Investigation Of Outer Membranes And Virulence Genes Of Pathogenic E.coli And A Novel Serotype Isolated From Infected Ducks

Escherichia coli(E.coli)are a common member of the intestinal normal flora,but some E.coli strains belonging to certain serotypes are pathogenic to human and animals. Pathogenic E.coli of animals may pose a serious threat to human health throughout the food chain.With the popularization of intensive raising duck,pathogenic E.coli is becoming an increasingly serious problem because of great economic losses in duck industry.Unfortunately,most of the researches concerning avian pathogenic Escherichia coli(APEC)were focused on E.coli originated from chickens,and only have a few of pathogenic characteristics of duck-origin E.coli been reported in the past years.The objectives of this study were to isolate pathogenic E.coli strains from different large-scale duck farms in southwest China,and to investigate their serotypes,outer membrane protein patterns(OMP)and virulence-related genes.A novel serotype O46 strain,which cause disease characterized by swollen leg,was isolated and its biological features were studied intensively in this study.The results of this research will provide a scientific data for clarifying the pathogenesis,and for controlling and preventing the diseases caused by these E coli.1.Isolation,identification and phylogenetic analysis of duck pathogenic E.coli from large-scale duck farms.Two hundreds and eighty two strains of pathogenic E.coli were isolated from septicemia ducks of large-scale duck farms in Sichuan,Chongqing and Yunan provinces in China.Two hundreds and ten out of the 282 isolates(74.45%)were determined for serotypes by using slide agglutination test and tube agglutination test.The results showed that the isolates were related to thirty seven serotypes.Serotypes O93,O78,O92 and O76 were predominant serotypes,accounting for 43.8%(96/210),and serotype O46,O32&O93 and serotype O60&O93 were mixed-serotype which were isolated from ducks for the first time.Twenty eight strains belonging to 14 serotypes were identified from 35 isolates from cloaca of clinically healthy ducklings,and O109 and O154,46.4%of the total,were predominant serotypes.Antibiotic sensitivity tests were performed for 207 isolates and these isolates showed serious antibiotic resistance to 29 selected antibiotics.The average antibiotic resistance rate was 28.83%while most of strains showed multi-resistance and 59.4%of swains were resistant against 10 to 18 antibiotics.Eighty two isolates belonging to 37 serotypes(8 strains from each prominent serotype and 50 strains from other serotypes)were selected and their pathogenicity was evaluated after injection into leg muscle of 7-day ducklings(0.2ml of 10~9CUF/ml each).The results showed that high-pathogenicity strains,middle-pathogenicity strains and low-pathogenicity strains accounted for 80.5%(66/82),17.1%(14/82)and 2.4%(2/82)of total isolates tested,respectively.It was also found that all strains belonging to predominant serotypes were high- or middle-pathogenicity strains,accounting for 84.4%(27/32)or 15.6%(5/32) of tested strains,respectively.Phylogenetic analysis was conducted according to the 16S rRNA gene sequences of E.coli from 14 typical pathogenic strains of duck and from other animals.The results demonstrated that phylogenetic tree divided into two clusters.The first cluster,having longer branch length,consisted of 3 duck-origin pathogenic strains while the second cluster included 11 duck-origin pathogenic strains and some strains from human and other animals.Among those 11 strains,6 strains had a close relationship with E.coli serotype O157 of human-origin(2 strains had a close relationship with O157 sakai strain),while the other 5 strains showed close relationship with E.coli isolates from pig,cattle,poultry,or E.coli K12 strain of human.It is also revealed that there is no direct correlation between O serotypes and phylogenitic relationship of strains.2.Determination of the outer membrane protein(OMP)patterns of pathogenic E.coli isolated from ducksThe outer membrane protein patterns(OMP)of 130 pathogenic E.coli isolates belonging to 34 serotypes from ducks were determined in this study.Among these 130 strains,53 isolates belonged to prominent serotypes such as O93,while 77 strains were in other 35 serotypes such as O46.The results revealed 4 different OMP patterns in all strains tested.62.3%(81/130)of strains were identified to be OMP-1 with 29 serotypes,including O93,O92,O78 and O76,suggesting OMP-1 was the main OMP.Subsequently,a primer pair for amplification of ompA gene was also designed according to sequence of the E.coli K-12 available in Genbank.The ompA gene were then amplified and sequenced from 9 prominent serotype strains and 1 serotype O46 strain.Sequence analysis revealed that the ompA gene of all 9 prominent serotype strains open reading frame(ORF)with a size of 1053bp,which encoded a 350 amino acid protein,pro-OmpA.The first 21amino acids encoded a signal peptide and the mature OmpA protein was composed of 329 amino acids. The ORF of ompA gene from O46 serotype strain was 1041bp in length with 12bp depletion at the site of 400bp～411bp compared to other serotype strains,and the mature OmpA protein contained 325 amino acids.It was also found that the nucleotide sequences of ompA gene were highly homological with an identity ranging from 95.8 to 100%among 10 pathogenic E.coli strains from ducks.Phylogentic analysis of OmpA from 10 isolates indicated that O46 serotype form a single branch while other 9 strains consisted of the second branch.The study also indicated that there is no direct correlation between serotype, OMP and OmpA phylogenetic relationship3.Analysis of virulence-related genes of duck pathogenic E.coli isolatesIn this part of the study,pesticin receptor gene(fyuA)within the highly pathogenecity