Virulence factors of avian Escherichia coli

A total of 45 strains of Escherichia coli isolates from chickens with colisepticemia were examined for virulence factors commonly found in pathogenic groups of E. coli. These strains were studied for the following: pathogenicity in 1-day-old chicks; toxin, hemolysin, and colicin production; cell invasiveness and adherence; hemagglutination for fimbriae detection; serum resistance; aerobactin production in iron-limited conditions; and plasmid content. The characteristics exhibited by virulent strains were invasion for HeLa and chicken fibroblast cells, serum resistance, colicin V, and aerobactin production. None of the isolates were toxigenic or positive in hemagglutination tests. The molecular genetic studies of the virulence factors by agarose electrophoresis showed that the plasmids of these strains are of high molecular weight.     

papA gene of avian pathogenic Escherichia coli

P fimbrial adhesins may be associated with the virulence of avian pathogenic Escherichia coli (APEC). However, most APECs are unable to express P fimbriae even when they are grown under conditions that favor P fimbrial expression. This failure can be explained by the complete absence of the pap operon or the presence of an incomplete pap operon in Pap-negative APEC strains. In the present study, we analyzed the pap operon, specifically the papA gene that encodes the major fimbrial shaft, to better understand the pap gene cluster at the genetic level. First, by PCR, we examined a collection of 500 APEC strains for the presence of 11 genes comprising the pap operon. Except for papA, all the other genes of the operon were present in 38% to 41.2% of APEC, whereas the papA was present only in 10.4% of the APEC tested. Using multiplex PCR to probe for allelic variants of papA, we sought to determine if the low prevalence of papA among APEC was related to genetic heterogeneity of the gene itself. It was determined that the papA of APEC always belongs to the F11 allelic variant. Finally, we sequenced the 'papA region' from two papA-negative strains, both of which contain all the other genes of the pap operon. Interestingly, both strains had an 11,104-bp contig interruptingpapA at the 281-bp position. This contig harbored a streptomycin resistance gene and a classic Tn10 transposon containing the genes that confer tetracycline resistance. However, we noted that the papA gene of every papA-negative APEC strain was not interrupted by an 11,104-bp contig. It is likely that transposons bearing antibiotic resistance genes have inserted within pap gene cluster of some APEC strains, and such genetic events may have been selected for by antibiotic use.     

Iss from a virulent avian Escherichia coli

No single characteristic of virulent avian Escherichia coli has been identified that can be exploited in colibacillosis detection protocols. Research in our lab suggests a strong association between the presence of an iss DNA sequence with an isolate's disease-causing ability. The study presented here focuses on the techniques used in the expression, purification, and characterization of avian E. coli Iss protein. In brief, iss was cloned into an expression vector, the construct was transformed into a protease-deficient E. coli, and expression was induced. The protein was expressed as a glutathione-S-transferase (GST) fusion and purified by affinity chromatography. The GST portion was cleaved from Iss, Iss was harvested by affinity chromatography, and the identity of Iss was confirmed by N-terminal sequencing. Currently, purified Iss is being used to prepare hybridomas for production of monoclonal antibodies with the goal of evaluating anti-Iss as a reagent for the detection of virulent avian E. coli.     

Vacuolating cytotoxin produced by avian pathogenic Escherichia coli

The purpose of this study was to determine whether avian pathogenic Escherichia coli produced cytotoxic activity. Culture supernatants of 20 E. coli strains isolated from cellulitis lesions in chickens, five E. coli strains from avian septicemia, five from swollen head syndrome, and five from the feces of healthy chickens were incubated with primary chicken embryo fibroblast (CEF) cells, primary chicken kidney (PCK) cells, a quail fibroblast cell line (QT-35), and four mammalian cell lines (human epithelioid cervical carcinoma, African green monkey kidney, Chinese hamster ovary, and human larynx epidermoid carcinoma). Cytotoxicity was observed with supernatants from the 30 avian pathogenic strains on the two primary chicken cells (CEF and PCK). The highest dilution of culture supenatant that induced cytotoxic changes in 50% of the cells was 1/64. Supernatants from the five strains from normal feces were noncytotoxic, and none of the supernatants was cytotoxic for the QT-35 or the four mammalian cell lines. The cytotoxic effect, which was observed as early as 2 hr after exposure of the cells, was maximal at 6 hr and was evident as vacuolation, morphologically indistinguishable from that previously reported for culture supernatants of Helicobacter pylori. Like the activity in H. pylori, the cytotoxicity of the avian pathogenic strains was destroyed by heating at 70 C for 30 min and by exposure to proteolytic enzymes and was retained by filtration with a 100,000 molecular weight cut-off ultrafilter. Supernatants of two vacuolating cytotoxin-positive cultures of H. pylori failed to induce vacuolation of the CEF and PCK cells but caused the characteristic vacuolation in HeLa and Vero cells. The observations suggest that avian pathogenic E. coli produce a cytotoxin that is similar to the cytotoxin of H. pylori but may be specific for avian cells.     

