries (Nidaullah et al.2017) including Pakistan. These live bird markets are served as wholesale markets and used as an important source of chicken consumed by the population on large scale. Live bird markets play an important role in the dissemination of Salmonella serovars to the environment and individuals (Sharma, et al., 2019). Many risk factors have been associated with the presence of  Salmonella  spp. at the retail outlets of live bird markets, including the size of live bird market, sources of chicken either from integrated or nonintegrated systems, storage conditions including temperature, geographical location and rearing types of chicken (Alali, et al., 2012; Jarquin, et al., 2015; Khan, et al., 2018; Tabo, et al., 2013; Yang, et al., 2011), At a worldwide level, foodborne illness is often related to the consumption of poultry meat and its products contaminated with pathogenic bacteria commonly from the retail shop level (Shafini, et al., 2017). In Pakistan, consumers prefer to purchase meat which has been slaughtered in front of them from retail shops. The important risk factors are contaminated knives, chopping boards, and tables and, contaminated hands of retailers. Poor sanitary and hygienic practices are prevalent in these retail outlets in developing Asian countries and are not monitored by regulatory authorities (Nidaullah, et al., 2017; Yang, et al., 2011). According to the author’s knowledge, there is limited data on the prevalence of non-typhoidal Salmonella in retail meat shops in Lahore. The present study was conducted to check the prevalence of non-typhoidal Salmonella at different steps of the poultry chain including sampling from transport vehicle vans, live bird market, and retail shops of nine towns of Lahore. Material and methods A cross-sectional study was conducted starting from February 2018 to August 2018 in District Lahore, Punjab. Samples were collected (n=720) from the live bird market located in Data Gunj Bakhsh Town and retail outlets of Lahore. Sampling was started from the live bird market by initially taking swabs samples (n=45) from transport vehicles. Hand swabs (n=33) were taken after the consent of the drivers. To avoid repetition, the name of the driver and the number of the vehicle were noted on the day of sampling. Sterilized cotton swabs were used for sampling the cages of vans and the hands of van drivers. Hand swabs were taken by swabbing the palms and fingers with sterilized cotton swabs dipped in 0.1% Buffered Peptone Water (BPW, Oxoid) (CM0509) (Okareh and Erhahon, 2015). Swabs were kept in test tubes containing 10 ml buffered peptone water. Forty (40) retail shops from the live bird market located in Data Ganj Bashkh town and 90 chicken retail outlets from 9 administrative zones of Lahore were selected. A total of 10 shops were selected from each administrative zone of Lahore including 9 towns. Four types of samples including chicken meat (n=262), chopping board swabs (n=130), cage swabs (n=70), and hand swabs of the retailers (n=180) were collected. Meat samples of slaughtered chicken including the thigh, chest meat, and chicken leg were collected. A minimum (approx. 225 gm) of meat was purchased and stored in sterilized polythene bags Sadiq et al. Non typhoidal Salmonellae in of Pakistan Veterinaria Italiana 2022, 58 (4), 425-434. doi: 10.12834/VetIt.2392.14037.1 427 Molecular identification of non- typhoidal Salmonella enterica by multiplex Polymerase Chain Reaction (PCR) A multiplex PCR was developed for the identification of common serovars of Salmonella enterica in Lahore. DNA extraction Four to five pure isolated colonies from fresh culture were first incubated in Lysogeny broth (Luria Bertani medium) (LB broth, Oxoid, CM0996) for 24 hours at 37ºC. From LB broth culture, one ml was taken in the Eppendorf tube and centrifuged at 10,000 rpm for 10 minutes. The supernatant was discarded, and the pellet was washed by adding 1 ml of ultrapure distilled water and again centrifuged at 10,000 rpm for 10 minutes. The supernatant was discarded, and 180 µL of 10X chelex resin (Bio Rad) were added. The suspension was mixed properly by vortexing. Eppendorf tubes were sealed with parafilm and placed in a water