| Clostridium perfringens(C. perfringens) can produce a variety of deadly exotoxins, which is easier to cause various diseases by C. perfringens infections in livestock and poultry, as well as people’s food poisoning, traumatic gas gangrene. The disease infected by C. perfringens seriously affects the people health, and also caused a certain losses to the breeding industry and public health. Therefore, it is very important to diagnose accurately the disease caused by C. perfringens. So far, the diagnosis methods of the disease caused by C. perfringens mainly include pathogen isolation and toxin detection of intestinal contents. The clostridial enteritis is mainly caused by intestinal infection. The bacteria itself does not always spread throughout the body. Hence, the bacteria cannot always be isolated from visceral organs. Mouse neutralization test of C. perfringens toxins to animal intestinal contents has gradually been replaced by the more sensitive enzyme-linked immunosorbent assay(ELISA). However, the effectiveness of ELISA result as diagnosis evidence has been questioned. Particularly, neither the detection of intestinal α toxin nor the isolation of a large amount of C. perfringens from the intestine can be the basis for diagnosis of the disease caused by C. Perfringens type A. Hence, there is an urgent need for a method to detect the pathogenic role of C. perfringens in order to confirm the diagnosis.C. perfringens α toxin is deadly exotoxin produced by various type, which is produced much more by C. perfringens type-A than the other toxin type. In this study, α toxin was produced efficiently by the main enterotoxigenic strains of C. perfringens type-A(NCTC 528) in anaerobic conditions, which is deteced by Enterotoxaemia ELISA kit, median lethal dose in mice(LD50) and SDS-PAGE in the cultures of supernatant. The experimental rabbits were infected of α-toxin produced in the cultures of supernatant by intraperitoneal injection and muscle injection, and then tissues of dead rabbits were collected timely within 72 h, including heart, lung, spleen, kidney, bladder, liver, stomach, small intestine and caecum, brain, cerebellum and the medulla. The tissues of dead rabbits were made into paraffin sections, which were used for subsequent histologic examination and immunohistochemistry of optimization. Results showed that Median lethal dose(LD50) of the α toxins in mice is 2-4.25/ m L, and the minimum lethal dose of it in rabbit is 2 m L, namely 38 times of LD50 in mice. By artificial injection, α-toxin invades organs with the blood circulation system causing the epithelial cell of stomach villi vacuoles degeneration, necrosis, epithelial cell of the small intestine and caecum necrosis, exfoliation and submucosa congestion, also causing hyperemia and hemorrhage of the related important solid organs, hemolysis of red blood cells, and acute injuries of the main mass cell degeneration, necrosis.Based on monoclonal antibody(MAb) 2A11 produced by anti-recombinant α-toxin of the hybridoma(antibody titer reached 1:102400), the MAb mediated immunohistochemistry was established and optimized by the test conditions of fixed, antigen repair, closed condition, MAb 2A11 dilution, HRP-Ig G and DAB staining. Results showed that the method could specifically locate α-toxin in the heart, lung, spleen, kidney, bladder, liver, stomach, small intestine, caecum, and oblongata of artificially infected rabbits. Especially α-toxin signal strength in the stomach, small intestine and kidney is stronger, which is mainly distributed in the cytoplasm of the cell. These results will provide reference for the future research in the pathogenesis of Clostridium perfringens α toxin.The cases of rabbit suspected clostridial enteritis outbreaks in the five large-scale rabbit farms in Shandong Province were diagnosed by immunohistochemistry established in this study, and combination of bacteriological cultivation and Multiplex PCR. As a result, 32 C. perfringens strains were isolated, with an isolation rate of 64%(32/50). All isolates were of toxin type A. As revealed of immunohistochemistry, α toxin was detected in at least one organ for 47(94%) out of the 50 rabbits with suspected infection, which was detected in the tissues of the kidney, liver, stomach, and small intestine. The results of this study showed that rabbit diarrhea outbreaking in the five large-scale rabbit farms in Shandong Province were highly correlated with C. perfringens infections. 94% of the rabbits with diarrhea suffered from clostridial enteritis, and 78.7% suffered from enterotoxemia caused by C. perfringens. The prevalent C. perfringens strains were of toxin type A. The C. perfringens α toxin positive rate detected in the tissues of rabbits with suspected clostridial enteritis by immunohistochemistry was significantly higher than the pathogen isolation rate. Therefore, this method can fill the gaps in routine diagnostic bacteriological cultivation, and provide a more reliable basis for the diagnosis of clostridial enteritis. |
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