Serogroup conversion of Vibrio cholerae.

Vibrio cholerae serogroup O1 can be detected in the environment in a viable but nonculturable form, whereas V. cholerae non-O1 cells can be readily cultured during interepidemic periods in geographical regions where cholera is endemic. In the present study, pure cultures of V. cholerae non-O1 cells contained O1 cells when examined by immune-fluorescence microscopy. Laboratory microcosms were used to examine the outgrowth of the O1 cells in cultures of non-O1 V. cholerae. One O1 cell per 10(6) non-O1 cells could be detected by direct fluorescent-monoclonal antibody staining but only after incubation of the non-O1 culture for 48 h. Individual O1 cells were not detected in cultures incubated less than 48 h. Hybridization study, using a polymerase chain reaction (PCR) amplified fragment of the O-antigen of V. cholerae O1 as a probe, revealed the existence of a homologous gene in a microcosm sample of V. cholerae non-O1 containing serogroup-converted cells. The mechanism by which O1 cells can occur in cultures of non-O1 V. cholerae most likely resulted from spontaneous mutation of gene(s) encoding the O-somatic properties and (or) chemical, physical, or biological changes in the environment inducing expression or repression of the controlling gene(s). These findings have important implications for the epidemiology of cholera and the environmental source(s) of toxin producing V. cholerae O1.

Detection of Vibrio cholerae O1 in the aquatic environment in Brazil employing direct immunofluorescence microscopy.

Culturing and immunofluorescence (FA) methods for detection of Vibrio cholerae O1 in samples collected from the aquatic environment at selected sites in Brazil were compared. Of the samples examined, 90% were positive for V. cholerae O1 by FA but none was positive by culture, although strains of V. cholerae other than O1 strains were readily isolated. Evidence for V. cholerae O1 being autochthonous to the aquatic environment of Brazil is presented. Furthermore, FA methods are recommended for cholera surveillance programmes directed at the natural environment.

Aeromonas jandaei and Aeromonas veronii dual infection of a human wound following aquatic exposure.

Exudate removed from an infection that developed below the left eye of a 10-year-old male following a previously inflicted wound after aquatic exposure was cultured and revealed two different Aeromonas spp. Further characterization showed that one strain was phenotypically identical to Aeromonas veronii, while the other strain was confirmed by DNA hybridization analysis to be Aeromonas jandaei sp. nov. This is the first report of these more recently described aeromonads, thus far rarely reported from clinical disease, occurring simultaneously in a human infection.

Enumeration of Vibrio cholerae O1 in Bangladesh waters by fluorescent-antibody direct viable count.

A field trial to enumerate Vibrio cholerae O1 in aquatic environments in Bangladesh was conducted, comparing fluorescent-antibody direct viable count with culture detection by the most-probable-number index. Specificity of a monoclonal antibody prepared against the O1 antigen was assessed and incorporated into the fluorescence staining method. All pond and water samples yielded higher counts of viable V. cholerae O1 by fluorescent-antibody direct viable count than by the most-probable-number index. Fluorescence microscopy is a more sensitive detection system than culture methods because it allows the enumeration of both culturable and nonculturable cells and therefore provides more precise monitoring of microbiological water quality.

The fate of enteric pathogenic bacteria in estuarine and marine environments.

Sufficient laboratory and field data are now available to hypothesize that enteric pathogens survive for very long periods of time in sea-water. In fact, these Gram-negative bacteria probably enter into dormancy, during which they remain viable and potentially virulent, yet are non-culturable when traditional bacteriological methods are employed. Increasing use of the world's oceans-for discharge of domestic wastes may result in public health problems in the future from the allochthonous human pathogens accumulating in the marine environment at disposal sites.

Coincident plasmids and antimicrobial resistance in marine bacteria isolated from polluted and unpolluted Atlantic Ocean samples.

