Rapid detection and identification of Clostridium chauvoei by PCR based on flagellin gene sequence.

We developed a one-step polymerase chain reaction (PCR) system that specifically detects Clostridium chauvoei. Oligonucleotide primers were designed to amplify a 516-bp fragment of the structural flagellin gene. The specificity of the PCR was investigated by analyzing 59 strains of clostridia, and seven strain of other genera. A 516-bp fragment could be amplified from all the C. chauvoei strains tested, and no amplification was observed by using DNAs from the other strains tested, including Clostridium septicum. Similarly, this PCR-based method specifically detected C. chauvoei DNA sequences in samples of muscle and exudate of obtained from mice within 12h of inoculation. In tests using samples of muscle or liver, the limit of detection was about 200 organisms per reaction. These results suggest that the one-step PCR system may be useful for direct detection and identification of C. chauvoei in clinical specimens.

Amplification of the 16S-23S rDNA spacer region for rapid detection of Clostridium chauvoei and Clostridium septicum.

Amplification of the 16S-23S rDNA spacer region by polymerase chain reaction (PCR) was used for the rapid detection of Clostridium chauvoei and C septicum. To assess its specificity, PCR was performed with total DNA from 42 strains of clostridia and three strains of other genera. PCR products specific to C chauvoei or to C septicum were generated from homologous cultures only. Clostridium chauvoer-specific or C septicum-specific amplicons were also generated from tissues of cows experimentally infected with C chauvoei or C septicum and in DNA samples from cows clinically diagnosed as having blackleg or malignant oedema. These results suggest that a species-specific PCR may be useful for the rapid and direct detection of C chauvoei and C septicum in clinical specimens.

Identification of the tetracycline resistance gene, tet(M), in Erysipelothrix rhusiopathiae.

This is the first report to demonstrate the presence of tet(M) in naturally occurring isolates of tetracycline-resistant Erysipelothrix rbusiopathiae, which causes swine erysipelas. The tet(M) gene was isolated from E. rhusiopathiae strain KY5-42. The nucleotide and the deduced amino acid sequence were 99% identical to the tet(M) gene from Enterococcus faecalis. The gene was necessary and sufficient for the expression of tetracycline resistance in Escherichia coli. The presence of the tet(M) gene in the 114 tetracycline-resistant E. rhusiopathiae isolates from diseased pigs was detected by the polymerase chain reaction assay. The specific amplified DNA fragment was obtained from all 114 tetracycline-resistant strains. It was suggested that the tet(M) gene was widely present in the field isolates of E. rhusiopathiae resistant to tetracycline.

Cloning and expression of a gene encoding the flagellin of Clostridium chauvoei.

Clostridium chauvoei is a causative agent of blackleg and the major protective antigen of the organism is the flagellar protein. Using an Escherichia coli expression library of the C. chauvoei Okinawa strain, we isolated the fliC gene encoding the flagellin protein. DNA sequence analysis revealed an open reading frame of 413 amino acid residues with a calculated molecular mass of 43819Da. Comparison of the sequence with those of flagellins from other bacteria showed considerable homology in the N-terminal and C-terminal domains. The glutathione-S-transferase (GST)-flagellin fusion protein and the purified FliC protein after removing the GST part with thrombin reacted with both polyclonal antisera and the non-protective monoclonal antibody (Mab), Mo-114. However, the protective Mab, Mo-41, which may recognize its conformational epitope, failed to react with both the GST-flagellin fusion protein and the purified FliC. Furthermore, the GST-flagellin fusion protein and the purified FliC induced very little protective immunity in mice. These results suggested that a conformation-dependent epitope play an important role in the development of immunity against blackleg.

Biological activities of lipopolysaccharides extracted from porcine vaccine strains.

Lipopolysaccharides (LPSs) were purified from Actinobacillus pleuropneumoniae serotype 2, Bordetella bronchiseptica and Haemophilus parasuis serotype 5, which were used for vaccine production in Japan, by the phenol-water procedure. In SDS-PAGE analysis, A. pleuropneumoniae LPS, as well as Escherichia coli LPS, demonstrated a typical ladder profile of a smooth-type LPS. On the other hand, B. bronchiseptica and H. parasuis LPSs lacked the ladder profiles. It was found that the biological activity of these LPSs was comparable to those of E. coli LPS in terms of activation of the clotting enzyme of Limulus amoebocyte lysate, mitogenic activity of mouse spleen cells, stimulation of TNF-alpha and nitric oxide production, but IL-6 production could hardly be observed in any LPS.

