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A J Guidry

Publications and source records attributed to A J Guidry.

At least 37 records · Page 2Linked to original sources
PubMed1994

Effect of whole Staphylococcus aureus and mode of immunization on bovine opsonizing antibodies to capsule.

Exopolysaccharide capsule is a major virulence factor of Staphylococcus aureus because it inhibits neutrophil recognition of antibodies to highly antigenic S. aureus cell wall. To circumvent this inhibition, two modes of immunization were tested for ability to induce anticapsular opsonins. Cows were immunized at drying off and boosted on d 14 and 28 by injection of Smith diffuse S. aureus plus dextran sulfate in the area of the supramammary lymph node or intramammarily. In cows immunized in the area of the supramammary lymph node, IgG1 and IgG2 sera antibody titers to capsule increased and remained elevated to the end of the study, 120 d postcalving. The IgM titers increased during the dry period but declined to preimmunization levels at calving. Response of serum IgG1 and IgM to intramammary immunization was similar to that with supramammary lymph node immunization, but more delayed and lower in magnitude. Antibodies of all four isotypes, IgG1, IgG2, IgA, and IgM, increased in dry secretions following immunization via lymph node. In cows immunized in the lymph node, IgG1 antibodies remained elevated throughout the study, but IgG2 antibodies dropped to baseline 15 d postcalving. In cows immunized intramammarily, only IgA antibodies increased significantly in lacteal secretions and remained elevated throughout the study. Immunization of cows in the lymph node during the dry period enhanced the ability of dry secretions and colostrum to support phagocytosis.

Analysis of Variance↗
PubMed1994

Bovine mammary teat and ductal epithelial cell cultures.

Bovine mammary epithelial cells from teat and ductal tissue were isolated at necropsy and were grown in culture. Cells were characterized by the presence of cytokeratin filaments, cell morphologic features, synthesis of milk proteins, esterase activity, DNA content, and growth patterns on polystyrene, fibronectin, laminin, collagen, and reconstituted basement membrane from the Engelbreth-Holm-Swarm murine sarcoma. Cultured teat and ductal cells stained intensely for cytokeratin and had similar morphologic features. Both cell types synthesized alpha-casein, beta-casein, alpha-lactalbumin, beta-lactoglobulin, and lactoferrin to variable degrees. Cell type and culture conditions did not affect the DNA content of the cells, as indicated by similar amounts of DNA in G0G1 and G2M phases of the mitotic cycle in cultured cells and in cells from freshly isolated mammary explants. Cells cultured on polystyrene, fibronectin, laminin, and collagen formed pavement-like cell monolayers suitable for cytotoxicity and bacterial adherence studies. Cells cultured on the reconstituted basement membrane from the Engelbreth-Holm-Swarm murine sarcoma formed three-dimensional structures closely resembling lactiferous ducts and alveoli, which could be used for studying lactogenesis and galactopoiesis. Freshly isolated cells and cultured cells were stored at -70 C or in liquid nitrogen. The latter storage method affected the cells less than did freezing at -70 C.

Animals↗
PubMed1993

Opsonization of Staphylococcus aureus by bovine immunoglobulin isotypes.

The ability of specific bovine Ig isotypes to enhance phagocytosis of Staphylococcus aureus by polymorphonuclear neutrophils was studied. Polymorphonuclear neutrophils were isolated from the blood of 14 lactating Holstein cows. Antibodies against S. aureus M10 were produced by two Holstein cows immunized via intramuscular injections and injections in the area of the supramammary lymph node with M10 emulsified in dextran sulfate. The IgG1, IgG2, and IgM were prepared from immune sera. Fluorescein-labeled, formalin-killed S. aureus M10 were opsonized with the respective isotypes prior to incubation with polymorphonuclear neutrophils. Percentage of polymorphonuclear neutrophils phagocytosing averaged 37.4, 1.1, 15.9, and 9.4% for immune sera, IgG1, IgG2, and IgM, using a M10: polymorphonuclear neutrophils ratio of 10:1; and 77.1, 1.8, 32.1, and 57.9 using a 40:1 ratio. When IgG1 was incubated with either IgG2 or IgM, phagocytosis was reduced to 10.0 and 5.0%, respectively, using the 10:1 ratio and 24.2 and 44.7%, respectively, using the 40:1 ratio. Significant variation occurred among cows in the ability of polymorphonuclear neutrophils to undergo phagocytosis independent of isotype and S. aureus M10: polymorphonuclear neutrophil ratio. These data show that IgG2 and IgM are opsonic for bovine polymorphonuclear neutrophils and that IgG1 inhibits the activity of both. These results will be helpful to determine immunization protocols to solicit synthesis of bovine IgM and IgG2 specific for S. aureus.

