摘要
目的:研究产气荚膜梭菌在淡水鱼消化道的分布及毒素型、进一步阐明鱼类产气荚膜梭菌的来源及产气荚膜梭菌的致病机理。方法:通过初步实验,自健康的活淡水鱼肠内容物中成功分离出产气荚膜梭菌,并建立了产气荚膜梭菌毒素型鉴定的三步PCR检测体系,在此基础上展开了对山东省主要湖(库)淡水鱼产气荚膜梭菌分布及毒素型调查,并将产气荚膜梭菌鱼类分离菌株α毒素全长基因进行克隆和核苷酸序列测定,与不同来源分离株进行同源性比较;进一步用Repetitive-Element PCR对同一采样环境的鱼源、泥源、禽源产气荚膜梭菌分离株进行基因相似性分析。本实验还进行了产气荚膜梭菌鱼源分离株对本动物及小白鼠的致病性试验,通过病理切片及免疫组织化学反应进一步阐明产气荚膜梭菌的致病机理。结果:自四大湖(库)淡水鱼及周围环境中共分离到产气荚膜梭菌141株,其中鱼源129株,泥源8株,鸭源4株;分离株主要为C型(102株,72.3%),其次为A型(35株,24.8%),B型(4株,2.8%)。C型和A型均携带cpe基因,而且β2毒素基因检出率较高。分离菌株α毒素全基因序列与A型畜禽源分离株及德国空气分离株的同源性为97.84-99.58%。Repetitive-Element PCR分析表明,同一采样湖(库)分离株基因相似性自45-100%,在每个湖区的分离株中,不同个体的鱼源分离株之间、鱼源与鸭源分离株之间、鱼源与泥源分离株之间均发现了相似性为92%甚至100%的产气荚膜梭菌分离株,表明产气荚膜梭菌在水体、在水体和周围环境间的传播。动物实验表明:产气荚膜梭菌鱼源分离株对本动物和小白鼠均有致病性,相同毒素型的鱼源分离株与标准菌株,稀释倍数相同时感染小白鼠,死亡率差异不显著;各型产气荚膜梭菌菌液与外毒素培养液对小白鼠的致死率差异不显著;相同剂量的菌液灌服与腹腔注射,小白鼠死亡率差异不显著,此结果肯定了产气荚膜梭菌经口感染的高效性。创新:本试验建立产气荚膜梭菌毒素基因检测的三步PCR体系,此体系克服了以往六重体系容易漏检β和肠毒素基因的缺点,与毒素的单基因PCR检测结果完全一致。应用该体系首次于淡水鱼产气荚膜梭菌分离株检测到α、β、ε、β2和肠毒素基因,首次在鱼检测到β、ε和肠毒素基因,首次在B型菌检测到β2毒素基因,从而验证了Tammy的推断:β2毒素可由各型菌产生。应用免疫组织化学方法证明产气荚膜梭菌毒素蛋白定位在肝、心、脑、脾、肺、肾等主质细胞的细胞质内,从而可以肯定产气荚膜梭菌感染动物可以形成毒血症,毒素侵害延髓的生命中枢以及心脏的传导系统是导致“猝死症”发生的原因。本研究共分七部分:
第一部分:淡水鱼产气荚膜梭菌的分离和初步鉴定
鲤鱼、鲢鱼、鲫鱼、鲶鱼、罗非鱼、黄鳝等肠内容物样品,厌氧环境下经过增菌、分离培养,选取血琼脂平板上典型的带双溶血环的菌落通过镜检、牛乳暴烈发酵试验及API 20A bioMerieux厌氧菌生化鉴定系统等细菌学方法,初步鉴定为产气荚膜梭菌,分离菌株通过α毒素基因(cpa)的PCR检测进一步确认,淡水鱼肠内容物中可以分离出产气荚膜梭菌,而肠壁分离结果为阴性。
第二部分:产气荚膜梭菌毒素基因三步PCR检测体系的建立
针对以往产气荚膜梭菌Multi-PCR检测体系容易漏检cpb、cpe基因的缺点,以A、B、C、D、E各型标准菌株为待检,建立了产气荚膜梭菌毒素型鉴定的三步PCR检测体系,即六种毒素基因的检测通过三次PCR反应完成, cpa、cpb2、etx、iA的四重PCR检测及cpb和cpe的单基因PCR检测,并通过特异性实验、扩增产物的核苷酸序列测定检验体系的可靠性,通过条件优化实验在各组分用量及反应温度方面进行优化,建立了最佳的反应体系。用此体系对实验一经PCR证实的产气荚膜梭菌分离株进行六种毒素基因(cpa、cpb2、etx、iA、cpb和cpe)检测,结果7株中有5株为C型、2株为A型,扩增产物核苷酸序列与Genebank上发表的同类序列Identity值在96%以上,排除了假阳性,也进一步验证了该方法的实用性。
第三部分:山东省四大湖(库)淡水鱼产气荚膜梭菌的分布及毒素型调查
对山东境内微山湖、雪野湖、东平湖及胜利水库采集鱼样、水样、泥样,首先测定水样大肠菌群MPN以确定水质受粪便污染情况,从而初步推测水体中产气荚膜梭菌存在的可能性,然后分离鱼肠内容物及泥样中的产气荚膜梭菌、三步PCR方法鉴定毒素型并且对扩增产物进行克隆和核苷酸序列测定,然后Blastn在线分析检出基因与GeneBank发表的相应毒素基因的同源性。本实验共分离到产气荚膜梭菌141株,其中鱼源129株,泥源8株,鸭4株;分离株主要为C型(102株,72.3%),其次为A型(35株,24.8%),B型(4株,2.8%), C型和A型分离株均检出β2和肠毒素基因,检出基因核苷酸序列(见附表)与GeneBank所发表的相应毒素基因同源性在96%以上。