bath for 20 minutes at 100ºC. Immediately after water bath, samples were chilled on ice for 2 minutes. Samples were centrifuged again at a maximum speed of 14,000 rpm for 10 minutes. The supernatant was used as template DNA for PCR. Template DNA was stored at -20°C until used. Multiplex PCR Multiplex PCR was developed for simultaneous identification of Salmonella serovar, Enteritidis, Typhimurium, Typhi, Dublin, and Salmonella enterica serovar Gallinarum biovar Gallinarum (Table I). For PCR, 25 µl of the reaction mixture was prepared to contain 1 µl (10 pmol) of each forward and reverse primer, 12.5 µl of Master Mix (Lucigen USA), 2 µl of template DNA and remaining nuclease-free water to make final volume. from retail shops. For the sampling of chopping boards and cages, sterilized swabs immersed in 0.1% BPW were used. A few samples containing fecal material and debris from the floor of the cages were also collected in sterilized polythene bags. All the samples were properly labeled and packed and immediately transferred to the laboratory of Epidemiology and Public Health within 24 hours for further microbiological processing. Isolation and identification For isolation and cultivation of Salmonella, ISO 6579 method was used. For meat samples, 25 gr of meat sample were weighed and suspended in 225 ml buffered peptone water for 24 hours at 37°C. For selective enrichment, two types of Salmonella selective broths were used. Firstly, 0.1 ml of BPW was inoculated into 10 ml of Rappaport- Vassiliadis  Enrichment Broth (RV broth, Oxoid, CM0669). The broth was incubated for 48 hours at 42°C. Secondly, one ml of BPW was inoculated in 10 ml of Tetrathionate broth (TT broth, Oxide CM0671). The broth was incubated for 18-24 hours at 37ºC. For culturing of Salmonella, three selective media were used. Three loops full (one loop for each media) of inoculum was used for inoculating Brilliant Green Agar (BGA, Oxoid, CM 0263), Salmonella Shigella agar (SS Agar, Oxoid CM0099) and Xylose Lactose Tergitol™ 4 (XLT 4, Oxoid CM1061). The plates were incubated for 18-24 hours at 37ºC. Three presumptive colonies (pink colonies with red background for BGA, round black centered for SS agar, and black centered with the pink background on XLT4) were selected and further streaked for purification on nutrient agar (Oxoid, CM0003). Suspect colonies showing morphological characteristics of Salmonella were further confirmed by the Triple sugar iron (Oxoid, CM0277) and urease tests (Oxoid, CM 057). Table I. Primer sequences for multiplex PCR for Salmonella serovar Enteritidis, Typhimurium, Typhi, Dublin, and Gallinarum biovar gallinarum. Target gene Sequence Amplification target Size bp Reference Sdf 1 TGTGTTTTATCTGATGCAAGAGG Serovar 304 (de Freitas, et al., 2010) TGAACTACGTTCGTTCTTCTGG Enteritidis Spy TTGTTCACTTTTTACCCCTGA Serovar 401 CCCTGACAGCCGTTAGATATT Typhimurium ViaB CACGCACCATCACACCG Serovar Typhi 738 AACAGGCTGTAGCGATTTAGG SeD_A1104 gene ACGCGAAATCTGATGGTCTT Serovar. Dublin 203 (Stegniy, et al., 2014) GCCCACCAGTTGTGAAAGGC SG0266 CCGCACAACACATCAGAAAG Serovar 97 AGCTGCCAGAGGTTACGCTG Gallinarum Non typhoidal Salmonellae in of Pakistan Sadiq et al. 428 Veterinaria Italiana 2022, 58 (4), 425-434. doi: 10.12834/VetIt.2392.14037.1 Figure I. Gel image of multiplex PCR to detect Salmonella. Cycling conditions Cycling conditions of the reaction comprised initial denaturation temperature of 95°C for 2 min; (ii) 30 cycles, with 1 cycle consisting of 1 min at 95°C, 1 min at 57°C, and 2 min at 72°C; and (iii) a final elongation step of 5 min at 72°C (Alvarez, et al., 2004). The PCR products were electrophoresed in 2.5% (wt/vol) D-1 agarose (Oxoid) and stained with 4 µl of ethidium bromide solution. In each PCR run, a non-template control was included to detect possible external DNA contamination. The gel was visualized in the gel Doc system (Syngene, USA). Figure II. Map showing prevalence of Salmonella in different towns of Lahore. Table II. Overall prevalence of Salmonella in chicken meat, environmental samples, and hand swabs. Sample type No of samples tested No of positive samples Percentage Chicken meat samples 262 46 17.56 Hand swabs of retailers 213 21 9.86 Cages of birds 70 6 8.57 Chopping