Clostridietm chauvoei causes blackteg, which is difficult to distinguish from the causative clostridia of malignant edema, Therefore, a single-step PCR system was devcloped i'or specific detection of C. chauvoei DNA using primers derivcd from the ]6S-23S rDNA spacer region and partial 23S rDNA sequences. The specit'icity of the single-step PCR system was demonstrated by testing 37 strains of clostridia and 3 strains of other genera, A 509 bp PCR product, whtch is a C. chauvoei-spccific PCR product, could be amplified from a]1 of tlte C. chauvoei strains tested, but not from the ether strains. Moreover, this single-step PCR system specifically detected C. chauvoei DNA in samples of muscle from miee 24 hr at'ter inuculation wiLh 1OO spores of C, chauvoei, and in clinieal inaterials from a cow affect ¢ cl with b]ttcklcg. Tltese results suggest that our single-gtep PCR system rnay be uscful for direct detection of C. chaLtvoei {n culture and in clinLca] materials from animals affected with bluck]eg. KEy woRDs: Ctostridium chauvoei, PCR, l6S-23S rDNA spacer region. . .. .. -. . -J, ;7lenMed, Sci, 62(]2):1275-]281,2000 Clostridittm chauvoei is an endospore-forming grarn-positive bacterium, which causes biackleg, a fatal disease of cattle, shcep, and other ruminants associated with sporecontaminated soil. Blackleg needs to be distinguished from other severe diseases with similar symptoms, such as `malignant edema' and anthrax caused by Closlf'idium sqptic"fn, Ctostridium novyi, Clostridiumpeij>'in.oens, Cl stridium sordellii or Bacillus anthracis, Typically, the diagnosis oC blackleg depends on obseryation of clinical symptoms, fbllowed by isolation and identification of the organism by immunological mcthods, such as immunofluorescence staining [1O]. Howcver, these diagnostic methods are time consuming and laborious, NucleicacidampHficationofaspecifictargetregionofthe bacterial genome by the pQlymerase chain reaction (PCR) is becoming widely used for detection and diagnostie purposcs [L8, 19]. The abjlity of PCR to amplify DNA specifHcally from Iow numbers ofbacteria, as well as its simplicity, rapidity and reproducibility, offers advantages over conventional methods for identification, Kuhnert et aL [1'4] reported a PCR systetn for distinguishing a chauvoei from C. scpticttm by usjng specific oligonucleotide primers for the 16S rRNA gene. This procedure is very effective in distinguishing C. chauvoei from C. septicum; however it requires restriction cnzyme digestion f the PCR products for definitiye d ntification of the organism, because the existenee of only small differences in the 16S rRNA gene sequences, results in the primers amp]ifying the 16S rRNA genes of both bacteria. PCR ainp]ification of the 16S-23S rDNA spacer region has been suggested as a target for a universa] bacterial identification and typing system [3, 9, 12]. Recently, it has been reported that species-specific primers to Actinobacitltts seminis were devcloped from the sequence of thc 1 6S-23S rDNA spacer region of rrnB for the identification and detection of A. seminis by PCR L1.1. In the present study, we determined three distinct ]6S-23S rDNA spacer regions and the partial 23S rDNA sequences of C chauvoei and dcsigned C. chauvoei.-specific primers for the detection of C. chativoei. To assess its appljcability as a rapid and re]iabte diagnostic method for clinical blackleg in rurninants, we evaluated our single-step PCR system using specimens from mice experimentaliy infected with (] chattvoeiandc]inicalmateriaisfromacowaffectedwithblackleg. MATERIALS AND METHODS Bact rial strains: Bacterial strains used are listed in Table L DIVA preparation: To isolate chromosomal DNA from bacteria, cegls in 1 ml of a late logarithmic phase culture were pelleted by centrifugation and used as the source of chromosomal DNA, To isolate the DNA samples from mice and a cow, approximatcly 50 mg of each of the vital organs was homogenated and resuspended in 3 mt of phosphate buffered saline (PBS), then ccntrjfuged at 80e x g for 10 min. The supernatant was centrifuged at 1O,OOO × g for IO min, The pellets were washed once with in 1.5 ml of PBS, and resuspended in 150 pt of sucrose-TES (25% rwlv] sucrose, 1O mM Tris-HCt, 1 mM EDTA, 150 mM NaCl) containing 4 mg/ml of lysezyme. The DNA extraction and purification was performed according to the methods of Takeuchi et al. [17]. DAL4 atmptijication and gel electrophoresis: PCR was pertbrmed in a reaction volume of 1OO ul containing 1 × PCR buffer (Mg2' free), 2.5 niM MgC12, O.2 mM dNTP mixture, 2.5 units of LA Taq DNA polymerase (TaKaRa LA Taq kit; TaKaRa Shuzo Co., Ltd., Kyoto, Japan), 1O pmol of each Japanese Society of Veterinary Science NII-Electronic Library Service apaneseSociety fVeterinaryScience ]276 Y, SASAKI ET AL, Table,1The list ef bacterial strains and results of PCR Bacterial strainsSource PCRresultsd) C. chauvoei ATCCIO092,,) ATCCI1956 ATCCI1958 ATCCI1957 ATCC19399 Oklnawa Chousen Souya Toehigi Jwate Ouita Yamanashi S4269A C6HC.

