Biomedical subjects

L B Corbeil

Publications and source records attributed to L B Corbeil.

At least 55 records · Page 3Linked to original sources

PubMed1991

Characterization of immunodominant surface antigens of Haemophilus somnus.

An immunodominant Haemophilus somnus outer membrane protein with an apparent molecular mass of 40 kDa on Western blots (immunoblots) of gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels was characterized because a monospecific antibody against this antigen was protective. This monospecific antibody was used for immunoaffinity purification of the antigen. The immunoaffinity-purified antigen reacted with a polyclonal antibody to the 40-kDa antigen but not with a monoclonal antibody (3G9) which reacted with the 40-kDa antigen in gradient gels. On 8 or 10% gels, the approximately 40-kDa antigen was resolved as two bands, a 40-kDa band which reacted with the protective monospecific polyclonal antibody (p40) and a band of lower molecular mass which reacted with monoclonal antibody 3G9. The latter antigen was designated p39. Both antigens were conserved in all H. somnus isolates tested. The specific antibodies were also used to detect cross-reacting antigens in other gram-negative bacteria. Antibody to p40 reacted with proteins of 55 to 28 kDa, with the greatest intensity shown among proteins from other members of the family Pasteurellaceae. Antibody to p40 was reduced by absorption with live H. somnus or other members of the family Pasteurellaceae, so the antigen appears to be surface exposed. Antibody to p39 only cross-reacted with a broad band (38 to 40 kDa) in Haemophilus agni. Since H. agni is not a bovine pathogen and since convalescent-phase serum from H. somnus-infected animals did recognize p39, the latter may be a good immunodiagnostic antigen, if the lack of cross-reactivity with antigens in other gram-negative bacteria is confirmed with a polyclonal antibody to p39. The cross-reactivity of antiserum to p40 with antigens of members of the family Pasteurellaceae and the ability of this antiserum to protect against H. somnus pneumonia indicate that p40 may be a useful vaccine antigen for H. somnus disease and perhaps even diseases caused by other members of the family Pasteurellaceae.

Animals↗

PubMed1991

Immunoglobulin binding by Tritrichomonas foetus.

A better method for diagnosis of bovine trichomoniasis is needed because culture is slow and somewhat lacking in sensitivity. Immunodiagnosis of Tritrichomonas foetus infection usually involves detection of antigen-antibody reactions with an anti-immunoglobulin conjugate. However, nonspecific immunoglobulin (Ig), bound to the surface of T. foetus, would also be detected by an anti-Ig conjugate and thus would interfere with the specificity of the immunoassay. The goals of this study were to define the binding of bovine immunoglobulins to T. foetus. To determine whether nonimmune binding of Ig to T. foetus occurs, we immunized rabbits with organisms that had been grown in medium containing normal bovine serum and vigorously washed three times with phosphate-buffered saline. The rabbit antiserum had similar titers to T. foetus and to normal bovine serum by enzyme-linked immunosorbent assay (ELISA). Furthermore, two bovine serum proteins were immunoprecipitated by the rabbit antiserum in an immunoelectrophoretogram. One of the serum proteins had a distribution characteristic of IgG2. The rabbit antiserum was then shown to react with purified bovine IgG and IgM by ELISA. Reactivity to IgG was greater. To identify the IgG subisotypes bound and to confirm nonimmune binding of Ig, we grew T. foetus in agammaglobulinemic fetal calf serum and reacted it with IgG1, IgG2, and IgM specific for dinitrophenol (DNP) in ELISA. The binding of IgG2 was greatest, that of IgG1 was next, and that of IgM was least. Little competitive inhibition by DNP was detected, indicating that binding of DNP-specific antibodies was predominantly nonimmune rather than antigen-specific Ig binding. We also demonstrated that T. foetus grew well in medium containing agammaglobulinemic fetal calf serum or serum made agammaglobulinemic by ammonium sulfate precipitation of Igs. This may overcome the problem of low specificity in diagnostic assays as a result of antigen with Ig bound by nonimmune mechanisms.

Animals↗

PubMed1991

Cleavage of proteins of reproductive secretions by extracellular proteinases of Tritrichomonas foetus.

