Distribution of NetB toxin in chickens infected with Clostridium Perfringens and cytotoxicity study of NetB toxin

Avian necrotic enteritis (NE) causes significant economic losses to the global poultry industry. Clostridium perfringens NetB toxin is a key virulence factor causing necrotizing enteritis in chickens, but the receptor of the toxin remains unknown, and the target organ of the toxin and its distribution in chickens are still unclear. To explore the distribution pattern and pathogenic mechanism of NetB toxin in the host, firstly in the study, by using a monoclonal antibody against NetB toxin, a specific immunohistochemical detection method and a double antibody sandwich method (DAS-ELISA) was established to dynamically monitor the distribution of NetB toxin in chicks after infection of Clostridium perfringens type G. The immunohistochemical results showed that NetB toxin was detected in the heart, lungs, spleen, kidneys, liver and small intestine 3 days after bacterial infection, the positive signals in the jejunal villi and lamina propria were the strongest. Toxins still could be detected in the liver and small intestine on the sixth day after infection. By the 14th day, only a small amount of toxin was detected in the intestinal tract. The DAS-ELISA test indicated that the concentration of NetB toxin in the serum was 10.37 ± 1.42 ng/mL 6 h after infection, and then decreased over time. Among the intestinal content samples, the toxin concentration in the jejunum contents was the highest, reaching 38.91 ± 2.36 ng/mL. Toxins in the contents of duodenal, ileum, jejunum and cecum could be detected 3 to 14 days after infection, but gradually decreased over time. Based on the above, this study conducted a treat test on chicken embryo hepatocytes using soluble NetB toxin expressed by Escherichia coli. The results indicated that the recombinant NetB toxin had a toxic effect on primary chicken embryo hepatocytes, leading to the release of lactate dehydrogenase, a decrease in cell activity, and destruction of cell morphology. The results of this study indicate that Clostridium perfringens NetB toxin not only causes pathogenic effects in the intestinal tract, but also is distributed in important organs such as the liver, spleen and kidney, and may cause pathogenic effects on them.