An immunoelectron microscopic study was performed to determine the distribution of antigenic components on particles of Chlamydia psittaci and infected cells using a number of monoclonal antibodies (MAbs). Of three anti-lipopolysaccharide (LPS) antibodies (4D5, A2 and 4G5), two antibodies (4D5 and A2) reacted with the surface of reticulate bodies (RBs) but not with that of elementary bodies (EBs). The other antibody (4G5) reacted with both EBs and RBs. Examination of infected cells in thin sections revealed that 4D5 and A2 combined with the membranes of both EBs and RBs. These results indicate that each LPS epitope localized at a different position in the chlamydial membrane. Most MAbs directed to protein antigens reacted on the surface of both EBs and RBs though 3E9 specific for the 90kDa and 50kDa protein components combined with RBs only.

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Vibrio parahaemolyticus Na2 and its pili were studied in relation to intestinal colonization. The isolated pili were adhesive to the intestinal epithelium. The adhesion of the organisms was blocked by masking the epithelial receptor with the purified pili, or by treating the organisms with anti-pilus antibody (Fab fraction). These results suggest that the pili of V. parahaemolyticus Na2 play an important role in the adhesion of the organisms to the rabbit intestine.

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A modified taurocholate-cefoxitin-cycloserine-fructose agar medium, pH 5.5, on which vegetative cells alone could grow, was newly devised for separate isolation of Clostridium difficile vegetative cells and spores from feces. The ratio of C. difficile-positive feces from healthy newborn infants younger than 10 days of the age was 30.8%, and 93.3% of feces from healthy infants older than 20 days were positive for C. difficile. C. difficile spores alone were detected in twenty-one samples (75%) of C. difficile-positive Twenty-eight specimens. Only 10.7% (3/28) C. difficile vegetative cells alone were detected. C. difficile spores alone were detected in one of nine healthy adults. These collective results offer potential explanations for high frequent isolations of C. difficile from newborn infants without occurrence of pseudomembranous colitis.

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A minocycline (MINO)-resistant mutant was isolated from Mycobacterium smegmatis strain Rabinowitschi. Polypeptide synthesis in the cell-free system prepared from the mutant was resistant to minocycline (MINO) because of alterated 30S ribosomal subunits. Upon two-dimensional gel electrophoresis, two proteins of 30S subunit were found to be altered. MINO resistance phenotype was transferred by mating to the recipient strain P-53. MINO resistance phenotype of a recombinant thus obtained was transferred by a different mating system to the recipient strain Jucho, once again. Ribosomal proteins of each of the donors, recipients and recombinants were analyzed and compared on 2-dimensional (2D) electrophoresis. Approximately 50 ribosomal proteins were observed in 70S ribosomes. Some proteins were differently electrophoresed in different strains. The 30S ribosomal subunits contained at least 19 proteins and 50S ribosomal subunits contained at least 23 proteins. Some proteins were easily washed off during dissociation of subunits in sucrose gradients. At least one protein (designated F) in both subunits was observed at the same position. One protein designated C in 30S subunits could be co-transferred to the recipient cells together with resistance phenotype at the frequency of 100% in the 30 recombinants examined so far. The other protein designated D in 30S subunits could be transferred at the frequency of 86-88%. Three other proteins in 50S subunits could be co-transferred to the recipient strain at a lower frequency. Minocycline resistance, therefore, could be mapped close to genes encoding the structure of ribosomal proteins in M. smegmatis.

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We prepared polyclonal antibody specific to Mycoplasma pneumoniae. Using this antibody, we developed a latex agglutination test (LAT) for detecting the organism in respiratory exudates as rapid diagnosis of M. pneumoniae infection. Further, LAT was compared with DNA-probe test (DP) which was the only commercially available test for the rapid detection of the organism. In LAT, both M. pneumoniae and M. genitalium give positive agglutination, but the titer of M. genitalium was significantly lower than that of M. pneumoniae. The detection limit of LAT was 2×10⁵CFU/ml and that of DP was 5×10⁴CFU/ml in vitro. It was considered that target molecules in LAT were accumulated in the pharyngeal portion of the patients, because of their long half-life at 37C. However, ribosomal RNA which was target molecule in DP was destroyed at 37C much sooner, and the accumulation could not be expected. Actually, positive rate in LAT was higher than that in DP among clinical specimens in which M. pneumoniae was detected by culture method. The procedure of LAT is much easier and more rapid than that of DP in which radioactive isotope is required. LAT could be the choice of test for rapid diagnosis of M. pneumoniae infection.