The effect of lipopolysaccharide (LPS) on humoral and cell-mediated immunity was assessed using LPS-sensitive C3H/HeN mice. A single injection of LPS significantly decreased the anti-sheep red blood cells (SRBC) antibody titers, but not the number of anti-SRBC antibody producing spleen cells. In contrast, double LPS injection did not significantly decrease the anti-SRBC titers and even increased the number of anti-SRBC antibody producing spleen cells. Similarly, single LPS injection significantly suppressed the swelling of the footpad, but double LPS injection caused milder suppression. These results suggest that a difference in the level and timing of exposure to LPS may influence the immune response to infection or vaccination.

We investigated the application of the bacterial endotoxin test for the quantification of the endotoxin contamination of various commercial porcine vaccines. In endotoxin-spiked samples, Freund's complete adjuvant and aluminum hydroxide gel adjuvant failed to interfere with the results of the endotoxin test, and both recovery ratios were within the permissible range mentioned in the Japanese Pharmacopoeia. At the various dilutions tested, none of the adjuvants in commercial porcine vaccines caused noteworthy interference in the test. In addition, none of the 39 samples of porcine vaccines approved in Japan induced an interfering effect in the endotoxin test. Our findings suggest that the bacterial endotoxin test using endotoxin-specific Limulus amoebocyte lysate (LAL) can detect endotoxin contamination in commercial porcine vaccines containing either oil or aluminum adjuvants.

The immune response of bovine monocytes-derived dendritic cells (DC) exposed to either live or killed Salmonella enterica serovar Typhimurium was compared. Both live and killed bacteria induced changes in morphology with distinctive formation of processes and up-regulation of the ability of DC to stimulate allogeneic T-cell proliferation. Also, both live and killed bacteria up-regulated the expression of MHC-I, MHC-II and CD80. However, live bacteria induced greater up-regulation of the expression of CD40 and CD86 than killed bacteria. Live bacteria also induced greater up-regulation of transcription for IL-6, IL-12 and GM-CSF than killed bacteria as measured by quantitative RT-PCR. These data suggest that blood-monocyte-derived DC may follow distinct maturation pathways following exposure to live or killed bacteria. These differences are likely to have consequences for the priming of the adaptive immune responses.

Exposing bovine dendritic cells (DC) and macrophages (MPhi) to Salmonella typhimurium at a ratio of 1 cell to 10 bacteria had a cytotoxic effect that was not evident with a ratio of 1000 cells to 1 bacterium. This lower dose was considered to mimic more closely the in vivo situation and a comparison was made with this model of the consequences of infection for MPhi and DC. DC infected with S. typhimurium up-regulated cell surface expression of major histocompatibility class I (MHC-I), MHC-II, CD40, CD80 and CD86. In contrast, infected MPhi did not exhibit detectable changes in expression of cell surface molecules, except for a marginal increase in CD40. mRNA transcription for tumour necrosis factor-alpha, interleukin (IL)-1beta, IL-6 and inducible nitric oxide synthase was up-regulated in both infected DC and infected MPhi, although mRNA transcription for granulocyte-macrophage colony-stimulating factor and IL-12p40 was up-regulated only in infected DC and for IL-10 was only in infected MPhi. Infected DC had an increased ability to stimulate both allogeneic and antigen-specific T-cell responses compared to non-infected controls. In contrast, infected MPhi showed an increased ability to induce allogeneic responses but this was less than seen for DC and no enhancement of ability to induce antigen-specific T cell responses was seen. Thus, in a low-dose infection model that does not result in the cytotoxicity of a substantial percentage of antigen presenting cells, bovine MPhi and DC respond differently to infection with S. typhimurium and this could have important implications for the development of the immune response.

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.

The partial sequences (1465 bp) of the 16S rDNA of Clostridium novyi types A, B and C and Clostridium haemolyticum were determined. C. novyi types A, B and C and C. haemolyticum clustered with Clostridium botulinum types C and D. Moreover, the 16S rDNA sequences of C. novyi type B strains and C. haemolyticum strains were completely identical; they differed by 1 bp (level of similarity > 99.9%) from that of C. novyi type C, they were 98.7% homologous to that of C. novyi type A (relative positions 28-1520 of the Escherichia coli 16S rDNA sequence) and they exhibited a higher similarity to the 16S rDNA sequence of C. botulinum types D and C than to that of C. novyi type A. These results suggest that C. novyi types B and C and C. haemolyticum may be one independent species generated from the same phylogenetic origin.

