- Volume 37, Issue 6, 1992

Serological evidence of infection with verotoxin-producing Escherichia coli (VTEC) was sought in 28 patients suffering from haemolytic uraemic syndrome (HUS) and 25 age- and sex-matched controls. ELISA was used to detect anti-lipopolysaccharide (LPS) antibodies to E. coli strains O157, O111, O26 and NCTC 10418, a non-VTEC strain, and Shigella dysenteriae O1. Sera from 19 of the HUS patients but from none of the 25 controls had significant antibody levels to the verotoxin-producing bacteria. Sera from 13 patients reacted with only one LPS of the four verotoxin-producing bacteria; sera from six reacted with more than one LPS antigen but not with LPS of E. coli NCTC 10418. Paired sera taken 2–3 weeks apart were obtained from 20 HUS patients; 14 of these had high levels of antibody in the acute phase sample. Analysis of antibody levels in the convalescent sera showed that one patient had an increase, one was unchanged and 12 patients had a decrease in antibody to the verotoxin-producing bacteria.

Extracellular high-mol.-wt material was separated from liquid cultures of Staphylococcus epidermidis. This material contained protein c. 20% w/w and polysaccharide c. 80% w/w. The polysaccharide was isolated by gel and ion-exchange chromatography and contained glycerol phosphate, glucose, N-acetylglucosamine, and d-alanine. Cell-wall teichoic acid was isolated from strain RP-62A and had a similar composition.

A protein (gal-FnBP), constructed by fusion of the genes encoding β-galactosidase of Escherichia coli and the binding domains of fibronectin-binding protein (FnBP) of Staphylococcus aureus was used. FnBP is a surface protein responsible for attachment of bacteria to extracellular matrix of various host tissues. Gal-FnBP is more stable and can be produced in larger quantities than native FnBP. The binding specificity of this fusion protein was established in a Western blot analysis. Treatment of gal-FnBP with formalin inactivated the binding capacity of the protein but immunogenicity was retained. Immunisation of mice with formalin-treated gal-FnBP resulted in high antibody titres against the fibronectin-binding part of this fusion protein. These antibodies were measured by their ability to block the specific binding of fibronectin to gal-FnBP in a blocking assay. Sera raised against formalin-treated gal-FnBP and non-treated gal-FnBP blocked this binding to 40 and 25% respectively, thereby indicating the usefulness of gal-FnBP as a vaccine component.

Plesiomonas shigelloides, an organism commonly found in water, is only rarely associated with diarrhoea in man. P. shigelloides serotype O:17 (SVC O1), which is antigenically similar to Shigella sonnei, was found to be neither virulent nor toxic for rabbits. Rabbits immunised by feeding with P. shigelloides (SVC O1) were completely protected against an oral challenge with 1010 cells of S. sonnei but non-immunised rabbits were not. P. shigelloides (SVC O1) may be a useful vaccine strain for shigellosis.

The analysis of ribosomal RNA (rRNA) gene patterns (ribotyping) has been used to differentiate strains within bacterial species. We used this method to investigate two outbreaks of campylobacter enteritis that occurred recently in Melbourne, Australia. The first outbreak involved seven patients although isolates from only five patients were available for typing. The second outbreak consisted of three patients infected with human immunodeficiency virus (HIV) on the same ward of a hospital. Analysis of the rRNA gene patterns revealed identical patterns for the isolates from five patients in the first outbreak, suggesting that these isolates were from the same source. However, ribotyping of the four isolates from the second outbreak showed three distinct ribotypes indicative of contact with unrelated sources. This study demonstrated that ribotyping is a useful, reliable and convenient typing scheme for epidemiological purposes.

Cytotoxic activity of culture supernates of Helicobacter pylori is manifested by vacuolation of mammalian cells in vitro. The formation and maturation of toxin-induced vacuoles in HeLa cells has been studied to examine the possibility that they are autophagosomal in nature. Observation by light microscopy revealed that vacuoles originate in a perinuclear position, increasing in number and size until cell degeneration and lysis occur after 48 h. Ultrastructural study of mature vacuoles indicated the presence of a bounding membrane with contents consisting of degenerate cytoplasmic components and acid phosphatase activity. Confocal fluorescence imaging demonstrated acridine orange accumulation in the vacuoles of toxin-treated cells, indicating an acidic intravacuolar pH. These features are characteristic of autophagosomes. In addition, the size of vacuoles in living, acridine orange-stained cells tended to be inversely proportional to fluorescence intensity. Fluid phase endocytic markers were observed only rarely within nascent vacuoles. Over the succeeding 24 h, labelling of most vacuoles with these dyes was observed. This, along with the observation of intravacuolar acid phosphatase activity, provides evidence that vacuoles communicate at some point during their development with endocytically derived compartments. These observations provide direct evidence for an autophagic origin of these structures.

