Investigation Of Clostridium Perfringens Toxin Types Isolated From Freshwater Fish In Four Lakes(Reservior) Of Shandong Province And The Study Of Pathogenicity

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. perfringensA 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 PCRRepetitive-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. perfringensTo 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.