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Background Clostridium perfringens (C. perfringens) is a gram positive, sporulating bacterium that is extremely pathogenic and responsible for a wide spectrum of anaerobic diseases in animals and humans. This bacterium is widely spread in the soil and the gastrointestinal tract of animals and is classified into five toxinotypes (A, B, C, D, and E) based on the production of one or more of the four main toxins (α, β, ε, and ι) 1 which are responsible for specific enterotoxemia in animals. 2 Enterotoxaemia is one of the most widespread lethal diseases in ruminants. This disease is indicated when a high level of toxins produced in the gut contents may be absorbed into the general circulation. 3,4 Typing C. perfringens strains is important, because different types of bacteria are associated with specific enteric diseases in animals. 5 Traditional methods have some limitations for bacterium typing. Serum neutralization test, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) have been previously used for the classification of C. perfringens, 5 among which PCR is the best choice for typing and sub-typing. Various PCR protocols have been established for the typing of C. perfringens isolates. 6 PCR has been applied in several studies and highlighted as a rapid and accurate method for the detection of low copy numbers of genes. This method is more accurate and faster than conventional methods. 7 Objectives To the best of our knowledge, there is a lack of data on the prevalence and typing of C. perfringens strains in Yazd ruminant farms. Therefore, we used PCR for typing toxigenic C. perfringens strains in this field. Materials and Methods Samples In total, 485 fecal and intestinal samples were investigated from the following species: domestic sheep (n = 206), goat (n = 129), and cow (n = 150). Collection and preparation of the samples was performed as previously described by Alimolaei, et al. 8 Bacterial strains were isolated from the samples and the isolated colonies were analyzed according to the shape, color, type of hemolysis, and gram staining smears as described by MacFaddin. 9 Biochemical identification of the catalase negative colonies presenting C. perfringens characteristics was performed as described

The alpha-toxin is one of the virulence factors of Clostridium perfringens for gas gangrene in humans and animals or necrotic enteritis in poultry. The C-terminal domain of this toxin ( cpa ) was synthesized and cloned into pT1NX vector to construct the pT1NX-alpha plasmid. This surface-expressing plasmid was electroporated into Lactobacillus casei ATCC 393, generating the recombinant L. casei strain expressing alpha-toxoid (LC-α strain). Expression of this modified alpha-toxoid was confirmed by SDS-PAGE, immunoblotting, and direct immunofluorescence microscopy. BALB/c mice, immunized orally by the recombinant LC-α strain, elicited mucosal and significantly humoral immune responses (p< 0.05) and developed a protection against 900 MLD/mL of the standard alpha-toxin. This study showed that this recombinant LC-α strain could be a promising vaccine candidate against gas gangrene and necrotic enteritis.