Cleavage of host defense proteins from reproductive secretions was investigated as a potential virulence mechanism for Tritrichomonas foetus extracellular proteinases. Three categories of susceptibility to digestion were found among the defense proteins tested. Cleavage of fibrinogen, fibronectin, and albumin occurred rapidly with more than 50% of these digested within 30 min. Lactoferrin, immunoglobulin G1, and immunoglobulin G2 were more than 50% digested after 4 h. Transferrin, immunoglobulin M, and immunoglobulin A were the most resistant to the Tritrichomonas foetus extracellular proteinases, since 50% or more of the parent molecule remained after 24 h. The responsible proteinases were classified as cysteine (thiol) proteinases because cleavage was inhibited by the cysteine proteinase specific inhibitor, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane and not by the serine proteinase specific inhibitor, phenylmethylsulfonyl fluoride. In addition, alpha 2-macroglobulin, but not alpha 1-antitrypsin, inhibits the action of the proteinases. The ratio of this naturally occurring inhibitor to the quantity of proteinases released may determine whether the above substrates are cleaved in vivo. Since these substrates are implicated in iron acquisition, cell adherence, and acquired immunity, Tritrichomonas foetus proteinases are likely to play a role in host-parasite interactions.

Albumins↗

PubMed1990

The effect of interferon-gamma intramammary administration on mammary phagocyte function.

The effect of recombinant bovine interferon-gamma on intramammary phagocyte function of mammary gland was studied in 4 Holstein cows (Study 1) and 7 Holstein cows (Study 2). Recombinant bovine interferon-gamma was intramammarily infused on day 6 of the dry period and phagocytes were collected from lacteal secretions and tested in vitro 24 hours later. Results from Study 1 indicate that phagocytosis of Escherichia coli and Staphylococcus aureus was significantly increased after than before interferon treatment. Similarly, the number of bacteria killed/active phagocyte was enhanced by treatment. Results from Study 2 suggested a trend towards increased production of oxygen dependent bactericidal components and increased killing ability by phagocytes exposed to interferon as compared to control phagocytes. These results from both studies suggest that intramammary infusions of bovine interferon-gamma can stimulate phagocyte function during the early phase of the dry period.

Animals↗

PubMed1990

Characterization of a 78-kilodalton outer membrane protein of Haemophilus somnus.

A 78-kilodalton (kDa) outer membrane protein (OMP) of Haemophilus somnus was one of the two antigens most consistently and most intensely immunoreactive in Western immunoblots of whole cells of H. somnus reacted with convalescent-phase serum obtained from cattle with experimental H. somnus pneumonia. This antigen was isolated by gel filtration chromatography of sodium dodecyl sulfate-solubilized OMP. Reactions of Western blots with bovine monospecific antiserum prepared against the 78-kDa antigen indicated that this 78-kDa OMP was present in each of 22 isolates of H. somnus obtained from cattle with pneumonia, thromboembolic meningoencephalitis, and abortion as well as from vaginal or preputial carriers. The 78-kDa OMP was also present in each isolate obtained weekly throughout the course of experimental H. somnus pneumonia in a calf. Monospecific antiserum to the 78-kDa OMP also reacted with proteins from closely related bacterial species in the family Pasteurellaceae but not with bacteria of 13 other genera. The 78-kDa OMP of H. somnus is of interest because it is surface accessible, highly conserved, immunogenic, cross-reactive with other members of the family Pasteurellaceae, and reactive with convalescent-phase serum which is passively protective against H. somnus pneumonia.

Animals↗

PubMed1990

Characterization of Tritrichomonas foetus antigens by use of monoclonal antibodies.

The specificity for and function of monoclonal antibodies against Tritrichomonas foetus were characterized. Four monoclonal antibodies generated by immunization of mice with live T. foetus were selected on the basis of enzyme-linked immunosorbent assay reactions. The approximate molecular masses of the predominant proteins were determined by Western blotting (immunoblotting). Monoclonal antibody TF3.8 recognized a predominant band at approximately 155 kilodaltons, whereas TF3.2 reacted with several bands. Monoclonal antibodies TF1.17 and TF1.15 recognized broad bands between 45 and 75 kilodaltons. The first two antibodies (TF3.8 and TF3.2) did not react with the surface of T. foetus, as determined by live-cell immunofluorescence, agglutination, and immobilization, whereas two other monoclonal antibodies (TF1.17 and TF1.15) did react with surface epitopes, as determined by these criteria. The latter two monoclonal antibodies also mediated complement-dependent killing of T. foetus and prevented of adherence of organisms to bovine vaginal epithelial cells. One antibody, TF1.15, also killed in the absence of complement. Since these functions are in vitro correlates of protection, the antigens recognized by these monoclonal antibodies may induce protective immunity.

Animals↗

PubMed1990

Molecular aspects of some virulence factors of Haemophilus somnus.

