Virulent Parental Strain Research Articles

Natural history of oral Salmonella dublin infection in BALB/c mice: effect of an 80-kilobase-pair plasmid on virulence.

BALB/c mice were infected orally with Salmonella dublin strains Lane and LD842, an isogenic derivative of the former that is avirulent because it was cured of its 80-kilobase-pair virulence plasmid pSDL2. Both strains colonized the intestine and invaded Peyer's patches with equivalent efficiency. However, the parent strain multiplied in mesenteric nodes and in the spleen; the plasmid-cured strain reached these organs, but the infection was low grade and remained relatively static until the mice developed active immunity and cured themselves. The histological response to plasmid-free strain LD842 was mononuclear, whereas the virulent parent strain produced abscesses. Sublethal irradiation of the mice before infection with strain LD842 prevented the mononuclear infiltrate in the liver and made the animals susceptible. Thus the virulence plasmid of S. dublin allows multiplication within the reticuloendothelial system and does not have any effect on the organism's ability to colonize the intestine or invade Peyer's patches.

Salmonella typhimuriumaroA mutants as carriers of the Escherichia coli heat-labile enterotoxin B subunit to the murine secretory and systemic immune systems

We investigated the ability of Salmonella typhimurium vaccines to deliver heterologous antigens to the systemic and secretory immune systems of the mouse, while retaining their immunogenicity against salmonellosis. S. typhimurium SL3261, an avirulent aroA mutant, or SL3261 carrying plasmid pBRD026, a pBR322 derivative encoding the gene for Escherichia coli LT-B were used to immunize BALB/c mice orally. Both immunizing strains invaded the mononuclear phagocyte system of the mice, grew slowly until approximately day 14 post-infection, and then were rapidly cleared. No salmonellae were detected in livers, spleens, mesenteric lymph nodes or Peyer's patches by day 42. Mice immunized with either strain and challenged orally with the virulent parent strain, SL1344, several weeks after clearing the immunizing organism, were protected against the lethal S. typhimurium infection. Mice infected with SL3261 (pBRD026) developed substantial levels of IgG and IgA anti-LT-B antibodies 14 days post-infection in both serum and gut samples. The sera neutralized the effects of LT in an in vitro Vero cell assay. Thus, aroA mutants of S. typhimurium can deliver a heterologous antigen from a different enteric pathogen to the murine systemic and secretory immune systems without altering their efficacy against salmonellosis.

Construction and characterization of deletion mutants of pseudorabies virus: a new generation of 'live' vaccines.

Various deletions were introduced into a cloned subgenomic fragment (BamHI-7), located in the unique short (US) region of the DNA from the virulent Northern Ireland Aujeszky-3 (NIA-3) strain of pseudorabies virus (PRV). In the cloned HindIII-B fragment, the MluI-BglII fragment was replaced by different MluI-BglII fragments of the deleted BamHI-7 clones. Transfection of the deleted HindIII-B fragments together with the HindIII-A fragment of either the NIA-3 or the non-virulent NIA-4 strain yielded replication-competent deletion mutants. The region in US in which sequences were deleted specified several mRNAs. Some of the mRNAs present in cells infected with NIA-3 were absent from cells infected with the deletion mutants, whereas other differently sized mRNAs were generated. The mutants were examined with respect to their biological properties in cell culture, mice and pigs. The results showed that the type of cytopathic effect induced in cell culture seemed to be determined by the UL region, using the mean time to death in mice as a parameter, markers for virulence were present in the US and UL regions and the introduction of deletions in US strongly reduced the virulence of PRV for pigs. Despite the impaired capacity of the deletion mutants to induce high titres of neutralizing antibodies in the serum, inoculation with mutants derived from NIA-3 prevented clinical disease in pigs upon challenge with the virulent parent strain. These deletion mutants provide a good basis for the production of bioengineered live PRV vaccines.

Vaccine potential of a live avirulent herpes simplex virus

We previously described a herpes simplex intertypic recombinant that is completely and specifically non-neurovirulent following intracranial inoculation. 1, 2 In this report the vaccine potential of this mutant (RE6) is analyzed. It is shown that RE6 replicates to wild type levels at the peripheral site of inoculation (the mouse footpad) but, unlike its virulent parental strains, does not replicate to detectable levels in lumbosacral ganglia. A single vaccination with RE6 is sufficient to completely protect mice against subsequent challenge with either HSV-1 or HSV-2. However, this agent establishes latent infections efficiently, and is fully neurovirulent for newborn mice.

