- Volume 25, Issue 2, 1988

The prevalence of antibody to human parvovirus B19 (anti-B19 IgG) in England and Wales was measured by an antibody-capture radioimmunoassay. Over 2000 sera were examined; 1422 from the general population, 374 from unselected children admitted to hospital and 300 from women attending an antenatal clinic. Waning levels of maternally-derived antibody were found in infants under 1 year old. In children 1–5 years old, 5–15% had anti-B19 IgG and this rose to 50–60% in older children, young adults and women of child-bearing age. In older people, the prevalence of anti-B19 IgG increased with age, rising to more than 85% in those over 70 years old.

Murine monoclonal antibodies and immune rabbit serum were raised against the rough mutant Salmonella minnesota strain R595. These antibodies were tested for their opsonic activity against the homologous strain and the smooth wild type S. minnesota by luminol-dependent chemiluminescence and a microscopic assessment of phagocytosis. Immune rabbit serum opsonised both strains. Treatment with normal rabbit serum inhibited the phagocytic uptake of S. minnesota R595. None of the monoclonal antibodies RE01 (anti-KDO), RE12 (anti-KDO) and RE23 (anti-lipid A) were opsonic. Unopsonised S. minnesota R595 stimulated marked chemiluminescence possibly because of its hydrophobic surface, but this was not reflected in increased uptake by phagocytic cells. Results obtained with luminoldependent chemiluminescence should be interpreted with caution when the opsonisation of rough bacterial strains or those with high surface hydrophobicity is being investigated.

The susceptibility of 76 clinical isolates of Enterobacteriaceae to ampicillin, piperacillin and gentamicin was assessed by ATP bioluminescence in a 4-h test. For most organisms tested (Escherichia, Klebsiella, Enterobacter and Serratia), there was good correlation with traditional MIC values estimated on 18-h cultures. However strains of Proteus mirabilis showed false resistance to the β-lactam agents with the ATP method; and concordance was achieved only after manipulation of the growth conditions. Our method is simpler than those described previously, though currently it is still labour-intensive and expensive.

Lysosomotropic weak bases impair in-vitro neutrophil functions including intracellular killing of Staphylococcus aureus strain 502a. To investigate whether prevention of phagosomal acidification could account for impaired microbicidal activity, a model phagosome was formulated with a freeze-thawed granule extract as a source of lysosomal enzymes and H2O2 as a source of toxic oxygen metabolites. The lysosomal extract alone killed Escherichia coli strain S15 efficiently at pH 5·5 and 7·0, but had little activity against S. aureus 502a. Sublethal concentrations of the two agents, when combined, acted synergically against either organism. Each organism was killed more effectively at pH 5·5 than at pH 7·0 by the lysosome extract-H2O2 combination, but the killing of E. coli was more rapid than that of S. aureus in the same conditions. These findings suggest that impairment of neutrophil antistaphylococcal activity by weak bases may be mediated by their ability to raise phagosomal pH, and that persistence of E. coli in similar conditions does not occur because the latter is killed by lysosomal constituents in a non-pH-dependent fashion.

The plasmid profiles of 112 clinical isolates of Pseudomonas aeruginosa were determined by two reproducible, rapid, plasmid screening methods. Plasmid DNA was present in 15% of isolates examined. Plasmids varied in size from 1·2 – 106 to 60·2 – 106 mol. wt. The dominant serotypes encountered were O:11 and O:4, which comprised 38% and 12% of strains, respectively. Four pyocin types (1, 10, 3 and 5) dominated (respective frequencies: 56, 15, 12 and 6%). Reproducibility of pyocin typing was distinctly inferior to both plasmid profiling and serotyping. Strains of identical serotypes could be further differentiated by dissimilar plasmid profiles. Serologically untypable or polyagglutinable strains were successfully characterised by plasmid profile patterns. Thus, plasmid profiling was shown to be a useful adjunct to serotyping for the epidemiological typing of P. aeruginosa.

