Study on the incidence of Salmonella enteritidis in poultry and meat samples by cultural and PCR methods

Aim: To study the incidence of S.enteritidis in poultry and meat samples by cultural and PCR methods. Materials and Methods: A total of 130 samples (25 each of chicken, mutton, poultry faeces, cloacal samples and 10 each of liver, spleen and kidney) collected from different sources were subjected to cultural and PCR methods for the presence of Salmonella and Salmonella enteritidis. Primers for invA and sefA gene were used for Salmonella and S.enteritidis respectively. Results: Out of 130 samples, 87 were positive for Salmonella spp. i.e. chicken-16(64%), mutton-12(48%), faeces-23(92%), cloacal swabs-23(92%), liver-5(50%), spleen and kidney samples-4(40%) each by PCR methods, whereas 77 were positive by cultural method i.e. chicken-14(56%), mutton-10(40%), faeces-22(88%), cloacal swabs-21(84%), liver-4(40%), spleen and kidney-3(30% each). Out of 87 positive for Salmonella by PCR method, 59(chicken-12, mutton-7, faeces-17, cloacal swabs-15, liver-3, spleen-2, kidney-3) were positive for S.enteritidis. High incidence of S.enteritidis (68%) in all the above samples are indicative of unhygienic conditions in poultry farms. Selective enrichment with Rappaport-Vassilidias (RV) broths and Tetrathionate (TT) broths were superior over Selenite-F (SF) and Selenite cysteine (SC) broths. Conclusions: High incidence of S.enteritidis was seen in most of poultry samples like chicken, kidney, liver and it's faeces than mutton, which was indicative of contamination of S.enteritidis is more prevalent in poultry farms.


Introduction
humanbeings includes diarrhoea, nausea, abdominal pain, mild fever, chills, vomition, prostration, headache, Salmonella is one of the most important pathogenic malaise etc. and the diarrhoea varies from thin vegetable genera implicated in food borne bacterial outbreaks soup like stools to a massive evacuation with accomand diseases both in developed and developing panying dehydration [8]. countries and constitute an important public health The number of organisms, to be swallowed problem [1]. Despite global improvements in public inorder to cause infection is rather small that is fewer health facilities, a marked increase in human than 10 [9] and it is a must for the livestock products to salmonellosis has been reported in many countries be tested for the presence of Salmonella due to it's including the European Union [2]. Outbreaks of potentially low infective dose [10]. The detection of Salmonella have been associated with wide variety of Salmonella in foods is problematic due to presence of foods especially those of animal origin [3]. In many fewer number of organisms, larger number of competing countries human salmonellosis is mainly due to microflora and due to injured organisms by different consumption of eggs followed by poultry, pork, beef, food processing methods [11]. The conventional and dairy products [4]. culture method, which is routinely used for isolation of S.enteritidis is the main cause of food borne Salmonella is time consuming, laborious and may not salmonellosis [5] and during the last 20 y, it has been a be suitable for viable but non culturable (VBNC) [10]. major causative agent of foodborne gastroenteritis in To overcome this drawback, immunological and humans [6]. There is increasing evidence suggesting genetic detection methods have been developed [12]. that the main source of human pathogens are poultry PCR method is rapid, specific and sensitive method products especially eggs [7]. The symptoms in for the detection of food borne pathogens [13]. Vet. World, 2012, Vol.5(9):541- 545 RESEARCH www.veterinaryworld.org Veterinary World, Vol.5 No.9 September 2012 541 To cite this article: 541-545, doi: 10.5455/vetworld.2012.541-545 Ramya P, Madhavarao T, Rao LV (2012) Study on the incidence of Salmonella enteritidis in poultry and meat samples by cultural and PCR methods, Vet World, 5(9): Study on the incidence of Salmonella enteritidis in poultry and meat samples by cultural and PCR methods In this study PCR method was used to detect Type Culture Collection (MTCC), Chandigarh was Salmonella spp. and S.enteritidis targeting invA and used as known positive strain in PCR analysis. About sefA genes respectively.
1.5 ml of enriched broths were taken in eppendorf tubes and bacteria were pelleted by centrifugation at

