- Volume 27, Issue 4, 1988

A murine monoclonal antibody (MAB AF-1) class IgM was raised to a soluble glucomannoprotein extract (GMP) of Candida albicans. Agglutination and indirect immunofluorescence assays with purified MAB showed that AF-1 was directed against a cell-surface epitope shared by C. albicans serotypes A and B, C. tropicalis, C. guilliermondii and C. viswanathii, but not by C. krusei, C. parapsilosis, C. kefyr (pseudotropicalis) and T. glabrata. Treatment with heat, protease or periodatetreated GMP and with other cell-wall extracts of C. albicans provided evidence that the epitope recognised by MAB AF-1 is carried by the polysaccharide moiety of cell-surface glucomannoprotein molecule(s).

To obtain monoclonal antibodies (MAbs) directed preferentially against the pathogenic phase of Candida albicans, mice were immunised with germ tubes of C. albicans serotype A, strain VW.32, killed by exposure to ultraviolet (UV) irradiation. Fusions were performed either by the standard chemical procedure with polyethylene glycol, or by electric discharge following linkage of the myeloma and lymphocyte cells with a Concanavalin A-mannoprotein bridge. The preliminary characteristics of one MAb obtained from each of these fusions are described. An IgM antibody (3B7) obtained from the chemical fusion reacted with a polysaccharide antigen that was heterogeneously distributed on both in-vitro and in-vivo forms of C. albicans. This MAb agglutinated different strains of C. albicans irrespective of their serotype. An IgG1 antibody (3G6) that had been obtained from the electric fusion was found to react in vitro with a proteinaceous antigen located only on the germ tubes of strain VW.32. However, MAb 3G6 displayed strong reactivity against all growth forms of C. albicans in vivo and reactivity extended to other strains.

Pernasal aspirate (PNA) was obtained from 543 children during a 6-month period when whooping cough was prevalent. Three tests for diagnosing pertussis were performed on the PNA: (a) examination of direct smears by immunofluorescence (IF) for Bordetella pertussis; (b) culture; and (c) estimation of B. pertussis-specific immunoglobulin-A antibody (P-IgA) by an enzyme-linked immunosorbent assay (ELISA). On clinical review, 395 children were assessed to have had pertussis (P children) and 148 children not to have had pertussis (non-P children). The non-P children comprised 66 admitted to hospital for acute respiratory infections and 82 outpatients suspected of having pertussis. Analysis of the results of the tests on the PNAs of the non-P children helped to assess the P-IgA test. The analysis showed that artificial immunisation against pertussis did not affect the antibody results, but that non-specific positive results occur requiring the labelling of many P-IgA results as ‘doubtful’. Among the 395 P children, 36% yielded positive cultures and more than half of these also had positive IF tests. The ELISA for P-IgA was positive in 24% of all the P children, equivalent to nearly 40% of the culture-negative P children. For the 148 non-P children, IF and culture-negative by definition, the P-IgA test was positive in 9%. The antibody test result was doubtful in 28% of the P children and in 40% of the non-P children. Estimation of P-IgA antibodies in PNA is a useful and economic complement to culture and IF in the diagnosis of pertussis. The occurrence of non-specific positive results makes evaluation of the clinical features of the child essential to the interpretation of the test.

For an animal model of infection to be useful in immunological studies it is necessary to establish that the surface antigens expressed by bacteria growing in vivo in the experimental infection mimic those expressed by bacteria in the human infection. In this study, chronic infection was induced by inoculating the lungs of rats with agar beads containing mucoid Pseudomonas aeruginosa. P. aeruginosa was obtained from the lungs 14 days after infection and studied without subculture. Several high-mol.-wt proteins were expressed in the outer membranes (OM) of the bacteria from the rat lungs which could be induced by cultivating the same isolate in iron-depleted conditions in vitro. The pattern of iron-regulated membrane proteins (IRMP) was similar to that obtained in an earlier study with another mucoid isolate of P. aeruginosa examined directly, without subculture, from the sputum of a cystic fibrosis patient. Immunoblotting with LPS-absorbed serum from infected rats and also with serum from CF patients showed that IgG in these fluids reacted with the IRMPs and other major OM proteins (OMPs) of P. aeruginosa. Antisera from rats immunised with whole cells of P. aeruginosa grown in iron-depleted media reacted with all the major OMPs of P. aeruginosa, including the IRMPs, confirming their immunogenicity.

