- Volume 24, Issue 1, 1987

Mice immunised by the oral or intraperitoneal route with a live aromatic-dependent strain of Salmonella typhimurium exhibited significantly less protection against oral challenge with 50 LD50 of an ovine isolate of S. typhimurium (12313) than when a bovine isolate with the same O antigens and phage-type as strain 12313 was used as the challenge organism. When challenged with 10 LD50, however, protection against both strains was significantly better than that obtained when mice were vaccinated with killed vaccines (heat-killed, acetone-killed or irradiated) even when the antigenic mass of the killed vaccine was increased by up to 500-fold in an attempt to compensate for the expected limited multiplication of the mutant organism. Sheep immunised with the live mutant strain by either the intramuscular or oral route were protected against oral challenge with the virulent ovine isolate of S. typhimurium; unimmunised sheep died of acute enteritis within 7 days, although there was no evidence of systemic invasion by the challenge organism. After challenge, immunised animals ate more food than the unimmunised controls and suffered only transient, mild diarrhoea. Serum antibody titres against O and H antigens measured by direct or antiglobulin tests were significantly higher in sheep immunised by the intramuscular route than in those immunised orally. Sheep in both immunised groups developed skin swellings within 30 min after intradermal inoculation with purified homologous lipopolysaccharide indicating development of immediate-type hypersensitivity, but only those immunised by the intramuscular route showed significant indurated skin swellings characteristic of delayed-type hypersensitivity 48 and 72 h post-inoculation. Thus it appears that protection against salmonellosis in sheep immunised by the intramuscular or the oral route may involve different mechanisms.

Antigenic relationships among type-1 fimbriae of 40 strains of Enterobacteriaceae representing 19 species and seven genera were determined by immuno-electronmicroscopy. Ten distinct antigenic groups were distinguished. Intra- and inter-generic relationships were observed although some antigenic groups were species-specific only. Antigenic relationships among type-1 fimbriae are more complex than have been reported hitherto.

The immunological responses of rabbits after intra-duodenal immunisation with live Vibrio cholerae organisms were studied in various body fluids. Serum, bile and intestinal samples were collected from rabbits at different times (1–8 weeks) after immunisation. Three different extracts from the small intestine were prepared. At first the small intestine was washed with saline (method A). Later, ultrasonic lysates were prepared from epithelial cells separated with citrate (method B) and of mucosal tissue above the muscularis layer (method C). All had agglutinating activities against V. cholerae strains belonging to both biotypes (classical and el tor) and both serotypes (Ogawa and Inaba). Levels in intestinal extracts, sera and bile of antibodies to somatic (lipopolysaccharide and cell-surface proteins) and secreted (cholera toxin and neuraminidase) antigens of V. cholerae were determined by an enzyme-linked immunosorbent assay. All contained antibodies to these antigens; although both IgA and IgG were present, IgA predominated. Serum and bile samples contained mainly IgG and IgA respectively. Immunoblotting studies demonstrated that the antisera contained antibodies to most cell-surface proteins and to cholera toxin. Cell-surface proteins appeared to be the major cross-reacting somatic antigens of V. cholerae.

Rabbits were solidly immunised by parenteral injection of purified Clostridium difficile toxin A such that they resisted an intravenous challenge with a normally lethal dose of toxin A. Ileal and colonic loops constructed in non-immune and immune animals received challenge injections of crude culture filtrate or purified toxin A of C. difficile. Protection of ileum was manifest after sufficient initial mucosal damage resulted in release of high levels of antitoxin A into the loop lumen of immune animals. There was less fluid accumulation in ligated ileal loops of immune than of non-immune rabbits. Less protection was observed when loops were challenged with crude culture filtrate containing toxins A and B than when challenged with purified toxin A. In-vitro studies with Ussing chambers yielded no evidence for tissue-localised immunity as judged by electrical responses and histology of toxin-treated tissue from non-immune and immune animals. No differences were found in the degree of epithelial damage, or volume or composition of fluid accumulating in colonic loops of non-immune and immune rabbits challenged with toxin A or crude culture filtrate. However, in colonic loops of immune rabbits there was no overt tissue-localised haemorrhage, whereas in those of non-immune rabbits tissue-localised haemorrhage was marked. In contrast to our findings with ileal loops, fluid accumulating in colonic loops was watery and contained substantially less total protein and (in immune animals) antitoxin A.