board samples 130 15 11.54 Transportation van sample 45 15 33.33 Total 720 103 14.31 Results Figure I showed results of multiplex PCR with five bands of Salmonella Enteritidis, Typhimurium, Dublin, Gallinarum and Typhi. Among the total (n=720) collected samples, 103(14.31%) were found to be positive for Salmonella enterica (Genus). The highest prevalence was found in transportation vehicle samples (33.3%), chicken meat samples (17.56%), chopping board samples (11.54%), hand swabs of retailers (9.6%) and cage samples (8.57%) (Table II). As the live bird market of Lahore is located in Data Gunj Bakhsh Town, the samples collected from Tollinton market with samples collected from retail shops in streets of Data Gunj Bakhsh Town were combined. Table III and Figure II present the area wise prevalence in different towns of Lahore. The highest prevalence was found in Samanabad Town (19%) Data Gunj Bakhsh Town (17.0%), Ravi Town (16%), Shalimar town (15.0%), Wahga town (15%), Alama Iqbal Town (12%), Aziz Bhatti Town (9%), and Gulberg Town (7%). Table IV shows the serovar distribution Sadiq et al. Non typhoidal Salmonellae in of Pakistan Veterinaria Italiana 2022, 58 (4), 425-434. doi: 10.12834/VetIt.2392.14037.1 429 Location of retail outlets Total number of shops tested Salmonella positive shops No of samples collected No of positive samples Percentage of positive samples Ravi Town 10 6 70 11 15.71 Shalimar Town 10 7 82 12 14.6 Aziz Bhatti Town 10 4 90 8 8.9 Data Gunj Bakhsh Town *50(10+40) 30 §195(75+120) ¶34(15+19) 17.43 Samanabad Town 10 8 70 13 18.57 Gulberg Town 10 2 58 4 6.90 Wahga Town 10 5 55 8 14.55 Alama Iqbal Town 10 6 60 7 11.67 Nishter Town 10 4 40 6 15 Salmonella serovars Total number identified Percentages Salmonella Typhimurium 37 35.92 Salmonella Enteritidis 26 25.24 Salmonella Dublin 15 14.56 Salmonella Gallinarum biovar gallinarum 9 8.74 Salmonella Typhi 0 0 Untyped 16 15.53 Total 103 100 Table III. Area prevalence of Salmonella in retail shops of towns of Lahore (Pakistan). Table IV. Salmonella serovar identification in retail shops of Lahore (Pakistan). * Total number of shops selected from live bird market and retail shops of Data Ganj Bukhsk Town. § Total number of samples selected from live bird market and retail shops of Data Ganj Bukhsk Town. ¶ Total number of Salmonella positive samples from live bird market and retail shops of Data Ganj Bukhsk Town. among the total isolated samples with the most common serovar being Salmonella Typhimurium (35.92%) then Enteritidis (25.5%), Dublin (14.56%), Gallinarum (8.74%), Typhi (0%), and untyped (15.53%). Discussion The present study was conducted for the first time to assess the burden of non-typhoidal Salmonella at the retail shops of Lahore (no study published earlier). Lahore is the largest city of Punjab province, Pakistan, and the fifth-largest city in South Asia. The land area of Lahore is 1772 square kilometer with a total population of approximately 12.18 million (2019 statistics). To meet consumer demands, there are more than 9,000 retail shops for red and white meat (Nisar, et al., 2018). According to the survey (Jalil, et al., 2013), the daily chicken meat consumption in Lahore is approx 361,000 kilograms. The chicken is supplied to the whole city through two major markets including the Tollinton market (shares 55% of total supply) and the Sheranwala market (shares 30% of total supply). The remaining 15% of chicken meat is sold by direct suppliers. The presence of Salmonella is a major hindrance to the export of poultry and poultry products from Pakistan as well as its zoonotic threat (http://www. livestockpunjab.gov.pk/LiveStockAdmin/uploads/ editor_files/per formance_report_-_directorate_ of_poultry_research_institute_oslln.pdf ). A limited number of studies on non-typhoidal Salmonella in poultry have been reported from Pakistan. The purpose of this research was to determine the presence of non-typhoidal Salmonella in the major live bird market (Tollinton market) and retail shops of nine administrative towns of Lahore. In the present study, the prevalence rate of Salmonella in raw chicken meat is 17.56% which is lower than that reported from all other studies conducted in different