Identification Of Avian Pathogenic Escherichia Coli Causing Black Provertriculus Of Broilers And The Biological Characterization Of Its OmpF And MetF Genes

Colibacillosis refers to any localized or systemic infection caused entirely or partly by avian pathogenic Escherichia coli (APEC), including colisepticemia, coligranuloma (Hjarre’s disease), air sac disease (chronic respiratory disease, CRD), swollen-head syndrome, venereal colibacillosis, and coliform cellulitis (inflammatory process), peritonitis, salpingitis, orchitis, osteomyelitis/synovitis (turkey osteomyelitis complex), panophthalmitis, omphalitis/yolk sac infection, and enteritis.Numerous surveys have been undertaken in many parts of the world to identify the E. coli serogroups most frequently associated with diseases in poultry. Variations occur across geographic regions, but in most studies, the common serogroups have been 01,02,035,036, and 078. Some outbreaks are consistently associated with a specific serotype,0111 causing mortality, septicemia, and polyserositis in egg-laying chickens, while some APEC do not belong to known serotypes or are untypeable.Since 2013, one disease outbreak has occurred in some AA broiler integrations in Jiangsu Province, Anhui Province, Shandong Province and Henan Province. The disease was not characterized by initial clinical signs until 3 days of age but, it appeared with rapid progression and sudden death in only several following days. Most affected AA or Cobb broiler flocks recovered progressively after they were 10 days old. However, all 1-day-old AA Cobb broilers were apparently healthy. The mortality was 3%～12%. The gross lesions of the affected/dead broilers were characterized at necropsy. The specific lesion of black proventriculus was observed in some dead AA or Cobb birds. After a few days the typical pattern of acute, fibrinoheterophilic polyserositis involving the pericardial sac, pleura, air sacs, and peritoneum becomes evident. As far as our knowledge, the disease has not been repoted as having black proventriculus.In this study, we isolated and identified an APEC with the uncommon 0142 serogroup, originating from 4-8-day-old affected/diseased Cobb broilers collected from four farms of one integration, and investigated the clonal distribution and virulence profiles of E. coli 0142 isolated from broiler breeders with black proventriculi. We used multilocus sequence typing (MLST) to identify their phylogenetic group and PCR-based virulotyping to characterize the APEC isolates collected from broilers with black proventriculi diagonosed at four different broiler breeding farms. To analyze the transcriptome of two APEC isolates (4d/9-1 proventrivular isolate and 4d/9-1 heart isolate) by specific DNA microarray, and new potential virulence genes of APEC 4d/9-1 0142 proventricular isolate will be discovered. For these new potential virulence genes, mutants will be constructed, and their virulence will be evaluated in the colonization and persistence assay in vivo. Based on molecular Koch’s postulates, new virulence genes will be confirmed. It will provide clues for pathogen-host interaction study in avian colibacillosis characterized with black proventriculus.1. Isolation, Identification, and Pathogenicity of O142 Avian Pathogenic Escherichia coli Causing Black Proventriculus and Septicemia in Broiler Breeders96 avian Escherichia coli isolates originated from dead birds, dead embryos, affected broilers, contaminated feed samples and air/dust from the farms in one corporation were isolated and identified by standard biochemical procedures. Among these E. coli isolates,88 were placed in determined serogroups,8 were unserogroupable. These isolates distributed in 16 serogroups and 80.68%(71/88) belonged to 4 O serogroups:0142,078,045 and 086, especialy, including 52.27%(46/88) 0142 isolates. Therefore the predominent serotype is the uncommon 0142 serogroup.The relative pathogenicity of 10 Escherichia coli isolates (4d/9-1 proventrivular isolate and 4d/9-1 heart isolate, respectively) with predominant serogroups from same broiler was evaluated in specific-pathogen-free (SPF) 1-day-old chickens and 1-day-old broilers. High mortality and black proventriculus are observed in experimental broilers challenged with APEC 4d/9-1 0142 proventricular and heart blood isolates by air sacs in accordance with the clinic cases. While in 1-day-old SPF chickens, the mortality is equal to that of experimental challenge broilers, the black proventriculus is not observed. Isolates mentioned above were highly pathogenic to both young broilers and SPF birds inoculated by air sacs while low pathogenic by orally inoculation. It implied that the major transmission route in clinical cases is respiratory tract rather than digestive tract. For histopathological examination, affected or dead birds are characterized by necrosis, exfoliation of proventricular epithelium. In diseased broilers, mucus hypersecretion is also observed in proventriculus while not in SPF chickens.In this study, APEC isolates were detected for 32 virulence genes by polymerase chain reaction (PCR), in which iutA,fimH,frzorf4, ompT and feoB were positive≥88, and more than 50% of the isolates habored cvaC, iroN, iucC, iucD, sitA, traT, irp-2, tsh, astA and neuC genes. Less than 10% of the isolates habored afa, papA, papG allele I, sfa, sfaS,fela, cdtB, vat, ace26 genes. The results of phylogenetic groups of APEC isolates showed that B2 phylogenetic group was the most common, accounting for 58.33% of the 96 APEC isolates, and D、A and B1 phylogenetic groups summing up to 11.46%,19.79% and 10.42%, respectively. Drug sensitivity test results demonstrated that the tested APEC isolates were highly or moderately sensitive to colistin sulphate, amoxicillin clavulanic acid, ampicillin, neomycin sulfate, florfenicol and apramycin sulfate. Most of the strains were highly resistant to ceftiofur sodium, tilmicosin and lincomycin hydrochloride. Samples with black proventriculus and without black proventriculus of different clinic died birds and experimentally challenged AA broilers were selected for heavy metalion content determination, mercury content was excessive in all tested tissues including