Isolation and prevalence of Salmonella from chicken meat and cattle milk collected from local markets of Patna , India

Aim: To evaluate the hygienic quality of raw chicken meat and raw milk sold in the local markets of Patna with reference to isolation of Salmonella and antibiotic resistance pattern of Salmonella against commonly used antibiotics. Materials and Methods: A total of 370 samples comprising of 228 chicken meat and 142 market milk samples were processed for isolation and serotyping, supplemented with molecular detection of isolates targeting invA gene of Salmonella. All the isolates were tested against commonly used antibiotics (13 nos). Results: Out of 370 samples, 23.7% (54/228) chicken meat and 7.7% (11/142) milk samples were found positive for Salmonella based on biochemical reactions. The serotyping of Salmonella isolates showed an incidence of 6.1% of Salmonella typhimurium, 2.6% of S. newport, 1.7% of S. gallinarum and 0.4% each of S. enteritidis, S. infantis and S. worthington in chicken meat; and 2.1% of S. typhimurium and 1.4% of S. newport in market milk samples. Polymerase chain reaction targeting invA gene showed positive presence of Salmonella in 18.42% chicken meat and 5.6% market milk samples. Conclusion: The antibiotic susceptibility test revealed the presence of multiple drug resistant Salmonella in chicken meat and milk. The present study indicates high prevalence of Salmonella in raw chicken meat and milk due to poor hygienic practices and therefore emphasizes the need for adopting these hygienic practices.


Introduction
Salmonella [7].Amplification of invA gene of Salmonella has been reported as a suitable target for PCR ampli-Salmonella enterica is a leading cause of enteric fication, with potential diagnostic applications [8].diseases in humans and animals with millions of illness Emergence of multidrug-resistant S. enterica serovars reported worldwide.The nontyphoidal Salmonella has been increasing and has become a major health serovars are predominantly associated with food of concern [1].One of the contributing factors for the animal origin such as eggs, milk, poultry, beef and pork widespread dissemination of multi drug resistant bacteria meat responsible for zoonotic transmission [1,2].The has been the indiscriminate prophylactic and theraincidence of salmonellosis has been reported in many peutic use of antimicrobial agents in food animals [9].developing countries including India, Egypt, Brazil Therefore the present study was aimed to study and Zimbabwe [3].S. enterica serovar Typhimurium the prevalence of Salmonella and also characterize the and S. enterica serovar Enteritidis are the most distribution of the drug resistant pathogen in chicken frequently encountered species from foods like meat and market milk of Patna, India.poultry, pork and beef products [4].Incidence of

Materials and Methods
Salmonella in chicken meat and milk has been reported by several workers using different methods and the Sample collection: A simple random method was frequency of detection ranges from 6.79% to 97.6% [5] adopted to collect a total of 370 samples constituting in chicken meat and 0.17% to 28.6% [6] in raw milk.fresh chicken meat (n=228) and milk (n=142) from Although, isolation of Salmonella by growth in a different vendors of Patna, Bihar between September, culture medium followed by serotyping is considered 2010 to March, 2013.The samples were maintained on as the gold standard for confirmation of Salmonella, it ice, transported to the laboratory and processed within is also time consuming and labour intensive.
1 hr of collection.Therefore techniques like PCR are increasingly Isolation and biochemical characterization: Ten being used for rapid detection and confirmation of grams of chicken meat and 0.5 ml of milk samples were used for pre-enrichment in buffered peptone water (BPW), at 37 °C for 18 hrs.One ml of pre-enriched broth was transferred into Selenite Cystine broth and further incubated at 37 °C for 24 h for enrichment.units), Teicoplanin (30 µg), Tetracycline (30 µg) or Selective plating was performed using Hektoen enteric Vancomycin (30 µg) (HiMedia, India).The isolates agar (HEA) (HiMedia, India) with overnight incubation were grown on autoclaved Mueller Hinton broths at 37°C.Typical black color colonies surrounded by (HiMedia, India) for 18 hrs at 37 °C.About 100 µl of narrow green margin on HEA were biochemically the inoculums was spread on Mueller Hinton agar tested by Indole (I), Methyl Red (M), Vogus Proskeur using sterile disposable L shaped spreader and antibiotic (Vi), Citrate (C), Triple Sugar Iron (TSI) and Urease discs were placed onto the plate using sterile forcep.Test.The colonies showing Salmonella specific IMViC The plates were incubated at 37 °C for 24 hrs and observed pattern (-+-+) were inoculated on TSI slant.
for zone of inhibition.The results were categorized as Furthermore, the colonies producing alkaline slant sensitive, moderately sensitive and resistant based on (pink) and acidic butt (yellow) with or without H S diameter of zone of growth inhibition corresponding to 2 production (blackening) were tested for urease different isolates.production on urea agar slant.All the urease negative

