Random amplified polymorphic DNA (RAPD) analysis was applied for molecular characterization of five Salmonella enteritidis strains from different avian sources and human cases of infection. A total of 16 primers were used and only five primers showed good discriminatory power for all five isolates. Dendrogram showed a common lineage among all five isolates. There was a close genetic relationship among isolates of eggs and human sources, while there was less pronounced homology among isolates of broiler meat and human sources. On the basis of results we have found that an endemic strain of S. enteritidis is prevalent between the poultry derived food and humans which gives us an insight to genetic diversity of S. enteritidis from these sources

E. YAQOOB, I. HUSSAIN AND S. U. RAHMAN

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Pakistan Vet. J., 2007, 27(2): 102-104. 102 MOLECULAR CHARACTERIZATION BY USING RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) ANALYSIS OF SALMONELLA ENTERITIDIS ISOLATES RECOVERED  FROM AVIAN AND HUMAN SOURCES  E. YAQOOB, I. HUSSAIN AND S. U. RAHMAN  Department of Veterinary Microbiology, University of Agriculture, Faisalabad- 38040, Pakistan  ABSTRACT    Random amplified polymorphic DNA (RAPD) analysis was applied for molecular characterization of five Salmonella enteritidis strains from different avian sources and human cases of infection. A total of 16 primers were used and only five primers showed good discriminatory power for all five isolates. Dendrogram showed a common lineage among all five isolates. There was a close genetic relationship among isolates of eggs and human sources, while there was less pronounced homology among isolates of broiler meat and human sources. On the basis of results we have found that an endemic strain of S. enteritidis is prevalent between the poultry derived food and humans which gives us an insight to genetic diversity of S. enteritidis from these sources.  Key words: Salmonella enteritidis, RAPD, dendrogram.   INTRODUCTION    Avian salmonellosis is of major economic concern in all phases of the poultry industry from production to marketing (Chansiripornchai et al., 2000). Poultry and poultry products, in particular chicken meat and eggs, are considered important sources of human infection with this pathogen (Rampling, 1993). Salmonella enterica subsp. enteica ser. enteritidis  (S. enteritidis) is a major cause of food-borne diseases (Brenner et al., 2000), and during the last two decades it has been isolated world wide in increasing numbers (Rodrigue et al., 1990; Herikstad et al., 2002).    Global surveillance data indicate that incidence of gastroenteritis caused by S. enteritidis has increased massively during the last decade. Among the S. enteritidis isolates, those assigned to phage type 4 (PT4) and phage type 8 (PT8) have been isolated predominantly from poultry and are most frequent cause of human salmonellosis. Similar increase in S. enteritidis infections has also been reported in Europe, where PT4 has emerged as the predominant phage type, spreading rapidly through both poultry and human populations and virtually replacing all other phage types (Rampling, 1993). In Pakistan, S. enteritidis is an endemic organism and its regular outbreaks occur due to poultry derived food (Mansoor, 1997). In the present study, molecular characterization of S. enteritidis strains isolated from different poultry and human sources was performed using Random Amplified Polymorphic DNA (RAPD) analysis.  MATERIALS AND METHODS    Five isolates of S. enteritidis kept at the Department of Veterinary Microbiology, University of Agriculture, Faisalabad, Pakistan were selected for the present study. These isolates were collected from eggs, broiler meat and human faecal samples during 2004-2005. All the isolates were previously confirmed and identified by the standard bacteriological and serological methods. Three isolates were from egg sources, 1 from boiler meat and 1 from human (Table 1). DNA from all isolates was extracted by the alkaline lysis (Phenol:Chloroform:Isoamyl) method (Silhavy et al., 1984).  