A questionnaire style survey on Pasteurella multocida infection was conducted at on 528 laboratories of hospitals. The questionnaire included the detection of Pasteurella multocida from sputum, the age distribution, underlying disease etc. of the patients.
In Japan, infectious diseases caused by Pasteurella multocida were found in 179 cases in 67 (26.0%) of the 258 hospitals. The incidence of the infections tends to increase, 18 cases in 1989, 25 cases in 1990, 18 cases in 1991 (Jan. Jul.). These 179 cases were broken down into 44 males (from 1 year old to 85 years old, mean 58.9 years old), 45 females (from 18 years old to 85 years old, mean 60.2 years old), and 90 patients whose sex was unknown. Underlying diseases were recognized in 72 cases. Diseases related to the respiratory organ were 61 (84.7%) cases. The cases of hemo-sputum had to be differentiated from malignant tumors Recurrent Pasteuralla multocida infections were recognized in 4 males and 4 females. And the 4 cases had been handling animals. There were only 26 cases who had contact with animals, the others were unknown. This result suggested that contact with animals may cause these infections.
Recently, the detection of Pasteurella multocida from the sputum tends to increase. The olderly patient with chronic pulmonary disease, who is handling animals must be educated on hygiene and zoonosis.
This study was aided by Grant-in-Aid for Encouragement of Young Scientists of the Ministry of Education, Science and Culture (No.03857348).

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The pathogenic factor of Vibrio cholerae that induces a severe watery diarrhea in humans is cholera enterotoxin (CT). We have earlier reported on the use of a specific polymerase chain reaction method (PCR) for confirmation of CT-production. In our results, a few CT-producing V. mimicus strains were detected by the method. Here we report on the PCR method using 2-primer sets in the same tube for differentiation of toxigenic V. cholerae (01 and non-01) and toxigenic V. mimicus.
One primer pair is for CT-gene (ctx), and the other pair is for the toxR gene which regulates the ctx gene of V. cholerae.
ToxR genes were detected in all CT-producing V. cholerae (both 01 and non-01). There were no isolates of the ctx gene positive and toxR gene negative in all V. cholerae strains. On the other hand, V. mimicus strain has not recognized toxR genes except one strain which is similar to the character of V. cholerae.
These results indicates that the CT-producing V. cholerae strains are regulated by the toxR gene, but the ctx gene of V. mimicus is controlled by another different genome from toxR of V. cholerae.

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To evaluate the bactericidal activity of povidone-iodine (PVI), synthetic mediums without the protein components were prepared. The stability of the available iodine in the synthetic mediums was compared with that in heart infusion broth (HIB; natural broth). The available iodine was remarkably decreased soon after addition of PVI in the HIB, but it was stable for 24 hr in the synthetic mediums. The growth of Escherichia coli and Salmonella enteritidis were found to be normal in the synthetic mediums. Minimal inhibitory concentrations (MICs) of PVI in the synthetic mediums were about one thousaundth of that in HIB, and the phenol coefficients (PCs) of PVI in the synthetic msHliums were about 30 times lower than that in the HIB.

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Twenty-five patients with uterine cervical cancer (Two cases stage 0; four cases stage Ia; five cases stage Ib; one case stage IIa; ten cases stage IIb; two cases stage IIIa; one case stage IVa) served as the subjects. The bacterial flora inside the cervical cancers was investigated using the optimal technique. There was mixed abnormal aerobic and anaerobic bacterial flora in all subjects.
The average number of bacterial species isolated from inside the cervical cancers was 6.3.
The predominant bacteria isolated were the aerobes, Staphylococcus haemolyticus, Streptococcus agalactiae, Escherichia coli and Gardnerella vaginalis, and Prevotella bivia. As the stages of cervical cancer progressed, G. vaginalis, B. distasonis and P. bivia were detected at higher rates and higher counts than other bacteria.
These findings suggest that there is a relationship between G. vaginalis, B. distasonis and P. bivia and the onset and growth of cervical cancer.

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We studied theusefulness of two sets of primers in macrophage infectivity potentiator gene sequence of Legionella pneumophila. PCR by 1st step primers produced 649 bp DNA bands and 2nd step primers 489 bp DNA bands. Two step PCR by these primers produced 489 bp DNA bands specific for L. pneumophila. Two step PCR detected 10 fg of pure DNA extracted from L. pneumophila, Philadelphia-1 strain. PCR sensitivity by these primers was superior to former primers reported by us using intratracheal aspirates collected from the patient with L. pneumophila serogroup 2 pneumonia.

