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Biomedical subjects

A J Winter

Publications and source records attributed to A J Winter.

At least 73 records · Page 4Linked to original sources
PubMed1984

Evaluation of lymphocyte blastogenesis for diagnosis of bovine brucellosis.

The lymphocyte blastogenesis test (LBT) was evaluated for its usefulness in the diagnosis of bovine brucellosis. Using a cell titration assay which measures the blastogenic response over a range of cell concentrations, peripheral blood lymphocytes (PBL) from various populations of cattle were tested with purified Brucella abortus porin proteins and with Brucella abortus soluble antigen (BASA). Cattle tested included groups infected with virulent B. abortus strain 2308, vaccinated with B. abortus strain 19, infected with Escherichia coli strain 0116:H31 (known to cause serological cross reactions with B., abortus), vaccinated with a bacterin of Staphylococcus aureus, and unimmunized controls. Pregnant heifers vaccinated with strain 19 could generally be distinguished from pregnant animals infected with virulent strain 2308 when LBT responses to porin were analyzed by comparing maximum delta counts per minute (the responses of the cell concentration at which there is the greatest difference between stimulation in the presence of antigen and without antigen). However, a group of nonpregnant heifers infected with strain 2308 either failed to react or reacted in an inconsistent fashion with porin or BASA. The lack of responsiveness in the LBT was accompanied by high concentrations of circulating agglutinins. False positive responses also occurred to porin, although much less frequently than to the crude preparation BASA. The use of nylon wool fractionated PBL did not provide an improved means for distinguishing Brucella infected from uninfected cattle. Based on these findings, we would not recommend the LBT with either of these antigens as a routine method for diagnosis. Lipopolysaccharide was extracted from B. abortus strain 2308 and tested for its role in inducing false positive responses. Although there exist components in B. abortus extracts which are mitogenic for bovine PBL, our data do not support such a function for purified Brucella LPS.

Animals↗
PubMed1983

Immune response to porin in cattle immunized with whole cell, outer membrane, and outer membrane protein antigens of Brucella abortus combined with trehalose dimycolate and muramyl dipeptide adjuvants.

The immune response of cattle to nonliving vaccines derived from Brucella abortus rough strain 45/20 was studied. Vaccines contained trehalose dimycolate and a derivative of muramyl dipeptide. N-acetylmuramyl-L-alpha-aminobutyryl-D-isoglutamine. A factorial experiment was designed to test the effects of type of antigen, quantity of antigen, and quantity of mineral oil on the immune response to porin. Muramyl dipeptide was kept constant at 5 mg per dose, and 1 part of trehalose dimycolate was incorporated for two parts of dry matter. Over a 10-week period, blastogenesis responses to porin were largest in cattle immunized with outer membranes; the highest antibody titers to the porin-lipopolysaccharide complex were achieved by immunization with detergent-extracted outer membrane proteins. There was no advantage in the use of 25, rather than 5, mg of any of the antigens, but antibody responses were improved by increasing the quantity of oil from 0.6 to 1.8 ml per dose. In other animals, blastogenesis and antibody responses were sustained at high levels longer than 3 months after two vaccinations with outer membrane proteins. Intradermal injection of porin evoked inflammatory reactions histologically consistent with delayed-type hypersensitivity. Cross-reactions in cases of delayed-type hypersensitivity occurred with porin derived from a smooth strain of B. abortus but were less extensive than in the blastogenesis test. The magnitude of the delayed-type hypersensitivity and blastogenesis responses induced by vaccination exceeded those observed after natural or experimental infections. No ill effects were observed after vaccination. These findings provide a basis for the use of trehalose dimycolate and muramyl dipeptide adjuvants in evaluating nonviable vaccines for bovine brucellosis.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1983

Nylon bead enzyme-linked immunosorbent assay for detection of sub-picogram quantities of Brucella antigens.

