Abstract: A total of 163 Salmonella sp. isolates representing 15 serotypes recovered from faecal samples of domestic animals (chicken, duck, goose and pig) in eastern China during 2008-2009 were tested for antimicrobial susceptibilities. S. Senftenberg, S. Typhimurium S. Pullorum and S. Enteritidis were the most prevalent serovars. Resistance was most often observed to carbenicillin (65.4%), followed by nalidixic acid (48.8%), tetracycline (46.9%), sulfafurazole (45.7%), ampicillin (43.2%), streptomycin (38.3%) and trimethoprim/sulfamethoxazole (33.3%). With regards to the source of isolates, chicken Salmonella sp. isolates displayed the highest rate of resistance being resistant to at least one antimicrobial (100%) followed by those recovered from pig (93.4%), goose (90.7%) and duck (80%). Serovars commonly showing Multidrug Resistance (MDR) to >9 antimicrobials were S. Enteritidis (55.6%), S. Pullorum (17.9%) and S. Typhimurium (17.2%). This study has revealed the prevalence and antimicrobial resistance patterns of Salmonella sp. in domestic animals in eastern China and provides the important information for better controlling these pathogens.

INTRODUCTION

Salmonella is a genus of bacteria that are a major cause of foodborne illness throughout the world. The reservoirs of Salmonella are considered to be domestic animals, particularly poultry and pigs and these organisms are easily isolated from the feces (Vo et al., 2006). These carrier animals likely play an important role in the spread of infection between herds and flocks and consequently serve as sources of food contamination and human infection (Carrique-Mas et al., 2008).

In China, during 1994-2005, a total of 57612 outbreaks of foodborne disease were reported and Salmonella was the most identified agent, accounting for 22.2% of illnesses (Wang et al., 2007). Salmonellosis in the food animal industry can be treated and controlled with antimicrobial therapy. However, the control of Salmonella infections is difficult because increasing antimicrobial resistance has been reported in animal and human species (Gebreyes and Thakur, 2005; M’ikanatha et al., 2010). Zhao et al. (2007) reported that 82% of 380 animal isolates of Salmonella were resistant to at least one antimicrobial drug, with 52% exhibiting resistance to five or more antimicrobial drugs. In China, there are few reports regarding the prevalance of Salmonella sp. in live animals and the antimicrobial resistance of the isolates (Liu et al., 2010). Therefore the objectives of the present study were to investigate the prevalance of Salmonella sp. in chickens, ducks, geese and pigs in the farm in eastern China and to characterize the antimicrobial resistance of the isolates.

MATERIALS AND METHODS

Sampling: The animal isolates were collected from chickens, ducks, geese and pigs (Table 1) in 7 provinces of in eastern China during the year 2008-2009. Faecal samples from healthy or sick animals were taken on farms. The animals sampled came from different flocks or herds. About 10 g of faeces was collected from each animal and placed in a sterile sampling bag, kept in an ice-box at 4-8°C and transported to the laboratory within 24 h. About 10-20 samples were taken at each animal farm. Samples were analysed at Jiangsu Key Laboratory of Zoonosis.

Isolation and identification of Salmonella: Faecal samples were inoculated into buffered peptone water (Difco) for enrichment at a ratio of 1 g faeces to 10 mL of broth. After incubation at 42°C for 18 h, the broth was inoculated onto desoxycholate hydrogen sulphide lactose and brilliant green agar plates (Hangzhou Microbial Reagent Co., Ltd., Hangzhou, China), each supplemented with 20 mg L-1 of novobiocin sodium (Hangzhou Microbial Reagent Co., Ltd.) and incubated at 37°C for 18 h.

Table 1:	Prevalence of Salmonella in domestic animals in eastern China

Candidate colonies were identified biochemically by triple sugar iron agar (Hangzhou Microbial Reagent Co., Ltd.) and lysine indole motility semisolid agar (Hangzhou Microbial Reagent Co., Ltd.). Identification of serovars was performed by slide agglutination tests (Hangzhou Microbial Reagent Co., Ltd.), according to the Kauffmann-White scheme. All of the isolates were stored in 25% glycerol at -80°C until use.