heart, liver, spleen, lung, kidney and proventricular of both diseased broilers without black proventriculus and died birds with black proventricular, while not excessive in those of the experimentally challenged AA broilers with black proventriculus and contaminated feed sample came from the feeder of diseased Cobb broilers. These results indicated that the mercury content did not contribute to the death and black proventriculus of the clinical and experimental infected broilers, but maybe contribute to the degree of black proventriculus.2. Comparison of Virulence Genes, Pathogenicity and Multilocus Sequence Typing of APEC Proventricular Isolates and Heart Blood Isolates Originated from Same BirdsThe median lethal dose (LD50)1 of APEC 4d/9-1 0142 proventricular isolate was 100 fold higher than that of APEC 4d/9-1 O142 heart blood isolate. In colonization and persistence assay carried out in 7-day-old broilers, bacterial load of APEC 4d/9-1 0142 proventricular isolate was about 10 fold higher than that of APEC 4d/9-1 O142 heart blood isolate in lung (P<0.01). and about 100 fold in heart blood, liver, spleen, kidney and proventriculus of inoculated broilers (P<0.001). Among the tested thirty-two common virulence genes of APEC, nearly same profile was found between proventricular isolates and heart blood isolates originated from same bird, except that APEC 4d/9-1 O142 proventricular isolate harbored the feoB gene while APEC 4d/9-1 0142 heart blood isolate not. Both APEC 4d/9-1 O142 proventricular isolate and APEC 4d/9-1 0142 heart blood isolate belonged to phylogroup B2, while by multilocus sequence typing (MLST), the former was assigned to ST131 and the latter was assigned to ST2704, respectively. The similarity of band profiles between two isolates was 97.3% in pulsed field gel electrophoresis (PFGE). The results of both MLST and PFGE showed that the genetic heterogeneity appeared between these two isolates although they came from the same died broiler.3. Screening of Distinct Expressed Genes between APEC 4d/9-1 Pro ventricular Isolate and Heart Blood Isolate Originated from Same Bird in SPF Chicken Challenge Model by DNA Microarray AssayBased on our previous experimental results, both APEC 4d/9-1 proventricular isolate and heart blood isolate were high pathogenicity. While the virulence of APEC 4d/9-1 O142 proventricular isolate was about 100 fold higher than that of heart blood isolate. Among the tested thirty-two common virulence genes of APEC, nearly same profile was found between the former isolate and the latter except that APEC 4d/9-1 O142 proventricular isolate harbored the feoB gene while heart blood isolate not. Futhermore, some reports have been proved no correlation between feoB gene with pathogenicity. This study is aimed to screen of distinct expressed genes between APEC 4d/9-1 proventricular isolate and heart blood isolate in SPF chicken challenge model by DNA microarray assay.The E coli whole genome Affymetrix oligo chip contained 10113 probes. According to multiple comparison, we screened related genes that had statistical significance (P<0.01) result showed that:the differential expression genes of APEC 4d/9-1 proventricular isolate and heart blood isolate between in vivo and in vitro were 1502 and 2260, respectively. While comparison of the differential expression genes between APEC 4d/9-1 proventricular isolate and heart blood isolate in vivo were 159 in total. Among 126 up-regulated genes, the most distinct up-regulated expressed genes were yehX, nrfG, lamb, ompF, gadB, ynjE, ygaM, yeiC, gltD, metF and 33 down-regulated genes, the most distinct down-regulated expressed genes were leuB, nhaR, sdaB, yghJ, ytfQ, ycdT, c2481, kpsM, C3689, ECs1505. The reliability of the microarray results was assessed by quantitative reverse-transcript polymerase chain reaction (qRT-PCR) analysis using selected 6 up-regulated distinct genes of yjhQ, C3292, nuoM, narH, metF, ompF and 4 down-regulated distinct genes of fimA, C0719, yjdB and yedV. For all genes except nuoM, similar distinct expression was determinconfirmed by qRT-PCR, and the level of correlation was well (r2= 0.9881). The results were in agreement with the data obtained by the microarray analysis. Gene ontology results demonstrated that some genes were related to cell part, binding, catalytic, transporter, cellular process and metabolic process, account for the largest portion. It implied that these distinct expressed genes were potential virulence-related genes and needed to be further studied. Besides specific genes, we also found 50 coding hypothetical protein genes, which were one of the key points to our further research.4. Construction of ompF and metF Mutant of APEC E491P and Evaluation of their Pathogenesis in ChickensOmpF is one of the most important porins and widely found in E. coli. The metF gene specifies methylenetetrahydrofolate reductase. This gene is located in the metJBLF gene cluster. Based on the above DNA microarray assay results, ompF and metF were chosen for deletion. The ompF and metF gene deletion mutant of APEC 4d/9-1 proventricular isolate (E491P) were created by the X Red recombinase method. The characterization of mutant strains and wild type strain were carried out. The results showed that the deletion of ompF significantly decreased the virulence of APEC E491P. The growth curves in LB results showed that the deletion of ompF and metF did not affect growth kinetics of APEC E491P, while the growth rate of AompF was slightly slower compared with the wild-type strain. Their abilities of resistance to serum have no significant difference among their parant strain E491P. The 50% lethal doses (LD50) of E491P, E491P△ompFand E491P△metF were 1038,1050 and 1040 CFU, respectively. The results of colonization showed that the E491P△ompF have significantly decreased colonization compared with the wild-type strain in all organs of chanllenged birds, while E491P△metF have not remarkable difference compared with the wild-type strain. Histopathological changes of heart and proventriculus in E491P△ompF challenged birds were less than those in E491P and E491P△ompF inoculated ones. It demonstrated that the deletion of ompF leads to attenuation of virulence of APEC E491P.