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During July to December in 1991, an epidemic of aseptic meningitis occurred in Gifu Prefecture in children. Epidemiological, virological and serological investigations were performed. The results are as follows:
1) Cases involved ranged from 0 to 13 years of age, and 75.9% of them were in the 3 to 7 year of age group.
2) Three types of enteroviruses were isolated from 83 of 133 cases (62.4%), and the echovirus type 30 was recovered from 71 cases (85.5%).
3) In 1991 with the cross neutralizing test between the prototype and isolated strains of echovirus type 30, a remarkable antigenic variation could not be found.
4) In serological studies, the positive rates of neutralizing antibodies to echovirus type 30 in infant sera aged 0-9 years in 1992-1993, was 51.9% with the prototype strain, but 34.0% with an isolated strain in 1991.

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Helicobacterpylori urease was recovered as a single peak by DEAE-Sepharose column chromatography and Sephacryl S-200 gel filtration. The purified urease was obtained by fast protein liquid chromatography using a Mono Q column. The purified urease preparation gave a single band in polyacrylamide gel discelectrophoresis.
Latex particles were sensitized with anti-urease immunoglobulin. The sensitized latex particles were agglutinated with the purified urease and by cell sonicates obtained from 55 strains of H. pylori which were isolated from the gastric mucosa of patients with gastric and duodenal disorders, while they did not react with those obtained from related bacteria known to be urease producers, such as Helicobacter mustelae and urease-positive “Campylobacter lari variants”, or by urease of some strains of Enterobacteriae. We have developed a specific and sensitive method for detecting the urease by using the reversed passive latex agglutination technique, in order to identify of the organism.

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To evaluate the host defense function in the elderly, chemotaxis and chemiluminescence (CL) were assessed as indicators of neutrophil function in healthy and bedridden subjects as well as in patients with bacterial infection. In addition, humoral host defensive factors were also investigated.
1) The differences in chemotaxis and CL between healthy elderly subjects and healthy adults were slight. Neutrophil function showed little decrease with aging.
2) Plasma fibronectin (PFN) values in bedridden patients were lower than in healthy edlerly subjects.
3) Chemotaxis and CL in patients in the acute phase of bacterial infection were higher than those of healthy elderly subjects regardless of whether these patients were bedridden or not. CL was slightly lower in bedridden patients than in non-bedridden patients. CL and body temperature were positively correlated in the acute phase of bacterial infection. Among the humoral defensive factors, PFN revealed lowered values in the acute phase of bacterial infection.
4) As the infection healed, chemotaxis and CL decreased, while PFN rose to normal. In the complement system, CH50 increased slightly as the infection healed.
From these results, we concluded that neutrophil function in elderly subjects, regardless of whether they were bedridden or not, is enhanced by infection. Responses to infection of bedridden patients were slightly weaker than those of non-bedridden subjects.

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We investigated the influence of granurocyte colony-stimulating factor (G-CSF) on bactericidal activities of macrophages and polymorphonuclear leukocytes (PMNs) from experimental pyelonephritis in leukocytopenic rats, in order to clarify the usefulness of G-CSF for opportunistic pyelonephritis.
We prepared three groups of experimental pyelonephritis, i. e., G-CSF administration group (group-1), cyclophosphamide (CPA) administration group (group-2), and CPA and G-CSF administration group (group-3). And we measured the active oxygen generation of peritoneal macrophages and PMNs in each group. On the other hand, we produced pseudomonal pyelonephritis in each group, and compared the survival rate of each group for 7 days.
G-CSF enhanced active oxygen generation of peritoneal macrophages and PMNs, significantly. Furthermore, G-CSF improved the survival rate of pseudomonal pyelonephritis in leukocytopenic rats.
These results indicated that G-CSF enhanced bactericidal activities of macrophages and PMNs in vivo, and prevents dissemination of infections.

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We investigated superoxide generation by polymorphonuclear leukocytes (PMNs) stimulated with various species of bacteria in order to clarify the species-specific immunocompromised status in the elderly.
We studied 66 elderly patients (ranged 66 to 89 year-old; mean 77-year-old) and 26 young healthy volunteers (ranged 25 to 3-year-old; mean 31-year-old) as a control group. The elderly patients were divided into two groups, i.e., the malignancy group and the benign group. Peripheral blood was prepared from each host and diluted. The activity of superoxide generation byPMNs was determined by means of chemiluminescence method. PMNs were stimulated with the standard stimulants; i.e., phorbol myristate acetate (PMA) and zymosan (ZY), and four bacterial species;i.e., Escherichia coli, Psudomonas aeruginosa, Staphylococcus aureus and Candida albicas. The activity of superoxide generation by PMNs in the malignancy group was significantly lower than that in the control group (p<0.05). Furthermore, the activity of superoxide generation by PMNs in the malignancy group and the benign group stimulated with C. albicans were significantly lower than those in the control group (p<0.05).
These results indicate that the elderly are in an immunocompromised status especially against fungus such as C. albicans.

