Biomedical subjects
Publications and source records attributed to C J Thorns.
The expression of the 987P fimbrial adhesin by strains of enterotoxigenic Escherichia coli was enhanced and, in some cases, restored by passaging the organisms through Craigie's tubes. In contrast, the fimbrial adhesins K99, K88 and F41 were not optimally expressed in this medium. The results suggest that Craigie's tubes should be used for the optimum expression of 987P.
A monoclonal antibody (Mab)-based antigen capture enzyme-linked immunosorbent assay (ELISA) was developed and standardised for the detection of the K99 adhesin of Escherichia coli in aqueous vaccines. A repeating epitope on the K99 antigen is first captured then detected using peroxidase-labelled antibody. The assay gave positive results with all the vaccines tested that were known to contain K99 and was specific, rapid and reproducible.
A fimbrialike structure expressed on the surface of Salmonella enteritidis was identified by using a monoclonal antibody (69/25) produced against intact S. enteritidis cells. Fimbriae were less than 5 nm in diameter and carried a protein consisting of subunits with a molecular weight of 14,300. No hemagglutinating activity associated with the fimbriae was detected. An epitope on the fimbrial antigen identified by monoclonal antibody 69/25 was expressed by all 58 S. enteritidis strains, 12 of 36 S. dublin strains, and a single strain of S. moscow examined. None of 169 other isolates tested from 17 salmonella serogroups expressed this epitope.
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A competitive enzyme-linked immunosorbent assay (CELISA) has been developed, standardized and compared with the toxin neutralization (TN) test performed in mice for the measurement of antibody responses in rabbits vaccinated with clostridial vaccines. In CELISA, sera were tested at a single dilution for their ability to compete with the reaction between a monoclonal antibody, which neutralizes epsilon toxin, and epsilon toxoid coated on to a solid phase. The results of the two tests correlated well. CELISA was specific, rapid, reproducible and simple to perform and offered an alternative to the TN test that reduced the requirement for experimental animals in the potency testing of clostridial vaccines.
A panel of monoclonal antibodies against fimbrial adhesins of porcine enterotoxigenic Escherichia coli were evaluated for the detection of enteric colibacillosis in paraffin-wax embedded sections of piglet small intestine. Using the immunoperoxidase technique, monoclonal antibodies were used to detect epitopes on the K99 adhesin and on the a and c regions of the K88 adhesin. However, monoclonal antibodies to the F41 and 987P adhesins failed to react in sections with organisms colonising the intestine of gnotobiotic piglets monoinfected with strains bearing those adhesins, whereas corresponding polyclonal antisera gave positive results. In contrast to apparent expression of all K99 organisms, only a proportion of organisms were identified by monoclonal or polyclonal antibodies as expressing K88. In some instances, failure of immunostaining was attributed to prolonged storage of tissue in formalin.
A monoclonal antibody that identifies a specific epitope on the F41 fimbrial adhesin was used in coagglutination and enzyme-linked immunosorbent assay (ELISA) tests, to identify successfully strains of Escherichia coli expressing the F41 adhesin. The antibody also bound to frozen sections of ileum from piglets infected with an F41 positive E coli demonstrating that the epitope is expressed in vivo.
Fifty murine monoclonal antibodies were produced against a purified preparation of K88ab antigen. Two monoclonal antibodies were selected for further studies together with a monoclonal antibody directed against the c region of K88 (5CA3). Monoclonal antibody K88-13 bound to all K88+ Escherichia coli examined, whereas K88-18 and 5CA3 bound to K88ab+ and K88ac+ strains, respectively. The monoclonal antibodies failed to react with K88+ E coli grown at 18 degrees C or with K88- E coli grown at 37 degrees C or 18 degrees C. Binding of monoclonal antibody K88-13 to K88 antigen was blocked by OK antisera to G7 (O8:K87 K88ab) and Abbotstown (O149:K91 K88ac), whereas absorbed antisera to K88b, c and d had no effect. Monoclonal K88-18 was inhibited by OK G7 antiserum and absorbed antiserum to K88b. One hundred and forty-nine strains of K88+ E coli representing seven somatic O groups were agglutinated by antibody K88-13; 142 of these, from at least six somatic O groups were agglutinated by 5CA3 and the remainder by K88-18. Monoclonal antibody K88-13 bound to cryostat sections of ileum from piglets infected with E coli strains Abbotstown and G7. K88-18 and 5CA3 bound only to sections from piglets infected with G7 and Abbotstown strains, respectively. It is concluded that monoclonal antibody K88-13 recognises an epitope on the a region of K88 while monoclonal antibody K88-18 is directed towards the b region. These monoclonal antibodies together with 5CA3 can be used to routinely identify K88+ E coli isolates.
Epitopes of antigens from a range of bacteria, parasites and fungi were identified by monoclonal antibodies as being similar to epitopes from mycobacterial antigens. Antibodies to many of these cross-reacting epitopes are present in normal mammalian and avian sera.