Production of recombinant porcine tumor necrosis factor alpha in a baculovirus expression system.

Porcine tumor necrosis factor alpha (TNF-alpha) was produced using a baculovirus system in Spodoptera frugiperda (SF21AE) cells. Cytotoxic activity was detected in supernatant of sonicated SF21AE cells infected with recombinant viruses. The recombinant protein was also demonstrated to be functionally active by its ability to cause apoptosis in Wehi 164 cells. Three distinct bands of 26, 17 and 14 kDa were revealed by Western blot analysis using anti-human TNF-alpha antibody. Moreover, the anti-human TNF-alpha antiserum significantly neutralized the cytotoxic activity of the supernatant of sonicated SF21AE cells infected with recombinant viruses.

Detection of Mycoplasma in avian live virus vaccines by polymerase chain reaction.

The polymerase chain reaction (PCR) was evaluated to detect mycoplasma contamination of avian live virus vaccines. The specificity of the primers showed that 34 strains belonging to nine species of avian mycoplasma DNA could be detected. The sensitivity of PCR to detect mycoplasma DNA was 10(0.2) colony forming units (cfu) of Mycoplasma synoviae and 10(0.7) cfu of Mycoplasma gallisepticum. When M. synoviae and M. gallisepticum were spiked into several avian live virus vaccines, PCR gave a positive reaction except for the avian pox and the avian encephalomyelitis vaccines which were prepared from organ homogenates. Short-term incubation of avian encephalomyelitis vaccines improved the sensitivity of PCR to detect both M. synoviae and M. gallisepticum. Therefore, PCR, combined with the short-term incubation, were shown to be most effective in detecting mycoplasma contamination in all of avian live virus vaccines.

Detection of mycoplasma DNA in veterinary live virus vaccines by the polymerase chain reaction.

We examined bovine, porcine, canine, feline, avian and mink veterinary live vaccines for the presence of mycoplasma DNA by the polymerase chain reaction including nested-pair primers. Mycoplasma DNA was detected in 22 of the 61 commercial veterinary vaccines (36.1%), although these vaccines did not contain any viable mycoplasma cells.

Effects of oil adjuvant on systemic response to Escherichia coli lipopolysaccharide in swine.

Intramuscular injection of 0.1 mg/kg of Escherichia coli lipopolysaccharide (LPS) mixed with Freund's complete adjuvant (LPS+FCA) in piglets mitigated the leukopenia and TNF-alpha and cortisol levels in the serum compared with that of LPS suspended in LPS-free saline. The endotoxin level in the serum of the LPS+FCA was remarkably reduced. These results suggest that the addition of oil adjuvant mitigate the systemic toxicity of LPS.

Effects of aluminum adjuvant on systemic reactions of lipopolysaccharides in swine.

In vivo effects of aluminum adjuvant on systemic reaction of bacterial lipopolysaccharide (LPS) in piglets were investigated. Intramuscular injection of 0.1 mg kg-1 of LPS added to aluminum hydroxide gel (LPS(+)AL) mitigated the leukopenia, trembling and serum levels of TNF-alpha and cortisol compared with the injection of LPS suspended in LPS-free saline (LPS(+)SALINE). The serum endotoxin levels were reduced remarkably but relatively long-lasting in the LPS(+)AL. The lethality in mice injected with LPS added to aluminum hydroxide gel was significantly reduced. Likewise, the Limulus activity of a test LPS was reduced by the addition of aluminum hydroxide gel or aluminum chloride.

Reversible expression of motility and flagella in Clostridium chauvoei and their relationship to virulence.

Clostridium chauvoei strain Okinawa produced spontaneous non-motile variants at an unusually high rate (approx. 10(-4) per generation) under normal conditions without mutagen. Revertants of non-motile variants were detected at a rate of approximately 10(-3). Biochemically, every variant corresponded well with the parental strain. By transmission electron microscopy, three of nine non-motile variants of strain Okinawa were found to be flagellate, while the other six were found to be aflagellate. These phenotypes were confirmed by Western blot analysis using monoclonal antibodies directed against the flagella of C. chauvoei. Moreover, the parental flagellate strain and non-motile flagellate variants were significantly more virulent in mice than non-motile, aflagellate variants. Our results demonstrated that phase variation in motility and flagellation occurs in C. chauvoei, and that the flagella are associated with the full expression of virulence.