Veterinary Practitioner Vol. 15 No. 1
June 2014
DETECTION OF YERSINIA ENTEROCOLITICA IN PORK SAMPLES BY AN INDIRECT ELISA Devan Arora1*, Y. Singh1, Ashok Kumar1 and Nitin Mehta2 Department of Veterinary Public Health and Epidemiology, College of Veterinary Sciences Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar-125 004, Haryana, India ABSTRACT An Indirect ELISA was standardized for detection of Y. enterocolitica organisms in pork. A total of 69 pork samples, 23 each from pig tongue, pig lymphoid tonsil and ground pork were analyzed using this Indirect ELISA. The Indirect ELISA was able to detect a minimum of 10 Y. enterocolitica organisms. Out of 69 samples, Y. enterocolitica was detected in 9 (13.04%) samples. More number of samples from pig lymphoid tonsils (6, 26.08%) as compared to pig tongue (2, 8.69%) and ground pork (1, 4.35%) were found positive by Indirect ELISA. Key words: Y. enterocolitica, pork, Indirect ELISA, pig lymphoid tonsils
Introduction Y. enterocolitica is a zoonotic pathogen widely distributed in nature that can cause acute gastroenteritis, mesenteric lymphadenitis, appendicitis and has emerged as an important food borne pathogen causing infection in human and animals (Bottone, 1999). Pigs are reported to harbour this organism in their throat and tonsils (Funk et al., 2000) and shed the organisms in faeces (Rasmussen et al., 1995). The organism has also been isolated from a variety of foods such as raw and pasteurized milk (Moustafa et al., 1983), beef (Inoue and Kurose, 1975), oysters (Toma and Lafleur, 1974), pork (Fredriksson Ahomma et al., 2006) and masala item of panipuri (Sathisbabu and Rati, 2003). Further, Y. enterocolitica is capable of multiplying at refrigeration temperature and in vacuum packed meats, milk and other food products which support its greater public health significance. Current methods for detection of Y. enterocolitica infection include conventional method of culture which gives less confirmatory diagnosis and modern molecular techniques viz. polymerase chain reaction (PCR) and. However, molecular techniques like PCR are quite expensive and cumbersome. Indirect ELISA has been successfully used in detection of foodborne pathogens from milk and meat (Ghatak et al., 2003). The present work describes the standardization of Indirect ELISA for detection of Y. enterocolitica from meat. Materials and Methods A total of 69 pork samples comprising 23 each from pig tongue, lymphoid tonsils and ground pork were collected aseptically from the local market and transported to the laboratory under cold conditions. Standardization of Indirect ELISA 1. Bacterial strain: Y. enterocolitica (MTCC - 861) IMTECH, Chandigarh. 2. Preparation of Y. enterocolitica antigen: Y.enterocolitica,
grown overnight in 100 ml TSA, cells collected by centrifugation, heat killed for 2 h in boiling water bath. Antigen was sonicated at 250 watt for 5 cycles of one min each, centrifuged and supernatant collected. Protein content was estimated as per standard procedure described by Lowry et al. (1951) and concentration was adjusted 4 mg/ml. The antigen was kept at -5oC till use. 3. Raising of hyperimmune serum against Y. enterocolitica: 3 New Zealand white rabbits were inoculated with protein antigen (1 mg/ml in NSS) with suspension emulsified in Freund’s Incomplete Adjuvant (FIA). Serum stored at - 20o C until used in Indirect ELISA. 4. Indirect ELISA: Indirect ELISA was standardized according to the method described by Ghatak et al. (2003) for Aeromonas spp. with slight modifications using whole cell culture of Y. enterocolitica standard strain. The optimum concentrations of antibody and conjugate were determined by chequer board titration using known negative and positive Y. enterocolitica sera in rabbit. 5. Development of indirect ELISA (Ghatak et al., 2003 with slight modifications): Indirect ELISA was standardized using ten fold serial dilution of Y. enterocolitica in carbonate buffer (101 -109 CFU/ml). Flat bottom 96-well polystyrene plates (MaxiSorp Nunc A/S, Roskilde, Denmark) were coated with 100 µl/well cell suspension PBS and incubated at 37o C for 2 h followed by overnight at 4oC. After washing plates with PBS-T, they were incubated for 2 h at 37o C with 100 µl/well of appropriate dilution of serum in PBS-T containing 3% skimmed milk. The plates were again incubated for 1 h at 37 o C with 100 µl/well appropriate dilution of antirab bit H RPO conj u gate. 100 µl/w ell O PD substrate solution was added to each well and incubated for 5 min. at 37 o C. The reaction was stopped with 100 µl/well of 1N H 2SO 4 and the absorbance was measured at 492 nm in a
1*
Correspondent and Present Address: Dr. Devan Arora, Department of Veterinary Public Health & Epidemiology, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar - 125 004, Haryana, India. Email:
[email protected]; 2Department of FST (Technology of Animal Foods), National Institute of Food Technology Entrepreneurship and Management (NIFTEM), Sonepat- 131028, Haryana, India. 173
Veterinary Practitioner Vol. 15 No. 1
June 2014
Table 1: Assessment of indirect ELISA for detection of Yersinia in spiked ground pork
Table 2: Detection of Y. enterocolitica by Indirect ELISA in pork samples
