Studies On The HPI Molecular Structures And Bacterial Ghosts Of E.coli Strains Causing Diarrhea In Calf From Farms In Northern China

The calf diarrhea, also called pullorum, is caused by Escherichia coli. The disease can occur with the sudden changes in temperature, and there is a high mortality rate, especially for 2-3 weeks calves. It has resulted in great economical loss to the cattle industry.The high pathogenicity island(HPI) plays an important role during the infection process of Yersinia, and because it contains the virulence gene cluster uptaking iron. HPI related virulence genes could spread between different species, animals and humans by horizontal transmission of plasmid. In recent years, HPI has been found in different species, including pathogenic E. coli, Klebsiella spp, Salmonella spp and other Enterobacteriaceae.Bacterial ghosts are the new vaccine, and most of them are generated by protein E of the phage Phix174 with forming the pore on the surface of Gram-negative bacteria, and contents are excluded through the pore resulting in the inactive and hollow shell-like "bacteria".Bacterial ghosts could enhance the immune response through the reserved natural bacterial surface antigens, and it has many advantages including simple preparation, safety, convenient transportation and multi-vaccine applications.In this study, the pathogenic E.coli were isolated from calves of diarrhea in six provinces and cities in northern. And we identified the O serotypes of them, detected the HPI genes of isolated strains, and cloned and analyzed HPI-related virulence gene sequences.One vaccine candidate strain was selected and prepared into bacteria ghosts.We analyzed part biological characteristics and immunogenicity of the bacteria ghosts.The results showed that140 strains were isolated from 420 samples of calves diarrhea(33.33%,140/420). All the isolated strains contained 15 serotypes, among them O14,O15,O101,O78 and O36 were dominant serotypes.Among the 140 isolated strains, 59 strains were HPI positive and the positive rate was 42.14%(59/140). And among the 59 strains, there were different positive rate for six major virulence factors: fyuA(49.15%, 29/59), asn_tRNA_intB(32.20%,19/59), irp3(42.37%, 25/59), irp5(23.73%,14/59), irp8(44.07%, 26/59) and ybtA(44.07%, 26/59).The homology between the virulence genes of different strains is high, and it may be related with collecting location and time of samples for part strains.There is no relationship between the number and species of HPI related virulence gene and the sample collecting location.The strains carrying five virulence genes(irp2, fyuA, irp3, irp8 and ybtA) accounted for 11.86%(7/59).In addition, we selected one strain with sensitivity to ampicillin, strong pathogenicity, characteristic of rapid growth, and advantage serotype(O15), and it was used as the candidate strain for bacterial ghosts and named as HBTN1311222.The bacteria ghost was constructed by the plasmid, and the lysis rate could reach to 99.877% after induced for 6h. And it could induce the humoral immune response and cellular immune response of mice, and produce good immune protection with rate of 80%.After challenge, the pathological changes of bacteria ghost group were lighter than other groups.These results will be helpful to understand the distribution and advantage serotypes of E.coli causing calves diarrhea in northern China and provide support to study the functions of HPI related virulence genes. In addition, it will lay the foundation for prevention of calves diarrhea and development of new vaccines.