毒力基因yqeH功能分析及对禽致病性大肠杆菌致病性的影响

【背景】禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)可引起禽类急性或亚急性感染,在近年新发现的大肠杆菌Ⅲ型分泌系统2 (Escherichia coli type III secretion system 2,ETT2)中,毒力基因yqeH对其致病性的影响尚不明确。【目的】探究yqeH在APEC致病过程中的作用,为后期深入研究ETT2致病机制奠定基础。【方法】利用Red同源重组技术构建yqeH缺失株ΔyqeH及其回复株CΔyqeH,通过运动性、生物被膜形成能力、抗逆性、抗血清杀菌能力等试验分析yqeH对APEC生物学功能的影响,并通过细胞黏附、侵袭试验、致病力测定及荧光定量PCR检测细胞炎性因子转录水平,探究yqeH对APEC感染宿主的影响。【结果】构建了缺失株ΔyqeH和回复株CΔyqeH;生物学特性试验结果表明,与野生株APEC81相比,缺失株ΔyqeH生物被膜形成能力、运动能力降低,对酸、碱、渗透压、氧化休克的耐受力降低,抗血清杀菌能力及致病力显著降低;与野生株APEC81相比,缺失株ΔyqeH对鸡气管黏膜上皮细胞的黏附及侵袭能...     

Suppression subtractive hybridization identifies an autotransporter adhesin gene of <Emphasis Type="Italic">E. coli</Emphasis> IMT5155 specifically associated with avian pathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> (APEC)

Background  

Extraintestinal pathogenic E. coli (ExPEC) represent a phylogenetically diverse group of bacteria which are implicated in a large range of infections in humans and animals. Although subgroups of different ExPEC pathotypes, including uropathogenic, newborn meningitis causing, and avian pathogenic E. coli (APEC) share a number of virulence features, there still might be factors specifically contributing to the pathogenesis of a certain subset of strains or a distinct pathotype. Thus, we made use of suppression subtractive hybridization and compared APEC strain IMT5155 (O2:K1:H5; sequence type complex 95) with human uropathogenic E. coli strain CFT073 (O6:K2:H5; sequence type complex 73) to identify factors which may complete the currently existing model of APEC pathogenicity and further elucidate the position of this avian pathoype within the whole ExPEC group.     

Common and specific genomic sequences of avian and human extraintestinal pathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> as determined by genomic subtractive hybridization

Background  

Suppression subtractive hybridization (SSH) strategy was used with extraintestinal pathogenic Escherichia coli (EXPEC) that cause avian colibacillosis (avian pathogenic E. coli or APEC) and human urinary tract infections (uropathogenic E. coli or UPEC) to determine if they possessed genes that were host and/or niche specific. Both APEC and UPEC isolates were used as tester and driver strains in 4 different SSHs in order to obtain APEC- and UPEC-specific subtraction fragments (SFs).     

DNA sequencing of a pathogenicity-related plasmid of an avian septicemic Escherichia coli strain

A 43-MDa conjugative plasmid isolated from an avian septicemic Escherichia coli (APEC) strain possessing genes related to the adhesion and invasion capacities of in vitro-cultured cells was sequenced. The results demonstrated that the 43-MDa plasmid harbors bacterial pathogenicity-related sequences which probably allow the wild-type pathogenic strain to adhere to and invade tissues and to cause septicemia in poultry. The existence of homology sequences to sequences belonging to other human pathogenic Enterobacteriaceae like Escherichia coli O157:H7, Shigella and Salmonella was also observed. The presence of these sequences in this plasmid could indicate that there is horizontal genetic transfer between bacterial strains isolated from different host species. In conclusion, the present study suggests that APEC strains harbor high-molecular weight plasmids that present pathogenicity-related sequences and that these are probably responsible for the pathogenicity exhibited by these strains. The presence of human pathogenicity-associated sequences in APEC conjugative plasmids suggests that these strains could represent a zoonotic risk.     