We have generated monoclonal antibodies (MABs) to staphylococcal enterotoxin B (SEB) in BALB/c mice. Five out of 20 clones which produce anti-SEB MABs have been characterised. Among them, three produce IgG1/k, one produces IgM/λ, and one apparently produces both IgG1/λ and IgM/λ MABs. The anti-SEB titres of ascites fluids range from 3200 to >819200 by ELISA. All of the MABs analysed thus far neutralise the mitogenic response of BALB/c splenocytes to a suboptimal dose of SEB. Also, the induction of suppressor cells by SEB in vitro is reversed by pre-incubating SEB with these MABs. Limited digestion with chymotrypsin, trypsin or Staphylococcus aureus V8 protease yields peptide fragments which have been tested by Western-blot analysis. MABs 1FD7 and 2GD9 are specific for the carboxy-terminal end of SEB, and have a similar, but not identical, binding epitope. MABs 2DA3 and 2HA10 bind to intact SEB but not to cleaved products, and are probably specific for antigenic determinants altered by the cleavage or by the denaturing conditions of the electrophoresis, or by both.

The number, frequency distribution and restriction enzyme fragmentation patterns of plasmids harboured by 163 methicillin-sensitive isolates of Staphylococcus aureus (MSSA) and 53 methicillin-resistant isolates (MRSA) were compared. Plasmids were demonstrated in less than half of the MSSA isolates; their frequency distribution did not differ from that predicted by a simple model of plasmid distributions. In contrast, all the MRSA isolates harboured plasmids, their distribution suggesting dissemination of a limited number of clones within the hospital. Among 72 MSSA isolates harbouring plasmids, 38 different restriction patterns were identified. There were fewer patterns among MRSA isolates; 11 were observed, and two predominant patterns accounted for 68% of those identified. These restriction patterns correlated with the presence or absence of aminoglycoside resistance. A multicopy plasmid of 2.6 kb was present in both MSSA and MRSA isolates that harboured more than one plasmid; it had the same restriction pattern irrespective of its source. The importance of these results in choosing a method of studying the spread of staphylococci is discussed.

Molecular variants of the serotype-specific plasmid (SSP) of Salmonella typhimurium (pSLT) were recognised in clinical and veterinary isolates by restriction enzyme fingerprinting. Three had undergone minor DNA rearrangements, whereas two had acquired resistance determinants to a wide range of antimicrobial agents including gentamicin, trimethoprim, tetracycline, streptomycin, ampicillin (Ap) and kanamycin (Km). One of the latter was the result of co-integrate formation with an IncX, conjugative R-plasmid that specified ApKm resistance. The co-integrate plasmid (pOG669) was incompatible with, and displaced, pSLT and its molecular variants. The restriction fingerprints of SSPs of S. enteritidis and S. dublin were compared with pSLT. All were related at the 35% level on the basis of a Dice coefficient of similarity. The SSPs of S. enteritidis and S. dublin were incompatible with the co-integrate plasmid pOG669. Whereas in S. enteritidis this resulted from incompatibility with the pSLT component (the SSP was compatible with the IncX component), the converse was found with S. dublin.

A total of 199 Shigella dysenteriae isolates resistant to one or more antibiotics and belonging to types 1, 2, 3, 4, 6 and 7 was examined by one-step broth mating with Escherichia coli K12 and, if non-conjugative, additionally by triparental crosses with the conjugative plasmids X and ◿. Of the S. dysenteriae type 1 (Shiga's bacillus) strains, 96% harboured conjugative plasmids. During 1974-79, isolates of Shiga's bacillus carried conjugative plasmids coding for ACSSuT (ampicillin, chloramphenicol, streptomycin, sulphonamide, tetracycline) resistance that transferred at low frequencies (>10−4). After 1980, however, about 50% of isolates of Shiga's bacillus with this resistance (R)-type carried conjugative plasmids that transferred at high frequencies (10°−10−2) and that expressed the ACT determinant only. The introduction of a new clone of Shiga's bacillus into Ethiopia in 1980 is suspected. Conjugative plasmids coding for SSuT resistance were detected in S. dysenteriae types 2, 3, and 4. Non-conjugative SSu determinants in S. dysenteriae type 3 were mobilised by conjugative plasmids X and ◿. R-type CSSuT in strains of types 2 and 7, and R-type ACST in type-3 strains were neither transferable nor mobilisable and are probably determined chromosomally.

Following natural or experimental primary infection, herpes simplex virus (HSV) becomes latent in sensory ganglia. Reactivation of latent virus may lead to recurrent disease. If HSV DNA remains stable during primary, recurrent and latent infections, that stability would enable us to trace the transmission of HSV from one individual to another. We inoculated mice in the ear pinna with HSV and collected virus at various intervals during primary infection. In mice surviving primary infections, recurrent disease was induced from which virus was isolated. Virus was also recovered from explanted dorsal root ganglia. Virus isolates were characterised by restriction endonuclease digestion and compared with the original inoculate(s). The data indicate that in all cases except two, the isolates from primary and recurrent infections remained identical to the original inoculates.