Sewage effluent and outfall confluence samples were collected at the Barceloneta Regional Treatment Plant in Barceloneta, Puerto Rico; outfall confluence samples at Ocean City, Md., were also collected. Samples from uncontaminated open ocean areas served as clean-water controls. Bacteria were enriched in marine broth 2216 amended with 1 microgram of one of a set of chemicals selected for study per ml: nitrobenzene, dibutyl phthalate, m-cresol, o-cresol, 4-nitroaniline, bis(tributyltin) oxide, and quinone. MICs of the chemicals were determined individually for all isolates. Bacterial isolates were evaluated for resistance to nine different antibiotics and for the presence of plasmid DNA. Treated sewage was found to contain large numbers of bacteria simultaneously possessing antibiotic resistance, chemical resistance, and multiple bands of plasmid DNA. Bacteria resistant to penicillin, erythromycin, nalidixic acid, ampicillin, m-cresol, quinone, and bis(tributyltin) oxide were detected in nearly all samples, but only sewage outfall confluence samples yielded bacterial isolates that were resistant to streptomycin. Bacteria resistant to a combination of antibiotics, including kanamycin, chloramphenicol, gentamicin, and tetracycline, were isolated only from sewage effluent samples. It is concluded that bacterial isolates derived from toxic chemical wastes more frequently contain plasmid DNA and demonstrate antimicrobial resistance than do bacterial isolates from domestic sewage-impacted waters or from uncontaminated open ocean sites.

Vibrio cincinnatiensis sp. nov., a new human pathogen.

A halophilic gram-negative rod was isolated from blood and cerebrospinal fluid collected from a 70-year-old male having no known contact with seafood or salt water. Positive biochemical tests included oxidase, sensitivity to 0/129, O-nitrophenyl-beta-D-galactopyranoside, lysine decarboxylase and fermentation of glucose, salicin, n-inositol, sucrose, L-mannose, L-arabinose, and arbutin. Negative tests included indole, ornithine decarboxylase, arginine dihydrolase fermentation of lactose, and production of gelatinase and urease. The DNA base composition was 45.0 mol% guanine plus cytosine. Numerical taxonomy indicated 70% similarity with known reference Vibrio sp. strains. The 5S rRNA sequence for this strain has been determined: 5'-U G C C U G G C G A C C A U A G C G U U U U G G A C C C A C C U G A U U C C A U G C C G A A C U C A G U A G U G A A A C G A A A C A G C G U C G A U G G U A G U G U G G G G U C U C C C C A U G U G A G A G U A G A A C A U C G C C A G G C A U-3'. Based on the phenetic, molecular genetic, and nucleic acid sequencing data, it is concluded that Vibrio cincinnatiensis represents a new species of the genus Vibrio sensu strictu (as defined by 5S rRNA sequencing results). On a basis of 5S rRNA comparative sequence analysis, the organism appears to share a recent common ancestor with V. gazogenes (98% homology) and close ancestry with V. mimicus, V. fluvialis, and V. metschnikovii.

The immediate-early enhancer element of herpes simplex virus type 1 can replace a regulatory region of the c-Ha-ras1 oncogene required for transformation.

A 0.8-kilobase SacI DNA fragment in the distal 5'-noncoding region of the c-Ha-ras1 oncogene hybridized to high guanine X cytosine sites of herpes simplex virus type 1 (HSV-1) DNA restriction fragments. Nucleotide sequence comparisons localized one of these sites to the intergenic region of HSV between the immediate-early genes coding for IEmRNA-3 and IEmRNA-4/5 that has enhancer-type activity. We tested the possibility that the HSV-1 enhancer and the upstream c-Ha-ras1 SacI fragment were functionally related by assaying for the capacity of recombinant plasmids in which the HSV-1 enhancer replaced the oncogene 0.8-kilobase SacI fragment to transform NIH/3T3 cells. Deletion of the 0.8-kilobase SacI fragment abolished the biological activity of c-Ha-ras1, but its replacement by the HSV-1 enhancer fully restored it. These results confirm the enhancer properties of the HSV-1 immediate-early intergenic region and suggest that c-Ha-ras1 sequences contained within the 0.8-kilobase SacI fragment plays a role in the transcriptional activation of the oncogene.

