Pathogenicity Of Human Shigella To Chicken And Comparative Study On Some Important Genes Of Chicken And Human Shigella

Bacillary dysentery is a kind of acute intestinal tract disease infected with and caused by shigella in the world and it is a public health problem especially in developing countries. Shigella can infect many animals besides human. 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 order to learn more about the molecular biology,pathogenicity and molecular mechanism of shigella infection ,especially shigella from chicken,so as to make it clear the origin and the public sanitation significance of shigella from chicken. In this paper,chicken Shigella boydii,human shigella boydii and flexneri were studied as follows.1. The chickens were intraperitoneally infected with different strains of shigella including chicken shigella boydii,human shigella boydii and flexneri with different dosages. The results showed that human shigella boydii and flexneri were pathogenic and lethal to 1-day-old chickens with a typical clinic sign of bloody dysentery.It indicated that human shigella may cause the disease of chickens with a definite morbidity and mortality post an experimental infection.2. Cohabitation test of chickens no-infected and infected with different dosages of human shigella flexneri. The result indicated that a morbidity of 20% was seen when the birds were orally inoculated with a dosage of 102 bacteria. After infection,various gross lesions appeared in each tested group of both infected and cohabited birds. The organism was re-isolated from each tested group of chickens except control ones. It proved human shigella not only infected chickens but also caused the horizontal transmission,and the virulence plasmid could not lost immediately in the process of transmission. It was also found the chickens could be the reservoir host of shigella,and the situation of chickens infected with shigella is similar to that of human,that is,the resistance against shigella is increased with age.This study have furnished indirect evidence for the origin of chicken shigella,and for the relation between human and chicken shigella. Moreover,the possibility of transmission between human and chicken shigella was proved. 3. The large virulence plasmid was successfully transfered from human shigella to chicken shigella by co-cultured in vitro,and the later has lossed the large virulence plasmid. Albino mice and chicken inoculated with different dosages of transfered-strain yielded an incidence up to 80% of infected chickens and 60% of infected mice without any death. The pathologic autopsy were 100%. The organism was re-isolated from each tested group of chickens except control ones. The culture characteristics of shigella changed after the virulence plasmids lossed. Most culture characteristics were restorable if shigella recover the virulence plasmids.The virulence plasmids may transfer from human shigella to chicken shigella in vitro,and the transfer-strain possessed certain stability and pathogenicity.From these results it was presumed that the virulence plasmid transfer would occur in the suitable external environment,if there are the virulence plasmids and the bacteria in the close category,and a new complete pathogenic strain would emerge.4. Resistance of chicken and human shigella boydi and human shigella flexneri to 12 conventional antibiotics were detected and three of them were found lack of resistance to quinolones. Two pairs of primers were designed to amplifying the gyrA gene and parC gene of three Shigellae strains respectively,and quinolone-resistant determining region(QRDR)within gyrA gene was analyzed. No mutations of base pair were found between the QRDR sequences of chicken and human Shigella boydii,which is consistent with the result of susceptibility test. In spite of no resistance of human Shigella flexneri to quinolones,a mutation of Ser83(TCG) to Leu(TTG) was found exsisting in the 83 site of gyrA gene,and just because of this,no resistance characteristics appeared,which is accordance to bacteria's resistance to quinolones that needs mutation of many sites in GyrA and ParC and other drug resistant mechanism. And a mutation of Ser58(AGC) to Ile(ATC) exsisted in the 58 site of parC gene,which is within the QRDR domain and different from common changes of the 80,84 amino acid. The result showed that the relationship between the mutation and the resistance to quinolones can't be determined. And its exact function in drug resistance of Shigella is unclear,which at least showed that the Shigella is in the variatation proceeding of drug resistance5. By using PCR assays,six fragments of copies of ipaH gene of chicken Shigella boydii were successfully obtained. And after the analysis of sequence homology,six fragments of copies in chicken Shigella boydii were found to have the highest sequence homology with that of American Shigella boydii CP001063,and have differences in that of Chinese Shigella boydii,which is inferior to that of CP001063 except for the fragment of ipaH₂. The result suggested the possibility that chicken Shigella boydii originated from human Shigella boydii. The genes of ipaB and ipaC of human Shigella boydii and Shigella flexneri were also cloned and sequenced respectively,and the result showed that the corresponding sequence homology of ipaB gene of human Shigella boydii(strain B)with that of published was 98.3%～99.9%,and the corresponding sequence homologies of ipaB gene and ipaC gene of human Shigella flexner(istrain F)with that of published one were 98.1%～99.5% and 97.9%～100.0% ,respectively. The study is a foundation for the following study of the expression of the IpaB and IpaC proteins and its molecular pathogenesis in Shigella. If the soluble IpaB and IpaC proteins were gained,and systemic characteristics,immunologic function,and also functions in vitro of them were studied,a better study of pathogenesis of Shigella can be obtained.This study privided the basis for the origin,pathogenicity and further molecular biology of both human shigella and especialoly chicken shigella.