Construction Of Mutants Of APEC E037 Strain And Identification Of Their Virulence

Colibacillosis refers to any localized or systemic infection caused entirely or partly by avian pathogenic Escherichia coli (APEC), including septicaemia, granuloma (Hjarre's disease), air sac disease, chronic respiratory disease (CRD), avian cellulitis, swollen head syndrome, peritonitis, salpingitis, osteomyelitis/synovitis, panophthalmitis, and omphalitis/yolk sac infection. Colibacillosis in mammals is most often a primary enteric disease, whereas colibacillosis in poultry is typically a secondary localized or systemic disease occurring when host defence has been impaired or overwhelmed. Collectively, infections caused by Escherichia coli (E.coli) are responsible for significant economic losses to the poultry industry in many parts of the world. The pioneer serological surveys indicate that the majority of avian septicaemic E.coli strains belong to a limited number of O serogroups (O1, O2, and O78). Analysis of the involvement of these potential virulence factors using experimental models of infection just began during the past few years. Nevertheless, all the actual roles of the known virulence factors are not fully elucidated and certain steps of the infection process have not been related to previously identified factors. Furthermore, the mechanisms by which avian pathogenic E.coli cause infection are largely unknown.It has been reported that APEC has many virulence factors, but no solitary factor can distinguish whether the strain is pathogenic or not pathogenic. Researchers are working hard on making clear the virulence factors, meanwhile, they transfer their attention to the isolation and identification of the new virulence factors. But many isolated strains don't have the virulence factors ever known, according to the pathogenic experiment of the APEC. The above-mentioned information indicates that the isolated strains have other virulence factors. It is significant to acquire new virulence factors of the APEC.In this reserach, we constructed defined mutants of APEC strain E037, isolated and identified some new genes related to the virulence of the APEC.1.Construction of defined mutants of APEC strain E037Suicide vectors for deletion of the tsh genes were constructed as follows. The 685-bp fragment of tshF and the 692-bp fragment of the tshR together with gentamycin gene were cloned into pUC18 which was named pUC18-tshFRGM. A resultant suicide vector containing the tshF-GM-tshR fragment was named pMEG375-tshFRGM. Mutant derivatives of strain E037 were generated by allelic replacements and were named E037 (?tsh) and E037 (?iro?tsh). The 50% lethal dose (LD50) of E037, E037 (?iro), E037(?tsh) andE037(?iro?tsh) in commercial 15 day-old chickens experimentally inoculated via intratrachea were 105.6CFU, 108.4CFU, 109.0CFU and 109.5CFU, respectively. In the chicken challenge model, the mutants were tested to determine the individual role of this system for virulence and persistence in chickens. Among 243 avian E.coli isolates, iro was present in 84.4% (205 strains), while 184 (89.8%), 18 (8.8%) and 3(1.5%) of 205 iro+ isolates were high, intermediate and low in pathogenicity, respectively. There were 167 tsh+ isolates while 146 (87.4%), 21 (12.6%) and 0 (0%) of them were high, intermediate and low in pathogenicity, respectively.2. Construction of mutants for two unknown genes of APEC strain E037 and evaluation of their virulenceTo identify unique DNA fragments associated with avian pathogenic Escherichia coli strains, suppression subtractive hybridization (SSH) technique was used. The genome of nonpathogenic E.coli K-12 strain MG1655 was subtracted from the genome of avian highly pathogenic strain E037 (serogroup O78) resulting in the identification of 17 specific fragments. Sequence homology analysis was done and four types of fragments were identified: plasmid sequences, phage sequences, sequences with known function and sequence without any currently known functions. In this study, we chose gene 88# and gene126#, which belong to sequence without any currently known functions, to construct the mutants. Suicide vectors of the gene 88# was constructed as follows. The 984-bp gene 88# was cloned into T-easy vector which was named T-88#. A resultant suicide vector containing the gene 88# was named pMEG375-88#. Mutant derivative of strain E037 was generated by allelic replacements and was named E037-1. The construction of the E037 -2 was in the same way as that of the E037 -1. In the chicken challenge model, the mutants were tested to determine the individual role of the system for virulence and persistence in chickens. Gene 88# and 126# seemed to be the potential virulence genes of APEC.