Problems associated with prophylactic use of erythromycin in 1566 staff to prevent hospital infection during the outbreak of pertussis.

BACKGROUND AND OBJECTIVE: Pertussis developed in Kagawa University Medical School and University Hospital in May 2007. To control the outbreak and prevent the infection of hospital inpatients, the Infection Control Team (ICT) carried out the prophylactic administration of erythromycin (EM) to hospital staff (1566 staff) who might be exposed to Bordetella pertussis. METHODS: An oral dose of 1000 mg/day EM was given for 10 days. To assess compliance and estimate the frequency of adverse effect, the ICT conducted a questionnaire survey. RESULTS AND DISCUSSION: Of 942 respondents (response rate: 60.2%), 264 (28.0%) experienced some form of EM adverse effects, of which the most commonly reported involved digestive organ symptoms, e.g. diarrhoea (15.6%), stomachache (7.5%), nausea (3.6%), epigastric distress (2.1%) and abdominal distention (1.8%). More importantly, 246 participants (26.1%) stopped taking the EM before completing 10 days because of perceived adverse effects. CONCLUSION: These results indicate that EM appears to cause adverse effects more frequently than reported in the package insert in Japan. The prophylactic use of EM for pertussis infection is recognized in the guideline of the Centers for Disease Control and Prevention. However, this study suggests that attention should be paid to EM non-compliance during a pertussis outbreak, which could extend the duration of the outbreak and increase the number of affected patients.

IFN-gamma-independent autocrine cytokine regulatory mechanism in reprogramming of macrophage responses to bacterial lipopolysaccharide.

Macrophages are now well recognized to have a critical role in both innate and acquired immunity. The sentinel macrophage function is highly regulated and serves to allow for intrinsic plasticity of the innate immune responses to potential environmental signals. However, the mechanisms underlying the dynamic properties of the cellular arm of innate immunity are poorly understood. Therefore, we have conducted a series of in vitro studies to evaluate the contribution of immunoregulatory cytokines, such as IFN-gamma, IL-10, and IL-12, in modulation of macrophage responses. We found that macrophages from IFN-gamma knockout (IFN-gamma(-/-)) mice exhibit only marginal LPS-induced TNF-alpha, IL-12, and NO responses, all of which can be fully restored in the presence of rIFN-gamma. Pretreatment with substimulatory LPS concentrations led to reprogramming of IFN-gamma(-/-) macrophage responses in a dose-dependent manner that manifested by an increased TNF-alpha and IL-12, but not NO, production upon the subsequent LPS challenge. These reprogramming effects were substantially attenuated and profoundly enhanced in macrophages from IL-12(-/-) and IL-10(-/-) mice, respectively, as compared with those modulated in macrophages from the congenic wild-type mice. LPS-dependent reprogramming was also fully reproduced in macrophages isolated from SCID mice after immunodepletion of NK cells. Our data strongly imply that cytokine (TNF-alpha and IL-12), but not NO, responses in macrophages may, at least in part, be governed by an autocrine IFN-gamma-independent regulatory mechanism reciprocally controlled by IL-10 and IL-12. This mechanism may serve as an alternative/coherent pathway to the canonical IFN-gamma-dependent induction of antimicrobial and tumoricidal activity in macrophages.

Phylogenetic positions of Clostridium novyi and Clostridium haemolyticum based on 16S rDNA sequences.

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.

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.

Rapid identification and differentiation of pathogenic clostridia in gas gangrene by polymerase chain reaction based on the 16S-23S rDNA spacer region.

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.

Differential host inflammatory responses to viable versus antibiotic-killed bacteria in experimental microbial sepsis.

Staphylococcus aureus killed during imipenem or ceftazidime chemotherapy in mice elicited an early release of tumor necrosis factor alpha (TNF-alpha) 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 in D-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-alpha antibody or 4 mg of dexamethasone per kg to these TNF-alpha-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-alpha response could also be demonstrated upon challenge with Escherichia 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-alpha, and possibly the Gram stain classification.

Rapid and direct detection of clostridium chauvoei by PCR of the 16S-23S rDNA spacer region and partial 23S rDNA sequences.

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.

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.

Limited use of cationic liposomes as tools to enhance the antiherpetic activities of oligonucleotides in vero cells infected with herpes simplex virus type 1.

We used commercially available cationic liposomes, lipofectin, DOTAP, and transfectam, to enhance the antiherpetic activities of phosphodiester oligonucleotides (D-oligos) or phosphorothioate oligonucleotides (S-oligos) targeted against immediate-early pre-mRNA4/5 of herpes simplex virus type 1 (HSV-1). With a 5-fold excess of S-oligos/D-oligos, formation of complexes with some of the S-oligos/D-oligos and the cationic liposomes could be visualized on agarose gel. A >5-fold excess of cationic liposomes enhanced the antiherpetic activities of Doligos, whereas there was not enhancement of the antiherpetic activities of S-oligos. As nuclear localization of D-oligos in the presence of cationic liposomes was not clear, we could not clarify the relation between antiherpetic activities of D-oligos and nuclear distribution of oligos. Subcellular distribution of S-oligos in the presence of lipofectin or DOTAP showed nuclear localization by confocal laser scanning microscopy. Transfectam had no effect on the nuclear distribution of S-oligos. These data showed that cationic liposomes would not be appropriate carriers to enhance the antiherpetic activities of S-oligos. Also, distribution of S-oligos into the nucleus does not necessarily enhance their biologic activity. Questions remain about the effectiveness of cationic liposomes in the enhancement of the antivirus activity of S-oligos.