第四部分:产气荚膜梭菌淡水鱼分离株与其它来源分离株的α毒素全基因序列分析
应用PCR技术,从C型产气荚膜梭菌淡水鱼分离株中扩增出产气荚膜梭菌α毒素全基因,并将其与pMD18-T载体连接、用DH5α感受态细胞转化,然后进行克隆和核苷酸序列测定。核苷酸序列分析证实,测序得到的α毒素全基因由1254 bp组成,包括1194 bp的转录区,自13位的ATG至2个连续的TAA,编码398个氨基酸,分子量为45KD,与目前已报道的其他α毒素一致,表明已成功克隆了α毒素全基因。C型产气荚膜梭菌淡水鱼分离株α毒素全基因序列与其他来源的A型产气荚膜梭菌α毒素全基因序列同源性为97.84～99.58%。
第五部分:Repetitive-Element PCR对产气荚膜梭菌基因相似性检测
为了研究同一湖(库)中不同来源及不同湖(库)同一毒素型产气荚膜梭菌的同源性,采用随机引物利用产气荚膜梭菌基因间重复一致序列的聚合酶链式反应(Repetitive-Element PCR)鉴定技术,扩增不同菌株的产气荚膜梭菌的DNA条带,形成聚类图谱,通过不同分离株的遗传相似性分析,确认同源性,基因相似性在90%以上的可视为起源相同的分离株。结果显示:同一采样湖(库)分离株基因相似性自45～100%,在每个湖区的分离株中,不同个体的鱼源分离株之间、鱼源与鸭源分离株之间、鱼源与泥源分离株之间均发现了相似性为92%甚至100%的产气荚膜梭菌分离株,表明产气荚膜梭菌在水体、在水体和周围环境间的传播。
第六部分:产气荚膜梭菌淡水鱼分离株对鱼及小白鼠致病性实验
针对未见产气荚膜梭菌引起鱼类疾病的报道,为确定产气荚膜梭菌淡水鱼分离株对本动物、哺乳动物的致病性特设计此实验。本实验以产气荚膜梭菌鲫鱼分离株为试验菌株,并以同型标准菌株为对照,用该菌的粗制外毒素对鱼类及小白鼠进行攻毒,Reed-Muench法计算毒素的半数致死量,同型试验菌株与对照菌株,稀释倍数相同时,对小白鼠的死亡率进行差异显著性检验。结果表明:产气荚膜梭菌鱼源分离株对本动物和小白鼠均有致病性,相同毒素型的鱼源分离株与标准菌株,稀释倍数相同时感染小白鼠,死亡率差异不显著。
第七部分:产气荚膜梭菌致病机理研究
以小白鼠为实验动物,以产气荚膜梭菌标准菌株A ( ATCC528 )、B(ATCC6121)、C(ATCC4989)、C(ATCC3180)和鲫鱼分离株DPJ12、DPJ4为试验菌株,进行菌液不同感染途径(灌服和腹腔注射)对小白鼠的致病性试验;同时制备的菌液与外毒素培养液灌服对小白鼠的致病性比较。在此基础上以C4989菌液灌服小白鼠,取24h后死亡小鼠的肝、心、脑、脾、肺、肾等器官为待检标本,分离产气荚膜梭菌菌体,然后以各器官组织提取的DNA为模板,用PCR方法检测其中的α毒素基因;肝、心、脑、脾、肺、肾、肌肉等器官制作组织切片,进行HE与免疫组织化学染色,观察病理变化并对器官中的毒素蛋白进行检测并定位。结果,各型产气荚膜梭菌外毒素培养液与菌液对小白鼠死亡率差异不显著,菌液灌服与腹腔注射对小白鼠致死性(死亡率)差异不显著,此结果肯定了产气荚膜梭菌经口感染的高效性。菌液和毒素灌服死亡小鼠呈全身出血性变化,肠粘膜脱落;在肝脏中可分离到产气荚膜梭菌菌体,其它器官未分离到;在肝、心、脑、脾、肺、肾等器官中均可检测到cpa基因与毒素蛋白,且毒素主要定位在主质细胞细胞质内。由此推断产气荚膜梭菌经口感染时能破坏肠粘膜上皮,进入血液循环,形成低菌血症与毒血症,毒素破坏主质细胞的细胞膜进入细胞质内,毒素侵害延髓的生命中枢以及心脏的传导系统是导致“猝死症”发生的原因。
