Epsilon toxin (ETX) is produced by toxinotypes B and D ofClostridium perfringens. It can induce lethal enterotoxemia in domestic animals, mainly in sheep, goats and cattle, causing serious economic losses to global animal husbandry. In this study, a novel and stable epsilon toxin mutant rETX^Y196E-C, obtained by substituting the 196th tyrosine (Y196) with glutamic acid (E) and introducing of 23 amino acids long C-terminal peptide, was determined as a promising recombinant vaccine candidate against enterotoxemia. After the third vaccination, the antibody titers against recombinant wild type (rETX) could reach 1:10⁵in each immunized group, and the mice were completely protected from 100 × LD₅₀(50% lethal dose) of rETX challenge. The mice in 15 μg subcutaneously immunized group fully survived at the dose of 500 × LD₅₀of rETX challenge and 80% of mice survived at 180 μg (1000 × LD₅₀) of rETX administration.In vitro, immune sera from 15 μg subcutaneously immunized group could completely protect MDCK cells from 16 × CT₅₀(50% lethal dose of cells) of rETX challenge and protect against 10 × LD₅₀dose (1.8 μg) of rETX challenge in mice. These data suggest that recombinant protein rETX^Y196E-C is a potential vaccine candidate for future applied researches.

Ricin is one of the most toxic and easily produced plant protein toxin extracted from the castor oil plant and it has been classified as a chemical warfare agent. Here, nanosecond pulsed electric fields (nsPEFs) at 30 kV/cm (pulse durations: 10 ns, 100 ns and 300 ns) were applied to inactivating ricin up to 4.2 μg/mL. To investigate the efficacy, cells and mice were tested against the ricin treated by the nsPEFs via direct intraperitoneal injection and inhalation exposure. Results showed that nsPEFs treatments can effectively reduce the toxicity of the ricin. Without the nsPEFs treatment, 100% of mice were killed upon the 4 μg ricin injection on the first day, however 40% of the mice survived the ricin treated by the nsPEFs. Compared to injection, inhalation exposure even with higher ricin dose required longer time to observe mice fatality. Pathological observations revealed damages to heart, lung, kidney and stomach after the ricin exposure, more pronounced for lung and kidney including severe bleeding. Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) and circular dichroism (CD) analyses revealed that although the primary structure of ricin was not altered, its secondary structures (beta-sheet and beta-turn) underwent transition upon the nsPEFs treatment.