Heterologous protection against alpha toxins of Clostridium perfringens and Staphylococcus aureus induced by binding domain recombinant chimeric protein.

Clostridium perfringens and Staphylococcus aureus are the two important bacteria frequently associated with majority of the soft tissue infections. The severity and progression of the diseases caused by these pathogens are attributed primarily to the alpha toxins they produce. Previously, we synthesized a non-toxic chimeric molecule r-αCS encompassing the binding domains of C. perfringens and S. aureus alpha toxins and demonstrated that the r-αCS hyperimmune polysera reacts with both the native wild type toxins. In the present report, we evaluated efficacy of r-αCS in conferring protection against C. perfringens and S. aureus alpha toxin infections in murine model. Immunization of BALB/c with r-αCS was effective in inducing both high titers of serum anti-r-αCS antibodies after three administrations. Sub-typing the antibody pool revealed high proportions of IgG1 indicating a Th2-polarized immune response. The r-αCS stimulated the proliferation of splenocytes from the immunized mice upon re-induction by the antigen, in vitro. The levels of interleukin-10 increased while TNF-α was found to be downregulated in the r-αCS induced splenocytes. Mice immunized with r-αCS were protected against intramuscular challenge with 5×LD100 doses of C. perfringens and S. aureus alpha toxins with >80% survival, which killed control animals within 48-72h. Passive immunization of mice with anti-r-αCS serum resulted in 50-80% survival. Our results indicate that r-αCS is a remarkable antigen with protective efficacy against alpha toxin mediated C. perfringens and S. aureus soft tissue co-infections.

Monoclonal antibodies against recombinant hemolysin BL complex of Bacillus cereus.

A three component complex system, designated hemolysin BL (HBL), is believed to be the major diarrheal toxin of Bacillus cereus. Identification of HBL toxin by immunoassay is advantageous over PCR as it detects the expressed form of the gene, thereby differentiating pathogenic strains from nonpathogenic strains. However, most of the immunoassays, like the BCET RPLA kit, are based on the utilization of polyclonal antisera, which show cross-reactivity at times with other Bacillus species. The use of monoclonal antibodies (MAbs) binding specifically to the B. cereus HBL toxin epitopes could be advantageous. To address the problems of non-specificity of the reported detection systems and toxicity of L(1) and L(2) components during expression, we made use of recombinant chimeric rHBL protein to generate murine monoclonal antibodies. From among the L(2) MAbs stabilized, immunoblotting analyses on B. cereus strains revealed nine MAbs to be directed against the hbl D encoded L(1) protein, two to the hbl A encoded B protein, and one with the hbl C encoded L(2) protein. When tested on a large number of B. cereus standard and other related Bacillus species, there was no cross-reactivity observed among the group of MAbs. The presence of HBL component toxins among the strains recovered from food and environmental sources was evaluated by these sets of MAbs and the results compared with that of PCRs for the individual HBL toxin gene components. The HBL toxin profile characterization of the strains by Western blot using MAbs almost matched with the PCR profiles. The MAbs reported here, therefore, can be of immense help in providing the B. cereus identification/detection reliably, rapidly, and at a relatively low cost.

Genotyping of Indian Yersinia pestis strains by MLVA and repetitive DNA sequence based PCRs.

India experienced two plague outbreaks in Gujarat and Maharastra during 1994 and then in the Shimla district of Himachal Pradesh during 2002. Yersinia pestis strains recovered from rodents and pneumonic patients during the 1994 outbreaks, pneumonic patients from the 2002 Shimla outbreak and rodents trapped on the Deccan Plateau during a surveillance activity carried out in 1998 were characterized by MLVA, ERIC-PCR and ERIC-BOX-PCR. MLVA genotyping of Indian Y. pestis strains revealed strains of 2 Orientalis, 1 Mediaevalis and 1 Antiqua genotypes distributed in three distinct branches corresponding to their biovar. The Orientalis genotype strains recovered from the 1994 outbreaks and 1998 surveillance activity clustered in one branch while the Antiqua biovar strains from the Shimla outbreak and the Mediaevalis strain recovered from a rodent trapped on the Deccan Plateau region during surveillance formed the other branches. The Orientalis Y. pestis strains recovered from rodents and patients from the 1994 plague outbreaks exhibited similar MLVA, ERIC-PCR and ERIC-BOX-PCR profiles and these were closely related to the Orientalis strains recovered from the rodents trapped on the Deccan Plateau. These data provide evidence for the possible linkage between the Y. pestis strains resident in the endemic region and those that were associated with the 1994 plague outbreaks. Mediaevalis and Antiqua biovars also were recovered from the environmental reservoir on the Deccan Plateau and from the pneumonic patients of 2002 plague outbreak. Therefore, as in Central Asian and African regions, Antiqua and Mediaevalis biovars seem to be well established in the Indian subcontinent as well. ERIC-PCR DNA fingerprinting delineated genotypes similar to those defined by MLVA. Thus ERIC-PCR appears to have the potential to be used as a molecular marker in the molecular epidemiological investigations of plague.