Pathotyping avian pathogenic Escherichia coli strains in Korea

To examine the genetic background of avian pathogenic Escherichia coli (APEC) that affects virulence of this microorganism, we characterized the virulence genes of 101 APEC strains isolated from infected chickens between 1985~2005. Serotypes were determined with available anti-sera and median lethal doses were determined in subcutaneously inoculated chicks. The virulence genes we tested included ones encoding type 1 fimbriae (fimC), iron uptake-related (iroN, irp2, iucD, and fyuA), toxins (lt, st, stx1, stx2, and vat), and other factors (tsh, hlyF, ompT, and iss). Twenty-eight strains were found to be O1 (2.0%), O18 (3.0%), O20 (1.0%), O78 (19.8%), and O115 (2.0%) serotypes. The iroN (100%) gene was observed most frequently followed by ompT (94.1%), fimC (90.1%), hlyF (87.1%), iss (78.2%), iucD (73.3%), tsh (61.4%), fyuA (44.6%), and irp2 (43.6%). The strains were negative for all toxin genes except for vat (10.9%). All the strains were classified into 27 molecular pathotypes (MPs). The MP25, MP19, and MP10 pathotypes possessing iroN-fimC-ompT-hlyF-iucD-tsh-iss-irp2-fyuA (22.8%), iroN-fimC-ompT-hlyF-iucD-tsh-iss (21.8%), and iroN-fimC-ompT-hlyF-iss (11.9%) genotypes, respectively, were predominant. Redundancy of iron uptake-related genes was clearly observed and some strains were associated with higher mortality than others. Therefore, strains with the predominant genotypes can be used for diagnosis and vaccine.     

Expression of hemagglutinin of Haemophilus paragallinarum serotype A in Escherichia coli.

The genomic DNA of Haemophilus paragallinarum (Hpg) serotype A strain 221 was cloned into vector plasmid pBR322. The recombinant plasmids were introduced into Escherichia coli (E. coli) strain C600. Subsequently, a total of 277 transformants were obtained. One, designated strain no. 6, expressed hemagglutination activity against chicken erythrocytes. Strain no. 6 contained the recombinant plasmid pNV102, and DNA of about 2.57 kb was inserted into pNV102. When strain no. 6 was cured of pNV102, the strain lost hemagglutination activity. When the cured strain was retransformed with pNV102, hemagglutination activity was restored. E. coli strain no. 6 reacted with monoclonal antibody specific to the hemagglutinin of Hpg serotype A in a dot-blotting analysis. Chickens immunized with the inactivated strain no. 6 produced the hemagglutination inhibition (HI) antibody, and chickens possessing the HI antibody showed protection against challenge exposure by Hpg strain 221.     

Virulence-associated genes and antimicrobial resistance among avian pathogenic Escherichia coli from colibacillosis affected broilers in Pakistan

