Search

ABSTRACTRecent epidemiological studies suggested that cpb2-positive Clostridium perfringensisolates are associated with gastrointestinal (GI) diseases in horses. These putative relationships, indicated by PCR genotyping, were tested in the present study by further genotyping and phenotyping of 23 cpb2-positive C. perfringensisolates from horses with GI disease (referred to hereafter as horse GI disease isolates). Our beta2-toxin (CPB2) Western blot analyses demonstrated that all of the tested isolates were unable to produce detectable levels of CPB2. However, Southern blot and nucleotide sequencing analyses identified intact cpb2open reading frames in all of our surveyed horse GI disease isolates. Furthermore, reverse transcriptase PCR and Northern blot analyses showed that cpb2genes in all of our surveyed horse GI disease isolates were transcriptionally active, i.e., an ~1.2-kb cpb2-specific mRNA was identified in total RNA from our surveyed isolates. The levels of cpb2mRNA in CWC245 (a high-CPB2-producing pig strain) and our surveyed horse GI disease isolates differed to such an extent (35-fold) that this difference could be considered as a major cause of the difference in levels of CPB2 production by CWC245 and horse GI disease isolates. This finding received further support from our observation that the complementing strain 106902(pMRS140), which produced significantly higher levels of mRNA than strain 106902, produced high levels of CPB2. Collectively, our results indicated that there is a positive correlation between cpb2transcription levels and the amount of CPB2 produced by a C. perfringenscell and that decreased transcription and/or message instability may be involved, at least in part, in the low CPB2 production noted for horse GI disease isolates in comparison to that noted for pig GI disease isolate CWC245.

ABSTRACTLive, attenuated bacteria are effective vectors for heterologous antigen delivery. However, loss of heterologous gene-bearing plasmids is problematic, and antibiotics and their resistance genes are not desirable for in vivo DNA vaccine delivery due to biosafety and regulatory concerns. To solve this problem, we engineered the first vaccine delivery strain that has no requirement for antibiotics or other selectable marker genes to maintain the recombinant plasmid. This model strain of Salmonella entericaserovar Typhimurium, SLDAPD, uses operator-repressor titration (ORT) technology, which requires only the short, nonexpressed lacOsequence for selection and maintenance. SLDAPD, recovered from the spleens and Peyer's patches of mice following oral inoculation, was shown to maintain a plasmid that, in contrast, was lost from parental strain SL3261. We also demonstrated successful application of this technology to vaccine development, since SLDAPD carrying a plasmid without an antibiotic resistance gene that expressed the Yersinia pestisF1 antigen was as efficacious in protecting vaccinated mice against plague as the parental SL3261 strain carrying an antibiotic-selected version of this plasmid. Protection of mice against plague by immunization with Salmonellaexpressing F1 has previously required two or more doses; here we demonstrated for the first time protective immunity after a single oral immunization. This technology can easily be used to convert any suitable attenuated strain to an antibiotic-free ORT strain for recombinant protein vaccine delivery in humans.

ABSTRACTAlthough Clostridium perfringensis recognized as an important cause of clostridial enteric diseases, only limited knowledge exists concerning the association of particular C. perfringenstoxinotypes (type A to E) with gastrointestinal (GI) diseases in domestic animals. Some C. perfringensisolates also produce the newly discovered beta2-toxin (CPB2). Recent epidemiological studies suggested that C. perfringensisolates carrying the gene encoding CPB2 (cpb2) are strongly associated with clostridial GI diseases in domestic animals, including necrotic enteritis in piglets and typhlocolitis in horses. These putative relationships, obtained by PCR genotyping, were tested in the present study by further genotyping and phenotyping of 29 cpb2-positive C. perfringensisolates from pigs with GI disease (pig GI disease isolates). PCR and restriction fragment length polymorphism analysis reconfirmed the presence of cpb2gene sequences in all the disease isolates included in the study. Furthermore, genotyping by pulsed-field gel electrophoresis analyses showed that the pig GI disease isolates included in this study all carry a plasmid cpb2gene, yet no clonal relationships were detected between the cpb2-positive pig GI disease isolates surveyed. Finally, CPB2-specific Western blotting demonstrated CPB2 expression by all of the cpb2-positive isolates surveyed. The CPB2 proteins made by five of these pig GI disease isolates were shown to have the same deduced amino acid sequences as the biologically active CPB2 protein made by the original type C isolate, CWC245. Collectively, our present results support a significant association between CPB2-positive C. perfringensisolates and diarrhea in piglets.

ABSTRACTProtective immunity against infection with Bacillus anthracisis almost entirely based on a response to the protective antigen (PA), the binding moiety for the two other toxin components. We cloned the PA gene into an auxotrophic mutant of Salmonella entericaserovar Typhimurium as a fusion with the signal sequence of the hemolysin (Hly) A gene of Escherichia colito allow the export of PA via the Hly export system. To stabilize the export cassette, it was also integrated into the chromosome of the live Salmonellacarrier. When S. entericaserovar Typhimurium with the chromosomally integrated PA gene was given intravenously to A/J mice, they developed high levels of antibody to PA. These mice were protected against intraperitoneal challenge with 100 or 1,000 50% lethal doses of B. anthracisstrain STI. This work contributes to the development of a Salmonella-based orally delivered anthrax vaccine.