Antimicrobial susceptibility testing: For the determination of antimicrobial susceptibility of the isolates, the disk diffusion methods were performed according to the Clinical and Laboratory Standards Institute (CLSI, 2006) standards. The following antimicrobials (Oxoid) were tested: amoxicillin (10 μg), ampicillin (10 μg), carbenicillin (100 μg), ceftriaxone (30 μg), cefotaxime (30 μg), gentamicin (10 μg), kanamycin (20 μg), streptomycin (10 μg), spectinomycin (100 μg), chloramphenicol (30 μg), tetracycline (30 μg), trimethoprim (5 μg), trimethoprime/sulfamethoxazole (1.25-23.75 μg), sulfafurazole (300 μg), ciprofloxacin (5 μg) and nalidixic acid (30 μg). Reference strains E. coli ATCC 25922 and Enterococcus faecalis ATCC 29212 were included as controls. For each isolate, the zone of inhibition around each disk was measured, after incubation at 37°C for 24 h.

RESULTS AND DISCUSSION

The percentage of Salmonella-positive samples was 4.8, 5.3, 10.7 and 9.9% for chicken, duck, goose and pig samples, respectively (Table 1). In Shanghai which is located in eastern China, the isolation rates of Salmonella sp. from faecal samples were 4.5% in 550 chickens between 2005 and 2006 (Liu et al., 2010).

The present results demonstrated similar Salmonella sp. isolation rates to this previously reported in China. The prevalence of Salmonella sp. in pigs in this study was lower than the 14.2% reprted by Murugkar et al. (2005). However, Kishima et al. (2008) showed that Salmonella sp. prevalence was 3.3% in pig faecal samples in 2004-2005. It is difficult to compare the results in other countries because there are variations in sampling methods and methods for isolation of Salmonella sp. (Kishima et al., 2008).

About 15 serovars were found among the 163 Salmonella isolates, including 4 serovars in chicken isolates, 6 serovars in duck isolates, 5 serovars in goose isolates and 9 serovars in pig isolates (Table 2). The predominant serovars (S. Senftenberg, S. Typhimurium, S. Pullorum and S. Enteritidis) accounted for about 75.5% of the isolates. S. Pullorum (84.9%) was the most common serovar among the 33 Salmonella isolates from chckens in this study as well as in previous studies (Liu et al., 2010). S. Pullorum, the causative agent of Pullorum disease is the most prevalent host-adapted pathogen in China (Pan et al., 2009). However, various researchers have documented the significance of S. Typhimurium (Cheong et al., 2007), S. Enteritidis (Van Duijkeren et al., 2002), S. Infantis (Ishihara et al., 2009) and S. Emek (Vo et al., 2006) in chickens in other countries. In duck, serovar Typhimurium, Newport and Saintpaul were predominant and represented 26.7, 20 and 20%, of the 15 duck isolates, respectively.

Ogasawara et al. (2008) also found S. Typhimurium (24%) were frequently isolated from ducks during 1999-2001 in Vietnam, but the serovar Newport and Saintpaul were not isolated. The predominant Salmonella serovars from duck eggs in Thailand were Typhimurium (23.3%), Cerro (17.7%) and Tennessee (12%) (Tran et al.., 2004). Thus, the ducks seem to be sensitive to S. Typhimurium. S. Senftenberg (66.7%) was the most prevalent serovar among 54 isolates originating from goose, followed by S. Typhimurium (18.5%) and S. Newport (11.1%). Few reports regarding the prevalance of Salmonella sp. in geese have been published. Trawinska et al. (2008) found that serovars Typhimurium (44.8%) and Enteritidis (29%) were predominant in the isolates of geese in 2001-2005 in Poland. Among the 61 pig isolates, S. Enteritidis (23%) and S. Typhimurium (21.3%) were the most common serovars, followed by S. Senftenberg (18%) and S. Derby (11.5%).