Several virulence factors of Haemophilus somnus have been investigated, but to a varying extent. It is known that some isolates are more virulent than others. Factors associated with virulence include attachment to vaginal epithelial cells, turbinate cells or embryos; serum resistance; growth stimulation by normal flora; interference with phagocyte function; Fc receptors; and toxicity for several bovine cell types. Lipooligosaccharide and Fc receptors have been investigated at the molecular level and studies are in progress to relate their molecular structure to virulence.

Animals↗

PubMed1989

Cross-reactivity of monoclonal antibodies to Escherichia coli J5 with heterologous gram-negative bacteria and extracted lipopolysaccharides.

A panel of eight murine monoclonal antibodies (MAbs) were produced against heat-killed Escherichia coli J5 and shown to react with J5 lipopolysaccharide (LPS) by enzyme-linked immunosorbent assay (ELISA). These antibodies were then assayed by a suspension ELISA for reactivity with up to 20 heterologous smooth or rough isolates of gram-negative bacteria, which were assayed after heat or formalin treatment, or as live cells. Extracted LPS from the same bacteria were tested for reactivity with the MAbs by direct ELISA. The MAbs demonstrated broad cross-reactivity with most heat-treated bacteria. In contrast, cross-reactivity of the MAbs with live or formalin-treated bacteria was limited almost exclusively to E. coli J5, Hemophilus species, or rough mutants of Salmonella minnesota. Reactivity with extracted LPSs and lipid A varied considerably depending on the MAb. Further, when Western blotting was used as the assay only four of eight MAbs reacted with J5 LPS, and none of the MAbs reacted with LPS from smooth S. minnesota or any of its rough mutants. Adsorption of the MAbs with acid hydrolyzed, boiled, or live E. coli J5 prior to ELISA of the MAbs with J5 LPS supported evidence that none of the MAbs were specific for lipid A and that reactivity was greater with boiled than with live cells. Thus, the cross-reactivity of antibodies to E. coli J5 LPS is dependent on the physical state of the bacteria or LPS used for assay, the assay used, and the specificity of the antibody.

Antibodies, Bacterial↗

PubMed1989

Immunoglobulin-binding activity among pathogenic and carrier isolates of Haemophilus somnus.

Nonimmune binding of immunoglobulin to whole bacteria was quantitated for North American isolates of Haemophilus somnus recovered from cattle with pneumonia, reproductive failure (abortion), or thromboembolic meningoencephalitis or from the vagina or prepuce of carrier cattle. Quantitative binding activity covered a wide range, with most pathogenic and carrier isolates demonstrating significant immunoglobulin-Fc binding. Isolates for which Fc binding was not detectable were recovered only from the prepuces of asymptomatic bulls. Expression of Fc-binding activity correlated with the presence of the 41,000-molecular-weight protein (41K protein) and 270K protein. Isolates that lacked Fc-binding activity did not possess 41K or 270K protein. A 33K protein was detected in isolates that lacked Fc-binding activity but not in isolates that bound Fc.

Abortion, Veterinary↗

PubMed1989

Adherence of Tritrichomonas foetus to bovine vaginal epithelial cells.

Adherence of Tritrichomonas foetus to bovine vaginal epithelial cells (VECs) in vitro was investigated with fresh washed bovine VECs and log-phase cultures of T. foetus. Observation under phase-contrast microscopy showed that T. foetus usually adhered first by the posterior flagellum and later by the body. Significantly more keratinized squamous epithelial cells were detected with attached parasites than nonkeratinized round epithelial cells. The optimal pH range for attachment was 6.0 to 7.5, with peak attachment at pH 6.5 for squamous VECs. Surface-reactive bovine antiserum to T. foetus prevented adherence to bovine squamous VECs. Inhibition of adherence occurred at nonagglutinating, nonimmobilizing serum dilutions. Antiserum fractions enriched for immunoglobulin G1 inhibited adherence, but fractions enriched for immunoglobulin G2 did not. The inhibitory antiserum was specific for several medium- to high-molecular-weight membrane antigens as detected in Western blots (immunoblots). The ability of surface-reactive antibodies to prevent adherence and to agglutinate and immobilize T. foetus indicates that they may be protective.

Animals↗

PubMed1989

Antibody response to Haemophilus somnus Fc receptor.

