Identification And Origin Analysis Of Shigella Boydii Isolated From Chicken Based On Molecular Marker

Shigella are Gram negative bacilli, and multiply within colonic epithelial cells, which can cause Shigellosis of human and animal. Shigella spp. can be subdivided into four serogroups namely S.sonnei, S.boydii, S.flexneri and S.dysenteriae. These species are subdivided into serotypes on the basis of O-specific polysaccharide of the LPS. There aren't obvious cross- protective among different serogroups or serotypes. Shigella spp. is an main enterobacterium of rhesus monkey causing bacillary dysentery. The significance of the organism in public health has been questioned since the first report of clinical case from chicken infected with shigella spp. by XU Lanju et al. in 2004. In this paper, Shigella boydii isolated from chicken was studied as follows.1. The genetic diversity among strains of Shigella boydii isolated from chicken, Shigella Dysenteriae, Enteropathogenic E.coli and S. Pullorum was analysed by random amplified polymorphic DNA (RAPD) with 6 random primers. The results shown that four of six 10 bp random primers were efficient to yield PCR product. Sixty four RAPD bands were identified and 3 of them were common in all four strains tested and 61 of them were polymorphic between any two strains. The polymorphic bands accounted for 95.3% of the total amplified bands. The primer P2 could be used to differentiate chicken Shigella boydii and S. Pullorum from other bacterial strains. The result also shown that the common rate of the amplified bands between Shigella boydii and Shigella dysenteriae was 67%, and that between Shigella boydii and Enteroinvasive E.coli was 44.4%. The amplified bands from Shigella and S. Pullorum were much different from each other when primer P6 was used, and this indicated the primer p6 could be used to differentiate different species. The heredity distance of 7 bacterial is 0.128-0.790 which was got by SPSS11.5. According to the heredity distance, 7 strains can be divided into 3 clustering groups. Chicken Shigella boydii and other two Shigella from human were in a same cluster. The sequences of PCR products and evolutionary relationship analysis showed that some PCR products included different genes though they were the same in size, which revealed that RAPD is a local method to a certain extent to differentiate bacteria species. The diversity origin of Chicken Shigella boydii was analysed on gene level, which confirmed Shigella evolved not with a single evolutionary origin. The phylogenetic tree constructed by these four genes revealed that the strain isolated from chicken maybe a new species or subspecies.2. Sequence analysis of 16S rRNA, gyrB, grpE, recA genes(accession nos. DQ229902, DQ995255, DQ995256, DQ995254) of Shigella was conducted and phylogenetic origins were analysised for the farther identification of chicken Shigella boydii. Results shown that the nearest homogeneity with chicken Shigella boydii is Shigella boydii isolated from human and the homogeneity is 99.6%～100%, even if almost all Shigella strains are distributed among E.coli strains. All sequences of four genes above from Shigella spp. strains are not identical to that from E.coli strains. On the other hand, recA gene and gyrB gene indicated a greater evolutionary divergence than 16S rRNA genes. It's concluded that using 16S rRNA and gyrB, grpE, recA gene sequences analysis may be a reliable, rapid, and low costs way for identification of Shigella strains. The phylogenetic tree constructed by these four genes revealed that the strain isolated from chicken maybe a new species or subspecies.3. According to the related sequences of Shigella deposited in the GenBank database, eight pairs of specific primers for eight housekeeping genes were designed and used to amplify thrB/thrC, trpC/trpB, purM/purN and mdh/argR genes of Shigella boydii successfully. The PCR products were cloned into PMD18-T vector and sequenced. The sequences reported in this paper have been deposited in the GenBank database (accession nos. DQ995264, DQ995265, DQ995261, DQ995263, DQ995260, DQ995258, DQ995259, DQ995257). By using the BLAST and Clustalx software, the sequences were analyzed and the phyligenetic tree for eight housekeeping genes in four regions of chromosome was established. Three clusters of Shigella strains were identified and nonetheless, are clearly within Escherichia coli. The locations of eight housekeeping genes in chicken Shigella boydii are identically distributed in four regions in chromosome, which are all contained in cluster 1. Chicken Shigella boydii probably originated from human Shigella boydii. It is estimated that the isolated strain maybe have evolved about 50,000-270,000 years.4. According to the related references, some specific primers for O antigen gene cluster and galF, gnd genes were designed and used to amplify O antigen genes cluster by using long PCR system. The results show O antigen gene cluster was amplified and cloned successfully. The recombinant plasmid was sequenced after identified and the sequences have been deposited in the GenBank database. On the other hand, the sequene of galF gene obtained in this paper was compared with the related sequences reported in GeneBank and phylogenetic tree were constructed. Results showed that Shigetta boydii isolated from chicken has the nearest homogeneity with Shigella boydii 4 isolated from human (CP000036), so Shigella boydii isolated from chicken is probably originated from Shigella boydii isolated from human. Some pathogenic of Shigella boydii isolated from chicken can be studied based Sequence of O antigen genes cluster.