Our research improves the evidence base on Salmonella epidemiology in commercially produced poultry in Bhutan by estimating the overall prevalence of Salmonella in broiler meat from the two major producer and supplier of broiler meat areas in Bhutan. Apart from our study only two other studies were available in the published literature from Bhutan on Salmonella in the animal sector; one in imported chicken meat and another in nationally produced beef(Dahal, Ellerbroek, & Poosaran, 2007; Dorjee, 2011). Our investigations identified two Salmonella serovars, S. typhi 17 (73.9%) and S. paratyphi B 6 (26.1%) and that among 23 Salmonella isolates, 60.87% were multidrug resistant and as high as 6 patterns of multidrug resistance were recorded. The detection of Salmonella in nationally produced broiler meat and the presentation of high proportion of multidrug resistant Salmonella indicates the need to further investments into the farm to fork approach to Salmonella control in Bhutan.
The Salmonella prevalence in raw broiler meat estimated in this study (13%) is comparable to that of Salmonella prevalence in imported chicken meat in Bhutan (Dahal et al., 2007) and to that reported prevalence of Salmonella in beef in Bhutan (15.8%) (Dorjee, 2011). Our results are also in line with studies conducted in Japan (Iwabuchi et al., 2011) and in Egypt (Hassanain et al., 2011) where a prevalence of 12.2% was reported in retail chicken products and broiler chicken respectively. However, our results are much lower to studies reported from other countries in the South Asian region, where the prevalence of Salmonella was estimated as high as 65.5% in frozen chicken meat (Parvin et al., 2020), and 38.7% in broiler farm (Mridha et al., 2020) in Bangladesh, 33.3% in chicken carcass (Balakrishnan, Sangeetha, & Dhanalakshmi, 2018) in India, and 57% in chicken meat in Nepal (Bohara, 2017) were reported. Also, in Vietnam and China, the prevalence of Salmonella in chicken carcasses was (48.7%) (Ta et al., 2014) and 43.3% (Wang et al., 2013) respectively.The varying prevalence of Salmonella in chicken meat between sampled commercial farms could be attributed to varying on-farm biosecurity measures applied and hygiene practices at the production system (Manoj & Singh, 2015). Other studies have shown that cleaning equipment and disposing of dead birds as significant risk factors for Salmonella detection in farms. The study by (Wang et al., 2013) concluded that the variation of prevalence of Salmonella between farms could be attributed to the farm chicken breed.
Differences in the prevalence between the study farms can be attributed to differences in bird density as higher bird density has been noted in previous studies to be associated with increased odds of detection of positive Salmonella (Donado-Godoy et al., 2012). In Bhutan, commercial poultry (broiler and layer) are reared in deep litter production system and depending on the size of the farms, farmers usually have more than two poultry shed and each shed has the capacity of more than 1000 birds. All five commercials’ farms had the farm size between 2,000–2,500 birds and follow all-in all-out system. The source of poultry could also pose a biosecurity risk for the introduction of Salmonella into a flock. In our study broiler day old chicks used by these farms were generally sourced in Bhutan and supplied by the National Poultry Research and Development Canter (NPRDC) which is run by the Department of Livestock under the Ministry of Agriculture & Forest, Bhutan. However, during periods of national shortage of DOCs, farmers resort to importing DOCs directly from hatcheries in India which are registered with BAFRA, Bhutan. The majority of the poultry farmers use feeds which are manufactured within the country, however, few poultry farmers procure feed directly from India. As per the farm biosecurity survey conducted by BAFRA in 2018, the poultry commercial farms had good farm biosecurity practices with clean water source, proper rodent control, proper fencing, proper documentation of visitor, treatment, feed and vaccination. In Bhutan, commercial broiler farms have a designated slaughter facility at the farm. The slaughter facility has minimum equipment for sticking, defeathering, evisceration and are carried out in same shed but separated by partition wall to avoid cross contamination.
In addition, to husbandry systems other factors could be implicated in the variation in prevalence observed in our study. As per (Wang, 2016), the difference between Salmonella prevalence in our study and others reported in the literature could be due to difference in the study design particularly number of samples collected, sample collection period, sample type, sampling procedures and method of detection used. Studies conducted in China, Greece and the USA described that prevalence and concentration of Salmonella in chicken was relatively higher in summer and spring than winter and autumn (Rodriguez et al., 2006; Zdragas et al., 2012; Wang, 2016).