Immunochemical characterization of pyrimidine kinase induced by varicella-zoster virus.

Thymidine kinase (TK) induced by varicella-zoster virus (VZV) was precipitated with ammonium sulphate and purified by Sephadex G-150, QAE-Sephadex and Blue Sepharose column chromatographies. The purified TK fraction also contained deoxycytidine kinase (dCK) activity and a 35000 mol. wt. (35K) polypeptide as a major component. The TK and dCK activities were both neutralized by anti-VZV serum. Antiserum to an extract of cells infected with a bromodeoxyuridine (BUdR)-resistant mutant virus contained no antibody to the viral TK and dCK activities or to the 35K polypeptide. Antiserum to the purified viral TK fraction was prepared and absorbed with a lysate of BUdR-resistant mutant virus-infected cells. The resulting absorbed antiserum (anti-vTK serum) neutralized the viral activities of both TK and dCK, and specifically immunoprecipitated a 35K polypeptide from the lysate of parental virus-infected cells, but did not immunoprecipitate any detectable polypeptide from cells infected with BUdR-resistant mutant virus. Anti-vTK serum stained mainly the nuclei of cells infected with the parental virus strain, but did not stain those infected with BUdR-resistant mutant virus by an indirect fluorescent antibody test. These results suggest that the 35K polypeptide produced in VZV-infected cells is responsible for the viral TK and dCK activities, and that the TK and dCK are mainly localized in the nuclei of infected cells.

Interaction of Pseudomonas solanacearum with Suspension-Cultured Tobacco Cells and Tobacco Leaf Cell Walls In Vitro

Attachment of radiolabeled Pseudomonas solanacearum cells to suspension-cultured tobacco cells and tobacco leaf cell walls was measured in vitro by a filtration technique that allowed separation of attached and unattached bacteria. An avirulent strain (B1) attached more rapidly to suspension-cultured cells than did the virulent parent strain (K60), and B1 attachment was less sensitive to inhibition by high ionic strength than was K60. Attachment of B1 bacteria to suspension-cultured cells and to leaf cell walls was comparable (50 to 70%), but only a small proportion (10 to 20%) of K60 bacteria attached to leaf cell walls under optimal conditions. With high bacterial populations (10 bacteria per ml), attachment of K60 to suspension-cultured cells was greatly reduced. Attachment of both strains was completely inhibited by pretreating bacterial cells with heat (41 degrees C) or azide and was partially inhibited by EDTA and kanamycin. The mechanism of attachment is not known, but ionic forces may be involved.

Canine distemper infection in mice: characterization of a neuro-adapted virus strain and its long-term evolution in the mouse.

The Onderstepoort strain of canine distemper virus (CDV) which has been adapted to newborn Swiss mice was used in a study in weanling mice. Intracerebral inoculation of newborn mice with either the parent or mouse-adapted virus strain led to mortality in 100% of the animals. In weanling mice the parent virus produced less than 10% mortality, whereas the mouse-adapted strain killed approximately 40% of the animals and this was not dose-dependent. Although a meningitis was observed in the surviving mice, the occurrence of viral antigens was less widespread in the brain of weanling mice than in the brain of newborn mice, and could not be detected more than 5 months after infection. In long-term experiments two phenomena were observed. At 4 to 6 months post-infection up to 30% of the mice became obese; however, no viral antigens were detected in the brains. At 13 to 17 months post-infection a number of mice became paralysed and virus antigen could be detected in the brain and lymph glands; however, infectious virus could not be isolated. These observations are discussed in relation to neurological infection and metabolic disease.

Immunogenicity of a low-virulence vaccinal strain against Salmonella abortus-ovis infection in mice.

Strain Rv6, a streptomycin-independent reverse mutant of Salmonella abortus-ovis, multiplied and disseminated in vivo to a lesser extent than the virulent parental strain 15/5, as determined by subcutaneous inoculation of two outbred strains of mice with different susceptibilities to salmonella infection. This lower virulence of strain Rv6 was not modified by 10 passages in susceptible mice. Vaccination with strain Rv6 induced a dose-dependent capacity of mice to control subsequent infection with the virulent parental strain. Induced resistance began as early as 3 days after vaccination and lasted at least 11 months. Its efficiency was controlled by different routes and doses of challenge. Strain Rv6 induced a greater protection than that conferred by heat-killed bacteria or by tested commercial killed vaccines.