Micro-enzyme-linked immunosorbent assay (Micro-ELISA) systems were developed and evaluated for the detection of circulating (free or immune-complexed) hydatid antigens in the sera of patients with hydatidosis, by employing monospecific antibodies to hydatid-specific antigens of 8-kDa and 116-kDa. Fifteen (75%) of 20 sera from patients with hydatidosis had both 8-kDa and 116-kDa antigens freely circulating in their sera while three and two samples, respectively, had only 8-kDa or 116-kDa antigen. All the surgically confirmed cases of hydatidosis had detectable levels of both 8-kDa and 116-kDa circulating immune complexes in glycine HCl-treated sera. However, none of the sera from control subjects (patients with cysticercosis, ascariasis, ancylostomiasis, hymenolepiasis, amoebic liver abscess or viral hepatitis) had any detectable level of either type of circulating specific antigen. These results suggest that the demonstration of either 8- or 116-kDa antigen(s) in free or immune-complex form could confirm the diagnosis of hydatidosis.

An evaluation of five ELISA methods for the diagnosis of acute toxoplasmosis was undertaken by comparing a laboratory-produced ELISA employing two recombinant Toxoplasma gondii polypeptides as antigen with a laboratory-produced ELISA and three commercially available ELISAs employing traditional parasite antigen preparations derived from whole tachyzoites. With a panel of 75 sera from patients who showed either serological and clinical evidence of acute toxoplasmosis, or of diseases caused by other infectious agents, or from patients who showed no evidence of infectious disease, the ELISAs gave overall positive predictive values of 81.6–100%, negative predictive values of 87.8–100%, sensitivities of 81.3–100%, and specificities of 83.7–100%. No two ELISAs gave identical results with all sera tested. In total, the ELISA based on the two recombinant T. gondii polypeptides appeared to be the most specific ELISA in this comparison, showing positive predictive values and specificities of 100% for all groups of patients tested. The overall negative predictive value for this ELISA was 87.8% and the sensitivity was 81.3%. Therefore, the ELISA based on recombinant antigen appears to be a promising advance in the serological diagnosis of acute toxoplasmosis.

Blood cultures were taken from 47 patients 1–2 min after dental extraction. These samples were tested by the radiometric Bactec 460 and Oxoid Signal systems for the detection of streptococcal and anaerobic bacteraemias. Streptococci were isolated from 19 (40%) patients and anaerobes from 15 (32%). In this study the Oxoid Signal system was significantly better for isolating oral anaerobes than the Bactec system; five isolates were obtained with the Bactec system and 14 with the Signal system. There was no significant difference in the isolation of streptococci between these two systems (Bactec 14, Oxoid Signal 10). The contamination rate was 4.1% for Bactec and 7.5% for the Oxoid Signal system.

Chancroid is a sexually transmitted diseased caused by A. Haemophilus ducreyi. The pathological manifestations of chancroid are unique among Haemophilus species and the virulence factors of H. ducreyi that account for these features have not been identified. Some of these virulence factors may be unique components of H. ducreyi, but attempts to identify H. ducreyi-specific components have been unsuccessful. Four polypeptides—A, B, C and D of 83, 77, 56 and 28 kDa, respectively—were identified with a panel of nine H. ducreyi-specific monoclonal antibodies (MAbs). Polypeptide C was one of the five major proteins in H. ducreyi and demonstrated micro-heterogeneity in SDS-PAGE. Polypeptides A, B and D were present in only small amounts in whole-cell lysates of H. ducreyi. The relative amounts of A and B varied, suggesting that they may be precursor molecules. The unique polypeptides C and D were not exposed on the surface. Polypeptide C was highly soluble and did not appear to be membrane-bound, whereas polypeptide D appeared to partition with the cytoplasmic membrane and was soluble in Sarkosyl. All four polypeptides appeared to be unique to H. ducreyi since MAbs directed against them did not cross-react with H. influenzae, H. parainfluenzae or Neisseria gonorrhoeae. The mol. wts of all of these polypeptides were conserved throughout 35 clinical isolates collected from 15 cities in eight countries and one reference strain of H. ducreyi that were tested. Based on the immune cross-reactivities, it was concluded that the four polypeptides belonged to a family of proteins with partial sequence homology that may have been derived from the same precursor polypeptide or the same transcription unit. A sequence relationship model for the polypeptides was proposed based on their cross-reactivities with the MAbs.

When whole cells (stationary phase) of Yersinia pestis strain EV76 were radiolabelled with Iodogen and 131I, 16 major and 10 minor surface-exposed outer membrane proteins (OMPs) were identified. Labelling with N-hydroxysuccinimidyl 6-biotinylamino-hexanoate (biotin X-NHS) resulted in a complex protein profile detectable after blotting and developing with peroxidase-conjugated avidin. Y. pestis cell fractionation revealed that biotin X-NHS labelled not only OMPs but also proteins of inner cell compartments. Therefore, radiolabelling was the more reliable technique for identifying the OMPs of Y. pestis.