Avian pathogenic Escherichia coli (APEC) causes colibacillosis that leads to high morbidity and mortality among poultry birds. To date, there is a lack of knowledge about virulence-associated genes (VAGs) and multidrug resistance of APEC isolates from Pakistan. In this study, we determined the VAGs and antibiotic resistance profiles of APEC isolates recovered from colibacillosis affected broilers in Faisalabad region of Pakistan. A total of 84 diseased and dead birds from different local broilers farms were collected and examined for the gross lesions of colibacillosis by conducting postmortem examination. Of these, APEC isolates were recovered from 75 (89.2%) birds. Antibiotic susceptibility tests against 11 antimicrobial agents showed the highest resistance against ampicillin (98.6%) followed by tetracycline (97.3%) and ciprofloxacin (72%). The presence of 11 virulence-associated genes (VAGs) was detected by multiplex polymerase chain reaction (PCR). Of the 75 APEC, 32 (42.6%) harbored > 5 VAGs. Most commonly found genes were increased serum survival (iss; 84%), iron transport (iutA; 74.6%), and colicin V (ColV; 60%). Twenty-two isolates (29.3%) were found to possess a combination of VAGs; iss, tsh, iroN, and iutA, in addition to other VAGs. To the best of our knowledge, this is the first report on the detection of virulence-associated genes and multidrug resistance among APEC isolates in Pakistan. In the future, the strains with the predominant set of VAGs can be used for colibacillosis diagnosis and as a potential vaccine candidate.

     

禽病原性大肠杆菌温度敏感性血凝素(Tsh)突变株的构建

运用基因重组方法将庆大霉素抗性基因(GM)连接到PCR扩增的tsh两端区域产生的2个目的基因片段之间,并共同插入到pUC18载体的多克隆位点中,构建出带GM标志的载体pUC18-tshFRGM,从中切下目的片段,再将之克隆到pMEG-375自杀性载体中,构建出自杀性载体pMEG375-tshFRGM,将突变载体转化到含tsh基因的受体APECE037株中,根据同源重组原理,筛选出tsh基因缺失的E037突变株。E037和E037(Δtsh)株的LD50分别为105.6CFU和109.0CFU,动物感染性试验表明,E037(Δtsh)株在内脏器官和血液中的感染能力和大肠杆菌病变程度均有了明显降低。     

禽致病性大肠杆菌生物被膜的形成及其影响因素

为确定影响细菌生物被膜(BF)的形成条件,本研究采用BF体外定性观察和定量粘附性检测两种方法对1株野生禽致病性大肠杆菌(E.coli)在不同环境(培养时间、培养基类型、引导载体类型、营养条件)下产生BF的差异进行了研究。结果显示野生禽致病性E.coli其宿主体外BF最适形成条件是培养时间为48h,培养基为5%TSB、引导载体为平底96孔聚苯乙烯微孔板(美国Corning Costar)。其生长周期为8h时开始起始粘附、24h形成若干微菌落、36h微菌落粘连、48h形成完整的BF、72h细菌脱落开始下一轮的BF生长。糖分和适量的无机盐都可以在一定程度上促进BF的形成和被膜内细菌的粘附性提高。该研究表明禽致病性E.coliBF的形成周期,并为抑制其形成提供了实验依据。     

Virulence factors of Escherichia coli serotypes associated with avian colisepticaemia

The current understanding of the pathogenesis of avian pathogenic Escherichia coli (APEC) in colisepticaemia is limited. This review discusses putative virulence determinants per se, such as a number of surface organelles including fimbriae and flagella; together with other factors such as iron sequestering mechanisms, which are involved in the survival of E. coli in the host rather than initiation of infection. It is concluded that avian colisepticaemia is a multi-factorial disease and that to date only a limited number of virulence factors of APEC have been thoroughly elucidated.     

Clonal relationships among avian Escherichia coli isolates determined by enterobacterial repetitive intergenic consensus (ERIC)-PCR

Forty-nine avian Escherichia coli isolates isolated from different outbreak cases of septicemia (24 isolates), swollen head syndrome (14 isolates) and omphalitis (11 isolates), and 30 commensal isolates isolated from poultry with no signs of illness were characterized by enterobacterial repetitive intergenic consensus (ERIC)-PCR technique and their serotypes were determined. The ERIC-PCR profile allowed the typing of the 79 isolates into 68 ERIC-types and grouped the isolates into four main clusters (A-D), with the omphalitis isolates being grouped with the commensals and separated from the septicaemia and swollen head syndrome. These results indicate that ERIC-PCR is a technique that could replace other molecular characterization techniques such as random amplification of polymorphic DNA (RAPD)-PCR and restriction fragment length polymorphism (RFLP), reinforce previous observations that omphalitis isolates are just opportunistic agents, and are consistent with many reports that specific genotypes are responsible for causing specific diseases. Most of the isolates were either nontypable or rough, supporting the need for alternative methods for typing these isolates.     