Complete nucleotide sequence of the attenuated poliovirus Sabin 1 strain genome.

The complete nucleotide sequence of the genome of the type 1 poliovirus vaccine strain (LSc,2ab) was determined by using molecular cloning and rapid sequence analysis techniques. The restriction fragments of double-stranded cDNA synthesized from the vaccine strain RNA were inserted into the adequate sites of cloning vector pBR322. Sequence analysis of the cloned DNAs revealed that the virion RNA molecule was 7,441 nucleotides long and polyadenylylated at the 3' terminus. When the nucleotide sequence was compared with that of the genome of the virulent parental strain (Mahoney), 57 base substitutions were observed to be scattered all over the genome. Of these, 21 resulted in amino acid changes in a number of viral proteins. A cluster of amino acid changes is located in the viral coat proteins, especially in the NH2-terminal half of the viral capsid protein VP1. These results may imply that the mutations in the VP1 coding region contribute to attenuation.

Relationships among virus spread, cytopathogenicity, and virulence as revealed by the noncytopathic mutants of Newcastle disease virus.

We have studied protein synthesis in cultured cells infected with the six noncytopathic (nc) mutants of the Australia-Victoria strain (AV-WT) of Newcastle disease virus and their plaque-forming revertants. Virus-specific polypeptides accumulated at 30 to 63% of wild-type levels in nc mutant-infected cells and between 66 and 175% of wild-type levels in revertant-infected cells. An exception was the L polypeptide, which accumulated in nc mutant-infected cells at only 5 to 20% of the levels found in wild-type infection. The reduced accumulation of the L polypeptide did not appear to be due to increased degradation of that polypeptide. A new polypeptide (X) accumulated instead of polypeptide P in cells infected with mutants nc4 or nc16 and in virions released from them. Peptide mapping identified X as an altered form of P. A revertant of mutant nc4 (nc4S1), which forms larger hemadsorbing spots, but still does not form plaques, accumulated P instead of the X polypeptide. Thus, a lesion in P can affect virus spread without affecting cytopathogenicity. Virions of mutant nc7 and two naturally occurring avirulent strains of Newcastle disease virus (NJ LaSota and B1-Hitchner) contained polypeptides (F(7) and F(A), respectively) related to, but migrating more rapidly than, F(0) in sodium dodecyl sulfate-polyacrylamide gels. As previously reported for avirulent strains, a brief treatment of nc7 virions with trypsin converted F(7) to F and increased infectivity. Similarly, culturing nc7-infected cells in the presence of trypsin facilitated fusion from within and viral spread from cell to cell. A plaque-forming revertant of nc7 still accumulated F(7) in virions, indicating that the lesions responsible for the F(7) and noncytopathic phenotypes are genetically separable. The virulent parental strain, AV-WT, exhibited a mean embryo death time of 42 h. Both the larger-spot-forming revertant of nc4 (nc4S1) and the small-plaque-forming revertant of nc7 exhibited a decrease in mean embryo death time (increase in virulence) from 74 to 63 h. A second-step, plaque-forming revertant derived from nc4S1 (nc4S1R1) exhibited a further decrease in mean embryo death time from 63 to 44 h. The results suggest that the F(A)-F(7) and X lesions affect the ability of virus to spread from cell to cell. In addition, these lesions appear to be genetically separable from those responsible for the noncytopathic phenotype. However, both types of lesions cause an extension of mean embryo death time and, thus, may be relevant to virulence in vivo.

Vaccinal properties of Salmonella abortus ovis mutants for streptomycin: screening with a murine model.

Streptomycin-dependent and streptomycin-independent reverse mutants of Salmonella abortus ovis were selected on medium with and without added streptomycin. Their in vivo properties were tested with a murine model. Reduced capacities of dissemination and multiplication of all mutant strains were evidenced by means of subcutaneous, intraperitoneal, and intravenous inoculations of two outbred strains of mice with different susceptibilities to experimental S. abortus ovis infection. A factorial immunization experiment demonstrated the effects of vaccinal strains and doses. It showed large differences among the mutants. First-time infection with the fully virulent parent strain excepted, the best protection was given by independent reverse mutants which had kept residual capacity of in vivo dissemination and multiplication. This screening partly determined the selection of strain Rv6 as a potential live vaccine.