Results and Discussion
isolates were considered as biochemically confirmed A total of 370 samples comprising of 228 raw and were submitted to National Salmonella and chicken meat samples and 142 raw milk samples were Escherichia centre, Central Research Institute Kasauli, processed for isolation and identification of Himachal Pradesh, India for serotyping.
Salmonella spp.by both growth enrichment on culture Optimization of PCR: A PCR protocol was standardized media and molecular techniques.Salmonella isolates targeting invA gene of Salmonella using vaccine strain were obtained from enriched samples by selective E2375 of Salmonella typhimurium.A 26-bp forward plating on HEA.Typical black colored colonies primer (5'-GTG AAA TTA TCG CCG CGT TCG GGC surrounded by narrow green margin were selected for AA3') and a 22-bp reverse primer (5' TCA TCG CAC biochemical characterization which showed the CGT CAA AGG AAC C 3') [10] were used to obtain a presence of Salmonella in 23.7% (54/228) and 7.7% 284 bp product.Amplification was carried out in a total (11/142) chicken meat and market milk samples, volume of 25 µl containing 10 pmols of each primer, 50 respectively.The serotyping of isolates showed the µM of each dNTP, 1.5 mM MgCl , 1 U Taq DNA 2 prevalence of Salmonella in 11.8% (27/228) of chicken polymerase, 1X PCR buffer and 5 µl template DNA.meat and 3.5% (5/142) of market milk samples.Among Template DNA was prepared by boiling and snap different serotypes of Salmonella, S. typhimurium was chilling method [11].A positive and negative control detected in 6.1% (14/228), S. newport in 2.6% (6/228), containing the template DNA from Salmonella S. gallinarum in 1.7% (4/228), S. enteritidis, S. infantis typhimurium vaccine strain E2375 and nuclease free and S. worthington in only 0.4% (1 each/228) of chicken water, respectively, was included in every experiment.meat samples, whereas, 2.1% (3/142) and 1.4% (2/142) The reaction condition was optimized with initial of S. typhimurium and S. newport, respectively were denaturation at 94 °C for 5 min followed by 30 cycles detected in market milk samples (Table-1).Our results 0 of denaturation at 94 °C for 1 min, annealing at 51 C are in agreement with the earlier findings reported from for 1 min and extension at 72 °C for 1 min.Finally, an different geographic regions [13][14][15][16][17].However, other additional extension was achieved for 5 min at 72 °C.
investigators have reported 6.7 to 97.6% prevalence of The PCR product was electrophoresed on a 1•5 % Salmonella in chicken carcasses and milk [5,18] which agarose gel at 100 V.The agarose gel was stained with further substantiate the present finding.Many of these -1 ethidium bromide (0•5 µg ml ) and visualized under serotypes have been reported earlier in cultures from gel documentation system (Biorad, USA).

Conclusion amplification of invA gene of Salmonella, which
The study concluded that non typhoidal comprises of 18.4% (42/228) and 5.6% (8/142) isolates Salmonella serotypes are prevailing in the poultry from chicken meat and milk samples respectively carcasses and milk and therefore act as a source of which is a higher distribution than that showed by human infection.The level of prevalence can be serotyping of isolates.The PCR assay also detected some of the isolates which had originally been detected reduced by adopting hygienic practices during poultry as negative in serotyping study.This is in concordance slaughter.The presence of multiple resistance in with the finding of Hamza [20].This may be attributed Salmonella isolates suggested that there is appreciable to the presence of rough mutant strains which lack the risk of infection to humans with multidrug resistant specific side chains responsible for 'O' specificity or Salmonella from consumption of unpasteurized milk some additional abnormalities of the core structure and undercooked chicken meat.

Authors' contributions
the study recommends that PCR may be used for rapid PK is a project leader and supervised the project.P K and sensitive detection of Salmonella supported by the and Anjay carried out bacterial isolation and molecular findings of Wang et al [7].
characterization.SK, SKB and SD collected samples Since food of poultry origin and milk are some of the most common sources of human salmonellosis, the and carried out ABST.All authors contributed in findings from this report may be correlated with the drafting and revision of the manuscript.All authors hygienic practices to reduce public health problem in read and approved the final manuscript.the area of study.Comparative investigation of prevalence and antimicrobial Indian J. Biotech., resistance of Salmonella between pasture and convention-12.Wayne, P.A. ( 2002 and 20.Eid, H.M.I. (2010) Rapid Detection of Salmonella in dairy Gyles, C.L. (1992) Amplification of invA gene sequence of cows using polymerase chain reaction.J. Am.Sci., 6: 31-37.Salmonella Typhimurium by polymerase chain reaction as a 21.Topley and Wilson's.(1990) Principles of bacteriology, specific method of detection of Salmonella.Mol.Cell.virology and immunity.In: Parker M T and Brian I (ed) th Probes, 6: 271-279.Systematic bacteriology 8 edn, Edward Arnold, Hodder 11.Nagappa, K., Tamuly, S., Brajmadhuri, Saxena, M.K. and And Stoughton Publishers, London, pp.475 -450.Singh, S.P. (2007) Isolation of Salmonella Typhimurium 22. Siemon, C.E., Bahnson, P.B. and Gebreyes, W.A. (2007) from poultry eggs and meat of tarai region of Uttranchal.