areas of Pakistan. The reported area wise prevalence of Salmonella in chicken meat was 38% in Hyderabad (Soomro, et al., 2010); (34%) in raw chicken meat in Kohat (Asif, et al., 2017); (30%) in chicken meat in Faisalabad (Akhtar, et al., 2010), and (18%) in raw poultry meat in Swat (Uddin, et al., 2018). The lower prevalence of Salmonella in Lahore (Punjab) as compared to other areas may be due to the role of the Poultry Research Institute (PRI, Punjab) in surveillance and Non typhoidal Salmonellae in of Pakistan Sadiq et al. 430 Veterinaria Italiana 2022, 58 (4), 425-434. doi: 10.12834/VetIt.2392.14037.1 control of poultry diseases at regional levels. The reported prevalence in raw chicken meat (17.56%) is also lower as compared to other Asian countries: 21-65.3% in Vietnam (Huong, et al., 2006; Nguyen, et al., 2016; Phan, et al., 2005; Ta, et al., 2014; Van, et al., 2007); 52.2% in China (Yang, et al., 2011); 48% in Malaysia (Abatcha, et al., 2018). The difference in the prevalence rate of Salmonella in Asian countries may be due to different farming practices, transport vehicles, slaughtering and processing environment, unhygienic practices at retail shops, and also could be due to differences in isolation methods (Sharma, et al., 2019). Moreover, in developing nations, the chicken meat is kept at room temperature as no cooling system is installed in wet meat shops and therefore it provides optimum survival conditions for Salmonella. The lack of strict hygiene-related laws in retail meat shops and unawareness among butchers are considered the two most important reasons for the increased prevalence of Salmonella. The purpose of hand swabbing was to determine the level of hygienic practices of the retailers and their involvement in the dissemination of pathogen to an uninfected chicken carcass. The contamination of the retailer’s hand can impose a greater risk for self-infection as well as the dissemination of pathogens to the family. In the present study, the frequency of the occurrence of Salmonella on the retailer’s hands was (9.86%) which is not comparable locally as no study has been conducted before to analyze the level of non- typhoidal Salmonella contamination on hands. Our reported prevalence varies from that reporte in other studies: 0%-14.29% on meat shop retailers hands in India (Sharma, et al., 2019; Suresh, et al., 2004); 23.3%-33.3% on hands in Ethiopia (Abdi, et al., 2017; Garedew, et al., 2015). Personal hygiene may be the reason for discrepancies in prevalence. Poor hand washing practices and use of single cloth for drying hands after every single slaughter are very common in retail shops of Lahore. Normally, there is one large bowl of water which is used repeatedly for washing of hands without any soap or sanitizer. A large piece of cotton cloth is also commonly seen which is used for drying hands. The prevalence of Salmonella in environmental samples was highest in transportation vans samples: 33.3% followed by chopping board samples; 11.54%, and cages; 8.57%. Transportation trucks are used to transport live birds from broiler farms to live bird markets and retail shops. These vehicles can be a continuous source of contamination to healthy flocks (Beach, et al., 2002; Ejo, et al., 2016; Gallegos Robles, et al., 2009; Hald, et al., 2000). The reported prevalence in Lahore (33.3%) is lower than reported in Korea (62.5%) from vehicles (Ha, et al., 2018), where the Salmonella prevalence in broiler transporting vehicles was highest in the whole food chain. The difference could be due to uncleanliness of floors of trucks and how frequently these vehicles are washed to reduce contamination. Chopping boards are used for cutting and chopping of meat after de-feathering and evisceration process. Retailers mostly clean the chopping board with a wet cloth after cutting off one carcass and this cloth is used repeatedly after every carcass cutting. The prevalence of Salmonella on chopping boards in the present study was 11.54%, which is higher than the 5.6% reported from Ethiopia (Garedew, et al., 2015) and lower than the 18.75% reported from India (Suresh, et al., 2004). The difference in prevalence could be due to the method of taking samples as