The amantadine-sensitivity of recombinant and parental influenza virus strains.

Several wild-type influenza A strains together with recombinants derived from these strains, were tested for sensitivity to amantadine using the in vitro techniques of inhibition in egg-bit culture and plaque reduction in MDCK cells. The results obtained were analysed with reference to the derivation of the recombinants. Susceptibility to amantadine was related to the gene coding for matrix protein, and these data are in agreement with previous reports of studies using other series of influenza viruses.

Characterization of hemadsorption-negative mutants of Mycoplasma pneumoniae.

Previously isolated mutants of Mycoplasma pneumoniae incapable of hemadsorption were characterized with respect to specific protein content, tracheal ring attachment capability, and virulence for both in vitro and in vivo model systems. Two-dimensional gel electrophoresis revealed both quantitative and qualitative differences between the protein complements of two different mutant strains and that of the virulent parent strain. Studies of mycoplasma attachment to hamster tracheal rings in vitro demonstrated that only one of these mutant strains still possessed the ability to attach to the respiratory epithelium via neuraminidase-sensitive receptors. Measurement of [3H]orotic acid uptake in mycoplasma-infected tracheal rings indicated that infection with the hemadsorption-negative mutants resulted in only slight reductions of ribonucleic acid synthesis, similar to levels observed for tracheal rings infected with an avirulent strain of M. pneumoniae. The virulence potential of the two mutant strains was further investigated by utilizing the hamster model system. Both mutant strains were rapidly cleared from the lungs of infected animals and produced little or no microscopic pneumonia.

Growth of influenza A viruses in hamsters.

The growth characteristics of four temperature-sensitive or cold-adapted recombinant influenza viruses and eight recombinant influenza viruses derived in other ways, together with the wild-type, parent viruses of these strains, were tested in hamster lungs and turbinates and in embryonated eggs at different temperatures for their replicative ability. The results showed that although the temperature-sensitive and cold-adapted recombinant viruses replicated to considerably lower titres than their wild-type parent virus strains in hamster lung and at 37 degrees C in embryonated eggs, no similar pattern of growth was observed for the group of A/PR 8 and A/Okuda recombinant influenza viruses studied in these systems. The hamster model is not therefore generally applicable as a marker for attenuated influenza virus vaccine strains.

Attenuated Live Fowl Cholera Vaccine I. Development of Vaccine Strain M3G of Pasteurella multocida

A live cholera vaccine was developed from a virulent avian septicemia strain of Pasteurella multocida serotype 1. The virulent parental strain was mutagenized with N-methyl-N'-nitro-N-nitroso guanidine. Mutants were selected that had either smaller colonies at 37 C or temperature sensitivity for growth at 41 C. Four small-colony mutants and 2 temperature-sensitive mutants were studied. All the mutants were avirulent for turkeys. Sixteen days after turkeys were vaccinated with each mutant, both the vaccinates and unvaccinated controls were challenge-exposed to virulent P. multocida of the homologous serotype and the heterologous serotype 3. Two of the small-colony mutant strains protected against both homologous and heterologous challenge. Suggested for a live cholera vaccine is P. multocida M3G, a small-colony-forming mutant, innocuous for both mice and turkeys and stable against reversion.

Correlation of pathogenicity and gene constellation of influenza A viruses II. Highly neurovirulent recombinants derived from non-neurovirulent or weakly neurovirulent parent virus strains

Mixed infections with various nonneurovirulent or weakly neurovirulent influenza A strains yielded recombinants that were highly neurovirulent for mice. A correlation was detected between gene constellation and neurovirulence of these recombinants. For the recombination pair FPV/A2-England the Pol 1 and Ptra genes had to be derived from the human strain; while the HA and/or M gene has to be from FPV in order to obtain highly neurovirulent recombinants. For the FPV/PR8 pair only the Pol 1 gene needs to be derived from PR8 while the HA gene had to be from FPV in order to get highly neurovirulent recombinants. The derivation of the other genes does not appear to be important in this respect. Recombinants between FPV and the A2-Singapore influenza strain do not exhibit significant neurovirulence suggesting that at least one parent strain should be adapted to grow in mice in order to obtain neurovirulent recombinants. In addition, there is a correlation between pneumovirulence and growth in mouse kidney cells, but neurovirulence for mice and pathogenicity for chickens was not correlated. The data presented here demonstrate in principle the possiblity of an increase in pathogenicity in recombinants derived from less or nonpathogenic parent viruses.