To characterize the bovine immune response to an Haemophilus somnus antigen known to be recognized by convalescent-phase serum, we studied isotypic antibody titers to the 270-kilodalton protein, which we had previously shown to be an immunoglobulin Fc receptor. With a modified immunodot procedure, an immune response was detected after experimental H. somnus abortion, experimental H. somnus pneumonia, or vaccination with commercial H. somnus vaccine, with the greatest titer found within the immunoglobulin G2 isotype. With protein A peroxidase conjugate, which detects primarily bovine immunoglobulin G2, we showed that cattle with H. Somnus disease could be distinguished from clinically normal carriers, culture-negative cattle, or cattle with disease due to Pasteurella haemolytica or P. multocida. Little cross-reactivity between the 270-kilodalton Fc receptor antigen and antigens from other gram-negative bovine pathogens was seen. Thus, this antigen may be a useful diagnostic antigen.

Abortion, Veterinary↗

PubMed1989

Pulmonary persistence of Haemophilus somnus in the presence of specific antibody.

Chronic experimental Haemophilus somnus pneumonia was produced in five 8- to 12-week-old calves to investigate host-parasite relationships in the respiratory tract. Calves were depressed and pyrexic and coughed intermittently for 3 days and then recovered except for sporadic coughing. Bacteria persisted in the lung for 6 to 10 weeks or more. Immunoglobulin G1 (IgG1), IgG2, and IgM but no IgA antibodies specific for H. somnus were detected in serum. Bronchoalveolar lavage samples contained detectable IgG1, IgG2, IgM, and IgA antibodies specific for H. somnus throughout most of the experiment. The kinetics of the isotypic antibody response against H. somnus in serum and bronchoalveolar lavage fluids differed, suggesting that both local and systemic antibody responses had occurred. Persistence of pulmonary infection for 10 weeks or more in the presence of antibody may be due to an inappropriate distribution of isotypes, toxicity of H. somnus for bovine macrophages, and perhaps other factors. Three of the calves were challenged with a 10-fold-higher dose of H. somnus at 10 weeks after the original inoculation. Immunity against H. somnus was indicated by the rapid clearance of bacteria from the lungs and the presence of minimal pneumonia at necropsy 3 days after bacterial challenge.

Animals↗

PubMed1989

Isotypic antibody responses in cattle infected with Haemophilus somnus.

Bovine antibody responses to Haemophilus somnus were compared on the basis of clinical and bacteriological findings. Serum IgG1 and IgM antibody titres were significantly increased in clinically normal cattle that were bacteriologically positive for H somnus from the nasal or vaginal mucosae compared with clinically normal, negative cows. IgG2 titres did not differ significantly between these two groups. However, IgG2 antibody was significantly higher in animals with H somnus disease (pneumonia or abortion) than in clinically normal cattle (whether bacteriologically positive or negative), while IgG1 and IgM titres did not differ between diseased and bacteriologically positive, clinically normal cattle. These antibody trends were duplicated in experimental H somnus abortion or pneumonia, with the greatest response occurring within the IgG2 subclass. Cattle vaccinated systemically with killed whole H somnus produced a predominant IgG2 response with minimal IgG1 and IgM responses. These results demonstrate that IgG2 antibody is consistently elevated in H somnus disease, and suggest that this response may be useful in discriminating diseased from asymptomatic cattle.

Animals↗

PubMed1988

Teat skin normal flora and colonization with mastitis pathogen inhibitors.

Isolates of bacteria from normal teats were used to attempt colonization of teats of dry cows or neonatal calves. Isolates for inoculation were chosen on the basis of ability to inhibit mastitis pathogens in vitro, with the ultimate goal of in vivo inhibition of mastitis pathogens at the teat surface. Three bacterial normal flora isolates (Corynebacterium xerosis, Bacillus sp. and Aerococcus viridans) persisted less than 10 days on the teats of dry cows. The fourth isolate, Staphylococcus hominis 1, was studied in greatest detail because studies characterizing the normal teat flora showed staphylococci to be the predominant flora. The S. hominis 1 isolated used for inoculation was an inhibitor of Gram-positive mastitis pathogens. It was a biotype not found on these teats prior to inoculation, thus facilitating identification of the inoculated isolate on sequential sampling. Colonization of newborn calves, before other bacterial flora became established, resulted in recovery of inoculated S. hominis 1 for an average of 51 days or longer. On dry cow teats it was detected for up to 28 days. On several occasions the inoculated S. hominis 1 was found in pure culture. Since many new infections occur during the dry period, the colonization of dry cow teats with S. hominis 1 organisms inhibitory for Gram-positive pathogens should be tested as an adjunct to other methods of mastitis prevention.

Animals↗

PubMed1988

Characterization of two Haemophilus somnus Fc receptors.