Materials and Methods
8000 rpm for 10 min and the supernatant was A total of 130 different mutton and chicken discarded. Fifty µl of sterile distilled water was added 0 related samples (25 samples each of mutton, chicken to the tubes and boiled in a water bath at 100 C for 10 meat, chicken faeces, cloacal samples of poultry and min and immediately snap chilled to release DNA. 10 each of chicken liver, spleen and kidney) were Then centrifuged at 13,000 rpm for 5 min and the collected from three retail markets and two slaughter supernatants containing DNA from respective houses (five replications each) in and around Hyderabad. samples were used as templates for PCR analysis. The samples were collected in the sterile containers Bacterial DNA amplification was done in 20 µl and transferred under cold conditions (with icepack) reaction mixture containing 2 µl of 10x Taq DNA H to the lab. Mutton, Chicken meat and chicken internal polymerase buffer containing 100 mM tris with p 9.0, organs (10 g) samples were preenriched in 90 ml of 500 mM KCl, 15 mM MgCl and 1% triton X-100), 2 2 buffered peptone water (BPW) in individual sterile µl of 10 mM dNTP mix, 0.9 U/µl of Taq DNA polythene bags homogenized thoroughly in a stomacher polymerase (Genei), 2 µl each of forward and reverse 0 for 3-5 min and incubated at 37 C for 16 h. Faeces and primer (4 pmol/µl) and 5 µl of crude bacterial cell cloacal swabs were inoculated in BPW in test tubes lysate. This mixture was made upto 20 µl using 0 (50ml) and incubated at 37 C for 16 h. After premolecular grade water. Amplification was done in enrichment 1 ml of each inoculum was transferred into thermal cycler following standardized conditions selective broths like Tetrathionate (TT) broth, ( Table-2). Selenite-F (SF), Selenite cysteine (SC) broths and 0.1  Enteric Agar (HEA) and differential agar like 5.
Spiking studies: To know the sensitivity of PCR All the enriched samples were subjected to PCR method for S.enteritidis, homogenized chicken was analysis for the presence of Salmonella spp. using inoculated at the rate of 250 cfu, 25 cfu, 2.5 cfu and primers specific to invA. The samples positive for 0.25 cfu per 10 g of chicken and transferred to pre-0 Salmonella by PCR method were further examined for enrichment media i.e. 90 ml of BPW, incubated at 37 C the presence of S.enteritidis strains using primers for 8 h and 16 h. After incubation, inoculum transferred specific for sefA gene (Table1

Results and Discussion
S.enteritidis in chicken might be due to not following hygienic methods in rearing, slaughtering and Out of 25 chicken meat samples, 14(56%) were marketing. positive for Salmonella by cultural method and Out of 25 mutton samples, 10(40%) and 12 (48%) 16(64%) by PCR method (Fig. 1). Out of 16 PCR were positive for Salmonella by cultural method and positives, 12 were positive for S.enteritidis by PCR PCR methods. Out of 12 PCR positives, 7 were ( Fig. 2) which accounts to 48 and 75% over total positive for S. enteritidis by PCR, which accounts to number of samples and positive samples for 28 and 58.3% over total number of samples and Salmonella by PCR respectively (results are shown in positive samples for Salmonella by PCR respectively.  [21] reported higher incidence (97.6%). The respectively. The incidence of S.enteritidis in mutton incidence of Salmonella by PCR method in the present samples by PCR method in this study (48%) is similar study (64%) was similar to the findings of Malkawi to the incidence (47.3%) reported by Malkawi and and Gharraibeh [22] and higher than the incidence (36.5%) reported by Uyttendaele et. al. [23]. The Gharraibeh [22]. incidence of S.enteritidis in the present study (75%) by The unhygienic slaughtering methods and PCR was less than the incidence (90%) reported by unhygienic environment in the sale shops of mutton might Wang et. al. [24] and higher than the incidence (5.6% be the reasons for higher incidence of salmonellosis.   were positive for S. enteritidis by PCR, which faeces than mutton, which was indicative of accounts 68 and 73.9% over total number of samples contamination of S.enteritidis is more prevalent in and positive samples for Salmonella by PCR poultry farms. Based on high incidence of Salmonella respectively. Low incidence (6.9% and 50%) of and S.enteritidis in mutton and poultry related samples Salmonella in poultry faeces by cultural method were in and around Hyderabad, strict hygienic and sanitary reported by Amini et. al. [20] and Mirmomeni et. al. procedures in rearing of livestock and poultry, [29] respectively than the present study (88%). The slaughtering and marketing of these products should incidence of Salmonella (92%) in this study by PCR be practiced. method is much higher than the incidence (1.8% and Author's contribution 66%) reported by Tamuly et. al. [30] and Carli et. al. [31] respectively. The incidence (73.91) of S. TMR participated in the preparation of experimental enteritidis in poultry faeces in the present study is less design and facilities of the research. P. Ramya, carried than the incidence (83.3% and 88%) reported by out the entire experiment. LVR helped in the analysis Salehi et. al. [32] and Mirmomeni et. al. [29] of the data. TMR, LVR and PR drafted and revised the respectively and higher than the incidence ( et. al. [20]. Salmonella infected poultry birds will void (2005). Salmonella enteric serotypes isolated from higher levels of organisms through faeces.