A filamentous phage was isolated from carrier strain AI-1841 of Vibrio cholerae O139 Bengal and thus was termed fs phage. The phage was measured to be approximately 1μm in length and 6nm in width. One end of the phage was slightly tapered and had a fibrous appendage. The plaques developed on strain AI-4450 of V. cholerae O139 were small and turbid. The phage grew in strain AI-4450 and reached a size of 10⁸ to 10⁹pfu/ml at 5hr after infection without inducing any lysis of the host bacteria. The group of phages attached on rod-shaped materials like fimbriae of this bacteria, with their fibrous appendages at the pointed end, were often found in the phage-infected culture. The anti-fimbrial serum effectively inhibited the infection of fs phage to the host strain AI-4450. We thus concluded that the phage can be adsorbed on fimbriae with a fibrous appendage on the pointed end of the phage filament.

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The role of CD14 in the phagocytosis and killing of microorganisms was investigated using macrophage-like cell lines, CD14-positive J774.1 cells and CD14-negative mutant J7.DEF.3 cells derived from J744.1 cells. The cells were infected with Salmonella typhimurium organisms of the smooth (S)-form LT2, mutant rough (R)-form TV148 or Staphylococcus aureus 248βH. At 30 or 180min incubation, the cells were washed and disrupted. Colony-forming units (CFUs) liberated from the disrupted cells were determined by quantitative cultivation, and the phagocytic index and killing rate were calculated. Both the phagocytic index and killing rate of J774.1 cells against LT2 organisms were greater than those of J7.DEF.3 cells. However, the index and rate of J774.1 cells against TV148 and 248βH organisms were similar to those of the J7.DEF.3 cells. The phagocytosis of LT2 organisms by J774.1 cells was partially inhibited by S-form LPS (S-LPS) and anti-CD14 antibody, but not by R-chemotype LPS (R-LPS). These results suggest that CD14 participates in the phagocytosis of S-form Salmonella.

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Diarrheal diseases are major causes of morbidity and mortality among children in developing countries. We have analyzed the causative agents of diarrhea in children under five years of age who resided in rural environments but attended a hospital in Malindi, a coastal town in Kenya. Bacterial diarrhea was found in 239 (27.7%) of 862 patients with diarrhea. Diarrheagenic Escherichia coli, including enteropathogenic, enterotoxigenic, and enterohaemorrhagic strains, was isolated from 119 (13.8%) patients, followed by Salmonella spp. (63 cases, 7.3%) and Shigella spp. (56 cases, 6.5%). Intestinal parasites were found in 109 (12.6%) of the patients. Entamoeba histolytica and Giardia lamblia were found in 67 (7.8%) and 42 (4.9%) of the cases, respectively. Rotavirus was found in 69 (16.1%) of 428 cases, a part of the 862 cases. Significant differences in age distribution were seen in diarrheal cases due to Campylobacter spp., G. lamblia, and rotavirus. No significant seasonal incidence of specific pathogens was found, but the number of diarrheal patients was significantly correlated to rainfall. Drinking water was contaminated with bacteria at concentrations ranging from 10³ to 10⁶CFU/ml in 98% of the households and by coliform bacteria at concentrations of 10² to 10⁵CFU/ml in 72% of the households. These results suggest that the main routes of infection may be contaminated drinking water and fecal-oral transmission of enteric pathogens. Consequently, we propose that the enhancement of hygienic practice through health education is a feasible control measure of diarrhea in the study area.

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A successful peptide vaccine for AIDS is desired to elicit T-helper and cytotoxic T lymphocyte responses besides neutralizing antibodies. The V3 loop peptide of HIV-1 has been shown to contain the principal neutralizing domain, one of the most immunodominant regions, having both B-cell and T-cell determinants. In this study, the tip of the V3 loop region was mutated from GPGR to GPGQ based on the sequence of Indian isolates (CKRKIHIGPGQAFYT). To further enhance the immunogenicity of this epitope, two delivery systems of immune stimulating complexes (ISCOMs) and liposomes were used to incorporate the peptide. Mice of differing haplotypes, H-2^b, H-2^d, H-2^k and H-2^s, showed no MHC restriction when immunized with these formulations. The IgG levels as assessed by ELISA were found to be significantly higher (P<0.05 to P<0.001) for even five-fold lower doses of the peptide in ISCOMs and liposomes as compared to the conventional alum-based preparation. The major subtype elicited was IgG2a/IgG2b, suggestive of a Th1-like response for all the formulations. Thus, it would appear that the same peptide incorporated in ISCOMs and liposomes selects a Th1 response and may therefore be important not only for neutralization but also for virus clearance.