the chopping board surface is porous and bacteria sometimes reside inside surfaces which is difficult to collect during swabbing (Ak, et al., 1994). Studies reported that uncleaned chopping boards are a potential source Salmonella cross-contamination (Zwe, et al., 2018). Cages are compartments mostly placed on one side or the backside of the shop. The floor surface of the cages is covered with droppings and it is cleaned occasionally before the entrance of the next batch of birds. According to previous studies, levels of shedding dramatically increased, while birds were confined in cages before slaughter (Corry, et al., 2002; Ha, et al., 2018; Hald, et al., 2000). A prevalence of 8.57% was reported from cages in the present study. Cage samples are considered under environmental samples and prevalence is in the range of that reported from the neighboring country India [7.94% from environmental samples (Sharma, et al., 2019)]. In our study, the area wise highest prevalence was found in Samanabad town (19%), followed by Data Gunj Bukh Town (17%), and Gulberg (6.9%). The reason behind the high prevalence in Samanabad and Data Gang Bukhsh is the high population congestion rate (22.72 and 25.33 persons/square meter, respectively). Conversely, Gulberg is a newly established area of Lahore with high literacy rate and elite class residents with congestion rate of 20 person/square meter (Joshua and Ali, 2011; Nisar, et al., 2018). The most prevalent serovar in our study was Salmonella serovar Typhimurium (35.92%), followed by S. Enteritidis (25.24%), S. Dublin (14.56%), S. Gallinarum biovar gallinarum (8.74%). All the samples were negative for Salmonella Typhi (0%) while the untyped isolates were 15.53%. Sadiq et al. Non typhoidal Salmonellae in of Pakistan Veterinaria Italiana 2022, 58 (4), 425-434. doi: 10.12834/VetIt.2392.14037.1 431 As both, Salmonella Dublin and Gallinarum are host specific serovars, the possible chances of occurrence may be due to infection of birds at farms or the environmental contamination of samples at retail shops. In our study, the predominant serovar was Typhimurium, a recent study reported a prevalence of 28.45% from farm samples from Faisalabad (Punjab) (Wajid, et al., 2019). No other study in Pakistan reported the prevalence of S. Typhimurium. The prevalence of Salmonella Enteritidis (25.24%) reported in our study was simlar to that reported from Kohat (23.3% (Asif, et al., 2017), but lower than reported in different districts of Punjab (31.34%), (Yasmin, et al., 2019). Conclusions This is the first baseline study in Lahore, Punjab for the prevalence of different Salmonella serovars. The high prevalence of zoonotic serovars in the retail shops of major city of Punjab is a major threat to public health. Food and Health authorities of Pakistan should implement appropriate control strategies to minimize the spread food borne zoonotic pathogens to consumers Ethical permission This study was approved by Ethical Review Committee for Animals and humans, University of Veterinary and Animal Sciences, Lahore. Acknowledgments Authors acknowledge the poultry retailers for their participation in study. The authors will also like to Mr. Mubashar Hanif laboratory assistant for help in laboratory work and Clerk, Miss Fakhra Sarwar for help in formatting of write up. Funding Statement The current research study was funded by project entitled “Molecular detection of emerging and remerging meat borne bacterial pathogens and evaluation of various control strategies” and HEC Indigenous PhD Scholarship (Awarded to Shakera Sadiq). The funding agency had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Non typhoidal Salmonellae in of Pakistan Sadiq et al. 432 Veterinaria Italiana 2022, 58 (4), 425-434. doi: 10.12834/VetIt.2392.14037.1 Abatcha M.G., Effarizah M.E. & Rusul G. 2018. Prevalence, antimicrobial resistance, resistance genes and class 1 integrons of Salmonella serovars in leafy vegetables, chicken carcasses and related processing environments in Malaysian fresh food markets. Food Control, 91, 170-180. Abdi R.D., Mengstie F., Beyi A.F., Beyene T., Waktole H., Mammo B., Ayana D. & Abunna F. 2017. 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