Clostridium perfringens (C. perfringens) is a Gram-positive anaerobic spore forming bacterium known to be the most widely-distributed pathogen in nature. It is commonly found in the environment and in the intestines of animals and humans as a member of the normal flora .This bacterium is still described in water and sediment samples. In fish, this bacterium was described in sea fishes such as cod, sardine, ratfish and English sole, but only the strains isolated from cod were identified to toxin type and all were type A. There are also a few publications on the occurrence of C. perfringens in freshwater fishes; in these studies, however, the bacterium was identified only to the genus level and not to toxin type. Information about C. perfringens toxin types and toxins in freshwater fish are rarely reported. In order to study the isolation rate and toxin type of Clostridium perfringens in freshwater fish, this experiment was performed.
The study consists of seven parts:
PartⅠ: Isolation and Toxin-type Identification of Clostridium Perfrigens from some Freshwater Fish Samples.
Fresh intestinal content and tissue samples of freshwater fish including Cyprinus carpio, Hypophthalmichthys molitrix, Carassius auratus, Silurus asotus and Monopterus albus caught from one water reservoir were examined bacteriologically for the occurrence of C. perfringens. Isolates were examined by polymerase chain reaction (PCR) for gene encoding the alpha toxin for further identification. C. perfringens could be isolated in intestinal contents and not the tissue samples, the alpha toxin gene was detected in all isolates identified bacteriologically.
PartⅡ: Establishment of PCR Assay for Detecting the six Lethal Toxins Genes (α,β,ε,ι,β2 and cpe) of C. perfringens
A new PCR assay was established to classify C. perfringens into toxin types and further sub-classification. This PCR assay was successful in detecting the toxin genes from standard strain and those isolated from freshwater fish. The new PCR assay needed three times PCR amplification: one forα,β2,ε,ιtoxin gene, one forβtoxin gene, and another for cpe. The result of PCR amplification was completely same as that for single toxin gene.