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The concentration of total IgA and the specificity and molecular size of IgA anti-human immunodeficiency virus (HIV) type-1 antibodies in plasma obtained from individuals at different stages of HIV infection were analyzed. The concentration of total IgA in the plasma was not decreased even in the late stage of HIV infection, in contrast with those of total IgG and IgM. The IgA anti-HIV antibodies differed to the IgG anti-HIV antibodies in their specificity as determined by Western blotting. The IgA antibodies mainly bind to Env glycoproteins. The IgA anti-HIV antibodies in plasma were detected between IgG and IgM by gel filtration, suggesting the presence of polymeric IgA anti-HIV antibodies. These results indicate that the production of non-specific IgA in plasma is enhanced by unknown mechanisms in every stages of HIV infection, and suggest that IgA anti-HIV antibodies in plasma which are possibly polymeric and have unique specificity may play an important role in HIV infection.

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Polymerase chain reaction (PCR) primers for O9 antigen (rfbE) and phase 1 fiagellin antigen (fliC) were designed for the rapid identification and detection of Salmonella serovar Enteritidis and Dublin. The rfbE primer pairs selectively amplified the rfbE region of group O9 Salmonella serovars. The fliC primer pairs amplified the DNAs of g, m and g, p-type fiagellar antigen; Salmonella serovar Enteritidis, Dublin, and Essen. However, DNA from flagellar-negative Salmonella serovar Gallinarum-Pullorum was also amplified. The sensitivity of PCR primer pairs was 10⁴CFU/assay by boiled DNA preparation and 10²CFU/assay by proteinase K-treated DNA preparation.

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Hyaluronidase (HAase) activity was detected in both a human pus sample and the culture supernatant of the only bacterial isolate from the pus, Streptococcus intermedius, using a zymographic technique. The optimum pH range for HAase activity was similar for both samples. Although the bands showing the strongest HAase activity of these samples differed from each other with respect to molecular size, both samples were equally inhibited by an antiserum raised against HAase of S. intermedius. These results suggest that S. intermedius may produce HAase in vivo as well as in vitro, and that this enzyme and/or its fragments may play an important role in host tissue degradation.

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Multicellular cells were efficiently induced in Staphylococcus haemolyticus by the addition of protease to exponentially growing cultures at 30C. Electron microscopy revealed the formation of tetrad-shaped multicells that were septated but not separated from each other. Incubation of the multicells with extract from the cells grown without protease resulted in a fourfold increase in the number of colony-forming units as compared with the untreated control. An electrophoretic analysis showed that protease caused a loss of cell wall-lytic activity of the cell, which possibly led to the formation of multicells through cessation of cross wall separation.

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An Escherichia coli R107 strain (O26 serotype) producing a Shiga-like toxin IIe variant (SLT-IIera) was isolated from the mesenteric lymph node of a freshly dead rabbit carcass. The entire structural gene for this SLT-IIera was cloned from chromosomal DNA by PCR using primers based on previously published slt-IIe sequences. Nucleotide sequence analysis indicated that the slt-IIera gene was very similar to slt-IIe (formerly called slt-IIv) from E. coli strains S1191 and 412; five and one nucleotide changes were detected in A and B subunits, respectively, which resulted in changes in amino acid sequences of the corresponding subunits by three and one residues. Recombinant SLT-IIera and SLT-IIe produced using an E. coli host-vector system showed similar cytotoxicity, suggesting that the variations in the structural gene of SLT-IIera have no significant effect on cytotoxic level.

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The aroA and aroD genes from Shigella dysenteriae type 1, encoding 5-enolpyruvylshikimate 3-phosphate synthase and 3-dehydroquinase, respectively, were cloned by polymerase chain reaction (PCR). Their nucleotide sequences were determined and predicted to code for 46kDa and 27.5kDa proteins, respectively. Protein expressed from these genes using the minicell system, corresponded to the size of the predicted protein products. The cloned genes were shown to be functional by complementation of Escherichia coli aroA^- and aroD^- mutants. The predicted amino acid sequences of the cloned aroA (427 amino acids) and aroD (252 amino acids) genes of S. dysenteriae type 1 were found to be highly homologous to the corresponding genes in other bacterial species, indicating the high conservation of these housekeeping genes. The use of the cloned aroA and aroD genes in the development of a vaccine strain against S. dysenteriae is discussed.

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Immunoelectron microscopy demonstrated antigenic heat-stable 120- to 130-kilodalton proteins (PS120) of spotted fever group (SFG) rickettsiae with antiserum against recombinant PS120 of Rickettsia japonica. In the case of R. japonica, a major part of the protein was shown to be localized outside the electron-lucent nucleoid-like region in the cytoplasm of the organisms. The other SFG rickettsiae represented a similar localization of the PS120 antigens cross-reactive to that of R. japonica. On the other hand, a typhus group rickettsia demonstrated no antigens cross-reactive to the PS120 of SFG rickettsiae.