Peritoneal polymorphonuclear neutrophils (PMN) from mice were tested for their ability to kill the yeast form of Blastomyces dermatitidis (Bd) in vitro and for their fungicidal mechanisms. PMN elicited from immune mice by the intraperitoneal injection of non-viable Bd (referred to as immunologically activated PMN or ActPMN) showed significantly enhanced fungicidal activity in comparison with PMN elicited with thioglycollate medium (ThioPMN) [means=44·7% (SD 12·8%) and 16·4% (SD 9·2%) killed; n= 14; p < 0·001]. Production of superoxide anion (O2 −) by ActPMN after stimulation with phorbol myristate acetate was enhanced in comparison with production by ThioPMN. Superoxide dismutase, which removes O2 −, inhibited ActPMN killing by 75% (p < 0·001) when added to cultures immediately before challenge with Bd (optimal concentration : 6000 U/ml). Sodium azide, which inhibits myeloperoxidase and scavenges singlet oxygen (1O2), and catalase, which breaks down hydrogen peroxide (H2O2), inhibited ActPMN killing by 64% (p < 0·001) and 52% (p < 0·001), with optimal concentrations of 1 mM and 10 000 U/ml, respectively. Two agents that both scavenge 1O2 and antagonise hypochlorous acid (HOCl−), histidine and tryptophan, were also powerful inhibitors of ActPMN killing. Quenchers of hydroxyl radical (·OH), dimethylsulfoxide and sodium benzoate, had less effect, and required higher concentrations. These data suggest that the enhanced killing of Bd by ActPMN involves one or more oxidative mechanisms, and that there is a prominent role for O2 −, either directly or as a precursor of other active oxygen species, a probable role for H2O2, and possible roles for 1O2, HOCl−, and · OH.

A partially-purified neuraminidase from the mucinase complex of Vibrio cholerae was used to prepare a specific anti-neuraminidase antiserum in rabbits. When the neutralising potency of this serum against V. cholerae neuraminidase was assessed in conventional tests, the enzymic activity, as measured by thiobarbituric acid, methoxyphenol-neuraminate and goblet-cell assays, apparently increased. These results are attributable to the presence of a sialylated glycoprotein substrate and small amounts of sialidase in the crude antiserum. However, a twice-purified DEAE-IgG fraction of the antiserum neutralised the enzymic activity of the V. cholerae neuraminidase.

Resistance to growth of Vibrio cholerae at the mucosa of blind intestinal loops developed in rats after intestinal or parenteral exposure to live organisms or other antigenic materials. Simultaneous serological studies suggested that neither serum vibriocidal activity nor intestinal mucus antibodies are likely to provide a direct test of antibacterial immune status. Challenge of rats 4 weeks after one dose of antigen may reveal a form of immunity that is not related to antibodies in the intestine and possibly is analogous to long-term immunity expressed in man following infection with the organism. This immunity has not been attributed to lipopolysaccharide (LPS) antigen and does not appear to involve flagella-associated antigens ; involvement of antigens other than LPS, such as protein antigens of the outer membranes of V. cholerae, has not yet been substantiated. Separation of monomeric sub-units of outer-membrane proteins by hydrophobic interaction high pressure liquid chromatography has revealed significant quantitative differences among preparations derived from the common serotypes of the organism. These differences may be sufficient to explain the better protection observed when homologous serotypes were used for immunisation and challenge in the long-term resistance model.

Six strains of Salmonella typhimurium (W118, TML, SL 1027, LT7, M206 and Thax 1) of different virulence were examined for the presence of antigens which react with antibodies to cholera toxin (anti-CT). A fluorescent-antibody-labelling technique employing anti-CT was used to analyse antigen expression. A rapid increase in the proportion of cells producing a CT-related antigen was demonstrated in cells in early log phase (1–4 h growth) followed by a rapid decline during mid-late log phase in each of the six strains. The nature of the CT-related antigen was analysed by immunoblotting using anti-CT. An antigen of mol. wt equivalent to a high-mol. wt species of CT B subunit was detected in polymyxin-B extracts of all strains but greater amounts were observed in the strains that we consider avirulent. Nothing equivalent to a CT A-related subunit was observed in any of the strains. The relatedness of the salmonella antigen to CT was limited. The high-mol. wt antigen was not disrupted in the denaturing conditions of SDS-PAGE; nothing was detected by enzyme-linked immunosorbent assays with either ganglioside or anti-CT as anchor.