Numerical taxonomy of phenanthrene-degrading bacteria isolated from the Chesapeake Bay.

Phenanthrene-degrading bacteria were isolated from Chesapeake Bay samples by the use of a solid medium which had been overlaid with an ethanol solution of phenanthrene before inoculation. Eighteen representative strains of phenanthrene-degrading bacteria with 21 type and reference bacteria were examined for 123 characteristics representing physiological, biochemical, and nutritional properties. Relationships between strains were computed with several similarity coefficients. The phenogram constructed by unweighted-pair-group arithmetic average linkage and use of the simple Jaccard (SJ) coefficient was used to identify seven phena. Phenanthrene-degrading bacteria were identified as Vibrio parahaemolyticus and Vibrio fluvialis by their clustering with type and reference strains. Several phenanthrene-degrading bacteria resembled Enterobacteriaceae family members, although some Vibrio-like phenanthrene degraders could not be identified.

Characterization of leader RNA sequences on the virion and mRNAs of mouse hepatitis virus, a cytoplasmic RNA virus.

Mouse hepatitis virus, which replicates in cytoplasm, contains leader RNA sequences at the 5' end of the virus-specific mRNAs. We have sequenced this leader RNA by synthesizing cDNA from a synthetic oligodeoxyribonucleotide primer (15-mer) that is complementary to the sequences at the junction site between the leader and body sequences of the mRNAs. The leader sequences on each mRNA have exactly the same size, which span approximately equal to 70 nucleotides. Leader cDNA fragments obtained from several mRNA species were sequenced and found to be identical. Computer analysis of the leader RNA sequences shows that they share extensive sequence homology with the long-terminal-repeat region of several mammalian sarcoma viruses, suggesting possible common functions. This is a novel case of spliced leader sequences in the mRNAs of a cytoplasmic virus. An identical leader sequence is also present at the 5' end of the virion genomic RNA. The leader RNA is thus probably encoded by the virion genomic RNA template and is fused to the different body sequences of the various mRNAs. Since conventional RNA splicing is not involved, a novel mechanism for fusing two noncontiguous RNA segments in the cytoplasm must be utilized during viral transcription. Several minor cDNA bands longer than the leader were also synthesized, suggesting the possible presence of partially homologous sequences in other parts of the genome RNA.

Further characterization of mouse hepatitis virus RNA-dependent RNA polymerases.

Two temporally and enzymatically distinct RNA-dependent RNA polymerase activities associated with membranes of the mouse hepatitis virus (MHV)-infected cells have been identified previously [Brayton et al., J. Virol. 42, 847-853 (1982)]. In this paper, the subcellular distribution and functions of these two polymerases were examined. Fractionation of the postnuclear membranes by sucrose gradient sedimentation showed that the early polymerase activity (detected at 1 hr p.i.) was homogeneous, while the late polymerase (6 hr p.i.) was associated with two distinct membrane fractions. The early polymerase synthesized a single RNA species of viral genomic size and negative sense. In contrast, the light peak of the late polymerase synthesized genomic-sized RNA of positive sense, while the heavy peak of the activity synthesized positive-sensed genomic and subgenomic mRNAs. These findings suggest that the light peak of the late polymerase represents a replication complex while the heavy peak represents a transcription complex. They also establish the essential features of the mode of replication of MHV.

Cytotoxic T lymphocytes recognize determinants on the BALB/c-H-2Ld molecule controlled by alpha 1 and alpha 2 but not alpha 3 external domains.

