Review of updated regulations and product license categories for veterinary vaccines in the United States.

The USDA continues to consider and implement regulatory pathways for evolving scenarios, needs, and technologies. The intent of this report is to make veterinarians and other users of veterinary biologics aware of recent regulatory additions and changes, particularly in the area of veterinary vaccines. These include new licensure pathways to increase product availability, standardization of labeling, and increased transparency regarding adverse event reports and the efficacy and safety studies accepted by the USDA for product licensure. This report did not undergo scientific peer review.

Detection of Clostridium sordellii strains expressing hemorrhagic toxin (TcsH) and implications for diagnostics and regulation of veterinary vaccines.

Clostridium sordellii is a Gram positive anaerobic bacterium that causes multiple disease syndromes in both humans and animals. As with many clostridial pathogens, toxins contribute to the virulence of C. sordellii. Two large toxins have been identified: a lethal toxin (TcsL) and a hemorrhagic toxin (TcsH) which are similar in structure and function to Clostridium difficile toxin B (TcdB) and toxin A (TcdA), respectively. While TcdA, TcdB, and TcsL have been extensively studied, relatively little is known about TcsH. This study elucidated the TcsH gene sequence using whole genome sequencing, compared the genotype with toxin expression of 52 C. sordellii strains, and examined the role of TcsH in batch release potency tests required for veterinary vaccines licensed in the United States and other testing utilizing WHO standard antitoxin. Data from this study will assist in future research to clarify the TcsH contribution to the pathogenesis of C. sordellii infections and may aid in the development of improved vaccines.

Erysipelothrix rhusiopathiae: association of Spa-type with serotype and role in protective immunity.

A collection of swine, fish, and cetacean Erysipelothrix rhusiopathiae strains representing 16 serotypes was analyzed for possession of the three currently recognized surface protective antigen (spa)-types: spaA, spaB, and spaC. Polymerase chain reaction (PCR) assays and Western blotting with a SpaA-specific monoclonal antibody demonstrated that spa-type is not confined to specific serotype groups. In particular, the spa-type of strains of aquatic origin was more variable than those of terrestrial origin, and possessed the distinct ability to express more than one spa. In a cross-protection study, mice immunized with an E. rhusiopathiae serotype 2 SpaA-type strain and challenged with various E. rhusiopathiae isolates were completely protected against strains exhibiting a single homologous spa, but variably protected against strains possessing a heterologous spa or those harboring more than one spa-type.

Evidence of the protective immunogenicity of native and recombinant Clostridium haemolyticum phospholipase C (beta toxin) in guinea pigs.

The immunogenic effects of the beta toxin of Clostridium haemolyticum were investigated in guinea pigs. Guinea pigs passively immunized with toxin-neutralizing monoclonal antibodies were protected from a 100 LD(50) spore challenge that was lethal to nonvaccinated controls. Guinea pigs actively immunized with varying doses of immunoaffinity-purified native beta toxin were similarly protected. In a third experiment, a recombinant toxoid was prepared from E. coli expressing the beta toxin gene. Guinea pigs immunized three times with recombinant toxoid also were protected against challenge. In each experiment, protection was correlated to the presence of anti-beta toxin antibodies in the serum. Taken together, these results indicate that a neutralizing antibody response to the beta toxin is a key component of protective immunity to C. haemolyticum in guinea pigs.

Using polyclonal and monoclonal antibodies in regulatory testing of biological products.

Polyclonal and monoclonal antibodies are often used in regulatory testing of biologicals (vaccines and related products). One of the most common applications for antibody-based immunoassays is as a batch release test. Batch release tests, whether they measure serological responses to vaccination or they quantify individual antigens by in vitro methods, must provide an acceptable estimate of potency of an individual batch of vaccine. Thus, due consideration must be given to the type of antibody used or quantified in such assays. Differences in specificity and avidity may affect the utility of an assay as an indicator of potency; case examples are given to illustrate these concepts. Concerns associated with antigen quantification assays (e.g., reagent denaturation upon binding to solid substrates, and interference from nontarget antigens or additives in a complex vaccine) are also discussed. International efforts to harmonize test methods in recent years have increased the importance of establishing standardized antibodies. Sources of such antibodies and issues associated with the ongoing availability of antibody supplies are described.

Cloning and molecular characterization of the beta toxin (phospholipase C) gene of Clostridium haemolyticum.

The phospholipase C (PLPC) gene from Clostridium haemolyticum was amplified using the polymerase chain reaction. Primers were selected from a consensus sequence of closely related clostridial PLPC genes and used to amplify an 871-base pair internal segment of the gene. The internal sequence was used to design nested primers that, together with adapter-specific primers, were used to amplify upstream and downstream sequences. The sequences of upstream and downstream segments were aligned with the internal segment to obtain the entire gene sequence. Primers were selected from the aligned sequence, and the entire gene was amplified, and the PCR product was inserted by ligatation into the pCR 2.1 plasmid. An open reading frame that encodes a 399-amino acid protein, containing a 27-amino acid signal sequence, was identified (GenBank Accession Number AF525415). The molecular weight of the active protein was 42869 Da. A 16-amino acid N-terminal sequence, determined by Edman degradation, exactly matched the putative amino acid sequence of the gene product. Together, N-terminal peptide sequencing and tryptic digestion followed by MALDI-ToF mass spectroscopy verified 48% of the amino acid sequences of the active beta toxin. Comparison of the nucleotide and amino acid sequences with Gene-bank databases demonstrated that the beta toxin of C. haemolyticum exhibits high homology with other bacterial PLPCs. The N-terminal portion of the beta toxin contains zinc-binding residues common to clostridial and other bacterial PLPCs, and it shows 34% homology to the N-terminal domain of bovine arachidonate 5-lipoxygenase. The C-terminal domain of the beta toxin protein shows considerable homology with the C-terminal domains of C. novyi type A PLPC, C. perfringens alpha toxin, C. bifermentens PLPC, although the percent identity between the N-terminal regions is much higher overall than that in the C-terminal domain.