Construction Of An IucD Mutant And A Ftsk Mutant Of Avian Pathogenic Escherichia Coli And Evaluation Of Their Pathogenicity

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 belongs to a limited number of O serogroups (O1, O2, and O78). In this study, the relationship between iucD gene or ftsk gene and the pathogenicity of APEC strain E058 was studied. Nevertheless, all the actual roles of the known virulence factors of APEC are not fully elucidated and certain steps of the infection process have not been related to previously identified factors. Furthermore, the mechanisms by which APEC cause infection are largely unknown.1. Construction of an iucD mutant of avian pathogenic Escherichia coli and evaluation of its pathogenicityThe expression of aerobactin counts for much to the pathogenesis of avian pathogenic Escherichia coli (APEC). iucA , iucB, iucC and iucD are involved in the aerobactin synthesis. Knockout mutant of iucD gene in APEC strain E058 was constructed and named E058iucD. The pathogenicity of the mutant and its parental strain was evaluated in chickens and chick embryos. The mutant was sensitive to the bactericidal activity of specific-pathogen-free (SPF) chicken serum. The iucD-defective mutant of E058 was out-competed by the wild-type strain both in vivo and in vitro. The 50% lethal doses (LD50) of E058, E058iucD for the 1-day-old SPF chickens challenged experimentally via trachea were 105.5 and 106.5 , respectively. The mortality rates of 35-day-old SPF chickens inoculated with wild-type strain E058 and mutant E058iucD were 50.0% and 16.7% (p<0.05), respectively. Chickens challenged with APEC E058 exhibited a typical signs and lesions of avian colibacillosis, while those inoculated with mutant E058iucD showed moderate airsaculitis, mild pericarditis and perihepatitis. Embryo lethality of wild-type E058 or mutant E058iucD was 60% or 35% (p<0.05) in 12-day-old chicken embryos. These results indicated that the IucD related virulence factor has a significant role in the pathogenesis of APEC strain E058.2. Construction of a ftsk mutant of APEC E058 and evaluation of its pathogenicity in chickensThe 709 bp fragment of ftsk1, 900bp fragment of the ftsk2 and the gene encoding kanamycin resistance were generated by PCR respectively. All of these fragments were cloned into pBSK vector, termed pSK-F2-Kan-F1. Fragment of F2-Kan-F1 amplified by PCR were transformed into APEC strain E058, and then ftsk knock-out mutant of APEC strain E058 were generated by allelic replacement and named E058ftsk. Southern blot showed two copies of Kanr gene were inserted into the genomic or plasmid DNA of APEC E058; RT-PCR showed that the insertion didn't have a polar effect on the upstream or downstream gene of ftsk; The growth rate of E058ftsk was slower than that of APEC E058. In chicken challenge model, the mutant was tested to determine the individual role of this gene for virulence and persistence in 1-day old SPF chicks. At 6, 12 and 24 hours after inoculation, the number of colony forming units (CFUs) recovered from the liver, spleen, lung and blood of chickens inoculated with E058ftsk were significantly less than those of chickens inoculated with the parent strain APEC E058 (p<0.05), respectively. All birds were died during 48 hours post-inoculation with E058, however, only 20% of the birds were died within the same period post-inoculation with E058ftsk. All data showed that E058ftsk had a reduced virulence in 1-day old chickens. It was hypothesized that ftsk may be an important virulence gene of APEC E058. The actual function of ftsk gene needs further studying.