Animals↗
PubMed1992

Immunoglobulins, lysozyme and lactoferrin in the teat and udder of the dry cow during endotoxin-induced inflammation.

Immunoglobulins (Ig) and antibacterial proteins like lysozyme and lactoferrin are components of the humoral defence against infections. Changes in Ig, lysozyme and lactoferrin concentrations during endotoxin-induced inflammation in the test cistern and udder quarter of the dry cow were studied. Surgical closure of the passage between teat and udder cisterns enabled studies of reactions in the teat cistern without interference of the mammary gland. After endotoxin infusion, IgG1, IgG2, lysozyme, and to some extent IgM, increased in the teats and udder quarters, and were positively correlated with changes in somatic cell counts. No significant changes were observed in IgA or lactoferrin. The origin and significance of Ig, lysozyme and lactoferrin in the bovine teat and udder are discussed. Ig probably originated both from serum and from local plasma cells, while leukocytes appeared to be the source of lysozyme during inflammation. Secretory epithelium appeared to be the source of lactoferrin. Support for this theory was the almost total absence of lactoferrin in teat cistern samples.

Animals↗
PubMed1991

Prevention of nonspecific binding of immunoglobulin to Staphylococcus aureus protein A in ELISA assays.

The Fc region of IgG of most mammals binds protein A on S. aureus resulting in high backgrounds when measuring specific antibodies to S. aureus in the ELISA. Removal of protein A from S. aureus or modification of the Ig Fc to prevent binding to protein A could affect specific antibody binding. We compared effects of blockage of Fc binding to protein A with purified protein A to trypsin removal of protein A from S. aureus, on specific antibody binding. When NMS was incubated without and with protein A (0 microgram, 50 micrograms, 200 micrograms and 400 micrograms) and high protein A Cowan I was the bound S. aureus antigen in the ELISA, absorbance OD405 was 0.769, 0.240, 0.224 and 0.210 +/- SE 0.026. When mouse Mab (IgG1, kappa) to bovine IgA was incubated without and with protein A (400 micrograms) prior to reaction with bovine IgA in the ELISA, absorbance was 0.645 and 0.639, indicating protein A had no effect on specific antibody binding. To determine the effect of trypsin on specific binding, Becker S. aureus was trypsin treated before linking it to microtiter wells. When Mab (IgM) to Becker (Nelles et al., Infect. Immun. (1985) 49, 14) was incubated with protein A (400 micrograms) before use in the ELISA, trypsin treatment of Becker resulted in reduced specific antibody activity (untreated Becker = 1.306, trypsin treated Becker = 0.331). These results suggest that purified protein A can be used to block nonspecific binding via Fc of Ig to S. aureus, thus avoiding trypsin denaturation of surface antigens.

Animals↗
PubMed1991

Effect of anticapsular antibodies on neutrophil phagocytosis of Staphylococcus aureus.

One of the major virulence factors of Staphylococcus aureus is development of an exopolysaccharide capsule in vivo, which inhibits recognition of antibodies to highly antigenic cell wall by neutrophils. To circumvent this inhibition, an attempt was made to produce anticapsular antibodies. Three cows per group were immunized in midlactation by injections in the area of the supramammary lymph node and intramuscularly and were boosted on d 14, 42, and 70 with three variants of Smith S. aureus: compact, unencapsulated; diffuse, rigid capsule; and diffuse large clearing, exceptionally large flaccid capsule using dextran sulfate as adjuvant. Serum agglutination and ELISA titers of cows immunized with diffuse and diffuse large clearing increased after immunization and after each boost and remained elevated to the end of the experiment at 112 d. Phagocytosis of diffuse and diffuse large clearing, measured by flow cytometry, was enhanced by immunization with either organism. No antibody response to capsule or enhanced phagocytosis of diffuse developed in cows immunized with compact. However, anticompact antibodies were opsonic for diffuse large clearing. These data show that bovine antibodies to S. aureus capsule are opsonic for bovine neutrophils and that capsule plays a role in inhibition of cell-wall opsonization of S. aureus.