Identification of a new Escherichia coli She haemolysin homolog in avian E. coli

Haemolysin is one type of virulence factor that assists in the pathogenesis of Escherichia coli. Currently, hemolytic activity in E. coli has been attributed to haemolysin genes found in either uropathogenic or enterohemorrhagic E. coli. Both haemolysins are classified as RTX toxins because they both have repeats in toxin domains and share similar operon organization, sequence homology, and mechanisms of action. Haemolytic avian E. coli isolates, however, lack either E. coli haemolysin gene. To investigate the avian E. coli haemolysin, a genomic library was made from an avian pathogenic E. coli. A haemolytic clone that was isolated was shown to contain homology with sheA, an E. coli K- 12 gene which causes haemolysis when present in high copy number. The cloned haemolysin gene, hlyE, lacked the conserved amino acid sequence and accessory genes common to all RTX toxins. DNA hybridizations and polymerase chain reaction amplifications showed that the nucleotide sequences homologous to hlyE were not present in a collection of three O157: H7 E. coli, five haemolytic canine uropathogenic E. coli, one haemolytic O26 E. coli, and three haemolytic avian pathogenic E. coli. Thus we have identified a new E. coli haemolysin distinct from the RTX haemolysins and have shown that some avian pathogenic E. coli possess a haemolysin with no apparent homology to hlyE or RTX haemolysins.     

Diversity of pilus subunits of Escherichia coli isolated from avian species

Pili from 69 avian isolates of Escherichia coli from six diagnostic laboratories in the United States were characterized. Three new pilus types were identified in addition to the three types previously described. A majority of the E. coli isolates (53.6%) examined expressed the classical type 1 pili.     

TonB is essential for virulence in avian pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC) strains have multiple iron-uptake systems that facilitate adaptation to iron-restricted environments and are believed to assist in colonisation of the host. These systems include several TonB-dependent transporters of ferri-siderophores encoded by the chromosome and the large virulence plasmid common to APECs. The tonB gene of the virulent APEC strain E956 was replaced with a selectable antibiotic resistance marker using Lambda Red recombinase mutagenesis. The phenotype of the ΔtonB E956 mutant was compared to the parent strain under various culture conditions and in chickens experimentally infected via the respiratory route. The mutant was resistant to streptonigrin, impaired in its ability to adapt to growth in iron-depleted medium and had greater tolerance of oxidative stress than the parental strain. The mutant was avirulent in chickens, did not affect the growth of chicks and colonisation was mostly limited to the trachea. This study has demonstrated that TonB is essential for virulence in APEC.     

禽大肠杆菌高密度发酵与溶氧关系的研究

利用GUJS—10C小型发酵罐分批培养禽大肠杆菌，通过平板计数、比浊计数的方法，对大肠杆菌高密度发酵中细菌生长与溶氧的关系进行分析。结果表明：溶氧的规律性变化能够反映大肠杆菌生长的规律，即在细菌生长的适应期细菌数量较少，耗氧量少，溶氧曲线最高；随着发酵时间的增加，细菌不断增多，耗氧量也随之增加，溶氧曲线下降；达到对数生长期，曲线最低；进入稳定期，曲线趋于平稳；到发酵后期，细菌进入衰老期，溶氧曲线略有上升，这时可终止发酵。     

IbeB is involved in the invasion and pathogenicity of avian pathogenic Escherichia coli

The ibeB gene in neonatal meningitis Escherichia coli (NMEC) contribute to the penetration of human brain microvascular endothelial cells (HBMECs). However, whether IbeB plays a role in avian pathogenic E. coli (APEC) infection remains unclear. Thus, this study was conducted to investigate the distribution of the ibeB gene in Chinese APEC strains and examine whether IbeB is involved in APEC pathogenicity. The ibeB gene was found in all 100 detected E. coli isolates with over 97% sequence homology. These results indicated that ibeB is a conserved E. coli gene irrelevant of pathotypes. To determine the role of ibeB in APEC pathogenicity, an ibeB mutant of strain DE205B was constructed and characterized. The inactivation of ibeB resulted in reduced invasion capacity towards DF-1 cells and defective virulence in animal models as compared to the wild-type strain. Animal infection experiments revealed that loss of ibeB decreased APEC colonization and invasion capacity in brains and lungs. These virulence-related phenotypes were partially recoverable by genetic complementation. Reduced expression levels of invasion- and adhesion-associated genes in ibeB mutant could be major reasons as evidenced by reduced ibeA and ompA expression. These results indicate that IbeB is involved in APEC invasion and pathogenicity.     