Avian disease at the Salton Sea

A review of existing records and the scientific literature was conducted for occurrences of avian diseases affecting free-ranging avifauna within the Salton Sea ecosystem. The period for evaluation was 1907 through 1999. Records of the U.S. Department of Agriculture, Bureau of Biological Survey and the scientific literature were the data sources for the period of 1907–1939. The narrative reports of the U.S. Fish and Wildlife Service's Sonny Bono National Wildlife Refuge Complex and the epizootic database of the U.S. Geological Survey's National Wildlife Health Center were the primary data sources for the remainder of the evaluation. The pattern of avian disease at the Salton Sea has changed greatly over time. Relative to past decades, there was a greater frequency of major outbreaks of avian disease at the Salton Sea during the 1990s than in previous decades, a greater variety of disease agents causing epizootics, and apparent chronic increases in the attrition of birds from disease. Avian mortality was high for about a decade beginning during the mid-1920s, diminished substantially by the 1940s and was at low to moderate levels until the 1990s when it reached the highest levels reported. Avian botulism (Clostridium botulinum type C) was the only major cause of avian disease until 1979 when the first major epizootic of avian cholera (Pasteurella multocidia) was documented. Waterfowl and shorebirds were the primary species affected by avian botulism. A broader spectrum of species have been killed by avian cholera but waterfowl have suffered the greatest losses. Avian cholera reappeared in 1983 and has joined avian botulism as a recurring cause of avian mortality. In 1989, avian salmonellosis (Salmonella typhimurium) was first diagnosed as a major cause of avian disease within the Salton Sea ecosystem and has since reappeared several times, primarily among cattle egrets (Bubulcus ibis). The largest loss from a single epizootic occurred in 1992, when an estimated 155000 birds, primarily eared grebes (Podiceps nigricollis), died from an undiagnosed cause. Reoccurrences of that unknown malady have continued to kill substantial numbers of eared grebes throughout the 1990s. The first major epizootic of type C avian botulism in fish-eating birds occurred in 1996 and killed large numbers of pelicans (Pelecanus occidentalis & P. erythrorhynchos). Avian botulism has remained as a major annual cause of disease in pelicans. In contrast, the chronic on-Sea occurrence of avian botulism in waterfowl and shorebirds of previous decades was seldom seen during the 1990s. Newcastle disease became the first viral disease to cause major bird losses at the Salton Sea when it appeared in the Mullet Island cormorant (Phalacrocorax auritus) breeding colony during 1997 and again during 1998.     

The genome sequence of avian pathogenic Escherichia coli strain O1:K1:H7 shares strong similarities with human extraintestinal pathogenic E. coli genomes

Escherichia coli strains that cause disease outside the intestine are known as extraintestinal pathogenic E. coli (ExPEC) and include human uropathogenic E. coli (UPEC) and avian pathogenic E. coli (APEC). Regardless of host of origin, ExPEC strains share many traits. It has been suggested that these commonalities may enable APEC to cause disease in humans. Here, we begin to test the hypothesis that certain APEC strains possess potential to cause human urinary tract infection through virulence genotyping of 1,000 APEC and UPEC strains, generation of the first complete genomic sequence of an APEC (APEC O1:K1:H7) strain, and comparison of this genome to all available human ExPEC genomic sequences. The genomes of APEC O1 and three human UPEC strains were found to be remarkably similar, with only 4.5% of APEC O1's genome not found in other sequenced ExPEC genomes. Also, use of multilocus sequence typing showed that some of the sequenced human ExPEC strains were more like APEC O1 than other human ExPEC strains. This work provides evidence that at least some human and avian ExPEC strains are highly similar to one another, and it supports the possibility that a food-borne link between some APEC and UPEC strains exists. Future studies are necessary to assess the ability of APEC to overcome the hurdles necessary for such a food-borne transmission, and epidemiological studies are required to confirm that such a phenomenon actually occurs.     