Agglutination Tests↗
PubMed1991

Encapsulation of coagulase-negative staphylococci of bovine origin.

Capsule expression was assessed in six coagulase-negative staphylococcal strains in serum-soft agar and by india ink and electron microscopy. Classification of strains as encapsulated by serum-soft agar and india ink methods differed. Staphylococcus chromogenes, Staph. hyicus, and Staph. simulans grew as diffuse colonies in serum-soft agar and unstained halos were detected in india ink preparations. Staphylococcus hominis and Staph. simulans grew as diffuse colonies in serum-soft agar but no unstained halo was seen in india ink preparations. Staphylococcus hyicus was the only strain that gave negative results with serum-soft agar and india ink assays. Conventional electron microscopy revealed the presence of capsular polysaccharides on the cell surface of Staph. chromogenes, Staph. hominis and Staph. hyicus. Conventional electron microscopic technique used to examine the surface of cells was detrimental to capsule structure. During dehydration the capsule collapsed and appeared as electron dense aggregates at the surface of cells. To confirm results of conventional electron microscopy and to visualize clearly the cell surface, encapsulated Staph. hyicus and unencapsulated Staph. simulans were observed after freeze-fracture and etching by scanning electron microscopy. The fibrous nature of capsular polysaccharides surrounding cells of Staph. hyicus were distinct and confirmed observation by conventional electron microscopy. A rapid transmission electron microscopic technique is described also for observation of capsule. Results of the rapid TEM method agreed with conventional TEM and SEM. The finding that coagulase-negative staphylococci isolated from bovine milk are capable of capsule production may be important when investigating pathogenicity of these micro-organisms.

Animals↗
PubMed1991

Immunogold labeling of stages of Cryptosporidium parvum recognized by immunoglobulins in hyperimmune bovine colostrum.

Ultrathin sections of mouse ileum infected with Cryptosporidium parvum were stained by immunogold techniques. Sections first were stained with polyvalent antibodies in whey from hyperimmune bovine colostrum (HBC), then stained by secondary antibodies in rabbit antibovine IgA, IgM, IgG1, and IgG2, and lastly labeled by goat anti-rabbit gold conjugate. Examination of the immunostained specimens by electron microscopy revealed that each bovine immunoglobulin isotype in the whey recognized antigens in meronts, merozoites, microgametocytes, microgametes, and macrogamonts. Based on these findings it is hypothesized that antigens in all stages of C. parvum provide targets of opportunity for the antiparasitic activity of HBC whey antibodies thereby accounting for its efficacy as an immunotherapeutic agent.

Animals↗
PubMed1988

Effects of Staphylococcus aureus mastitis on bovine mammary gland plasma cell populations and immunoglobin concentrations in milk.

Bovine mammary tissue and milk samples were examined to determine effects of chronic Staphylococcus aureus mastitis on the humoral immune response. Parenchymal and teat end tissues from lactating bovine mammary glands were stained immunohistochemically to determine distribution of immunoglobulin (Ig) G1-, IgG2-, IgA-, and IgM-producing plasma cells. Numbers of all Ig-producing plasma cells tended to be higher in tissues from S. aureus infected quarters compared with controls, but most differences were not statistically different. Numbers of IgG1-producing plasma cells at the Furstenberg's rosette area of infected quarters were significantly (P less than 0.05) higher than uninfected quarters. There were no significant differences in concentrations of Ig isotypes in milk from S. aureus infected and uninfected quarters. Data suggest that the antigenic effect of chronic S. aureus infection on the humoral immune response of the bovine mammary gland is minimal. Persistency of S. aureus infection may result, in part, from suboptimal stimulation or immunosuppression of the mammary immune system.

