- Volume 28, Issue 2, 1989

Cell walls were isolated from stationary-phase cultures of Candida albicans grown at 25°C or 37°C, in iron-depleted and iron-sufficient conditions. Proteins solubilised from cell-wall fractions were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Approximately 40 protein bands were detected by Coomassie blue staining in all wall extracts, regardless of temperature or other growth condition. Sera from patients with oral or systemic candidosis, from whom the isolates were obtained, and pooled normal human serum were examined for the presence of IgG and IgM antibodies to cell-wall proteins by Western blotting. Patient sera recognised more antigens than pooled normal human serum. In particular, an antigen of 44 kda was detected by IgG antibodies in the sera of patients and two antigens of 41 and 14 kda were detected by their IgM antibodies when the sera were used as probes against walls from iron-depleted cells, but not from iron-sufficient cells, grown at 25°C. Two antigens of 45 and 40 kda were detected by IgM antibodies in the sera of patients tested against walls from iron-depleted but not from iron-sufficient cells grown at 37°C. IgG antibodies did not distinguish between these wall preparations from cells grown at 37°C. These results suggest that the specific cell-wall proteins induced during growth in iron-depleted conditions, as well as other proteins, were immunogenic and were recognised by the patients’ antibodies.

Formalin-killed cells of Pseudomonas aeruginosa strain M-24 elicited an antibody-independent protective effect against P. aeruginosa infection in mice. The effect was observed as early as 6 h after administration and 100% protection was obtained by 48 h. The protective effect could not be attributed to the production of specific antibody. In M-24-treated mice, the bacteria in the peritoneal cavity, blood and liver were eliminated 12 h after P. aeruginosa infection. This suggested that the protective effect was due to enhanced bacterial elimination. The percentage of macrophages in the peritoneal cavity was increased after M-24 administration. Furthermore, the enhanced bacterial elimination was abrogated by treatment of mice with 60Coirradiation or carrageenan. These findings suggest the involvement of macrophages in the enhanced bacterial elimination observed. The chemiluminescence of peritoneal exudate cells from M-24-treated mice was markedly increased when compared with that of cells from untreated mice. The ability to kill P. aeruginosa in vitro was also greater in macrophages from mice treated with killed M-24 than in cells from proteose-peptone-treated mice. The M-24-treated mice showed enhanced nonspecific protection against infection with lethal doses of P. aeruginosa, Escherichia coli or Listeria monocytogenes. However, susceptibility to LPS in mice was not increased by M-24 treatment. These results suggest that macrophage activation without increasing LPS susceptibility was responsible for the antibody-independent protection induced by killed M-24.

The intracellular and extracellular protease and elastase contents of Pseudomonas aeruginosa were studied in relation to the age of the culture. The intracellular protease and elastase content of the organisms decreased as the culture grew older, whereas extracellular protease and elastase greatly increased 24 h after incubation commenced, suggesting that host tissue injury by P. aeruginosa infection may increase in proportion to the duration of tissue colonisation.

DNA in infected urines from 81 patients with urinary tract infection was hybridised directly with a non-radioactive DNA probe specific for bacterial genes coding for TEM-type β-lactamase. The results were assessed by means of a computerised image analysis system and compared with those obtained following isolation of the infecting organism, conventional sensitivity testing and isoelectric focusing (IEF) procedures for the detection of TEM-type β-lactamase. Of the 27 ampicillin-resistant gram-negative organisms isolated in pure culture from the urines, 14 were shown by both hybridisation and IEF to carry a gene for TEM β-lactamase production. Only four discordant results were obtained: three “false positive” direct hybridisation results, one due to urine pigmentation, and one, possibly, to a TEM β-lactamase gene which was not being expressed, and one “false negative” result due to insufficient cell numbers in the urine. The system is capable of screening large numbers of samples and is applicable to any gene for which a suitable DNA probe is available.

In the first study, genital carriage of Haemophilus parainfluenzae was investigated in 103 women and 292 men attending a clinic for genitourinary medicine. In a second study, pairs of urethral and throat swabs were studied in 279 men. The vaginal carriage was 2%, urethral 12.4% and throat 13.3%. Biotype 2 was found to be a genital type and throat carriage of this biotype was significantly associated with its concomitant urethral carriage. Biotypes 1 and 3 were mainly found in the throat. Biotype 2 was significantly more likely to be resistant to ampicillin, tetracycline and sulphonamide but biotype I was significantly more likely to be resistant to trimethoprim.