禽致病性大肠杆菌IMT5155 疑似毒力基因在人源及动物源大肠杆菌中的分布

[目的]检测禽致病性大肠杆菌IMT5155自分泌黏附素基因等具有代表性的疑似毒力基因在不同来源大肠杆菌中的分布,为进一步研究其致病机理提供依据.[方法]采用PCR和Dot blot,检测疑似毒力基因在不同地区(101株大肠杆菌中国分离株和121株大肠杆菌德国分离株)、不同来源(人源、禽源及猪源)大肠杆菌中的分布,并分析其和大肠杆菌系统进化分群的关系.[结果]自分泌黏附素基因B11等11个疑似毒力基因在禽致病性大肠杆菌中分布率较高,阳性率分别为:A1 36.4%(32/88)、A8 53.4%(47/88)、A1063.6%(56/88)、B1137.5%(33/88)、F3 59.1%(52/88)等,且疑似毒力基因主要存在于大肠杆菌B2进化群中.值得注意的是,D1、E9和F11基因片段在新生儿脑膜炎大肠杆菌中有较高的分布率,分别为60%(6/10)、80%(8/10)和90%(9/10),而在新生儿脑膜炎大肠杆菌中未检测到B11基因.[结论]自分泌黏附素B11等疑似毒力基因与禽致病性大肠杆菌关系密切,但疑似毒力基因D1、E9和F11与新生儿脑膜炎大肠杆菌密切相关,提示禽致病性大肠杆菌可能是新生儿脑膜炎大肠杆菌的毒力基因储库.     

双组分系统rcsC基因影响禽致病性大肠杆菌的致病性及相关生物学特性

【目的】双组分系统Rcs感受外界环境变化,并调控细菌的适应性及生存等。本文探讨Rcs双组分系统传感器激酶RcsC对禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)相关生物学特性及致病性的影响。【方法】采用Red同源重组的方法构建rcsC基因缺失株,并利用互补质粒构建互补株,然后比较野生株、基因缺失株与互补株的生长特性、运动性、生物被膜、凝集沉淀能力、致病力及毒力基因转录水平的差异。【结果】rcsC基因缺失不影响APEC的生长速度,然而,缺失RcsC导致APEC的运动能力升高、生物被膜形成能力降低和凝集能力增强。凝集试验结果显示rcsC基因有助于APEC的凝集沉降。细胞黏附入侵结果表明,rcsC在APEC侵袭DF-1细胞过程中发挥作用,而对黏附能力无影响。动物感染试验结果表明rcsC基因缺失能显著降低APEC的毒力。荧光定量PCR检测结果表明,rcsC基因缺失株中ompA、aatA、fyuA和luxS基因的转录水平均显著降低,而fimC和tsh基因的转录水平显著升高。【结论】RcsC参与调控APEC的运动性、生物被膜形成、凝集沉降和致病力。     

pagP基因对禽致病性大肠杆菌抗菌肽抗性和致病性的影响

【目的】禽致病性大肠杆菌(Avian pathogenic Escherichia coli,APEC)不仅严重影响全球的养禽业,对人类公共健康也造成巨大的潜在威胁。pag P基因在细菌的抗菌肽抗性和致病性方面发挥重要作用,但关于pag P基因在APEC中的功能尚不清楚。本文构建禽致病性大肠杆菌pag P基因缺失株,对缺失株的抗菌肽抗性和致病性进行研究。【方法】利用Red重组系统构建APEC的pag P基因缺失株,然后利用回复质粒构建回复株。研究pag P基因对细胞黏附与入侵、生物被膜形成能力、外膜渗透性、抗菌肽敏感性、致病性等方面的影响。【结果】成功构建pag P基因缺失株和回复株,抗菌肽抗性试验发现pag P基因缺失株对多粘菌素B、鸡β-防御素2(AVBD2)的敏感性显著增加(P0.01),致病性试验结果表明pag P基因缺失株的毒力显著降低(P0.01)。【结论】APEC的pag P基因对AVBD2的敏感性和APEC的致病性密切相关,为深入研究pag P基因的功能及调控作用奠定了基础。     

Biofilm formation by avian Escherichia coli in relation to media, source and phylogeny