An indirect sandwich enzyme-linked immunosorbent assay, using antibody covalently coupled to nylon beads, has been adapted for the detection of Brucella antigens. Optimum conditions were achieved by incubation of 1 ml of reaction mixture with a single bead, and by minimizing nonspecific interactions through the use of beads coated with purified bovine antibodies, preabsorption of third layer rabbit antibodies with normal bovine serum, and treatment of beads with normal goat serum before addition of the goat anti-rabbit enzyme conjugate. Beta-galactosidase was selected for use with clinical samples primarily because of low levels of endogenous enzyme in bovine leukocytes. Use of a fluorogenic substrate enhanced sensitivity 20-fold. Under these conditions, 100 fg of solubilized crude lipopolysaccharide or 8 to 10 Brucella cells was detectable in a fixed volume of 1 ml. A system was also devised for concentrating antigen which permitted ready detection of 2 pg of lipopolysaccharide in a volume of 50 ml (40 fg/ml). Attempts to detect lipopolysaccharide in the presence of concentrated serum or plasma were unsuccessful, but 10 brucellae added to a suspension of leukocytes from 100 ml of normal bovine blood were easily measured.

Animals↗
PubMed1982

Outer membrane proteins of Brucella abortus: isolation and characterization.

Outer membrane proteins were derived from one rough and four smooth strains of Brucella abortus by sequential extraction of physically disrupted cells with N-lauroylsarcosinate and dipolar ionic detergent. Extraction of outer membrane proteins was ineffective, however, without predigestion with lysozyme. Three groups of proteins were present and could be separated in their native state by sequential anion-exchange chromatography and gel filtration. Membrane proteins contained substantial quantities of tightly adherent lipopolysaccharide which could be reduced but not eliminated by extraction of cells with trichloroacetic acid before disruption. Group 2 proteins, apparently trimers in their native state, gave rise to 43,000- and 41,000-molecular-weight bands after complete denaturation in sodium dodecyl sulfate. They were antigenically identical among all the strains, showed close resemblance in amino acid composition to each other and a general similarity to OmpF of Escherichia coli, and are proposed to be the porins of B. abortus. Group 3 proteins occurred as 30,000-molecular-weight bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, although additional bands were frequently observed in this region. In none of the strains did group 3 proteins manifest heat-modifiable characteristics. Proteins of different strains bore a high degree of similarity to each other in amino acid composition, except in methionine, isoleucine, tyrosine, and histidine. Differences occurred consistently in amino acid composition between group 2 and 3 proteins, and some of these correspond to differences between OmpF and OmpA. Group 2 and 3 proteins were antigenically distinct from each other, but the principal group 3 antigens were shared among all the strains. Despite the lack of heat modifiability, perhaps influenced by adherent lipopolysaccharide, group 3 proteins are proposed as counterparts to OmpA. Most of the group 1 proteins, minor components, were physically associated with those of group 3 unless in sodium dodecyl sulfate. Group 1 proteins produced a major band at 94,000 and exhibited heat modifiability. No evidence was found of a low-molecular-weight lipoprotein in the outer membrane of B. abortus, but this is not taken to exclude its occurrence.

Amino Acids↗
PubMed1982

Mycoplasma bovigenitalium in the upper genital tract of bulls: spontaneous and induced infections.

Mycoplasma bovigenitalium, was isolated from vesicular gland secretions of a large proportion of clinically normal pubescent bulls. Infections established at this site in 1 to 2 year old bulls sometimes persisted for over a year, induced a local immune response, and were associated in some animals with unacceptably low motility of sperm following freezing of semen for artificial insemination. Inoculation of as few as 1000 cfu directly into the vesicular gland sufficed to establish infection but the naturally occurring syndrome could not be successfully reproduced by this method. Intratracheal injection of the organism produced infection of the upper genital tract in one of two bulls. The ELISA test was more sensitive than the IHA test in detection of antibodies to M. bovigenitalium in serum or vesicular secretions, but its usefulness was limited by extensive cross reactions with other Mycoplasma species.

Animals↗
PubMed1981

Immune response in the bovine mammary gland after intestinal, local, and systemic immunization.

