Legislation concerning the safety assessment and labelling of foodstuffs has been implemented in many countries. Consequential to a number of cases of food adulteration reported globally, a fast and reliable detection method for the food traceability is required in ensuring effective implementation of food legislation in a country. In this study, PCR-RFLP technique based on cyt b gene has been tested for its suitability for these purposes. This method combines the use of a pair of universal primer that amplifies a 359 bp fragment on the cyt b gene from meat muscle DNA and restriction enzyme analysis. Analysis of experimental beef frankfurter, minced beef, pork frankfurter and pork cocktail samples demonstrated the suitability of the assay for the detection of the beef (Bos taurus) and pork (Sus scrofa), but not applicable for some processed food, particularly detection of mackerel (Rasterelliger brachysoma), sardine (Saedinella Fimbriata) and tuna (Thunnus tonggol) origin in canned food. Commercial frauds through species mislabelling or misdescribed were not detected. The assay is demonstrated applicable for routine analysis of meat traceability of foodstuffs and legislation purposes, if sufficient availability of detectable mtDNA in the foodstuffs is ensured.
Cholera caused by the O139 serogroup still remains a public health concern in certain regions of the world and the existing O1 vaccines do not cross-protect cholera caused by this serogroup. An aminolevulinic acid (ALA) auxotroph vaccine candidate against the O139 serogroup, designated as VCUSM2, was recently developed. It was found to be immunogenic in animal model studies but showed mild reactogenic effects due to the presence of two intact copies of Vibrio cholerae toxin (CTX) genetic element. In the present study we have modified the ctx operon by systematic allelic replacement methodology to produce a mutant strain, designated as VCUSM14. This strain has two copies of chromosomally integrated and mutated ctxA gene, encoding immunogenic but not toxic cholera toxin A subunit (CT-A). The amino acids arginine and glutamic acid at position 7th and 112th, respectively, in CT-A of VCUSM14 were substituted with lysine (R7K) and glutamine (E112Q), respectively. Two copies of the ace and zot genes present in the ctx operon were also deleted. Cholera toxin-ELISA using GM1 ganglioside showed that the both wild type CT and mutated CT were recognized by anti-CT polyclonal antibodies. VCUSM14 produced comparatively less amount of antigenic cholera toxin when compared to the VCUSM2 and Bengal wild type strain. VCUSM14 did not elicit fluid accumulation when inoculated into rabbit ileal loops at doses of 10(6) and 10(8) CFU. The colonization efficiency of VCUSM14 was one log lower than the parent strain, VCUSM2, which can be attributed to the ALA auxotrophy and less invasive properties of VCUSM14. VCUSM14, thus a non-reactogenic auxotrophic vaccine candidate against infection by O139 V. cholerae.