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Mouse anti-endotoxin monoclonal antibody (E5) is now under clinical trial (PhaseII) in Japan by assessing clinical findings and plasma endotoxin levels. We attempted to evaluate an inhibitory effect of E5 on hypotension induced by endotoxin and TNF-α. Systolic blood pressure wa measured with a programmable sphygmomanometer using the tail-cuff method. The mixture of endotoxin (E. coli O111: B4, Sigma, 2μg/mouse) and recombinant mouse TNF-α(Genzyme, 8000 units) were injected into mice (ddY, 6-8-weeks-olds), and the change of blood pressure was observed for 3 hrs. The mixture significantly decreased the blood pressure to about 70% of the control. E5 (20μg/mouse), which was injected 10 min before, significantly abrogated the effect ect of endotoxin and TNF-α. The hypotension induced by the mixture was definitely inhibited by the injection of E5 (50, 100μg/mouse), injected 10 min later, or the injection of E5 (100μg/mouse) 30 min later. We previously found that E5 prevents the lethality induced by a concomitant injection of endotoxin, TNF-α and IL-2. Theseresults suggest that E5 effective for inhibition of endotoxin activity in vivo.

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From May 1990 to August 1991, 36 patients admitted to the Department of Internal Medicine in a medical school hospital with hematological malignancies or solid tumors, developed respiratory tract colonization with Pseudomonas cepacia. Sixteen (44.4%) of these patients developed pneumonia, and four (11.1%) died of respiratory failure due to P. cepacia pneumonia. Extensive survey of the hospital environment as well as equipment showed that nebulizer devices used by the patients for inhalation were contaminated with P. cepacia. Phenotypic characteristics, (production of hemolysin and extracelluler enzymes [lipase, lecithinase and protease]), the Analytical Profile Index 20 NE pattern, and the pattern of DNA fingerprinting by pulse-field gel electrophoresis in clinically isolated strains and strains derived from nebulizer devices were compared. The strains of P. cepacia obtained from patients in the Department of Internal Medicine were indistinguishable from each other and also from those isolated from nebulizer devices, but were different from those isolated from patients in other departments at the same time. These results demonstrated that the outbreak of P. cepacia respiratory colonization in immunocompromised patients was a nosocomial acquisition, and probably occurred by transmission through contaminated nebulizer devices.

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A 64-year-old male was admitted to Oita Medical University Hospital because of primary lung cancer. After the anticancer chemotherapy, the patient developed a high fever. His chest X-ray showed diffuse reticular shadows bilaterally, which were considered to be a drug induced pneumonia, then pulse therapy with corticosteroid was performed. With this treatment, the diffuse reticular shadows disappeared. One month later, however, a severe interstitial pneumonia developed, and the patient died of respiratory failure. Autopsy findings demonstrated cytomegalovirus (CMV) and Pneumocystis carinni pneumonia.
The polymerase chain reaction (PCR) for CMV in mononuclear cells in the blood was positive through out his hospitalization. In the sera, however, it was negative at the time of admission, thenb ecame positive 10 days before the onset of pneumonia. In addition, the PCR for P. carinni on the swab became positive as well.
These results imply that PCR can be used for diagnosis of CMV and P. carinii infections from sera or swabs.

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We report here a case of miliary tuberculosis with tubercular meningitis in which the polymerase chain reaction (PCR) amplification method was useful for its rapid diagnosis and follow up. A 70 year old male was hospitalized for further examination and treatment of diffuse small nodular shadows on the chest X-ray. After receiving antimicrobial therapy shadows still remained and he gradually lost visual acuity. He had no meningeal signs, and no remarkable finding on cranial CT. Cerebrospinal fluid examination showed increased cell number with predominantly lymphocytes. Cranial MRI (Gd DTPA) showed lateral ventricular ependymitis. Pulmonary tuberculosis and secondary tubercular meningitis were suspected, but we failed to detect microorganisms from the cerebrospinal fluid, gastric juice, sputum, and urine by the conventional method. However, by the polymerase chain reaction amplification method specific DNA fragments of Mycobacterium tuberculosis complex was detected from the cerebrospinal fluid, gastric juice, bronchoalveolar lavage fluid and serum. The final diagnosis of miliary tuberculosis with tubercular meningitis was established. We administered antitubercular drugs and observed the clinical course. He recovered and the polymerase chain reaction showed negative consequences in all samples.
The judgement of PCR and the clinical course were compatible and parallel with the clinical ourse.

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Patients infected with human immunodeficiency virus (HIV) are highly susceptible to various bacterial infections, especially, Streptococcus pneumoniae. Encapsulated bacteria such as S. pneumoniae, Hemophilus influenzae and Klebsiella pneumoniae are known to be resistant to phagocytosis by neutrophils and macrophages unless cell-wall-specific antibodies are present. It is suggested, therefore, that the serum of HIV-infected patients is deficient in opsonic activity against these bacteria. In this study, we compared the serum opsonic activity of the patients with that of healthy normal persons by using the luminolenhanced chemiluminescence (CL) method. As CL assay is a very sensitive and reliable way for determining the opsonic activity of serum, the method can be used to quantify the opsonic activity of sera from HIV-infected patients.

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