Animals↗
PubMed1988

Production and characterization of monoclonal antibodies to bovine beta 2-microglobulin.

In an attempt to isolate monoclonal antibodies specific for bovine lymphocytes, spleen cells from mice immunized with bovine lymphocytes were fused with the mouse myeloma cell line SP-2/0. The resulting hybridoma cell lines were tested for reactivity with bovine lymphocytes, polymorphonuclear neutrophils, RBC, gamma-globulin, kappa-casein, beta-casein, alpha-S1-casein, and beta 2-microglobulin (beta 2m) and with beta 2m from rabbits, goats, and human beings. None of the clones secreted anti-bovine lymphocyte-specific antibody. However, 4 secreted monoclonal antibodies to bovine beta 2m. They also reacted with beta 2m from rabbit, goat, and human being. One monoclonal antibody also was found to be reactive with bovine immunoglobulin. Monoclonal antibodies to beta 2m could serve as a tool to (1) explore the homology of the beta 2m molecule among various species, (2) examine the relationship of beta 2 m with the constant region of the immunoglobulin molecule, (3) quantitate bovine beta 2m in various body fluids and major histocompatibility antigens on cell surfaces, (4) help characterize those antigens in cattle, and (5) be used for tissue typing of those antigens.

Animals↗
PubMed1988

Relationship between immunoglobulin concentrations in milk and phagocytosis by bovine neutrophils.

Using bovine neutrophils and radio-labelled Staphylococcus aureus, skim milk samples taken at 4 stages of lactation from the 4 mammary quarters of 48 cows were used in an in vitro phagocytosis assay. Immunoglobulin (Ig) isotype concentrations in the milk samples were estimated by use of an ELISA procedure. To determine associations of Ig concentrations with phagocytosis, correlations, simple regressions, and partial regressions were calculated. Simple correlations were computed between each Ig isotype and phagocytosis percentage for each lactation number stage of lactation category. Ranges of these correlations for IgM, IgG1, IgG2, and IgA were 0.33 to 0.60; -0.16 to 0.43; 0.04 to 0.46; and -0.30 to 0.36, respectively. Correlations for concentrations of IgG2 and IgM with percentage of phagocytosis tended to be slightly higher for samples from older cows, in contrast to the correlations calculated for IgA and IgG1. Multiple regression of percentage of phagocytosis calculated simultaneously on concentrations of the 4 Ig isotypes in the sample indicated that IgM, followed by IgG2 and IgA, was most closely associated with phagocytosis. Partial regression calculated on concentration of IgG1 was not significant. Addition of bacteriologic status of the quarter and somatic cell concentration in the milk sample did not increase accuracy of predicting percentage of phagocytosis, compared with use of Ig concentrations alone. These results supported the attribution of unique modes of action to IgM and IgG2 in promoting phagocytosis by neutrophils.

Animals↗
PubMed1988

In vitro depression of bovine lymphocyte function by treatment of cultured bovine lymphocytes with physiologic concentrations of hydrocortisone.

The effect of hydrocortisone (hydrocortisone sodium succinate) on bovine lymphocyte blastogenesis in response to Staphylococcus aureus antigens and phytohemagglutinin was measured in vitro. Lymphocytes isolated from the blood of cows were treated for 6 to 8 days with physiologic hydrocortisone concentrations known to be inducible by environmental stress (10 ng/ml), acute clinical mastitis (25 ng/ml), or adrenocorticotropin treatment (45 ng/ml). All 3 concentrations of hydrocortisone caused a depression (P less than 0.01) in lymphocyte blastogenesis in response to phytohemagglutinin and S aureus antigen extract. Hydrocortisone concentrations as low as 10 pg/ml caused a depression in the lymphocyte blastogenic response to phytohemagglutinin. Marked variation existed among cows in the normal response of their nontreated lymphocytes and in the degree of depression of lymphocyte function after the in vitro treatment with hydrocortisone. Macrophage depletion experiments showed that the suppressive effect of hydrocortisone was not mediated by induction of suppressor macrophages. The data suggest that T-cell function was impaired directly by hydrocortisone treatment.