An IgM immunosorbent agglutination assay (ISAGA) was compared with a standard ELISA IgM test for the diagnosis of congenital toxoplasmosis. It was more sensitive, detecting all of five mothers of infected babies whereas the IgM ELISA was positive in two of three mothers tested at delivery and neither of two mothers referred 10–12 months after delivery. Five women infected in a previous pregnancy had IgM detectable by ISAGA in a subsequent pregnancy. The assays were comparable when sera from patients with past infection were tested or following toxoplasma-associated miscarriage or abortion. Four cord sera from congenitally-infected babies were positive by the ISAGA but only three of these were positive by ELISA for IgM. The ISAGA also detected IgM in another four sera from congenitally-infected babies referred late (10–18 months old); none were IgM positive by ELISA. The increased sensitivity of the ISAGA is an improvement in the diagnosis of congenital toxoplasmosis.

Staphylococcus aureus strain MC31 showed pseudodiffuse growth in serumsoft agar and reacted with immune rabbit sera to strains Smith diffuse (capsular type A), NS58D (type B) and NS41D (type C) but not with strain NS68D (type D) in serum-soft agar. Immunisation of mice with 300 μg of cell-surface polysaccharide extracted from strain MC31 protected against lethal infection by strain MC31 and the strains of capsular types A, B and C. Immune rabbit serum prepared against strain MC31 passively protected mice against challenge infection with the homologous strain, but approximately 30 times more anti-MC31 serum was required to protect against infection with the strains of capsular types A, B and C. Absorption of the passive protective activity of immune sera raised against the three capsular type strains required at least 10 times the quantity of MC31 cell-surface polysaccharide than the quantity of cell-surface polysaccharide from the homologous capsular strain. Electronmicrographs of strain MC31 treated with ferritin-labelled antisera to the three capsular strains showed only small amounts of ferritin granules around the cell wall.

A receptor binding the Fc region of equine immunoglobulin G (IgG) has been isolated from a heat-extracted preparation of a clinical isolate of Streptococcus zooepidemicus. This Fc receptor has a Mr of 45 x 103 and was occasionally seen as an apparent trimer of Mr 130 x 103. Antibodies prepared in horses against the receptor could be adsorbed to and eluted from whole live bacteria, confirming the surface location of this protein. Another 11 isolates of S. zooepidemicus from horses with pneumonia, abscesses or endometritis were tested for Fc-receptor activity. Although the Mr of the Fc receptors varied among isolates, their antigenicity was conserved. Thus, the Fc receptor is an attractive candidate for application in the diagnosis of, or protection against, infections with S. zooepidemicus.

The genes encoding two protein antigens of Leptospira interrogans serovar pomona were cloned and expressed in Escherichia coli. Rabbit antisera raised against the cloned proteins, designated p12 and p20, were used to identify the antigens in Western blots of disrupted leptospiral cells. The proteins p12 and p20 were conserved within the genus Leptospira and were not detected in Leptonema illini. Although both proteins were present in leptospiral outer envelope preparations they did not elicit the production of agglutinating or opsonising antibodies.

To evaluate the long-term recoverability of bacterial enteropathogens, two freezing conditions (deep-freezing at – 70°C and liquid nitrogen) and three preservation media (Cary-Blair, Amies, and buffered glycerol-saline) were tested. These were compared with storage in containers with no preservation medium and refrigeration at 4°C. At 4°C, viability of organisms could not be consistently maintained beyond one month; Cary-Blair medium generally gave the best results and storage without preservation medium was the least efficient. Storage in liquid nitrogen and deep-freezing effectively preserved all organisms except Campylobacter jejuni for the entire period of study (12 months). There was no difference between the various preservation media, or between storage with or without medium. Storage in preservation medium was superior to storage without such supplement for C. jejuni. We conclude that most enteropathogens survive in faecal specimens for as long as 12 months when stored at very low temperatures (-70°C) whether or not preservation media are used.