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Murine hybridomas secreting monoclonal antibodies to Mycobacterium bovis were produced and three soluble antigens were identified using radioimmunoassays and immunoblotting from polyacrylamide electrophoresis gels. Antibody MB3 (IgM, k chain) reacted with 20-100 kDal antigens produced by all mycobacterial strains examined while antibody MB5 (IgG2a, k chain) identified a 29.8 kDal antigen detected in field isolates of M. bovis and M. bovis strains Vallée and AN5. There was insignificant binding to M. bovis BCG, M. tuberculosis, M. microti, M. africanum, M. avium or M. paratuberculosis. Monoclonal antibody MB17 (IgA, k chain) reacted with a 17.4 kDal antigen present in M. bovis, M. tuberculosis and M. microti. Absorption of monoclonal antibodies with antigens from different species of Mycobacterium confirmed the specificities of MB3 and MB5 but the binding of MB17 was inhibited to some extent by all the extracts examined. The antigen identified by MB3 was present in purified protein derivative (PPD) from M. bovis, M. paratuberculosis and M. avium but antigens identified by MB5 and MB17 were not detected in these reagents.
All the K99+ Escherichia coli grown at 37 degrees C stained strongly with a peroxidase labelled K99 monoclonal antibody using a direct immunoperoxidase staining procedure. There was no reaction when these bacteria were cultured at 18 degrees C or when K99- E coli were grown at either temperature. The binding of the monoclonal antibody to K99 antigen was inhibited by OK antisera to heterologous K99+ E coli but OK antisera to E coli producing adhesins other than K99 were without effect. Using the slide agglutination test the reactions of the monoclonal antibody were identical to those of a polyclonal antiserum to K99 when both were used in parallel to examine 100 K99+ E coli from at least 10 somatic O groups and 1308 K99+ E coli from at least 82 different somatic O groups submitted for routine serological typing in England or the, USA. The monoclonal antibody reacted with K99+ E coli in cryostat sections of the ileum from a piglet infected with E coli strain B44 (O9: K30, K99, F41) but there was no reaction with similar material from piglets infected by E coli strains 1751 (O101: F41), X177/81 (O9: K103, 987P) or Abbotstown (O149: K91, K88ac).
Monoclonal antibodies (MoAb) produced against a sonicated antigen extract of Mycobacterium bovis were examined for their ability to bind to 10 common tissue antigens. The binding patterns of MoAb MB2, MB3, MB7, MB11 and MB13 indicated that each recognized different epitopes. MoAb MB5 and MB17 failed to bind to any of the tissue antigens. MB3 reacted strongly with normal tissue from M. bovis infected guinea pigs and ferrets but there was no reaction with MB5 and MB17. These specificities were confirmed by reciprocal absorption experiments. It is concluded that Mycobacterium species contain epitopes that are also present in host tissue antigens.
The effect of heat killed Mycobacterium bovis on the phytohaemagglutinin (PHA) response of normal peripheral blood leucocytes from ferrets was dose dependent. Concentrations of 10(6) organisms ml-1 and below enhanced the response, whereas more than 10(6) organisms ml-1 suppressed it. This suppression correlated with cytotoxicity of M bovis for ferret peripheral blood leucocytes. Three environmental strains, M phlei, M smegmatis and M aurum were not cytotoxic and did not affect the PHA response but were found to be mitogenic at high concentrations.
The enterotoxigenic Escherichia coli strains 1676, 1706, 1751 and KEC96a, which do not produce fimbrial adhesive antigens of the K88, K99 or 987P antigen type reacted both in vitro and in vivo with antiserum to F41 fimbriae in an indirect immunofluorescent antibody technique. Antiserum used to demonstrate material B, an adhesive antigen thought to mediate the adhesive and mannose-resistant (MR) haemagglutinating properties of E. coli strains 1676, 1706 and 1751, reacted in vitro with an F41+ strain. The antiserum also inhibited the MR haemagglutinating activity of F41 antigen and gave an anionic precipitation line in immunoelectrophoresis experiments with an extract containing F41 antigen. The MR haemagglutinating properties of an antigen extract containing material B from E. coli strain 1706 was neutralized by antiserum to F41 fimbriae and by OK antisera to E. coli strains that produce both F41 and K99 fimbriae. These sera also gave an anionic precipitation line with the MR haemagglutinin from E. coli strain 1706 and the MR haemagglutinin gave a line of identity with F41 in gel diffusion experiments with antiserum to F41 fimbriae. OK antisera to K99+ F41- bacteria and OK antisera to K88+ bacteria and 987P+ bacteria did not react with this haemagglutinin. Transmission electron microscopy on the ileum of newborn gnotobiotic piglets infected with E. coli strain 1706 showed irregular, poorly defined filamentous material surrounding some,though not all, bacteria but regular fimbrial structures were not visible.(ABSTRACT TRUNCATED AT 250 WORDS)
Three badgers exposed to conjunctival instillation of Brucella abortus strain 544 developed a mild subclinical infection accompanied by the production of agglutinating and complement fixing antibodies. Six months after inoculation, the infection could not be detected in one badger and was confined to lymphoid tissue in the other two. No cell mediated immune response to B abortus antigens could be detected by in vitro lymphocyte transformation tests or intradermal tests for delayed hypersensitivity during the observation period.