The immune response in mammary glands of cattle was measured after intestinal, local, and systemic immunization with T4 bacteriophage. Nonlactating pregnant cows were immunized by infusions into the intestine or mammary gland and by subcutaneous injections in the region of the prescapular or external inguinal lymph nodes. Titers of antibodies of different isotypes were measured in serum and in lacteal secretions by enzyme-linked immunosorbent assay, and numbers of cells producing antibodies of each isotype were determined in lacteal secretions by the Jerne plaque assay. Substantial increases in immunoglobulin G subclass 1 (IgG1) and IgG2 antibody titers were detected in serum and lacteal secretions of animals immunized through an intestinal fistula. IgM and IgA antibody responses were low or undetectable. Low numbers of IgA and IgG1 plaque-forming cells were occasionally detected. It is proposed on the basis of these data that migration of antigen-stimulated IgG lymphoblasts, and perhaps of antigen, to spleen and peripheral lymph nodes may be dominant events after intestinal immunization of ruminants. This is consistent with the predominance of serum-derived IgG antibodies in colostrum and milk. Intramammary infusion of antigen gave rise to increases in antibody titers in all classes which were greater not only in lacteal secretions but also in blood serum than with either systemic route used. There was clear evidence from relative antibody titers for local synthesis of antibodies, principally IgA and IgG1, in the immunized glands. Comparison of IgA titers in secretions from the immunized glands with those in serum also suggested that locally synthesized IgA antibodies may have contributed in some measure to serum titers. Local synthesis in both immunized and nonimmunized glands was also reflected by the presence of increased numbers of IgA and IgG1 plaque-forming cells. It is hypothesized that antibody-forming cells responsible for local synthesis originated in lymphoid tissue within the mammary gland or from peripheral lymph nodes, depending upon the route of immunization.

Animals↗
PubMed1981

Decreased motility of bull spermatozoa caused by Mycoplasma bovigenitalium.

Mycoplasma bovigenitalium mixed with bull semen in egg yolk-citrate buffer and held at 5 C caused a highly significant time- and dose-dependent depression in sperm motility. Mycoplasma adhered to a majority of spermatozoa, principally to the acrosome, but also the the midpiece and tail. This may reflect the basis for a naturally observed condition in young bulls in which genital mycoplasmosis is associated with low sperm motility.

Animals↗
PubMed1980

Antibody-producing cells in bovine lacteal secretions after local immunization.

The indirect Jerne plaque assay was used to determine the presence of antibody-forming cells in lacteal secretions of 2 cows inoculated in the mammary gland with T4 phage. Two adjacent mammary glands of 2 nonlactating 7-months pregnant cows were inoculated by intramammary injection 4 times at 3- to 5-day intervals. The presence of plaque-forming cells (PFC) was assessed in each quarter beginning on postinoculation day 8 and at 4- to 14-day intervals thereafter. Amounts of antibodies in serum and secretions were measured by an indirect hemagglutination test. The PFC were detected in secretions from all quarters of both cows between postinoculation days 8 and 32. Concentrations of PFC fluctuated within quarters during the course of the experiment but no relationship was evident between numbers of PFC in a quarter and its inoculation status. The use of monospecific antiglobulin sera at 1 sample-collection period revealed that cells synthesizing immunoglobulin (Ig)G and IgA antibodies were predominant in lacteal secretions. Antibody amounts in serum and secretions rose after inoculation, and titers in secretions were markedly higher in most instances. Antibody-forming cells were thus demonstrated to accumulate in the mammary gland after intramammary inoculation. The presence of antibody-forming cells in non-inoculated glands may have been the result of antigen transfer among quarters, but was considered more likely to have resulted from systemic migration of antigen-stimulated cells with migration into all quarters, regardless of inoculation status. Antibodies in lacteal secretions may have accumulated through a combination of local synthesis and selective transport from the bloodstream.

Animals↗
PubMed1978

Evaluation of a transport medium for campylobacter (Vibrio) fetus.