Animals↗
PubMed1987

The application of hybridoma technology to the study of bovine immunoglobulins.

Studies are described in which hybridoma technology is used to produce a variety of reagents for the characterization and manipulation of the bovine humoral immune system. Selected members of a set of murine monoclonal antibodies (MAb) specific for each of four major isotypes of bovine Ig constant regions, one specific for anti-bovine Ig constant regions as well as one specific for anti-bovine light chains are discussed. Interspecific fusion of bovine lymphocytes with the established mouse cell line, SP2/0 was used to produce a collection of stable hybridomas among which were found secretors of bovine IgG1, IgG2, IgM, IgA and bovine light chain. Interspecific fusion of SP2/0 with lymphocytes from a multiparous Holstein four days post immunization with Streptococcus agalactiae yielded MAb with specificity for the immunizing antigen. One of these hybridomas, LHRB 19.17, which displayed a particularly stable secretory phenotype, was used as an immunogen for the production of a library of murine monoclonal anti-idiotype antibodies. Competitive antigen binding analysis showed that 15 of the 24 anti-LHRB 19.17 idiotype antibodies isolated blocked the binding of the idiotype to its nominal antigen and so were candidates for evaluation as antigen mimics. Some of the ways in which monoclonal anti-idiotypes in particular, and monoclonal in general, might be of use in problems of animal disease are discussed.

Animals↗
PubMed1987

In vitro sensitization and stimulation of bovine lymphocytes with encapsulated or nonencapsulated Smith strain of Staphylococcus aureus.

An in vitro immunization procedure was developed, whereby bovine blood and mammary gland lymphocytes were isolated, and their blastogenic responses (measured by [3H]thymidine uptake) to sensitization and subsequent stimulation with selected antigens were determined. Capsular extracts of Staphylococcus aureus, encapsulated S aureus, and nonencapsulated S aureus were used as test antigens. Differences were observed between kinetics of the secondary response to encapsulated and nonencapsulated S aureus. The secondary response to nonencapsulated S aureus peaked in 48 hours, whereas the secondary response to the encapsulated S aureus peaked in 72 hours. The delayed peak response to encapsulated S aureus was observed only when encapsulated S aureus was used for sensitization of lymphocytes, regardless of whether the encapsulated or nonencapsulated strain was used for stimulation. Sensitization of lymphocytes with the nonencapsulated strain and stimulation with the encapsulated strain did not alter the kinetics of the secondary response of cells sensitized with the nonencapsulated strain. Seemingly, T and B cells were responsive to in vitro immunization with S aureus antigens.

Animals↗
PubMed1986

The association of extrinsic bovine IgG1, IgG2, SIgA and IgM with the major fractions and cells of milk.

The association of purified, iodinated bovine immunoglobulins with the various fractions of whole milk and with cell-free skim milk and with two different mammary cell populations was studied. Associations in whole milk were concentration independent over a 6-fold range and revealed that SIgA and IgM were 5-fold more prevalent in milk fat than IgG1 and IgG2; the concentration of IgM and SIgA was 3-fold and 2-fold higher in fat than whey, respectively. A significant proportion of the IgM, ca 20% and to a lesser extent IgG2, ca 10%, were found in association with the casein pellet. Greater than 85% of the IgG2, greater than 90% of the IgG1, approximately equal to 80% of the SIgA and 70% of the IgM were found in milk whey. Isoelectric precipitation of casein significantly reduced the amount of IgM which associated with fat. When labelled Igs were incubated with milk leukocytes alone, only SIgA and IgM became significantly associated with them. However, when 10(6) cells were added, the amount of SIgA and IgM in the casein-cell pellet was not additive, although the increase for IgM was significant. These Igs also associated with the casein pellet of cell-free skim milk. When whole milk was used, the milk fat competed with cells and casein for association with SIgA and IgM. Homogenization of the fat layer from normal milk with or without added cells, caused significant release of the Igs which sedimented in the pellet.

Animals↗