EPIDEMIOLOGY AND MOLECULAR CHARACTERlZATION OF INDIGENOUS SALMONELLA ISOLATES FROM POULTRY

Abstract

Salmonellosis ranks among the most frequently occurring food borne disease encountered with heavy economic loses for the food and poultry industries. Nontyphoidal salmonellae are recognized as important source of human salmonellosis in developed as well as developing countries, with an annual incidence of 1.3 billion cases and 3 million deaths. Poultry associated Salmonella infection has emerged as a major public health problem during the last decade. The surveillance studies conducted by the consulting group of WHO reflects the world-wide distribution of salmonellae as zoonotic disease with variable incidence from on country to another. A progressive increase in the prevalence of non-typhoidal salmonellae has been evident in Pakistan, however, precise information describing . the microbiological and molecular analysis of indigenous strains for epidemiological hypothesis has never been published so far. The zoonotic and economic importance of the disease, therefore, underscores the need to generate base line data on the epidemiology and molecular characterization of indigenous Salmonella isolates. The analysis of 1747 random samples collected through 1994-1997 from 12 different sources and 56 different locations in the Northern Punjab area (Pakistan) showed that 162(9.27%) were positive for salmonellae. The isolation prevalence in poultry tissues was 86(8.04%) and in various allied sources, it was 76(11 .20%). The prevalence of motile serovars was 111(6.31/%) while non-motile was 51(2.91%). Serotyping and phage typing revealed that S. enteritidis PT4 and S. albany were the exclusively isolated serovars from studied sources with isolation prevalence of 48 (43 .24%) and 63(56.35%), respectively. All isolates were susceptible to at least 9 of the 14 antimicrobial agents tested and highly resistant to SMX whereas S. albany isolates showed additional resistance to Kanamycin and tetracycline. Microdilution MICs revealed that baci tracin (MIC range 12.5 -;:::100).!g/ml), Kanamycin (MIC range 3. 125- 50).!g/ml and 12.5 - ~ 1 OO~Lg/ml) , and tetracycline (MIC range 6.25 - 100~l g/ml and 25- ;::: 100).!g/ml), all had very poor activity against serovar Enteritidis and Albany, respectively. These findings suggest the limited therapeutic potential and low typeability of these strains by using these antimicrobials. Plasmid profile analysis showed that 45(93%) of the S. enteritidis isolates harboured a single plasmid of 55kbp, while the remaining tlu-ee (7%) possessed a 90kbp plasmid with an additional plasmid of approximately 2.1 kbp. The digestion of plasmid DNA with HindlII confirmed the structural homogeneity of all 55kbp and that of 90kbp virulence plasmids. Hybridization with the virulence gene probe showed that SpvB/SpvC genes were located on HindIII fragment of 3.6kbp in each of the two types of virulence plasmids. All S. albany isolates included in the study w~re plasmid free. Ribotyping based on the detection of RFLP's of rRNA genes carrying Pvull fragment of the chromosomal DNA was performed by Southern blot hybridization experiment using an enzymatically labelled gene probe, which recognized 5S-16S-23S rRNA gene. This probe has previously been used for typing of many other salmonellae such as serovar Typhimurium, Enteritidis, and Dublin. An identical hybridization pattern was observed with PvuII digested whole cell DNA of both S. enteritidis and S. albany isolates consisting of seven and nine hybridizing fragments ranging in size from 2.1- 12.3kbp and 2.1-11.4kbp, respectively. Southern blot hybridization experiment with Salmonella specific insertion sequence IS200 and Pstl digested whole cell DNA of S. enteritidis showed conserved IS200 profiles and carried their IS200 copies on PsI! fragments of 4.5kbp and 3.9kbp. Non of the S. albany isolates included in the study carried an insertion sequence of IS200 type. Such data suggest the limited potential of rRNA gene and Salmonella specific IS200 element for epidemiological analysis of both of these serovars. The results of macrorestriction analysis strongly depend on the choice of the restriction enzyme. Therefore, two different restriction enzymes, Spe I and Xba 1, which proved to be useful for the differentiation of isolates of S. typhimuriul11, S. enteritidis, S. dublin, S. choleraesuis and S. hadar, were chosen. PFGE analysis of S. enteritidis isolates with both Xbal and SpeI digested whole ce ll DNA revealed the presence of three different fragment patterns, consisting of 9-15 bands and 15-20 bands in the range of 48-533kbp with discriminatory indices of 0.0520 and 0.667, respectively. The comparative use of these two suitable enzymes increased the discriminatory power (0.843) of this method. The PFGE analysis of S. albany isolates revealed two different fragment patterns with Xbal and identical pattern with Spel endonuclease digestion within the same range as described for S enteritidis. The index of discrimination for S albany isolates was 0.479. On the basis of PFGE analysis, seven genomic groups were assigned to S enteritidis and two to S albany. PFGE proved to be superior in its discriminatory values to other molecular methods such as plasmid profile analysis, ribotyping and IS200 typing for these serovars. The observation that epidemiologically unrelated Salbany isolates were almost indistinguishable, even by using molecular methods suggested a strong clonal relationship among S albany isolates