Identification of Alpha Toxin in Iranian Clostridium novyi Isolates from Slaughterhouse Samples

Background: Clostridium novyi type B bacteria cause infectious gangrene in the liver (black disease) of sheep and goats.This bacterium produces a cytotoxin called alpha toxin.The spores of this bacterium enter the body of the animal through the digestive system and penetrate the liver.If the necessary conditions for bacterial growth are provided, including hypoxia and liver damage, such as the migration of parasite larvae, latent spores are activated, multiply, and secrete alpha and beta toxins.These toxins spread from the affected areas of the liver, are absorbed into the bloodstream, enter the vital organs of the body, and eventually lead to the death of the animal.Infectious gangrene in the liver is associated with economic losses, including the costs of sheep deaths, depreciation of affected farms, and health problems of infected carcasses.It is timeconsuming to diagnose and isolate the cause of the disease with conventional methods; thus, there is a need for a simple and fast method to isolate and diagnose C. novyi.Therefore, this study was conducted for the molecular identification of alpha toxin in Iranian isolates of C. novyi from sheep liver.Methods: In this study, 62 liver samples suspected of infectious liver gangrene (with necrotic and parasitic areas) were collected from Iranian slaughterhouses (Marand 5, Ahvaz 8, Shahryar 13, and Ilam 36 samples) during the last three months of 2018 and in the first three months of 2019.To isolate the bacteria, pieces from different parts of the liver were first separated and smeared, and the supernatant was collected as an extract by centrifugation.The pieces harvested from the livers were cultured in chopped liver broth and incubated under anaerobic conditions.For molecular confirmation, the DNA of the tissue extract samples was extracted using the phenol-chloroform method, and the DNA of the cultured samples was extracted using a kit.The isolates were investigated by the polymerase chain reaction (PCR) using alpha toxin-specific primers.Isolates in which the alpha toxin gene of Clostridium difficile was detected were amplified based on the 16S rDNA sequence using specific primers.To isolate bacteria, positive samples were cultured in liver broth and placed under anaerobic conditions.To destroy non-spored bacteria (cleaning bacteria), the medium was heated at 100 C for 10 min.Then, the extract was cultured on a blood agar medium, and the hemolytic colonies were cultured in a rich fermenter medium to increase growth and toxin production.Bacteria were evaluated using Gram staining to control Gram-positive and Gram-negative bacteria and by malachite green staining to determine the shape and type of spores.The alpha toxin gene was sequenced in positive isolates, and the nucleotide sequences of the alpha toxin gene in these isolates were compared with the equivalent sequences of isolates and vaccine strains that were previously sequenced and registered in GenBank.Results: After cutting suspicious pieces of the liver with parasite contamination, parasite eggs were observed under a microscope.After incubation under anaerobic conditions, bacterial colonies with irregular shapes and unclear margins (polymorph) appeared on the agar medium and grew in the egg yolk agar medium, and Gram-positive, rod-shaped, endospore-forming bacteria were identified by staining.By performing PCR for the alpha toxin, a band was created in the range of 609 bp, and it was determined that seven samples belonged to C. novyi.PCR results using primers related to the alpha toxin gene present in type B C. novyi showed that out of 62