Chicken embryo lethality assay for determining the virulence of avian Escherichia coli isolates

Multiple isolates of Escherichia coli from clinical cases of colibacillosis and E. coli from the intestinal tracts of normal broilers at slaughter were assayed by the embryo lethality test to determine their virulence. The assay was repeated five times in order to establish reproducibility and determine the statistical parameters of the test. This study showed that the inoculation of approximately 100 colony-forming units in the allantoic cavity of 12-day-old embryos discriminated between virulent and avirulent E. coli isolates. Gross lesions included cranial and skin hemorrhages in addition to encephalomalacia in embryos inoculated with virulent isolates. Abnormalities were observed by microscopic examination of the heart, brain, and liver in embryos inoculated with virulent isolates. Analysis of data indicated that the length of the test should be 4 days. In the virulent group, day 2 postinoculation had the most significant death patterns. Sample size calculations indicated that 11 embryos are sufficient for the assay. On the basis of death rates, isolates considered to be avirulent had an embryo death rate of <10%, moderately or secondary pathogens had a 10%-29% death rate, and virulent isolates had a death rate of >29%. An important aspect of this assay is the accessibility of good-quality fertile embryonated eggs.     

Cloning and sequencing of the iss gene from a virulent avian Escherichia coli

Control of colibacillosis is important to the poultry industry. We have found that the presence of a gene for increased serum survival, iss, is strongly correlated with Escherichia coli isolated from birds with colibacillosis. Therefore, the iss gene and its protein product, Iss, are potential targets for detection and control of avian colibacillosis. The iss gene was amplified from a virulent avian E. coli isolate and sequenced. The sequences of the gene and the predicted protein product were compared with those of iss from a human E. coli isolate and lambda bor. The iss gene from the avian E. coli isolate has 96.8% identity with the iss gene from the human E. coli isolate and 89.4% identity with lambda bor. The Iss protein from the avian isolate has 87% identity with Iss from the human isolate and 90% identity with Bor. The low identity between the two Iss proteins is because of a frame-shift in their respective coding sequences. In sum, iss from this avian E. coli isolate is very similar to iss from a human E. coli isolate, but because of a frameshift mutation in the coding sequence of iss from the human E. coli isolate, Iss proteins from avian and human E. coli isolates have only 87% identity. The strong association of iss with E. coli isolated from birds with colibacillosis, suggests that this sequence be studied for its value as a marker or target to be used in colibacillosis control.     

禽致病性大肠杆菌唾液酸酶siaK1基因缺失株的构建及其生物学特性分析

禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)是危害养禽业发展的重要病原之一。本试验利用Red同源重组技术构建APEC IMT5155唾液酸酶基因sia K1缺失株和互补株,系统比较其生物学特性的差异。生长曲线测定表明,缺失株比野生株和互补株生长速度加快,而野生株和互补株的生长速度无明显差异;生物被膜形成能力测定表明,sia K1缺失导致细菌生物被膜形成能力显著减弱,而互补株可恢复至野生株水平,说明sia K1基因缺失可影响禽致病性大肠杆菌的生长速度及生物被膜的形成。本研究为进一步探讨sia K1基因功能奠定了基础。     

Occurrence of enterotoxigenic Escherichia coli in calves with acute neonatal diarrhoea

Enterotoxigenic Escherichia coli (ETEC) were isolated from 16.4% of diarrheic calves of up to 30 days old. Of 1-2-day-old calves nearly one half (45.0%) harboured ETEC, while this was the case with only 4.9% of 8-day-old calves. Among 206 9-30-day-old calves just three were found to harbour ETEC.