Haemophilus somnus expresses two types of receptors that bind to the Fc region of bovine IgG, IgA and IgM. In this study, the relationship between these two types of Fc receptors is characterized. The high molecular mass receptors (350, 270 and 120 kDa) were secreted into the culture medium and were also in the insoluble protein fraction of the culture medium. The 41 kDa Fc receptor, which is a major outer-membrane protein, was only present in the insoluble protein fraction. Peptide mapping of the two types of Fc receptors suggests that the 41 kDa receptor is related to the high molecular mass receptor complex. Disulphide linkage is unlikely to be the mechanism of association of the 41 kDa receptor with the high molecular mass receptors since reducing agents had no effect on separating the individual receptors. Although the 41 kDa receptor is a major protein in the outer membrane of H. somnus, it does not react with convalescent bovine sera in Western blots. In contrast, convalescent bovine sera reacts intensely with the high molecular mass receptors in Western blots.

Bacterial Proteins↗

PubMed1988

Non-immune immunoglobulin binding by "Haemophilus somnus".

In-vitro culture of Haemophilus somnus in liquid or solid media supplemented with bovine blood or serum resulted in non-immune binding of immunoglobulin (Ig) by the organism. This binding was independent of the antigen-combining site of the Ig molecule, since binding of an IgG preparation specific for the hapten dinitrophenol was unaffected by the presence of the homologous antigen. Quantitative comparison of the binding of Ig fragments Fab and Fc demonstrated that the non-immune binding occurred in the Fc region of bovine IgG. The isotypes of Ig that became bound to H. somnus included both bovine IgG subclasses (IgG1 and IgG2), which were bound equally, and bovine IgM.

Animals↗

PubMed1988

Isolation and characterization of Fc receptors from Haemophilus somnus.

Receptors that bind the Fc region of bovine immunoglobulin (Ig) have been isolated from the culture supernatant of Haemophilus somnus by chromatography on a Sepharose 4B column. One receptor with a relative molecular weight of 41,000 weakly binds both bovine IgG subclasses, IgA and IgM, while three high molecular weight receptors (350,000, 270,000, and 120,000) strongly bind bovine IgG2, IgA, and IgM. All four Fc receptors are antigenically related and the 41,000 receptor appears to be a subunit of the high molecular weight receptors. In addition to bovine Ig, the purified 270,000 Fc receptor strongly binds horse IgG, rabbit IgG, pig IgG, cat IgG, dog IgG, and sheep IgG. The receptor also reacts weakly with mouse, rat, chicken, human, and guinea pig IgG and does not bind goat IgG. Fc receptors from 19 H. somnus isolates were compared. Variations in the molecular weight of the 41,000 protein were demonstrated among preputial isolates from asymptomatic carriers, but all other isolates appeared to have identically migrating proteins.

Epitopes↗

PubMed1988

Cloning and expression of genes encoding Haemophilus somnus antigens.

A genomic library of Haemophilus somnus 2336, a virulent isolate from a calf with pneumonia (later used to reproduce H. somnus experimental pneumonia), was constructed in the cosmid vector pHC79. The gene bank in Escherichia coli DH1 was screened by filter immunoassay with convalescent-phase serum, which reacted with several outer membrane antigens of H. somnus. On Western blotting (immunoblotting) of immunoreactive colonies, five clones were found to express proteins which comigrated with H. somnus surface antigens. Three clones (DH1 pHS1, pHS3, and pHS4) expressed both a 120-kilodalton (kDa) antigen and a 76-kDa antigen, one clone (DH1 pHS2) expressed only the 76-kDa antigen, and the fifth clone (DH1 pHS5) expressed a 60-kDa antigen. The 120-kDa and 76-kDa antigens were found internally, whereas the 60-kDa protein was detected in the DH1 pHS5 culture supernatant as membrane blebs or insoluble protein. Both the H. somnus 120-kDa antigen and the recombinant 120-kDa antigen had immunoglobulin Fc-binding activity. Restriction endonuclease mapping demonstrated that the genomic DNA inserts of clones expressing the 76-kDa antigen shared a common 28.4-kilobase-pair region, and the three clones also expressing the 120-kDa antigen shared an additional 7.0-kilobase-pair region. The restriction endonuclease map of pHS5, which expressed the 60-kDa antigen, was not similar to the maps of the other four plasmids. Since these three H. somnus antigens reacted with protective convalescent-phase serum, the recombinants which express these proteins should be useful in further studies of protective immunity in bovine H. somnus disease.

Antigens, Bacterial↗