Plasmid Content and Tumor Initiation Complementation by Agrobacterium tumefaciens IIBNV6

Avirulent strains IIBNV6 and NT1, derived from virulent strains of Agrobacterium tumefaciens, were tested for their ability to enhance tumor initiation (complement) on coinoculation with tumorigenic strains. Strain NT1, cured of the Agrobacterium virulence plasmid, failed to complement when inoculated with its virulent parental strain or with other virulent strains. Strain IIBNV6, however, complemented with all virulent strains tested. Attachment to host wound sites by both strain IIBNV6 and the virulent strain was essential for this effect. Inoculation of the tumorigenic strain at different times on leaves previously inoculated with IIBNV6 showed that the capacity to complement is lost during the period between 4 and 8 h after IIBNV6 inoculation. The rate of tumor appearance obtained with an inoculum containing IIBNV6 and a virulent auxotrophic strain was characteristic of the appearance rate obtained with prototrophic bacteria. Evidence is summarized which suggests that strain IIBNV6 can induce tumors when supplied with a substance produced or induced by a virulent bacterium at a separate site. A deoxyribonucleic acid plasmid about 40% the size of the Agrobacterium virulence plasmid was obtained from strain IIBNV6. We propose that this plasmid accounts for the ability of strain IIBNV6 to complement and that it contains part of the genetic information necessary for tumor initiation.

Ultrastructural Localization of Sucrases in Streptococcus mutans GS-5 and an Extracellular Polysaccharide Mutant: a Comparative Cytochemical and Immunocytochemical Study

Electron microscopy and cytochemical and immunocytochemical procedures were used to study the ultrastructural distribution of sucrase enzymes in two strains of Streptococcus mutans. In a strongly adherent and virulent parent strain, GS-5, most of the invertase and fructosyltransferase activities were demonstrated extracellularly or bound to the cell surfaces. Intracellularly, enzymatic sites were detected near the plasma membrane on the periphery of the nucleoid and central mesosome. In GS-511, a mutant of diminished virulence and adherence, most of the enzymatic activity was not located on the cell surfaces, but was found away from the cell walls and associated with extracellular polysaccharides. Intracellularly, GS-511 manifested the same distribution of invertase and fructosyltransferase as did GS-5; however, the close association of these enzymes with the plasma membrane was not shown in GS-511. In both strains, extracellular areas near regions associated with cross wall formation appeared to show localized concentrations of these sucrases. Antibodies against partially purified glucosyltransferase (GTF) enzymes from GS-5 were used to localize GTF by immunocytochemical techniques. Indirect ferritin localization procedures showed that the extracellular and cell-bound GTF enzymes were distributed in similar locations as the fructosyltransferase and invertase enzymes. By absorption of the antiserum with whole GS-511 cells, the location of extracellular GTF and surface antigens unique to GS-5 was demonstrated. The dramatically reduced levels of cell-bound sucrase activity in GS-511 indicates the significant role of these enzymes in adherence and cariogenicity.

An attempt to cultivate mycobacteria in simple synthetic liquid medium containing lecithin-cholesterol liposomes.

An attempt was made to cultivate mycobacteria in a simple synthetic liquid medium containing lecithin-cholesterol liposomes. This lipid complex showed a marked growth-promoting effect on the submerged growth of M. tuberculosis and M. bovis. The role of lecithin as nutrient was suggested. The bacillary growth in such environment retained good viability, strong acid-fastness, and high virulence in mice. An avirulent strain of tubercle bacilli, H37Ra, did not respond to lecithin-cholesterol liposomes unlike the parent virulent strain, H37Rv. However, this was not a general rule for virulence, as a highly virulent strain of M. bovis (Ravenel) and an attenuated strain (BCG) both grew well in the presence of lipsomes. Lipid analysis showed that cholesterol in the liposome medium was esterified to some extent during the bacterial growth. It was discussed that the culture in the liposome-containing medium may present an experimental model for the study of interaction between mycobacteria and the macrophage membrane.

BORIC ACID TOLERANT VIBRIO CHOLERAE: BIOLOGICAL AND PHYSICAL PROPERTIES

Two boric acid tolerant variants were developed by passage in boric acid containing media from a mouse virulent parent Vibrio cholerae strain. The variants were stable, exhibited increased acid resistance, were less virulent to mice, but protected against challenge with the mouse virulent parent strain.