AIMS: To assess the abilities of 105 avian pathogenic Escherichia coli (APEC) and 103 avian faecal commensal E. coli (AFEC) to form biofilms on a plastic surface and to investigate the possible association of biofilm formation with the phylotype of these isolates. METHODS AND RESULTS: Biofilm production was assessed in 96-well microtitre plates using three different media, namely, M63 minimal medium supplemented with glucose and casamino acids, brain-heart infusion broth, and diluted tryptic soy broth. Avian E. coli are highly variable in their ability to form biofilms. In fact, no strain produced a strong biofilm in all three types of media; however, most (75.7% AFEC and 55.2% APEC) were able to form a moderate or strong biofilm in at least one medium. Biofilm formation in APEC seems to be mostly limited to nutrient deplete media; whereas, AFEC are able to form biofilms in both nutrient deplete and replete media. Also, biofilm formation in E. coli from phylogenetic groups B2, D and B1 was induced by nutrient deplete conditions; whereas, biofilm formation by members of phylogenetic group A was strongest in a rich medium. CONCLUSIONS: Biofilm formation by APEC and phylotypes B2, D and B1 is induced by nutrient deplete conditions, while AFEC are able to form biofilms in both nutrient rich and deplete media. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to investigate biofilm formation by a large sample of avian E. coli isolates, and it provides insight into the conditions that induce biofilm formation in relation to the source (APEC or AFEC) and phylogenetic group (A, B1, B2 and D) of an isolate.     

Effects of avian malaria on male behaviour and female visitation in lekking blue‐crowned manakins

Avian malaria, the infection by blood parasites of the genus Plasmodium, can reduce host fitness not only through mortality, but also by impairing the expression of sexual selection traits. Although different studies highlight the association of parasitism with a decrease in host reproductive success, few studies have addressed the role of parasites in honest signalling by lekking species. Hence, it is still uncertain which fitness components are affected by parasites in these species. We investigated whether avian malaria is associated with a decrease in mating behaviour of male blue‐crowned manakins Lepidothrix coronata and whether it affects female visitation in leks of a population in the central Amazon. Through behavioural observations, we estimated the rates of total male activity and social interaction, as well as the frequency of female visits at individual perches. We then examined if individuals were infected with Plasmodium spp. using molecular techniques. Avian malaria was associated with a decrease in male mating behaviour in each lek, and mating behaviour correlated with female visitation. Although rates of social interaction were not correlated with avian malaria among males, we observed that interacting with several individuals within a lek may be advantageous for males, as they also vocalized and displayed more, thus increasing their chances of being visited by females. Although female visitation was not associated with avian malaria in individuals or leks, it is still possible that female visitation is indirectly affected by avian malaria through the latter's effects on male activity. We suggest a role for male activity as an honest sexual signal for females. Thus, male display rate could be used by females as cue for the probability of a male being infected.     

DNA sequence of a ColV plasmid and prevalence of selected plasmid-encoded virulence genes among avian Escherichia coli strains

ColV plasmids have long been associated with the virulence of Escherichia coli, despite the fact that their namesake trait, ColV production, does not appear to contribute to virulence. Such plasmids or their associated sequences appear to be quite common among avian pathogenic E. coli (APEC) and are strongly linked to the virulence of these organisms. In the present study, a 180-kb ColV plasmid was sequenced and analyzed. This plasmid, pAPEC-O2-ColV, possesses a 93-kb region containing several putative virulence traits, including iss, tsh, and four putative iron acquisition and transport systems. The iron acquisition and transport systems include those encoding aerobactin and salmochelin, the sit ABC iron transport system, and a putative iron transport system novel to APEC, eit. In order to determine the prevalence of the virulence-associated genes within this region among avian E. coli strains, 595 APEC and 199 avian commensal E. coli isolates were examined for genes of this region using PCR. Results indicate that genes contained within a portion of this putative virulence region are highly conserved among APEC and that the genes of this region occur significantly more often in APEC than in avian commensal E. coli. The region of pAPEC-O2-ColV containing genes that are highly prevalent among APEC appears to be a distinguishing trait of APEC strains.     