Table 1:  Distribution of collected samples on the basis of sources Source  Total No. of samples  Strain designation Eggs 3  E3 E12 E29 Broiler 1  B15 Human 1  H9 Total 5    The concentration of total genomic DNA was measured by spectrophotometer (CECIL, CE 2021, 2000 series) at 260 nm wavelength. Quality of DNA was checked by running 5 µl DNA on 0.8% agarose gel prepared in 0.5X TBE buffer. Furthermore, DNA quality was checked by measuring the ratio of absorbance (A260/A280); more than 1.8 indicates the    Pakistan Vet. J., 2007, 27(2): 102-104.   103purity of DNA free from contaminants like proteins or phenol.  RAPD-PCR   Sixteen different primers (Genelink Company) were used for fingerprinting S. enteritidis isolates. The 25 µl PCR cocktail contained: 3 mM MgCl2, 100 µM each of dATP, dCTP, dGTP, dTTP, 0.2 µM primer, 15 ng of genomic DNA and 1 unit of Taq polymerase. The thermolcycler (Eppendorf Mastercycler, USA) was programmed for 5 minutes initial denaturation at 95°C, 1 minute denaturation at 37°C and 2 minutes extension, 72°C for 40 cycles and final extension at 72°C for 10 minutes.    The PCR reaction products (10 µl each) were loaded on 1.2% agarose gel (Gibco BRL, Gaithersburg, Md.) containing 0.5 µg/ml ethidium bromide, at 80 V in 0.5X TBE buffer for approximately 1.5 hours. Two lanes of 1 kb ladder (Fermentas) were added in each gel for reference.  RAPD data analysis    The data obtained from amplification products by primers were used to estimate genetic similarity among different isolates on the basis of shared amplification products (Nei and Li, 1979). The RAPD patterns were scored on the basis of presence or absence of band. The similarity coefficients were utilized to generate dendrogram by using UPGMA (Unweighted Pair Group Method of Arithmetic means) through the programme, Popgene Version 1.31 (Microsoft windows based Freeware for population Genetic Analysis).  RESULTS    Out of 16 primers used, 8 were found totally non polymorphic and produced no polymorphic band. While out of the remaining 8 primers, only 5 primers namely: A-10 (5′-GTGATCGCAG-3′), A-13 (5′-CAGCACCCAC-3′), A-16 (5′-AGCCAGCGAA-3′), C-02 (5′-GTGAGGCGTC-3′) and C-15 (5′-GACGGATCAG-3′) provided good discriminatory power among different isolates of S. enteritidis. Primer C-02 provided unique results among all the five polymorphic primers (Fig. 1).  No amplification products were seen in reaction performed in the absence of primer or in the absence of DNA template.   The dendrogram obtained from RAPD data showed a single lineage pattern (Fig. 2). There was less genetic variation among isolates of poultry eggs and humans. However there was about 28.57% genetic relatedness among poultry eggs and broiler sources; and 73.47% genetic relatedness was found between human and layer sources (Table 2).             M       A        B       C      D        E       M   Fig. 1: Amplification of five RAPD products by Salmonella enteritidis isolates with primer C-02. Lane A = B15,  Lane B = E3,  Lane C = E29,  Lane D = H9, Lane E = E12, Lane M = DNA Marker (1 kb)                               0.2   0.3   0.4   0.5   0.6   0.7   0.8   0.9   1.0  Fig. 2: Dendrogram among five isolates of Salmonella enteritidis generated through RAPD data using UPGMA method.   DISCUSSION  Salmonella enteritidis is among the major etiological agents of the human gastroenteritis and is also most frequently isolated from poultry products. To our knowledge, the present manuscript is the first molecular study of S. enteritidis isolates in Pakistan to look for insight into the genetic diversity of strains that have been circulating locally between the human and poultry.    Pakistan Vet. J., 2007, 27(2): 102-104.   104  In this study, maximum numbers of amplified loci were obtained using primer C-02 sequence code (5'-GTGAGGCGTC-3') that is nine times repetition was found in the entire genome of S. enteritidis (Fig. 1). Similar results have been found by Hilton et al. (1996), Chansiripornchai et al. (2000) and Mare et al. (2001). The same primer C-02 was found unique for all isolates as it produced a unique common locus in all isolates. The genetic similarity matrix of RAPD data for 5 isolates was constructed based on coefficient of similarity utilized for the construction of dendrogram using the UPGMA, as described by Chansiripornchai et al.  (2000). Dendrogram was highly branched, suggestive for a genetically diverse population (Fig. 2). Similar results have been described by Millenman et al. (1996), Chansiripornchai et al. (2000) and Mare et al. (2001). The diversity in phylogenetic tree proved that the field isolates were distinctive with regard to biochemical profiles and serotyping.    The phylogenetic tree constructed from RAPD data showed a single lineage pattern, which indicated that all isolates are descendents of a single species of Salmonella enterica. The differences in the percent similarity could be the result of strain variation, as described by Hilton et al. (1996). On the basis of phylogenetic analysis of field isolates, S. enteritidis were found 28.57 to 73.47% similar among each other. There was very little (28.57%) genetic homology of isolates taken from the broiler sources, suggesting that there is genetically less related strain circulating in broilers. While more closely related (73.47%) strains are circulating between layers and humans.  Acknowledgements    Technical assistance to conduct PCR and RAPD analysis was provided by the Center for Agriculture Biochemistry and Biotechnology (CABB), University of Agriculture, Faisalabad. We are thankful for technical assistance of Mr. Ahsan Iqbal Rao and Mr. Usman Khan.  REFERENCES  Brenner, F. W., R. G. Villar, F. J. Angulo, R. Tauxe and B. Swaminathan, 2000. Salmonella nomenclature. J. Clin. Microbiol., 38: 2465-2467. Chansiripornchai, N., P. Ramasoota, A. Bangtrakuknonth, J. Sasipreeyajan and S. B. Svenson, 2000. Application of randomly amplified polymorphic DNA (RAPD) analysis for typing avian Salmonella enterica subsp. enterica. FEMS Immunol. Med. Microbiol., 29: 221-225. Herikstad, H., Y. Motarjemi and R. V. Tauxe, 2002. Salmonella surveillance: a global survey of public health serotyping. Epidemiol. Infect., 129: 1–8. Hilton, A. C., J. G. Banks and C. W. Penn, 1996. Random amplification of polymorphic DNA (RAPD) of Salmonella: strain differentiation and characterization of amplified sequences. J. Appl. Bacteriol., 81: 575–584. Mansoor, A., 1997. Molecular characterization of Avian Salmonella isolates. M.Phil. Thesis, Quaid-i-Azam University, Islamabad, Pakistan. Mare, L., L. M. Dick and M. L. Vanderwalt, 2001. Characterization of South African isolates of Salmonella enteritidis by phage typing, numerical analysis of RAPD-PCR banding patterns and plasmid profiles. Int. J. Food Microbiol., 64: 237-245. Millenman, Y., M. C. Lesage-Descauses, J. P. Lanfont and E. Chaslus-Dancla, 1996. Comparison of random amplified DNA analysis and enterobacterial repetitive intergenic consensus-PCR for epidemiological studies of Salmonella. FEMS Immunol. Med. Microbiol., 14: 129-134. Nei, N. M. and W. Li, 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci., 76: 5269-5273. Rampling, A., 1993. Salmonella enteritidis five years on. Lancet., 342: 317–318. Rodrigue, D. C., R. V. Tauxe and B. Rowe,  1990. International increase in Salmonella enteritidis: a new pandemic. Epidemiol. Infect., 105: 21–27. Silhavy, T. J., M. L. Berman and L. W. Enquist, 1984. Experiment with gene fusion. Cold Spring Harbor Lab. Cold Spring Harbor, New York, USA, pp: 138-139. Table 2: Similarity matrix of five Salmonella enteritidis isolates obtained from RAPD markers  Strains   B15  (Broiler) E3 (Eggs) E29 (Eggs) H9 (Human) E12 (Eggs) B15   1        E3 0.4490  1       E29   0.2857  0.6327  1    H9  0.4286 0.7347  0.5714 1   E12  0.3673 0.6735  0.4286 0.6939 1 

Pakistan Veterinary Journal

MOLECULAR CHARACTERIZATION BY USING RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) ANALYSIS OF SALMONELLA ENTERITIDIS ISOLATES RECOVERED FROM AVIAN AND HUMAN SOURCES

MOLECULAR CHARACTERIZATION BY USING RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) ANALYSIS OF SALMONELLA ENTERITIDIS ISOLATES RECOVERED FROM AVIAN AND HUMAN SOURCES

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