Clostridium chauvoei causes blackleg, which is difficult to distinguish from the causative clostridia of malignant edema. Therefore, a single-step PCR system was developed for specific detection of C. chauvoei DNA using primers derived from the 16S-23S rDNA spacer region and partial 23S rDNA sequences. The specificity of the single-step PCR system was demonstrated by testing 37 strains of clostridia and 3 strains of other genera. A 509 bp PCR product, which is a C. choauvoei-specific PCR product, could be amplified from all of the C. chauvoei strains tested, but not from the other strains. Moreover, this single-step PCR system specifically detected C. chauvoei DNA in samples of muscle from mice 24 hr after inoculation with 100 spores of C. chauvoei, and in clinical materials from a cow affected with blackleg. These results suggest that our single-step PCR system may be useful for direct detection of C. chauvoei in culture and in clinical materials from animals affected with blackleg.

In cattle, sheep, and other ruminants, clostridial myonecrosis (gas gangrene) is mostly caused by Clostridium chauvoei, C septicum, C novyi and C sordellii. A polymerase chain reaction (PCR) system using common primers designed from multiple alignment of the 16S rRNA and 23S rRNA genes of Clostridium species was developed to identify pathogenic clostridia. The PCR was performed with total DNA from 26 strains which included seven different Clostridia species. These bacteria were differentiated at species level by the different PCR product patterns. To characterise the 16S-23S rDNA spacer regions of these clostridia further, most PCR products of these bacteria were sequenced. The smallest PCR products of each bacterium represented the fundamental 16S-23S rDNA spacer region; larger PCR products of each bacterium were caused by insertion sequences, i.e. tRNA gene sequences. The authors' observations indicate that the PCR patterns of the 16S-23S rDNA spacer regions have the potential to be used as an identification marker of pathogenic clostridia in gas gangrene.

ABSTRACTStaphylococcus aureuskilled during imipenem or ceftazidime chemotherapy in mice elicited an early release of tumor necrosis factor alpha (TNF-α) into the systemic circulation. This response was coincident in time with an increase in leukocyte-endothelium adhesive interactions in the microvasculature. Equivalent responses were not observed without the antibiotic treatment (imipenem or ceftazidime). Protective efficacy of the same antibiotic treatment was markedly diminished ind-galactosamine-treated mice compared to controls; e.g., it dropped from 2,000-fold to 70-fold with 4 mg of imipenem per kg given at the time of challenge. Nevertheless, protection was quantitatively restored upon concurrent administration of neutralizing anti-TNF-α antibody or 4 mg of dexamethasone per kg to these TNF-α-hypersensitive mice. Importantly, protection afforded by dexamethasone was not seen when the animals were challenged with viable organisms but without the concurrent administration of antibiotic. An early TNF-α response could also be demonstrated upon challenge withEscherichia coli, but in this instance, neither the timing nor the magnitude of that response was influenced by treatment with these antibiotics. We conclude from these studies that the inflammatory response to viable versus killed bacteria may differ markedly depending on the particular bacterium, host sensitivity to TNF-α, and possibly the Gram stain classification.

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.

Escherichia coli were incubated in mouse whole blood ex vivo supplemented with beta-lactam antibiotics that possessed preferential affinities for penicillin-binding proteins (PBPs). After 4 h, viable bacteria were undetectable in the presence of any of the 3 antibiotics tested, whereas significant increases in colony-forming units were detected in samples not treated with antibiotics. Differential levels of endotoxin in platelet-rich plasma were detected using the limulus amebocyte lysate assay, according to differential antibiotic affinities for the various PBPs. Levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in antibiotic-treated cultures after 8 h of incubation correlated well with the levels of endotoxin at 4 h (r = .96, P < .0001 for TNF-alpha; r = .91, P = .0002 for IL-6). These data indicate that differential affinities of beta-lactam antibiotics for PBPs affect both endotoxin and cytokine responses ex vivo in mouse blood and correlate with in vivo protective efficacy of these antibiotics in gram-negative experimental models.