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The genomic DNA fragment which contains ribosomal RNA (rRNA) genes for Treponema phagedenis was cloned into bacteriophage vector lambda EMBL3. A restriction map of the fragment was constructed and the organization of the rRNA genes was determined. The fragment contained at least one copy of the 16S, 23S and 5S sequences and the genes are arranged in the order 16S-23S-5S. Southern hybridization using radiolabeled rRNA gene probes to genomic DNA from T. phagedenis strain Reiter and T. pallidum strain Nichols showed that these organisms have two radioactive fragments which hybridize to the probes in their genome. These results suggest that both pathogenic and non-pathogenic strains of Treponema may carry at least two sets of rRNA genes on their chromosomes.

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Serum samples from healthy adults in four geographic/ethnic groups (Ghanaian Blacks, Malaysian Chinese, Malaysian Indians and United States Caucasians) were tested under code for antibodies to human herpesvirus-6 (HHV-6). The prevalence and titer of HHV-6 antibody in the healthy Ghanaians were significantly higher than in the Malaysian Chinese; United States Caucasians and Malaysian Indians had intermediate prevalence and titer of antibodies. Thus far, no specific differences in HHV-6-associated diseases have been noted between geographic/ethnic groups with these marked variations in antibody patterns.

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Coliphage WPK was originally isolated from sewage in Kiel, Germany, because its plaque diameter continued to expand for days. Electron microscopy revealed an isometric capsid with dimensions of 54nm between opposite apices, and a short, noncontractile tail 16nm long, placing phage WPK into morphogroup C1. The nucleic acid of phage WPK was linear double stranded DNA. The host ranges of phages WPK and T3 were identical. Of ten E. coli strains tested for host range, two were resistant and of eighteen other Enterobacteriaceae only four were susceptible. Seven gram-negative species which are not members of the Enterobacteriaceae were refractory. However, there were differences in plaque morphology and plaque expansion between the two phages. Phage T3 plaques expanded for at least seven days on E. coli B only, while phage WPK plaques expanded for at least seven days on four strains of E. coli. The buoyant density of WPK, determined by isopycnic density gradient centrifugation in CsCl, was 1.508g/ml which was significantly different than that of T3 at 1.493g/ml (P<0.05). Phage-encoded proteins were examined for each phage using [³⁵S]methionine incorporation, SDS-PAGE, and autoradiography. Of thirty proteins identified in phage WPK and twenty-eight in phage T3, only fourteen were of the same size in both. We concluded that phage WPK was distinct, but related to T3.

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For identifying virus-specific antigens of Marek's disease virus (MDV), monoclonal antibodies (MAbs) against strain Md5 of serotype 1, which is known to be a very virulent MDV (vvMDV), were isolated. Fifty-eight hybridoma clones that secreted MAbs against vvMDV were obtained. Of these MAbs, 36 gave positive reactions in an immunofluorescence (IF) test, and 22 gave positive reactions on enzyme-linked immunosorbent assay (ELISA). None of these MAbs gave positive reactions in both the IF test and ELISA. Of the MAbs that gave positive reactions in the IF test, 33 clones reacted with MDV1-specific epitopes, the other three reacting with MDV1-HVT intertypic epitopes. None of the clones reacted with MDV1-MDV2 intertypic epitopes. Three virus-specific polypeptides were identified by radioimmunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or immunoblotting. These polypeptides were recognized by 12 MAbs giving positive reactions by IF, but by none of those giving positive reactions by ELISA. In addition, size heterogeneity of the MDV1-specific phosphorylated polypeptides in the MDV1 strains was shown using the MAbs against Md5.

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Studies were undertaken in mice using immune-stimulating complexes (ISCOMs) or micelles prepared from envelope glycoproteins of human influenza virus (PR8) and matrix (i.e., ISCOM skeleton without incorporated antigen). Electron microscopic studies showed that ISCOMs, in contrast to micelles, have a remarkable affinity for cell membranes and seem to rapidly promote their own internalization by cells to which they adhere. PR8 ISCOMs, but not matrix nor micelles, significantly increased the expression of membrane Ia by peritoneal mononuclear leucocytes 24hr after intraperitoneal immunization.

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