The two Salmonella serovars detected in our study were S. typhi and S. paratyphi B. Among two serovars, the S. typhi was found more prevalent 73.9% (17 isolates) than S. paratyphi B 26.1% (6 isolates). S. typhi and S. paratyphi B were also recovered from chicken carcass in Egypt (El-Aziz, 2013), Indonesia (Kusumaningrum and Dewanti-Hariyadi, 2012), and India (Murugkar et al., 2005), broiler farm in Bangladesh (Akond et al., 2012; Barua et al., 2013), and eggs in Nepal (Singh et al., 2010). Both the Salmonella serovars fall in typhoid subcategory of Salmonella meaning it infects a very narrow range of hosts including humans (Hendriksen et al., 2011; Maka & Popowska, 2016) and are usually associated with higher number of fatal cases (Wang et al., 2019). These serovars do not present poultry as a reservoir but rather an indication of contamination during the slaughter processes at different points of the production line (Newell et al., 2010). The predominant Salmonella serovar reported in Asia region were S. typhimurium (15.34%) and S. enteritidis (69.84%) in China in chicken meat (Zhu et al., 2017), S. typhimurium (15.38%) in Bhutan in imported chicken (Dahal et al., 2007), and S. typhimurium (22.2%) in chicken meat in Nepal (Singh, Yadav, Singh, & Bharti, 2010). A meta-analysis study on the worldwide epidemiology of Salmonella serovar in animal based food reported S. typhimurium as the most prevalent and disseminated serovar worldwide (Ferrari et al., 2019). The other serovar reported globally were S. pullorum, S. gallinarum, S. enteritidis, and S. enteric in chicken (Ozbey and Ertas, 2006; Rayamajhi et al., 2010; Singh et al., 2010; Mahmud et al., 2011; Ta et al., 2014; Al-Salauddin et al., 2015).
Our results also indicate a high proportion of Salmonella isolates were sensitive to gentamycin 73.91% (17 isolates) and streptomycin 56.52% (13 isolates) but high resistance to tetracyclines 95.65% (22 isolates), trimethoprim 86.96 (20 isolates) and amoxicillin 65.20 (15 isolates). Our study findings were in consistent with the findings of (Murugkar et al., 2005) which labelled doxycycline, ampicillin, amoxicillin and tetracycline resistant to Salmonella isolates but, sensitive to norfloxacin, enrofloxacin, gentamicin and ciprofloxacin. In the Asia region, high resistance of Salmonella was reported for tetracycline (97.14%) and chloramphenicol (94.28%), in chicken farm in Bangladesh (Alam et al., 2020; Mridha et al., 2020), trimethoprim sulfamethoxazole (70.3%) and tetracycline (54.3%) in chicken meat in Myanmar (Moe et al., 2017), sulphonamide compounds (98.9 %) and tetracycline (96.9 %) in chicken carcass in Nepal (Goncuoglu, Ormanci, Uludag, & Cil, 2016). The emergence of high resistance of Salmonella to tetracycline, amoxicillin and trimethoprim antimicrobials in the study may be contributed to indiscriminate use of these antimicrobials in poultry production as growth promotion, prophylactic and therapeutic purposes (Nourouzi, Mirzaii, & Norouzi, 2004) and Bhutan is no exception. As per the drug distribution report of Bhutan, 2018, the tetracycline trimethoprim was largely distributed for the poultry use. The consistent reports of high resistance of Salmonella to tetracycline, trimethoprim and amoxicillin antimicrobials in several studies conducted globally raise serious public health concern since these antimicrobials are used as first line drugs for human and animals. Our study also observed that 95.65% (22 isolates) of the Salmonella isolates showed resistance to one or more antibiotics with six antibiotic resistance patterns and 60.87% of multidrug resistance. A study conducted by (Dahal et al., 2007) in imported frozen chicken in Bhutan, also reported Salmonella resistance to one or more drugs antimicrobials namely; nalidixic acid (96.15%), amoxicillin (11.54%) and cephalexin (5.77%). A widespread multidrug resistance of Salmonella was reported in chicken eggs in Nepal (Singh et al., 2010), broiler chicken at slaughter house in China (Zhu et al., 2017) and broiler farm in Bangladesh (Mridha et al., 2020). Recognizing the role of meat in Salmonella transmission to humans added by reports of high prevalence of Salmonella and multidrug resistance from the two main producer and supplier of broiler meat areas in Bhutan, altogether, it raises serious public health concern which require immediate action.
The findings of our study need to be interpreted in light of the fact that within the five commercial broiler farms that fulfilled the study’s inclusion criteria, we collected samples from the same batch of birds in the farm to get the required sample size. While we employed a cluster sampling weight to adjust the sample size calculation, birds in the batch are usually raised in same production system.
In this study we present the first estimates of Salmonella prevalence and antibiotic resistance profiles in nationally produced broiler meat in the dominant broiler production and supply areas in Bhutan. These findings should serve as an important resource for national Salmonella control program managers to plan the design of farm-level surveillance to help reduce the human risk of Salmonellosis at the country level.