Table 2:	Distribution of Salmonella serovars in animals of eastern China
aIsolates from diseased animals

Table 3:	Antimicrobial resistance of Salmonella isolates by domestic animals in eastern China

In several countries, S. Typhimurium and S. Derby are the predominant serovars in the isolates from pigs (Boonmar et al., 2008; Davies et al., 2004). Ogasawara et al. (2008) showed that the predominant serovars of pig isolates were S. Javiana (30.8%) and S. Derby (15.4%) in 1999-2001. Murugkar et al. (2005) also showed that S. Enteritidis was frequently found in 44% (11/25) of the pig isolates in 2003-2004. Thus the predominant serovars of Salmonella sp. found in pigs in China were similar to those observed in other countries.

Higher resistance rates were observed against carbenicillin (65.4%), nalidixic acid (48.8%), tetracycline (46.9%), sulfafurazole (45.7%), ampicillin (43.2%), streptomycin (38.3%) and trimethoprim/sulfamethoxazole (33.3%). Resistance rates to ceftriaxone, ciprofloxacin and cefotaxime were <5% (Table 3). Chicken and pig are two important food animals in China and >90% of isolates in chicken were resistant to ampicillin, carbenicillin, sulfafurazole and nalidixic acid and higher than the results for Salmonella sp. isolated from chickens in USA (Zhao et al., 2007).

The prevalence of resistance of pig isolates to each of the antimicrobials ampicillin, carbenicillin, sulfafurazole, nalidixic acid, tetracycline, streptomycin and trimethoprim/sulfamethoxazole was high and these prevalences ranged approximately from 50-80%. A recent study reported that 38 Salmonella sp. isolates from pork samples obtained in China were resistant to tetracycline (53%), sulfafurazole (61%), ampicillin (26%), trimethoprim/sulfamethoxazole (47%), streptomycin (32%) and nalidixic acid (29%) (Yang et al., 2010). Chicken Salmonella sp. isolates displayed the highest rate of resistance to at least one antimicrobial (100%), followed by those recovered from pig (93.4%), goose (90.7%) and duck (80%) (Table 4).

Table 4:	Multidrug resistance observed among Salmonella isolates obtained from animals

Over three-quarter of the chicken isolates (75.7%), 45.8% of pig isolates, 29.6% of goose isolates and 20% of duck isolates showed resistance to 4-9 antimicrobials (Table 4).

Furthermore, approximately 23% of pig isolates and 18% of chicken isolates were resistant to >9 antimicrobials. A high percentage of multiple antimicrobial resistant Salmonella sp. from various food animals was also reported by other researchers (Ogasawara et al., 2008; Zhao et al., 2007).

As animals are a main reservoir of Salmonella and the use of antimicrobials in food animals for therapy, prophylaxis and growth promotion accelerates the emergence of antimicrobial resistant pathogens, it is not surprising that an increased number of human salmonellosis cases are caused by foodborne antimicrobial resistant Salmonella (Foley and Lynne, 2008).

In the present study, the prevalence of S. Enteritidis, S. Pullorum and S. Typhimurium resistance to >9 antimicrobials was 55.6, 17.9 and 17.2%, respectively (Table 5) which was in agreement with previous observations among Salmonella isolated from retail meats and food animals (Pan et al., 2009; Yang et al., 2010) but differed from some other reports (Liu et al., 2010; Zhao et al., 2007).

Table 5:	Multidrug resistance observed among Salmonella serovars obtained from animals

CONCLUSION

In conclusion, the high prevalence of Salmonella in domestic animals in eastern China and the level of antimicrobial resistance has raised the concerns and constituted a real threat to public health. More detailed epidemiological studies including molecular characterization would be of great interest to allow better control of this pathogen.

ACKNOWLEDGEMENTS

This research was supported by grants from National Key Technology R and D Program (Nos. 2007BAD40B01, 2009BADB9B01), National Nature Science Foundation of China (No. 30871860), the 863 program (No. 2006AA10A206), the Government of Jiangsu Province (No. BK2008011) and Qing Lan Project.