禽沙门菌诱导宿主免疫应答的特性

沙门菌病(Salmonellosis)是全世界最普遍的食源性疾病之一,不仅对养殖业造成经济损失,还对人类安全构成威胁。禽沙门菌感染肠道后,可诱导肠上皮细胞表达多种TLRs和炎症反应的发生,在分泌的趋化因子作用下免疫效应细胞迁移到感染部位。细菌通过肠上皮细胞屏障后被巨噬细胞或树突状细胞吞噬,其中巨噬细胞是沙门菌的主要定殖场所。天然免疫系统将抗原递呈给淋巴细胞后,机体能够在2–3周内通过以Th1为主的免疫应答清除在肠道和深层组织中的沙门菌。而宿主特异性血清型鸡白痢沙门菌从肠道侵入后,在肝脾和其他器官中定殖,进而引发全身感染。早期感染阶段不会引起肠道炎症反应,主要诱导以Th2为主的免疫应答,而Th1型应答相对较弱,有利于鸡白痢沙门菌在机体内的持续存在和感染。本文围绕禽沙门菌的致病机理和免疫应答特性进行阐述,尤其对鸡白痢沙门菌免疫逃逸和持续载菌的特性进行深入分析,为禽沙门菌病的防控提供新靶标和新见解。     

Persistence of Pasteurella multocida in wetlands following avian cholera outbreaks

Avian cholera, caused by Pasteurella multocida, affects waterbirds across North America and occurs worldwide among various avian species. Once an epizootic begins, contamination of the wetland environment likely facilitates the transmission of P. multocida to susceptible birds. To evaluate the ability of P. multocida serotype-1, the most common serotype associated with avian cholera in waterfowl in western and central North America, to persist in wetlands and to identify environmental factors associated with its persistence, we collected water and sediment samples from 23 wetlands during winters and springs of 1996-99. These samples were collected during avian cholera outbreaks and for up to 13 wk following initial sampling. We recovered P. multocida from six wetlands that were sampled following the initial outbreaks, but no P. multocida was isolated later than 7 wk after the initial outbreak sampling. We found no significant relationship between the probability of recovery of P. multocida during resampling and the abundance of the bacterium recovered during initial sampling, the substrate from which isolates were collected, isolate virulence, or water quality conditions previously suggested to be related to the abundance or survival of P. multocida. Our results indicate that wetlands are unlikely to serve as a long-term reservoir for P. multocida because the bacterium does not persist in wetlands for long time periods following avian cholera outbreaks.     

酰基高丝氨酸内脂合成酶LasI对禽致病性大肠杆菌生物学特性的影响

【背景】禽致病性大肠杆菌(Avian pathogenic Escherichia coli, APEC)是禽类主要病原菌之一,群体感应(Quorumsensing,QS)系统可通过信号分子调控其生物学特性。在APEC中信号分子AHL对其生物学特性的影响目前尚不清楚。【目的】研究信号分子AHL对APEC生物学特性的影响。【方法】将含铜绿假单胞菌酰基高丝氨酸内脂合成酶(Acyl-homoserine-lactone synthase,lasI)基因的表达质粒转化至APEC菌株DE17中,构建重组菌株DE17-lasI,利用LasI在DE17中合成AHL。比较野生株和重组菌株产生AHL信号分子、生长特性、生物被膜形成能力、运动性以及耐药性等生物学特性的差异;运用Real-timePCR技术,比较野生株和重组菌株中与生物被膜形成、运动性以及毒力因子相关基因的转录水平。【结果】对重组菌株AHL信号分子检测表明,DE17-lasI能够产生AHL信号分子,与野生株DE17相比,DE17-lasI生物被膜形成能力和运动性显著降低(P0.01),但其生长特性和耐药性无显著变化(P0.05);Real-time PCR检测结果表明,重组菌株的毒力因子fimH转录水平上调了58.8倍,而ompA、iss分别下调了95.4%、77.3%。与生物被膜形成相关基因agn43下调了75%,鞭毛合成基因flhA下调了80.8%。此外,AHL受体sdiA的转录水平上调了19.8倍。【结论】转化lasI至APEC中,能促进其在APEC中合成信号分子AHL,并显著影响APEC的部分生物学特性,为进一步探讨AHL型群体感应系统对APEC的调控作用提供参考。     

