Identification and antimicrobial susceptibilities of 21 strains of Salmonella from poultry

A total of 21 Salmonella strains were isolated from clinical cases in Yangzhou, China, and the surrounding areas from Feb 2011 to Dec 2011. All isolates were screened by PCR amplification for InvA gene, which is specific to the Salmonella genus. Thus, twenty-one Salmonella were identified from the specimens. The isolates belonged to 6 serovars of Salmonella. Both Salmonella typhimurium and Salmonella pullorum were the dominant serovars isolated. Results of susceptibility test show that a large percentage of Salmonella were resistant to doxycycline (90.5%), polymyxin B (85.7%), streptomycin (85.7%), lomefloxacin (85.7%), and nalidixic acid (81.0%). The high resistance among these strains from infection animals suggested that essential measure such as infection control and drug rational use need to be taken.


INTRODUCTION
Salmonella is a heterogeneous species of Gram-negative enterobacteria and is a commensal of the intestine of many animal species.Salmonella a pathogen of significant importance in poultry, can cause septicemia and enteritis.Salmonella includes more than 2500 different serotypes (Dunkley et al., 2009;Foley and Lynne, 2008;Popoff et al., 2001).Salmonella pullorum and Salmonella typhimurium are the most prevalent serovars in the worldwide domestic birds (chichen, duck, geese), and may cause significant disease or death in poultry (Pan et al., 2010;Tizard, 2004).Contaminated poultry products are important sources of human Salmonella infections.Moreover, S. typhimurium is the most frequently reported serovars associated with human food-borne illnesses (Fearnley et al., 2011).Serovars S. typhimurium is most prevalent in geese and ducks.Occurrence of the same Salmonella serovars in humans, animals and food might indicate their epidemiological links (Hoszowski et al., 2012;He et al., 2011;Pan et al., 2010).
Antibiotic-resistant Salmonella are becoming increasingly of concern due to indiscriminate use of antibiotics in animal feeds as growth promoters and therapeutic agents, is a further threat to human and animal health (Forshell and Wierup, 2006).Therefore, resistant strains make it more difficult to treat patients with severe infections.For instance, S. typhimurium DT104 is a high rate of resistance to particular antimicrobials among veterinary isolates in Europe, North America, and Asia (Chiu et al., 2006;Futagawa-Saito et al., 2008;Gebreyes et al., 2004;Graziani et al., 2008;Hur et al., 2011;USDA, 2006).The present study was carried out to determine the prevalence of Salmonella serovars in bird infections including chickens, ducks, and geese.All isolates were also examined for antibiotic resistance.

Bacterial strains of isolation and culture conditions.
Individual clinical specimens, including liver, ovaries and other organs, were collected from 45 cases of poultry affected by Salmonellosis in Yangzhou and the surrounding areas from February 2011 to December 2011.Most of the animal sampled showed typical clinical signs including septicemia and enteritis.The strains were isolated from the sample, and grown on blood agar (LB containing 5% [v/v] defibrinated sheep's blood) at 35°C for 24 h.The subcultures bacteria were streaked onto Luria-Bertani (LB) agar (Oxoid) and MacConkey agar (Shanghai China Academy Of Sciences Shanghai Hexapod Technology Development Co., Ltd.) and incubated at 37°C for 24 h.

Amplification of InvA gene
The InvA gene of the isolated strains was used for Salmonella PCR detection, and amplified enzymatically with the forward primer-5′GTGAAATTATCGCCACGTTCGGGCAA3′ and reverse primer -5'TCATCGCACCGTCAAAGGAACC-3' according to Rahn (1992) and Malorny (2003).A negative control tube with Escherichia coli S11701 identified and a positive control tube with Salmonella S6702 confirmed by Key Laboratory of Animal Infectious Diseases of Ministry of Agriculture, Yangzhou University, China, DNA were included on each occasion.PCR was performed with the initial denaturation of 3 min at 95°C, followed by 30 cycles of denaturation at 94°C for 45 sec, annealing at 58°C for 45 sec, extension at 72°C for 1 min and final extension at 72°C for 8 min.The expected size of amplicon was approximately 284 bp, and visualized by agarose gel electrophoresis on a 1% agarose gel.

Biochemical tests and Salmonella serovars
The ability to ferment glucose, mannitol, lactose, sucrose, adonitol, and maltose in the presence of omithine decarboxylase, lysine decarboxylase, and urease activity, and the ability to produce indole, diacetyl, and hydrogen sulfide were performed in conventional tube media for Enterobacteriaceae (Hangzhou Microbial Reagent Co., Ltd, China) at 35°C for 24 h.Salmonella serovars was identified by slide agglutination according to the Kauffmann and white scheme.

Antimicrobial susceptibility tests
Antimicrobial susceptibility tests were done on pure, 1-day-old cultures of all the Salmonella isolates, using the Kirby-Bauer disk diffusion test and antimicrobial susceptibility slip (Hangzhou Microbial Reagent Co., Ltd, China), according to the Clinical and Laboratory Standards Institute (CLSI, 2006) standards.

Salmonella identification
All isolates tested positive using the culture method and positive control 284 bp band on electrophoresis (Figure1).

DISCUSSION
Salmonellosis is an important cause of disease and death in poultry and contaminated poultry products pose a significant health hazard to humans (Tizard, 2004).In this study, 21 Salmonella had been isolated form diseased or Salmonella isolated from infected animal cases (chickens, ducks, and geese) were resistant to doxycycline (90.5%), polymyxin B (85.7%), streptomycin (85.7%), lomefloxacin (85.7%), nalidixic acid (81.0%), gentamycin (52.4%) and norxacin (52.4%).However, Pan et al. (2010) observed that Salmonella species isolated from faecal samples of healthy domestic animals (chickens, ducks, geese and pigs) were resistant to nalidixic acid (48.8%), and streptomycin (38.3%).The higher resistance rates of Salmonella strains isolated in this study suggests that the strains might have originated from treatments where antimicrobials have been utilized.A low level of resistance to chloramphenicol and furazolidone was found which may be due to their banned use for some time in domestic animals.
This study demonstrated that S. typhimurium is the common serovars in geese and a majority of isolates was resistant to multi-drug.S. typhimurium is also the major cause of human salmonellosis via contaminated food, becoming increasingly multi-drug resistance (Torpdahl et al., 2013;Cavallaro et al., 2011;Wójcik et al., 2012).Therefore measures to reduce Salmonella infection and antimicrobial usage are important to animals.

Table 2 .
Antimicrobial resistance rates of Salmonella isolates.