Each of nine different toxigenic strains of Clostridium difficile was administered orally to groups of hamsters pre-treated with clindamycin and housed individually in sterile isolator boxes. Faecal pellets and caecal contents from well, diarrhoeic, moribund and freshly dead animals were analysed for C. difficile and toxins A (enterotoxin) and B (cytotoxin), and tissue obtained when animals were killed was examined histologically. Not all strains were equally virulent in this model. Four strains of C. difficile killed all animals within 48 h and are designated as highly virulent for hamsters. These strains were clinical isolates from three cases of disease in man and one case in a hamster. Five strains caused death of some animals but only after 5 and up to 13 days and are designated as less virulent for hamsters. These strains were isolated from asymptomatic infants (2) and household pets (2), and from the environment (1). The surviving test hamsters were killed after 14 days and, in most cases, were colonised by C. difficile, though levels of toxins A and B in caecal contents were low. None of the cultures used for challenge was capsulate or hydrophobic. There was no correlation between virulence and production of toxins A and B in vitro in tryptic-nitrate broth. With two strains examined, there was a correlation between virulence and toxin A (but not toxin B) production in caecal emulsions derived from clindamycin pre-treated hamsters. Caecal contents from the majority of moribund and freshly dead animals had quantities of toxin A sufficient to cause disease or death if given orogastrically. Toxin B was not produced in a fixed ratio with toxin A. The data support the view that high virulence of C. difficile is determined by efficient disease-inducing colonisation of the gut and the ability to generate, rapidly, high levels of toxin A in vivo.

The hydrophobicity–hydrophilicity of various strains of Staphylococcus has been studied by a technique involving partitioning of the cells between aqueous and hydrocarbon phases. S. aureus was typically hydrophobic, and to a greater degree in stationary-than in exponential-phase cultures. Mutants that lacked teichoic acid, protein A or coagulase production were hydrophobic, indicating that none of these factors was responsible for hydrophobicity. The presence of a capsule rendered strains hydrophilic. Thus, determination of hydrophobicity may be a useful additional test for capsulation. However, a non-capsulate S. aureus strain was hydrophilic. Trypsin treatment converted strains from hydrophobic to hydrophilic. Isolated bacterial cell wall preparation, either crude or purified, and peptidoglycan were hydrophilic. These results indicate that the determinant of hydrophobicity is a protein or protein-associated molecule localised at the cell surface of the organism, i.e., a component of either the cell wall, cell membrane, or both. Twenty-five strains of twelve coagulase-negative species were examined and most (18) were hydrophobic, again indicating that protein A is not a major determinant of hydrophobicity in these staphylococci. Four of seven hydrophilic strains were capsulate; three strains of S. sciuri were hydrophilic but non-capsulate.

The production of toxic shock syndrome toxin-1 (TSST-1) was studied in batch and continuous culture of Staphylococcus aureus strain 1169 in a carbohydrate-free chemically defined medium (CDM). In continuous culture oxygen- and arginine-limitation were required for steady-state TSST-1 synthesis. Aeration suppressed toxin synthesis. The amount of TSST-1 per mg dry weight (specific toxin) at dilution rates from 0·05 to 0·15 h–1 was inversely proportional to the dilution rate. Protease activity increased with increasing dilution rates. In batch culture, TSST-1 began to accumulate in the medium towards the end of the exponential phase of growth, after a critical cell mass was attained. Maximum specific toxin production was observed in medium with an initial pH between 6·5 and 7·0. Growth and toxin synthesis took place in anaerobic conditions when CDM was supplemented with pyruvate and uracil. The Mg++ concentration had no effect on the specific toxin in anaerobic conditions. In aerobic conditions, specific toxin increased c. 23-fold as the Mg++ concentrations increased to 0·4 mm. Further increases in the Mg++ concentration resulted in a reduction in specific toxin.

Group B streptococci produce both a pigment and a haemolysin. The requirements of group B streptococci for the formation and release of pigment and haemolysin are similar and have been examined to extend observations on the relationship between the two products. The amount of pigment and haemolysin extractable from actively metabolising washed-cell suspensions of group B streptococci varied with the atmosphere of incubation, the pH at which the extraction was carried out and the presence of Mg2+ ions. Both pigment and haemolysin were produced in significant amounts in all phases of the growth cycle. When conditions were established for obtaining maximum yields of haemolysin, its production correlated closely with pigment yields, but pigment did not function as a carrier for haemolysin. Formation of pigment, but not of haemolysin, increased in the presence of trimethoprim or higher concentrations of glucose. The composition of pigment produced in different conditions differed qualitatively and different strains of group B streptococci formed pigment of different appearance, suggesting that group B streptococcal pigment is composed of several different substances.

In a Limulus amoebocyte lysate micro-test system, visual differentiation between positive and negative reactions was obtained by the addition of a small drop of fresh, defibrinated sheep blood to the reaction mixture. This method has greatly facilitated end-point identification in a series of dilutions of endotoxin.