【作者】 刘红英；

【导师】 王川庆；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2008， 硕士

【摘要】 菌痢是由志贺菌引起的一种在全球范围流行的急性肠道传染病,尤其是发展中国家的一个重要的公共卫生问题。志贺菌除可感染人引起痢疾外,还可造成多种动物感染、发病。2004年许兰菊等首次发现鸡志贺菌感染病例,但其与人志贺菌之间的关系尚不清楚。为进一步了解志贺菌尤其是鸡鲍氏志贺菌的分子生物学特性、致病性及其致病性的分子机制,以便弄清鸡鲍氏志贺菌的来源及其在公共卫生方面的意义,本文进行了下列研究并取得预期结果:1.用不同剂量的鸡鲍氏、人鲍氏和人福氏志贺菌通过腹腔注射人工感染1日龄雏鸡。结果发现后两者均能引起雏鸡发病和死亡,且有拉血痢的典型症状出现。证明人志贺菌可人工感染雏鸡并引起发病死亡。2.用不同剂量的人福氏志贺菌对雏鸡做同居感染试验,结果显示102个菌即可以引起雏鸡20%的发病。各人工感染试验组和同居自然感染雏鸡均有不同程度的病变发生,剖检病变率达到100%。从各试验组雏鸡体内都回收到了原接种的菌株。由此证明人志贺菌不仅可以感染雏鸡还能造成水平传播,并且在此过程中其毒力大质粒不会很快丢失。此外还发现鸡体可以做为志贺菌的储存宿主,而且志贺菌对鸡的感染与对人的感染存在相类似的情况,即随着年龄的增长对志贺菌的抵抗能力增强。本研究不仅为鸡志贺菌的来源及人-鸡志贺菌之间的关系提供了间接证据,而且证明人禽之间存在交叉感染的可能性。3.将人福氏志贺菌的毒力大质粒与丢失此质粒的鸡志贺菌通过体外培养的方式,可使该质粒转移到丢失自身毒力大质粒的鸡志贺菌中。用不同剂量的毒力大质粒转移菌株通过口服和腹腔注射分别人工感染雏鸡和小白鼠,结果显示感染雏鸡最高发病率为80%,小鼠最高为60%;虽均无死亡,但剖检病变率达100%。从试验动物体内分别回收到原接种菌株。在培养过程中发现志贺菌丢失毒力大质粒后其培养特性发生较大改变,重新获得毒力大质粒则能恢复其大部分培养特性。本研究首次发现人志贺菌毒力大质粒可以在体外转移给鸡志贺菌,且新生成的转移菌株具有一定稳定性和致病性。据此推测在适宜的外界环境中只要有毒力大质粒和丢失毒力大质粒的相近病原菌存在,两者就可能会发生质粒转移,产生一株新的完整的致病菌株。4.通过药敏试验检测了鸡和人鲍氏志贺菌、人福氏志贺菌对12种常用抗生素的耐药情况,发现三株志贺菌均未产生对喹诺酮类药物的耐药性。根据已发表的序列设计了两对引物,分别扩增三株志贺菌的gyrA基因和parC基因,并对其中的耐喹诺酮类决定区（QRDR）进行分析。发现鸡鲍氏和人鲍氏志贺菌的QRDR区的序列没有发生任何碱基的突变,这与药敏试验结果相吻合。而人福氏志贺菌的gyrA基因中却存在83位Ser83（TCG）→Leu（TTG）的突变,由于只有该位点发生了突变,所以还没有出现耐药性表征,这与细菌对喹诺酮类耐药需同时存在GyrA和ParC多个位点突变或其它耐药机制的观点一致;同时在其parC基因中存在58位Ser58（AGC）→Ile（ATC）的突变,该突变点虽与常见的80、84位氨基酸的改变有所不同,却仍在QRDR区域之中。虽然暂时还无法根据这一结果来判断该突变与喹诺酮类耐药性之间的关系,但至少表明该菌株正处在耐药性演变进程之中。其在志贺菌耐药性形成中的确切作用还有待进一步研究。5.利用PCR技术成功地获取了鸡鲍氏志贺菌ipaH基因的6个拷贝片段,并对其进行了序列同源性分析,发现鸡鲍氏志贺菌与美国报道的人鲍氏菌株CP001063在6个拷贝片段的核苷酸序列比较中同源性都是最高的;与国内报道的人鲍氏菌株CP000036在6个拷贝片段的核苷酸序列同源性比较中虽然存在差异,但与除ipaH₂之外的5个片段的同源性仅次于CP001063。这一结果更进一步显示了鸡鲍氏志贺菌起源于人鲍氏志贺菌的可能性。此外还选择了人鲍氏和福氏志贺菌的ipaB和ipaC基因为研究对象,对其进行克隆测序分析。结果表明人鲍氏志贺菌（B株）的ipaB基因与已发表的相应序列的同源性为98.3%～99.9%,ipaC基因与已发表的相应序列的同源性为97.8%～100.0%;人福氏志贺菌（F株）的ipaB基因与已发表的相应序列的同源性为98.1%～99.5%,ipaC基因与已发表的相应序列的同源性为97.9%～100.0%。这些结果为以后进一步研究IpaB和IpaC蛋白的表达及其在志贺菌致病过程中的分子机制奠定了基础,如果能够得到可溶的IpaB和IpaC蛋白,研究其系列化特性及免疫学功能,并进行体内外功能研究,就可以更好的研究志贺菌的发病机制。本研究为志贺菌尤其是鸡鲍氏志贺菌的来源、致病性及其更进一步的分子生物学研究提供了一定的理论依据。更多还原

