GENOMIC DIVERSITY OF THE CLOSTRIDIUM PERFRINGENS ISOLATES FROM HEALTHY AND ENTEROTOXEMIA AFFECTED LIVESTOCK USING MLVA METHOD

Clostridium perfringens is a component of the normal gut microbiota in humans and animals. However, it can become pathogenic under certain conditions, causing serious intestinal and systemic diseases. Enterotoxaemia is one of these diseases causing significant mortality in livestock annually. Analysis of genetic diversity or relatedness of isolates with molecular typing methods can be helpful for understanding the epidemiology of these infections and observing population structures of these bacteria. The aim of this study was to perform molecular typing of Clostridium perfringens isolates obtained from both healthy and clinically suspected enterotoxemic livestock in Fars Province using Multiple Locus Variable Tandem Repeat Analysis (MLVA) method. Collection of previously isolated and preserved Clostridium perfringens along with new collected isolates from healthy and diseased livestock were evaluated for toxin type and some virulence genes including cpe, tpeL, netB, and β2. Genetic typing of isolates was done using specific primers for amplification of eight Variable Number Tandem Repeat (VNTR) loci. PCR was done and electrophoresis banding patterns of fragments were analyzed by BioNumerics software. The results of typing revealed 71 distinct MLVA profiles from 74 strains. The strains analyzed included 27 clinical isolates, 43 non-clinical isolates, three vaccine strains, and the reference strain 13 obtained from the GenBank database. Clostridium perfringens strain 13 sequence was used as a reference strain and was evaluated for comparison of sequences for each locus. Clustering of isolates according to the minimum spanning tree showed 5 different clusters, excluding three orphan isolates. Clinical and non-clinical isolates were scattered in all clusters and no defined cluster related to health status, date of isolation, kind of host and place of isolation was detected. The total diversity index of our typing method was estimated to be 0.99. Overall MLVA molecular typing method shows high genetic diversity among Clostridium perfringens isolates.