We have shown that cytotoxic T lymphocytes (CTL) raised in H-2d mice use H-2Ld but not H-2Dd or H-2Kd antigens as restricting elements in lymphocytic choriomeningitis virus (LCMV) and vesicular stomatis virus (VSV) infections. To localize the regions of H-2Ld protein recognized by CTL, we constructed a recombinant H-2Ld/Dd gene encoding a hybrid antigen with alpha 1 and alpha 2 external domains of H-2Ld and alpha 3, transmembrane and cytoplasmic domains of H-2Dd. The recombinant gene was transfected into mouse cells and the hybrid molecules were characterized serologically, biochemically and functionally. In all assays, H-2Ld/Dd molecules were recognized by LCMV-and VSV-specific H-2Ld-restricted CTL in a manner similar to that of wild-type H-2Ld antigens. Analogous results were obtained with alloreactive CTL. Hybrid antigens containing the alpha 3 domain of H-2Ld fused to alpha 1 and alpha 2 domains of a Qa-2,3 region-encoded antigen were not used as restricting elements by LCMV-specific CTL. These results suggest that H-2Ld-restricted CTL directed against LCMV and VSV recognize determinants controlled by the alpha 1 and/or alpha 2 domains of the H-2Ld molecule.

Studies on the mechanism of RNA synthesis of a murine coronavirus.

The mechanism of viral RNA replication in mouse hepatitis virus (MHV)-infected cells was studied by oligonucleotide mapping of every mRNA. We discovered that an oligonucleotide, No. 10, was localized at the 5'-end of every mRNA, and was not colinear with the sequences of the virion genomic RNA. This result indicates that all of the mRNAs contain a leader sequence which is joined to the body sequences of the mRNAs. We have also studied the structure of the replicative intermediate (RI) RNA in the MHV-infected cells. This RI RNA consists of a single species corresponding to the MHV genomic RNA. No subgenomic RI structures were detected. Furthermore, the nascent RNA chains in the RI structure contained the leader sequences, suggesting that the leader RNA was not added to the mRNA post- transcriptionally , but rather, it was probably synthesized independently and then used as a primer for the synthesis of mRNAs. We have also shown that the poly (A) sequences in the MHV genome were transcribed from the poly (U) sequences present in the negative-strand template. The RNA polymerases involved in the MHV RNA synthesis were also characterized. The early polymerase synthesizes a single negative-stranded, full-length RNA. The late polymerases could be separated into two activities, one synthesizing positive-stranded genomic RNA, and the other synthesizing genomic as well as subgenomic RNAs. Thus, the replication and transcription functions of MHV could probably be separated. A plausible model of MHV replication is presented.

Expression and function of transplantation antigens with altered or deleted cytoplasmic domains.

Two mutants of the class I gene encoding the H-2Ld transplantation antigen have been constructed. In one mutant the cytoplasmic domain of the class I molecule has been altered by deletion of 24 of the 31 C-terminal residues, and in the second the C-terminal 25 residues of the cytoplasmic domain have been replaced with a unique sequence of 19 amino acids. These mutant class I genes have been transferred into mouse L cells by DNA-mediated gene transfer. Both mutant genes are expressed at normal levels on the cell surface, and they have charge properties and sizes consistent with the introduced alterations. These mutant Ld molecules can serve as target antigens for allogeneic cytotoxic T cells and as restricting elements for virus-specific cytotoxic T cells. These results show that the 24 residues replaced or deleted from the carboxy terminus of the class I molecule are not required for its transport to or integration in the plasma membrane, nor for its function as a target antigen or a restricting element during T-cell-mediated cytotoxicity.

New selective plating medium for isolation of Vibrio vulnificus biogroup 1.

A new plating medium (VV agar) has been developed as an alternative to thiosulfate-citrate-bile salts-sucrose (TCBS) agar for the isolation of Vibrio vulnificus. Salicin (2% wt/vol) is employed as the source of carbohydrate, with potassium tellurite (0.0005% wt/vol), crystal violet (0.00015% wt/vol), oxgall (0.8% wt/vol), and a pH of 8.6 to inhibit growth of gram-positive and gram-negative organisms other than V. vulnificus. Because strains of V. vulnificus do not strongly ferment salicin in VV agar, a pH indicator has not been included in the medium. Growth of V. vulnificus appears on VV agar as large grey colonies with black centers. Other non-Vibrio strains which grow on the medium produce smaller colonies and fail to take up tellurite. VV agar has proved to be more effective than TCBS agar in inhibiting members of the Enterobacteriaceae as well as gram-positive cocci. Only Vibrio strains capable of utilizing salicin grow well on VV agar. Recovery and growth of V. vulnificus are superior on VV agar, compared with TCBS agar.