Enhancement of anti-herpetic activity of antisense phosphorothioate oligonucleotides 5' end modified with geraniol.

We have previously shown that antisense phosphorothioate oligonucleotide (SON) targeted against immediate early (IE) pre-mRNA5 of the herpes simplex virus type I (HSV-I) possessed potent anti-herpetic activities in vitro system. However, anti-herpetic activities of SON were not still efficient enough. Lipophilic compounds have been often conjugated with antisense oligonucleotide to enhance the biological activity. In this study, we selected geraniol as a lipophilic compound and newly synthesized SON bearing 5' terminal geraniol (geranyl-SON) toward IE pre-mRNA 5 of the HSV-1 to enhance the anti-herpetic activity. Geraniol is a olefinic terpene alcohol which is found in many essential oils. It possesses lipophilic characteristic. It is thought to be absorbed in tissue. Geraniol enhanced the anti-herpetic activity of SON with less cytotoxicity in a sequence specific manner. Terminal modification with geraniol did not affect binding affinity with complimentary DNA. Cytoplasm distribution of geranyl-SON was confirmed by confocal microscope. While some of the geranyl-SON was seen in the nucleus, unmodified SON had a punctate distribution in the cytoplasm with little in the nucleus. These results suggested that geranyl modification enhances anti-herpetic activity by changing the subcellular distribution of the oligonucleotides. Consequently geraniol-modifica-tion could provide new means for the efficient delivery of oligo-nucleotides.

Correlation of antibiotic-induced endotoxin release and cytokine production in Escherichia coli-inoculated mouse whole blood ex vivo.

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.

Spectral distortion properties of the integral pulse frequency modulation model.

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.

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.

Intratracheal infection of chickens with Salmonella enteritidis and the effect of feed and water deprivation.

The tissue distribution of Salmonella enteritidis in intratracheally inoculated chickens and the effect of deprivation of food and water on tissue distributions of the bacteria have been investigated. Seven-week-old specific-pathogen-free chickens were inoculated intratracheally with 10(2), 10(5), or 10(8) cells and orally with 10(5) cells. The intratracheally inoculated organisms entered the blood stream immediately after inoculation and produced generalized infection. Infection by the intratracheal route resulted in colonization of S. enteritidis in the cecum that was similar to infection by the oral route. The tissue distribution of S. enteritidis was markedly affected when chickens were deprived of food and water for a short time, demonstrating an increased susceptibility of chickens to S. enteritidis infection. This suggests that stresses such as food and water deprivation are one of of the causes of the rapid dissemination of S. enteritidis among chickens in poultry houses.

In-vivo induction of apoptosis in murine lymphocytes by bacterial lipopolysaccharides.

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.

Lipopolysaccharide-induced apoptosis in swine lymphocytes in vivo.

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.

Mutagenic evaluation of primaquine, pentaquine and pamaquine in the Salmonella/mammalian microsome assay.

The 8-aminoquinolines, primaquine, pentaquine and pamaquine, were investigated for mutagenic activity in Salmonella typhimurium strains TA100, TA98, TA97 and TA102 in the rat liver microsomal activation system. Primaquine and pentaquine induced mutations in TA97 in the presence and absence of S9 mix. Pamaquine was mutagenic to TA98 only in the absence of S9 mix.

Comparison of the pathogenicity for chickens of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum.

Twenty-two strains of Erysipelothrix rhusiopathiae, 14 strains of Erysipelothrix tonsillarum, and four other strains representing two serovars were examined for virulence in 30- to 40-day-old specific pathogen-free chickens. Some of the chickens that were given strains of serovars la, 2, 5, 6, 8, 9, 15 or 21 of E. rhusiopathiae showed signs of disease, lesions, bacteraemia or recovery of the bacterium. In contrast, there were no clinical signs, pathological lesions or bacterial isolations in chickens inoculated with the E. tonsillarum or other strains. The study thus confirmed that strains of some serovars of E. rhusiopathiae are pathogenic for chickens, but that E. tonsillarum strains are not pathogenic and should be omitted from potential causes of erysipelas in chickens.

Induction of chromosome aberrations by pyrimethamine in cultured Chinese hamster (CHL) cells.

Induction of chromosome aberrations was investigated in cultured Chinese hamster cells treated with pyrimethamine. Pyrimethamine without metabolic activation strongly induced structural chromosome aberrations in a dose-dependent manner. Aberrant metaphase cells occurred at a frequency of 80%, when cells were treated at 1.6 microgram/ml for 48 h.