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Direct detection of Escherichia coli O157 and foodborne pathogens associated with bloody diarrhea were achieved using polymerase chain reaction (PCR) after the preparation of DNA from stool specimens using the microspin technique. PCR was compared with cultivation and toxin production tests with respect to the efficiency of detection of each pathogen; E. coli O157, Vibrio parahaemolyticus, Salmonella serovar Enteritidis and Campylobacter jejuni. Detection of some or all of the above pathogens in clinical stool specimens was achieved using PCR. The minimum number of cells required for the detection of the above pathogens by PCR was 10¹CFUs/0.5g of stool sample. PCR was completed within 6hr. The above pathogens were also detected in cultivation and toxin production tests. Partial purification of the template DNA using the microspin technique was essential for the elimination of PCR inhibitors from the DNA samples. This PCR method is an accurate, easy-to-read screening method for the detection of Shiga-like toxin producing E. coli O157 and enteropathogens associated with bloody diarrhea in stool specimens.

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Sixty-eight sera from the acute, recurrent, and provoked types of female genital herpes were compared for the seroprevalence of herpes simplex virus (HSV) types 1 and 2 by immunodot assay using HSV glycoprotein G. In the HSV-1-isolated patients, no HSV-2 antibodies were detected, whereas in the HSV-2-isolated patients, HSV-1 seroprevalence was 9% for the acute type, 89% for the provoked type (P< 0.005), and 55% for the recurrent type (P<0.05). The natural history of female genital herpes and the possible protective role of pre-existing antibodies in preventing the acquisition or clinical manifestation of a subsequent HSV infection are discussed.

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The 5'-untranslated genomic region of the pestivirus strain Europa, originated in human leucocytes and previously identified as bovine diarrhea virus (BVDV), was amplified by reverse transcription-PCR and sequenced. Analyses based on primary nucleotide sequence homology and on secondary palindromic sequence structures characteristic to genotypes revealed that this human isolate should be assigned to a novel genotype of pestivirus, type Ic. This newly emerged genotype was related to, but distinguishable from the three known BVDV genotypes, Ia, Ib and II. Three other bovine field isolates of BVDV originated from Germany were also found to belong to this new genotype Ic. Within pestivirus genotype Ic strains, the overall nucleotide sequence homology was 95-96%, and 88-92%, 88-90% and 77-79% with the other BVDV genotypes Ia, Ib and II, respectively. With the strains from border disease virus (genotype III) and hog cholera virus (genotype IV), homologies were less than 75%.

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Sho-saiko-to (SST), a traditional Kampo medicine, has been examined for its inhibitory effect on human immunodeficiency virus type 1 (HIV-1) replication in peripheral blood mononuclear cells (PBMCs). SST alone moderately inhibited HIV-1 replication at a concentration of 25μg/ml. When SST was combined with zidovudine (AZT), lamivudine (3TC) or AZT plus 3TC, SST enhanced the anti-HIV-1 activity of 3TC. In contrast, SST slightly enhanced the anti-HIV-1 activity of AZT plus 3TC but did not enhance the activity of AZT alone. These results suggest that the combination of SST and 3TC has potential as a chemotherapeutic modality of HIV-1 infection.

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Antigenic domain 1 (AD-1), located between amino acids 608 and 625 of human cytomegalovirus (CMV) gB protein, is the major domain recognized by neutralizing antibodies. Amino acids 552 to 630 are essential for the binding of neutralizing antibodies. We developed an enzyme-linked immunosorbent assay (ELISA) to detect antibodies against a fusion protein containing amino acid residues 549 to 644 of the gB polypeptide and maltose binding protein (MBP). Of 180 seropositive samples, 106 (58.9%) showed positive immuno-reactivity against the fusion protein. None of the seronegative samples reacted with the fusion protein. Among 57 seropositive individuals typed for HLA, subjects with HLA-DR9 had a higher positive rate against the fusion protein (13/14=92.9%) than those without HLA-DR9 (25/43=58.1%). In addition, subjects with HLA-DR15 had a lower positive rate against the fusion protein (7/16=43.3%) than those without HLA-DR15 (31/41=75.6%). Mean OD values of HLA-DR15-positive individuals were significantly lower than those of HLA-DR15-negative individuals. Thus, among CMV-infected individuals, HLA-DR9 may be associated with responders for neutralizing antibodies and HLA-DR15 may be associated with non/low-responders.

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