Natural killer cell activity during mouse hepatitis virus infection: response in the absence of interferon.

The ability of the JHM3 strain of mouse hepatitis virus (MHV) to induce natural killer (NK) cells was examined. Infection of C57BL/6 (B6) mice with this virus resulted in the augmentation of natural cytotoxicity against YAC-I target cells in the absence of a detectable interferon response. The cells responsible for this increased cytotoxicity were sensitive to complement-mediated lysis with an anti-Q-5 reagent but not with a Thy 1.2 antiserum, indicating that they possess an NK-like surface phenotype. Although variation in the NK response of individual B6 mice following JHM virus infection was found, even the animal with the most responsive NK cell population had no detectable interferon in the spleen. This finding contrasted with observations with an unrelated virus (lymphocytic choriomeningitis virus) and a serologically related strain of MHV. Infection with both of these viruses induced augmented NK cell activity and interferon responses. In addition, we found that neither the ability to mount an augmented NK cell response nor preferential lysis of virus-infected targets correlated with resistance or susceptibility to JHM virus infection.

Statistical evaluation of a quality control method for isolation of pathogenic Vibrio species on selected thiosulfate-citrate-bile salts-sucrose agars.

The recovery of Vibrio cholerae, Vibrio fluvialis, Vibrio parahaemolyticus, and Vibrio vulnificus, employing eight strains of each species, was studied by using four brands of thiosulfate-citrate-bile salts-sucrose (TCBS) agar prepared according to manufacturers' instructions and following a standardized procedure. A standardized broth inoculum of each strain was placed on duplicate plates of each brand of TCBS agar and also on tryptic soy agar (Difco Laboratories) containing 1% (wt/vol) NaCl, the latter serving as the control. Plates were inoculated in a sequence designed to compensate for bias associated with multiplication of the bacteria during the inoculation procedure. Colony counts and quality of growth were recorded after incubation for 18 h at 35 degrees C. The comparison procedure was repeated four times at weekly intervals. Data were analyzed by using an analysis of variance model. The recovery and quality of growth of each species varied significantly on the different brands of TCBS agar. Significant variability was also identified for some components of the inoculation procedure. Modifications of the inoculation procedure are suggested to minimize sources of variance. A simplified statistical procedure, based on the t test, is described for media quality control for laboratories routinely isolating pathogenic Vibrio spp.

Murine coronaviruses: isolation and characterization of two plaque morphology variants of the JHM neurotropic strain.

Two plaque-size variants of the neurotropic JHM strain of mouse hepatitis virus have been isolated from the virus stock after eight serial passages in suckling mouse brain. One variant, JHM-DL, produces large plaques, while the other, JHM-DS, produces small plaques in tissue culture. DS replicates more slowly, has a lower virus yield in vitro, and is less virulent for mice than DL. They also differ in their pathogenicity for mice: JHM-DL infection results in acute encephalomyelitis while JHM-DS infection results in demyelination. Oligonucleotide fingerprint analysis of the RNA genomes of these two variants revealed that they had almost identical genetic sequences. Each variant, however, had a unique oligonucleotide spot not found in the other. The unique spot of the large plaque variant, JHM-DL, was localized at approximately 3 to 5 kb from the 3' end, while the JHM-DS unique spot was mapped at 14 to 15 kb from the 3' end of the genome. We have further shown that these oligonucleotide changes are not correlated with the plaque morphology. These two viruses may be useful for studying the molecular basis of virus-induced demyelination.