PartⅢ: Investigation of Clostridium perfringens Toxin Types Isolated from Freshwater Fish in four Lakes(reservoir) of Shandong Province.
C. perfringens strains were isolated from samples of freshwater fish, mud and duck from the four major fish raising area in Shandong Province, e.g. Wei-shan, Xue-ye, Dong-ping Lake and Shengli- Reservoir. The new PCR amplification assay showed that: in 141 isolates, 102 strains (72.3%) were C. perfringens toxin type C, 35 strains (24.8%) were toxin type A, and 4 strains (2.8%) were toxin type B (α,βandεtoxin positive). In addition, the genes encodingβ2 toxin and enterotoxin were found in both type A and type C strains. The genes encoding for iota-toxin were not found in any isolate.
PartⅣ: The Homology Analysis of Complete alpha Toxin Gene Amplified from C. perfringens Freshwater Fish Isolates and Those Isolated from other Living Environments.
The complete alpha toxin gene was amplified from C. perfringens freshwater fish isolates by PCR, the PCR products were separated by electrophoresis on a 1.5 % agarose gel and purified with the Agarose Gel DNA purification Kit, purified PCR products were ligated into the PMD18-T vector and transformed into E. coli DH5αfor sequencing. The nucleotide sequences of detected toxin gene were compared with the toxin gene of strains isolated from other living environments using the DNAMAN computer software to reveal the origin of C. perfringens. the cpa 1254 gene was identified and sequenced to be consisting of 1194bp encoding 398 amino acid residues.After being analyzed by DNAMAN computer software, the cpa 1254 gene was known to be 97.84-99.58% consistent with the toxin gene of strains isolated from other living environments.
Part V: The Homology Analysis of Freshwater Fish -associated Clostridium perfringens Isolates by Repetitive-Element PCR
Repetitive-Element PCR analysis was used to analyze the homology of the isolates from one sampling lake, the results showed that the strains isolated from different fish samples, mud samples and duck samples had the same genetic distance, in all, the genetic distance of the strains isolated from the same lake was 45～100%. So it was concluded that the C. perfringens could spread in water environment.
PartⅥ: Testing for the Pathogenicity of Freshwater Fish -associated Clostridium perfringens Isolates to Freshwater Fish and Mice.
To study the pathogenicity of freshwater fish-associated C. perfringens isolates to freshwater fish and mice, the pure toxin cultures were grown in Godan buffer for 4 hours firstly, then harvested by centrifugation at 10,000rpm for 30 min for further purification deposited by 60% (NH4)2SO4 , purified toxins were inoculated into freshwater fishes and mice finally. Results showed that they have the pathogenesis to the two type animals, and the death rate of mice inoculated with the same toxin type standard strains was not significant different compared with those isolated from freshwater fish.
PartⅦ: Studying on the Pathogeny of C. perfringens
To make the pathogenicity of C. perfringens clear, the alpha toxin gene was detected from the inoculated mice tissues of hepar, cor, encephalon, spleen, pulmones and ren, and immunohistochemical location of C. perfringens toxins in organ tissues of mice was surveyed too. It showed that alpha toxin gene could be detected in hepar, cor, encephalon, spleen, pulmones and ren, and the positive cells with the toxin protein could be found in all these organs using the method of immunohistochemistry. So the probable cause of sudden death of animals infected by C. perfringens lies in the fact that the vital centre in medulla oblongata was harmed by toxins.
This was the first report of C. perfringensα,β,ε,β2 and enterotoxin were detected in freshwater fish, and ofβ,εtoxins were detected in fish species in general, and theβ2 toxin was detected in strains of type B too. Data may play an important role in public health risk evaluations, as these freshwater fishes are important fish species exploited for human consumption.
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