Effects of historical and contemporary factors on global patterns in avian species richness

Aim  The aim of this study was to determine how regional and historical factors influence global patterns in avian species richness.
Location  Global.
Methods  Using a comprehensive data set including 710 World Wildlife Fund terrestrial ecoregions covering nearly all the land surface of the Earth, avian species richness was compared among six biogeographical regions after accounting for sample area, elevational range and climate. Analysis of variance and multiple regressions were used. Spatial autocorrelation in model residuals was accounted for.
Results  Significant effects of region on avian species richness were found in nearly all comparisons between biogeographical regions.
Main conclusions  Regional and historical processes have played a role in regulating large-scale avian species richness patterns across the globe. Avian species richness in different regions of the world cannot be accurately predicted by a single global model. Avian species richness in areas of similar environments may differ substantially between regions, and thus avian species richness in one biogeographical region cannot be predicted using the richness–environment relationship derived from the data of another biogeographical region, even one with similar environments.     

Receptor binding profiles of avian influenza virus hemagglutinin subtypes on human cells as a predictor of pandemic potential

The host adaptation of influenza virus is partly dependent on the sialic acid (SA) isoform bound by the viral hemagglutinin (HA). Avian influenza viruses preferentially bind the α-2,3 SA and human influenza viruses the α-2,6 isoform. Each isoform is predominantly associated with different surface epithelial cell types of the human upper airway. Using recombinant HAs and human tracheal airway epithelial cells in vitro and ex vivo, we show that many avian HA subtypes do not adhere to this canonical view of SA specificity. The propensity of avian viruses to adapt to human receptors may thus be more widespread than previously supposed.     

Use of lambda Red-mediated recombineering and Cre/lox for generation of markerless chromosomal deletions in avian pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC) are bacteria associated with extraintestinal diseases in poultry. A method to generate markerless deletions of APEC genome is described. Lambda Red recombination is used to introduce a LoxP cassette (loxP-rpsL-neo-loxP) containing the rpsL gene for streptomycin sensitivity and the neo gene for kanamycin/neomycin resistance into the APEC genome, with attendant deletion of a desired chromosomal gene. The loxP sites are incorporated into primers used to amplify the rpsL-neo marker during the construction of the LoxP cassette, making the method rapid and efficient. The cassette is specifically integrated into the fiu gene or intergenic region 2051-52, and the Cre/lox system is used to remove the marker, hence deletion of the drug-resistance genes. The results demonstrate that the Cre/lox system can successfully be used to generate markerless deletions in APEC, and rpsL counter-selection can be used to select the deletions so that one does not have to pick and test to find the desired product.     

Multi-species patterns of avian cholera mortality in Nebraska's Rainwater Basin

Nebraska's Rainwater Basin (RWB) is a key spring migration area for millions of waterfowl and other avian species. Avian cholera has been endemic in the RWB since the 1970s and in some years tens of thousands of waterfowl have died from the disease. We evaluated patterns of avian cholera mortality in waterfowl species using the RWB during the last quarter of the 20th century. Mortality patterns changed between the years before (1976-1988) and coincident with (1989-1999) the dramatic increases in lesser snow goose abundance and mortality. Lesser snow geese (Chen caerulescens caerulescens) have commonly been associated with mortality events in the RWB and are known to carry virulent strains of Pasteurella multocida, the agent causing avian cholera. Lesser snow geese appeared to be the species most affected by avian cholera during 1989-1999; however, mortality in several other waterfowl species was positively correlated with lesser snow goose mortality. Coincident with increased lesser snow goose mortality, spring avian cholera outbreaks were detected earlier and ended earlier compared to 1976-1988. Dense concentrations of lesser snow geese may facilitate intraspecific disease transmission through bird-to-bird contact and wetland contamination. Rates of interspecific avian cholera transmission within the waterfowl community, however, are difficult to determine.