Gene expression studies
CLOSTRIDIUM perfringens is one of the important causes of food borne illness. The organism is an anaerobic, spore forming bacterium which is abundant in the environment and intestine of humans and animals.
Virulence of this organism is relied upon production of multiple major and minor toxins. Toxinotype B of Clostridium perfringens produces alpha, beta and epsilon among major toxins.
Alpha (á) toxin is responsible for lecithinase activity whereas, beta (â) and epsilon (å) toxin forms channels and pores in the infected cells which results in necrosis.
The higher concentration of proteins in intestine causes increased production of beta toxin. Besides, epsilon toxin is a classified potential bio-war and bioterrorism agent by the US Centre for Disease Control (CDC).
For control of disease like lamb dysentery caused by this organism, vaccination is very important.
For large scale vaccine production, characterization of indigenous isolates followed by in-vitro culturing on commercially available growth media is salient landmarks.
Infections even after vaccination require insights on expression of major toxin genes of Clostridium perfringens toxinotype B to assess the effect of in-vitro manipulation.
This study was funded by Technology Development Fund (TDF) program of Higher Education Commission (HEC), Islamabad, (Project # TDF-02-028) entitled “Preparation and evaluation of multivalent vaccine for Clostridium Perfringens using locally characterized toxinotypes” awarded to Prof.
Dr Aftab Ahmad Anjum, Institute of Microbiology, Faculty of Veterinary Sciences, University of Veterinary and Animal Sciences, Lahore, Pakistan.
Moreover, the intellectual and moral support was also provided by Prof.Dr. Yung Fu Chang, Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America (USA) to complete this task in time.
For this purpose, current study was planned and executed to isolate Clostridium perfringens toxinotype B from lamb dysentery cases on the basis of biochemical profile molecular testing.
Confirmed bacteria were optimized for higher toxin production under various physical and chemical parameters.
Highest level of alpha, beta and epsilon toxins was produced in cooked meat medium at 37°C and 6.0 pH as tested by Multiscreen AgELISA Enterotoxaemia®.
For gene expression studies, the RNA was isolated from Clostridium perfringens toxino type B under optimized conditions followed by DNA synthesis, amplification and nucleotide sequencing of major toxins.
The variation in toxin nucleotides was evaluated by online bioinformatics tools named PolyPhen-2®, PhD SNP®, PROVEAN®, SIFT®, MUpro® and I-Mutant 3.0®.
Furthermore, online bioinformatics tools named I-TASSER®, PSIPRED® and InterPro Scan® were used to evaluate 3-D structure and models of toxins.
It was verified that most of the reported mutations of this study had non-hazardous effect on the toxins produced with standard structures.
In conclusion, cooked meat medium can be used to culture Clostridium perfringens toxin type B for industrial scale vaccine production.
Moreover, in-vitro manipulation of this toxino type doesn’t cause marked effect on normal functioning and antigenicity of major toxins as indicated by gene expression studies.