Concentration of Y. enterocolitica in spiked pork Cfu/gm Zero 108 107 106 105 104 103 102 10 1 OD 0.060 0.196 0.176 0.172 0.166 0.148 0.145 0.126 0.122 0.069 ELISA value >2 >2 >2 >2 >2 >2 >2 >2 >2 2 was taken as positive. 6. Assessment of Indirect ELISA (Ghatak et al., 2003): (a). Experimental Inoculation of Y. enterocolitica in Meat: 5 g presterilized pork samples were spiked with different concentration of Y. enterocolitica (10-10 9 cfu/ml) and simultaneously seeded with other bacteria (Salmonella and E. coli) at a concentration of 104 cells/ml and then enriched with 45 ml MRB and incubated at 25oC for 24 h. After incubation cells were pelleted by centrifugation at 1000 rpm for 2 min for resting the meat debris. The supernatant collected, heat killed in boiling water bath for 2 h, centrifuged at 5000 g for 15 min at 4o C. Finally, cells were suspended in 1 ml carbonate buffer (pH 9.6) and Indirect ELISA was performed as per procedure described above. 7. Detection of Y. enterocolitica in field pork samples by indirect ELISA: The sample was prepared for Indirect ELISA test by 5g pork sample homogenized aseptically with 45 ml MRPB and incubated at 25oC for 24 h. Rest of the procedure kept same as followed in case of detection of Y. enterocolitica from spiked ground pork. In each plate known Yersinia negative and Yersinia positive pork sample were included as controls. All the field samples used for detection of Y. enterocolitica were inoculated in duplicate wells. A positive to negative ratio of >2 was taken as positive. Results and Discussion Standardization of indirect ELISA: The Indirect ELISA was standardized with the help of chequer board titration. A 1:2000 and 1:160 dilutions of anti-rabbit goat HRPO conjugate and hyperimmune serum were found optimum, respectively and were used subsequently. When the indirect ELISA was applied for detection of Y. enterocolitica organisms in experimentally inoculated ground pork with varing concentrations (10-108 ) of organisms per gram pork followed by enrichment in MRB for 24 h of incubation then it was found that as minimum as 10 organisms per gram of pork could be detected (Table 1). It is well known that Y. enterocolitica obligately feeds on lymphoid tissue, so it can be easily parasitic on pig throat tonsils, which are rich in lymphoid tissue (Horter et al., 2003). Pigs (mainly their tonsils) are assumed to be the main source for pathogenic Y. enterocolitica because the pig is so far the only animal species from which pathogenic strains have
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Nature of sample No. of samples No. of samples showing (No.) examined ELISA value > 2 (%) Pig tongue (23) 23 2 (8.69) Lymphoid tonsil (23) 23 6 (26.08) Ground pork (23) 23 1 (4.35) Total (69) 69 9 (13.04)
frequently been isolated (Lindblad et al., 2007). The results of Indirect ELISA showing detection of Y. enterocolitica organisms in field pork samples are shown in Table 2. Out of 69 pork samples, Y. enterocolitica organisms were detected in 9 (13.04 %). Maximum number of samples from pig lymphoid tonsils, 6 (26.08 %) was found positive by Indirect ELISA as compared to 2 (8.69 %) and 1 (4.35 %) from pig tongue and ground pork, respectively. The results of present study are strengthen by the findings of Fredriksson-Ahomaa (2009) who reported the higher prevalence of Y. enterocolitica in lymphoid tonsils (44%) as compared to other sample on application of real-time PCR. However, Fredriksson-Ahomaa (2008) reported prevalence of Y. enterocolitica in tonsils of Swiss wild boars up to 24%, 18% and 5% by real-time PCR, PCR and culture techniques respectively. These findings clearly indicate that lymphoid tonsils may be the major source of for human being. Thibodeau et al. (2001) have suggested that Indirect ELISA may be used for primary screening of food samples. Thus, this test may be used in conjunction with isolation of causative agent and Indirect ELISA can be used for primary screening of food samples. However further detailed investigations are needed taking more number of samples into consideration for detection of Y. enterocolitica infection. References Bottone, E.J. (1999) Microbes Infect. 1: 323-333. Funk, J.A. et al. (2000) J. Food Prot. 63: 395-399. Fredrikksson Ahomaa, M. et al. (2006) Int. J. Food Microbiol. 119: 207-212. Fredriksson-Ahomaa, M. et al. (2008) Detection of enteropathogenic Yersinia in 94 Swiss wild boars. 1st National Conference on Yersinia. 25-26.7.2008. Braunschweig, Germany. Fredriksson-Ahomaa, M. et al. (2009) Int. J. of Food Microbiol. 135: 199-202. Ghatak, S. et al. (2003) J. Vet. Pub. Hlth. 1: 17-24. Hochel, I. and J. Skvor, J. (2007) Folia Microbiol. 52 (5):511–518. Horter, D.C. et al. (2003) Anim. Hlth Res. Rev. 4: 143-155. Inoue, M. and Kurose, M. (1975) Jpn. J. Vet. Sci. 37: 91-93. Lindblad, M. et al. (2007) J. Food Prot. 70: 1790-1797. Lowry, Ï. Ç. et al. (1951) J. Biol. Chem. 193 265-275. Moustafa, M.K. et al. (1983) J. Food Prot. 46: 276-278. Rasmussen, H.N. et al. (1995) J. Appl. Bacteriol. 78: 563-568. SathisBabu, H.N. and Rati, E.R. (2003) J. Food Sci. Tech. 40: 303305. Thibodeau, V. et al. (2001) Vet. Microbiol. 82: 249-259. Toma, S. and Lafleur, L. (1974) Appl. Microbiol. 28: 469-473.
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