Gram-negative and Gram-positive bacteria have been compared with respect to lethal effects when each is administered to normal and D-galactosamine-sensitized mice, both with and without concomitant dexamethasone treatment. In the case of Escherichia coli, the extent of sensitization by D-galactosamine treatment (10,000-fold) and the relative magnitude of the corresponding protection with dexamethasone (150-fold) are both consistent with an expected significant role of LPS in production of TNFα that then mediates lethal toxicity. With Staphylococcus aureus, however, marginal sensitization by D-galactosamine (5-fold) and a corresponding lack of dexamethasone protection indicate a reduced role for TNFα as a lethal mediator. In vitro comparisons of TNFα release from E. coli and S. aureus stimulated peritoneal macrophages (100-fold difference) add further support to this conclusion. Endotoxin hypo-responsive mice (C3H/HeJ) infected with E. coli are not protected by dexamethasone. Each of these comparisons indicate that the contribution of TNFα to the pathophysiological manifestations of experimental sepsis may vary substantially even among extracellular bacteria and, correspondingly, that differential dexamethasone protection may serve a discriminatory function for the potential involvement of this cytokine.

The integral pulse frequency modulation (IPFM) model has been used for the following two purposes. First, it has been utilized to verify the correspondence between the spectral structure of autonomic input and the estimated spectrum of heart rate variability (HRV), relying mainly on the theoretical work of Bayly (1968). Second, the IPFM model provides a framework for evaluating how precisely the proposed method of HRV analysis could estimate the input spectral structure. However, the appropriateness of the IPFM model for both purposes has not been examined sufficiently in realistic situations. In this paper, the spectral structure of the pulse train generated by the IPFM model is theoretically derived for an input signal containing multiple frequency components. This is a more general condition than the single sinusoidal input signal used earlier. In accordance with the theoretical results, the magnitude of the spectral distortion is computed for a pair of varied frequencies, considering the corresponding coefficient of variation of interpulse intervals. Results show that the distortion could be nonnegligible under practical values of the coefficient of variation. Such distortion may well affect the spectral structure in the wide frequency range. This study suggests that the spectral structure of HRV should be interpreted carefully, taking the above distortion properties into account, even though the IPFM model appears to be established as a mechanism mediating between autonomic input and heart rate variability.

The results from our laboratory summarized in this review support the importance of endotoxin release from the Gram-negative microbe as a requirement for the full manifestation of the biological activity of the lipopolysaccharide (LPS) macromolecule. They further suggest that LPS is the most important bacterial component released from Gram-negative microbes treated in vitro with cell-wall active antibiotics in terms of biological activity. The use of several experimental mouse models of infection confirm that mice treated with the reversible hepatotoxin, D-galactosamine, are sensitive to both endotoxin lethality and to infection with Gram-negative microbes. Infected LPS-sensitive animals can be protected if they are treated with antibiotics, but the degree of protection (e.g. 5-10-fold) is significantly less than that observed when infection-sensitive but LPS-insensitive mice are similarly treated (500-fold protection). Additional levels of protection in infected, antibiotic-treated mice can be observed with antiendotoxin agents (e.g. polymyxin B-dextran, monoclonal antibody). Finally, antibiotics with equivalent minimal inhibitory concentrations for Escherichia coli, but differing in their mode of action and endotoxin releasing potential in vitro, also differ in their protective efficacy in vivo to reduce mortality in infected animals. Collectively, these data provide evidence to support a role for microbe-derived endotoxin, released from the microbial surface following treatment with cell-wall active antibiotics, in the pathogenesis of experimental Gram-negative sepsis.

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.

The in vivo effects of bacterial lipopolysaccharide (LPS) on the immune systems of piglets were investigated. Intravenous injection of 0.5 mg of LPS per kilogram of body weight induced apoptosis, which was characterized by nuclear chromatin condensation and fragmentation and a ladder formation of nucleosomal DNA in lymphocytes both in the cortex of the thymus and in the germinal centers and paracortical areas of mesenteric lymph nodes at 24 h postinjection. The levels of endotoxin, tumor necrosis factor alpha, and cortisol in serum increased, generally according to the dose of LPS. These findings suggest that LPS can induce in vivo apoptosis of lymphocytes in piglets and support the notion that cytokine and endocrine responses may play an important role in LPS-induced apoptosis in the immune system.

The effect of bacterial lipopolysaccharide (LPS) on the lymphoid organs in C3H/HeN and C3H/HeJ mice was investigated. In C3H/HeN mice, LPS induced apoptosis, characterised by morphological nuclear condensation and DNA fragmentation resulting in thymic atrophy. Similar but less severe changes were also observed in the spleen and lymph nodes. In C3H/HeJ mice, only a slight depletion of lymphocyte numbers was observed in the lymphoid organs. The plasma endotoxin levels were dependent on the LPS dose regardless of mouse strain. On the other hand, the plasma TNF-alpha levels were significantly elevated in C3H/HeN mice 1 h post-injection and the time course of plasma corticosterone concentration correlated well with the development of apoptosis. These findings suggest that TNF-alpha and corticosterone may play an important role in LPS-induced apoptosis of lymphocytes.

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.