| Escherichia coli is ubiquitous bacteria in the natural environment.And it is also ubiquitous in the digestive tract of warm-blooded animals. E.coli is Gram negative bacteria of medium size. Studies have shown that certain serotypes of E.coli are pathogen, and certain serotypes of E.coli are unvirulent as animal intestinal probiotics.And some other E.coli are conditional pathogenic bacteria.They don’t cause diseases in general, but cause diseases in conditions such as poor sanitation, stress and low immunity. Presently, the researches mainly focus on detecting whether iss gene(increased serum survival gene) is presented and the results of detecting are used as an E.coli virulent indicator. But the experiment also shows that, iss gene exists in almost all E.coli. Therefore, further study of E.coli iss gene and pathogenicity is important for scientific prevention and treatment of this disease. 72 strains of avian E.coli had been detected for iss gene by one step PCR, nested PCR and real-time fluorescence quantitative PCR were tested. Test results of nested PCR showed that there were 69 strains of E.coli having iss gene.And the other 3 strains were also detected by real-time fluorescence quantitative PCR. Experimental results showed that iss gene was prevalent in E.coli, but the the transcription level of different strains varied.This study sequenced iss gene from 69 strains of E.coli. Aligning the sequences with the other 19 strains of E.coli, there was a total of 16 different kinds of gene sequence and 10 animo acid sequence, named type 1 to type 16 and form 1 to 10 separately. After comparing, it was found that the difference between different sequences was very small and the location of the mutation was relatively fixed. However, most of the amino acid sequences did not change, a fact shows that the iss gene was highly conservative. Phylogenetic tree analysis showed that the 16 genes were mainly divided into 2 branches. Among them, strains of sequence type15 and strains of sequence type 16 were in the same branch and sequence type1-14 strains were in one branch. Evolutionary tree of amino acids showed that the strains were mainly divided into two major branches. Among them, strains of form 3 and form 5 were in the same branch and strains of the other sequences were in the other branch. 30 strains of E.coli were tested by chicks lethality assay to measure the virulence. Transcription level of iss gene was tested by real-time fluorescent quantitative PCR. The results showed that the logarit hm of relative copy numbers was positively correlated with virulence. The analysis of statistical software showed that there was a positive correlation between the logarithm of copy numbers, serum resistance and virulence. There was a significant positive correlation between the logarithm of copy numbers and dead numbers of chicks(p=0.000<0.01) and the correlation coefficient was 0.769. There was a significant positive correlation between the logarithm of copy numbers and living rates of E.coli(p=0.000<0.01), and the correlation coefficient was 0.662. There was a significant positive correlation between living rates of E.coli and dead numbers of chicks(p=0.011<0.05), and the correlation coefficient was 0.456. This showed that the higher the transcription level of iss gene was, the stronger the resistance ability of E.coli to serum, and the stronger pathogenic ability was. The results of this study proved that the iss gene in E.coli was ubiquitous, and its correlation with virulence depended on transcription level. The more relative copy numbers there were, the more virulent the strain was. And there was a positive correlation between the transcription level and serum resistance as well. Research of this subject provided the reference for the study of other virulence genes of E.coli, and provided experimental data and theoretical basis for the further study of pathogenic factors of E.coli. |
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