islands(HPI),iron regulatory protein gene(irp),type 1 pilus essential protein gene(fimC), the pap pilus structural protein gene(papA)and serum tolerance associated gene(iss)of the 210 pathogenic E.coli strains isolated from ducks showing clinical signs(clinical isolate) and the 28 E.coli strains isolated from clinically healthy ducklings by cloacal swabs(cloacal isolate)were determined by using polymerase chain reaction(PCR) combined with nucleic acid sequence analysis.The results showed that fyuA,irp2,fimC, papA and iss were detected in 41.0%,43.8%,92.9%,97.6%,and 96.7%of clinical isolates, and in 21.4%,25.0%,92.9%,100%and 92.9%of cloacal isolates,respectively. Statistically,there were no significant difference(P＞0.05)between clinical isolates and cloacal isolates for iss,fimC and papA gene detection.For papA gene detection,there were significant differences(P＜0.05)between the clinical isolates and isolates from other hosts such as chickens,swine and humans.The gene of fyuA was widely distributed in ducks and its detection rate in ducks showing clinical signs was significantly higher (P＜0.05)than that in clinically healthy duckling.There was a close positive correlation between the fyuA gene and the pathogenicity of E.coli.Furthermore,the gene of fyuA was closely related with the specific serotype of O78.The detection rate of fyuA,irp2,fimC,iss and papA2 in ducks showing clinical signs(37.6%,79/210)was significantly higher(P＜0.01)than that in clinically healthy ducklings(14.3%,4/28).4.Molecular epidemiology of Shinga toxin genes in E.coli from ducks.Four sets of primers were designed for stx1,stx2,hlyA and eaeA according to the sequences published in Genbank,and a multiplex PCR assay was established for rapid detection of shiga toxin producing E.coli.This PCR method was able to detect 4 virulence factors specifically and with a sensitivity of 10~3CFU/ml of pure bacterial culture.Two hundreds and ten of clinical isolates and 28 of cloacal isolates were detected by the multiplex PCR.The results showed that 6 strains of the 210 clinical isolates were stx1 positive,4 strains were hlyA positive and 1 strain was eaeA positive.The positive rate of stx1,hlyA and eaeA were 2.85%(6/210),1.90%(4/210)and 0.47%(1/210),respectively. Among the 210 clinical isolates studied,5 strains belonged to genotype stx1~+,3 strains belonged to genotype hlyA~+ and 1 strains belonged to genotype stx1~++hlyA~++eaeA~+, accounting for 2.38%(5/210),1.43%(3/210)and 0.47%(1/210),respectively.All clinical isolates were str2 negative while all cloacal isolates were negative for all interested genes in this study.Sequencing analysis of all positive PCR products found hlyA gene have 100% identity with that of EHEC O157:H7 Sakai strain in Genbank,while stx1 and eaeA have high homology with that of STEC.In this study,non-O157 STEC strains carrying stx1, hlyA and eaeA,and belonging to serotypes O158,O60,O78,O36,O77,O46,O137 and O192,were isolated from sick ducks.This inferred that E.coli strains from duck-origin may be an important problem in public health.5.Identification of a serotype O46 strain from ducks characterized by swelling leg and establishment of experimental pathological model.A duck-origin E.coli strain,SYW004,was isolated from a flock of sick ducks with clinical signs characterized by acute death and swollen leg,and proved to be serotype O46 after morphological investigation,biochemical study and serotype analysis.16S rRNA gene sequencing revealed that SYW004 strain had a close relationship to the human shigella and E.coli O157:H7(99～99.5%identity)but a far relationship with E.coli of chicken.Detection of 9 virulence genes demonstrated that this isolate carded 6 virulencerelated genes including hlyA,fyuA,irp2,fimC,papa and iss,suggesting it is an EHEC carrying multiple virulence genes.The SYW004 strain showed high pathogenicity in ducklings.Experimental model of infection closely resembled the natural infection.Autopsy,histopathologic and ultra-structural pathological observation showed that the major pathological features were characterized by wide fibrinous inflammation in the serosa.Hyperemia,hemorrhage and inflammatory cell infiltrate were observed in heart,liver,spleen,lung,kidney,bursa of Fabricius,small intestine,pancreas and brain.Degeneration,necrosis or apoptosis were found in renal tubular epithelial cells,liver cells,cardiac muscle cells and intestinal epithelial cells.Lymphocytes decreased in bursa of Fabriscius.E.coli was observed on ultra-thin sections of heart,lung,spleen,liver and small intestine under electron microscope.The target cells were lymphocytes,phagocytes,intestinal epithelial cells and cardiac fiber.6.Infection pattern of E.coli O46 isolate in an experimental model in ducksTo investigate the distribution of the O46 strain isolated in this study,an immunohisto chemical assay was developed with O46-specific antiserum and used to detect the bacteria of different organs at different times after experimental infection with the O46 isolate.The results showed this assay have good specificity.Samples of liver,spleen and kidney from ducks infected with O46 isolate showed positive results by this assay,while liver samples from dead ducks infected with E.coli O78,duck riemenella nantepestifer,duck plague virus,duck-origin avian influenza virus,salmonella or pasteurella multocida showed negative.With the advantages of direct,sensitivity and specificity,this assay could be used for accurate antigen localization.The study also indicated that the O46 isolates have wide tropism while heart,lung,spleen,kidney and intestine were found to be the main target organs.The bacteria were detected first in heart while bacteria could not be detected in trachea.Those findings provided important information for the in vivo distribution and proliferation of the O46 strain and accumulated useful knowledge for pathogenesis of this isolate.