【Abstract】 Bacillary dysentery is a kind of acute intestinal tract disease infected with and caused by shigella in the world and it is a public health problem especially in developing countries. Shigella can infect many animals besides human. The significance of the organism in public health has been questioned since the first report of clinical case from chicken infected with shigella spp. by XU Lanju et al. in 2004. In order to learn more about the molecular biology、pathogenicity and molecular mechanism of shigella infection ,especially shigella from chicken,so as to make it clear the origin and the public sanitation significance of shigella from chicken. In this paper,chicken Shigella boydii、human shigella boydii and flexneri were studied as follows.1. The chickens were intraperitoneally infected with different strains of shigella including chicken shigella boydii、human shigella boydii and flexneri with different dosages. The results showed that human shigella boydii and flexneri were pathogenic and lethal to 1-day-old chickens with a typical clinic sign of bloody dysentery.It indicated that human shigella may cause the disease of chickens with a definite morbidity and mortality post an experimental infection.2. Cohabitation test of chickens no-infected and infected with different dosages of human shigella flexneri. The result indicated that a morbidity of 20% was seen when the birds were orally inoculated with a dosage of 102 bacteria. After infection,various gross lesions appeared in each tested group of both infected and cohabited birds. The organism was re-isolated from each tested group of chickens except control ones. It proved human shigella not only infected chickens but also caused the horizontal transmission,and the virulence plasmid could not lost immediately in the process of transmission. It was also found the chickens could be the reservoir host of shigella,and the situation of chickens infected with shigella is similar to that of human,that is,the resistance against shigella is increased with age.This study have furnished indirect evidence for the origin of chicken shigella,and for the relation between human and chicken shigella. Moreover,the possibility of transmission between human and chicken shigella was proved. 3. The large virulence plasmid was successfully transfered from human shigella to chicken shigella by co-cultured in vitro,and the later has lossed the large virulence plasmid. Albino mice and chicken inoculated with different dosages of transfered-strain yielded an incidence up to 80% of infected chickens and 60% of infected mice without any death. The pathologic autopsy were 100%. The organism was re-isolated from each tested group of chickens except control ones. The culture characteristics of shigella changed after the virulence plasmids lossed. Most culture characteristics were restorable if shigella recover the virulence plasmids.The virulence plasmids may transfer from human shigella to chicken shigella in vitro,and the transfer-strain possessed certain stability and pathogenicity.From these results it was presumed that the virulence plasmid transfer would occur in the suitable external environment,if there are the virulence plasmids and the bacteria in the close category,and a new complete pathogenic strain would emerge.4. Resistance of chicken and human shigella boydi and human shigella flexneri to 12 conventional antibiotics were detected and three of them were found lack of resistance to quinolones. Two pairs of primers were designed to amplifying the gyrA gene and parC gene of three Shigellae strains respectively,and quinolone-resistant determining region（QRDR）within gyrA gene was analyzed. No mutations of base pair were found between the QRDR sequences of chicken and human Shigella boydii,which is consistent with the result of susceptibility test. In spite of no resistance of human Shigella flexneri to quinolones,a mutation of Ser83（TCG） to Leu（TTG） was found exsisting in the 83 site of gyrA gene,and just because of this,no resistance characteristics appeared,which is accordance to bacteria’s resistance to quinolones that needs mutation of many sites in GyrA and ParC and other drug resistant mechanism. And a mutation of Ser58（AGC） to Ile（ATC） exsisted in the 58 site of parC gene,which is within the QRDR domain and different from common changes of the 80、84 amino acid. The result showed that the relationship between the mutation and the resistance to quinolones can’t be determined. And its exact function in drug resistance of Shigella is unclear,which at least showed that the Shigella is in the variatation proceeding of drug resistance5. By using PCR assays,six fragments of copies of ipaH gene of chicken Shigella boydii were successfully obtained. And after the analysis of sequence homology,six fragments of copies in chicken Shigella boydii were found to have the highest sequence homology with that of American Shigella boydii CP001063,and have differences in that of Chinese Shigella boydii,which is inferior to that of CP001063 except for the fragment of ipaH₂. The result suggested the possibility that chicken Shigella boydii originated from human Shigella boydii. The genes of ipaB and ipaC of human Shigella boydii and Shigella flexneri were also cloned and sequenced respectively,and the result showed that the corresponding sequence homology of ipaB gene of human Shigella boydii（strain B）with that of published was 98.3%～99.9%,and the corresponding sequence homologies of ipaB gene and ipaC gene of human Shigella flexner（istrain F）with that of published one were 98.1%～99.5% and 97.9%～100.0% ,respectively. The study is a foundation for the following study of the expression of the IpaB and IpaC proteins and its molecular pathogenesis in Shigella. If the soluble IpaB and IpaC proteins were gained,and systemic characteristics,immunologic function,and also functions in vitro of them were studied,a better study of pathogenesis of Shigella can be obtained.This study privided the basis for the origin、pathogenicity and further molecular biology of both human shigella and especialoly chicken shigella.更多还原