A quantitative assessment was performed on a serum enrichment medium for Campylobacter (Vibrio) fetus, to test its effectiveness as a transport medium. Abundant yields of C fetus were obtained with an inoculum size of 10(4), even after keeping the medium in air at 22 to 23 C for 4 days prior to incubation at 37 C under microaerophilic conditions. The organism could be isolated from the medium kept in air at 22 to 23 C for at least 2 days, with an inoculum as small as 100 organisms. It was concluded that this formulation comprises a convenient and effective transport medium for field use. Transit time should not exceed 3 days and, ideally, should be restricted to less than or equal to 2 days. At least duplicate vials of transport medium should be inoculated with each sample.

Campylobacter↗
PubMed1978

Microcapsule of Campylobacter fetus: chemical and physical characterization.

The antiphagocytic antigen (antigen [a]) comprising the microcapsule of a strain of Campylobacter fetus subsp. intestinalis has been purified from culture supernatants by ammonium sulfate fractionation and free-flow electrophoresis. Antigen [a] is a glycoprotein containing about 4% carbohydrate consisting of hexose, pentose, and methylpentose. The composition of the protein was typical of bacterial extramural structural proteins in its low content of basic, aromatic, and sulfur-containing amino acids. The protein had a high content of aspartic acid, threonine, glycine, and alanine. Antigen [a] had an Rf of 0.33 on polyacrylamide gel electrophoresis and a molecular weight calculated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 98,000. In contrast to its free form in culture supernatants, antigen [a] in vesicles derived from sheared cells appeared to exist in a complex with lipopolysaccharide. This complex could be dissociated by ethylenediaminetetraacetic acid or by ethylenediaminetetraacetic acid plus Triton X-100. A mutant strain that lacked a microcapsule, when incubated with soluble antigen [a] in a calcium medium, became agglutinable by monospecific [a] antiserum and showed an additional structural layer similar in appearance to the microcapsule on its cell wall. Points of similarity are emphasized between antigen [a] of C. fetus and the outer structural protein of the taxonomically related Spirillum serpens.

Agglutination↗
PubMed1977

Blastogenic transformation by lipopolysaccharide of blood leukocytes from immunized but not normal cattle.

Escherichia coli lipopolysaccharide produced blastogenic transformation in whole-blood leukocytes from heifers that had been infected and immunized with Campylobacter fetus, but not in cells from control animals. This suggests that lipopolysaccharide dose not function as a B-cell mitogen in cattle and that its stimulation of cells from immunized animals occurred through another mechanism.

Animals↗
PubMed1976

Antibody-mediated immobilization of Campylobacter fetus: inhibition by a somatic antigen.

Immobilization tests were conducted on a wild-type strain of Campylobacter fetus subsp. intestinalis and on a mutant lacking an antiphagocytic cell surface component. Highly effective immobilization of the mutant, both as single cells and clumps of cells, was produced with an antiserum containing antibodies specific for the flagellar hook and filament and for the O antigen. Damage to flagellar hooks after reaction with this antiserum was observed only with cells of the mutant. Single-cell immobilization of the mutant was also produced with an antiserum specific for a heat-stable somatic antigen which was distinct from the O antigen and was exposed on the cell surface only of the mutant. Minimal immobilization of the wild strain was brought about by either of these antisera. It was shown also that O antibodies had no effect on the motility of either the wild strain or mutant. These findings indicate that antibody-mediated immobilization may be brought about by effects on the flagellar hook or cell body, as well as on the flagellar filament. Furthermore, the protection from immobilization afforded the bacterium by the antiphagocytic surface structure suggests a dual function for this virulence factor in the infected animal.

Animals↗
PubMed1975

Bovine veneral vibriosis: antigenic variation of the bacterium during infection.

Host parasite relationships in the female genital tract were studied in bovine venereal vibriosis by investigating agglutinin production and alterations in superficial antigens of the bacterium during the course of infection in two heifers. Cervicovaginal mucus (CVM) steamed cell agglutinins were shown to appear earlier and remain at consistently higher levels than whole-cell agglutinins. Whole-cell agglutinin titers fluctuated much more than steamed cell titers, suggesting possible changes in whole-cell antigens. Marked antigenic variation was demonstrated in successive monthly CVM isolates from the two heifers by agglutination tests with rabbit antisera of various specificities. Some changes in CVM antibody specificity during the infection were noted also. Antigenic variation in the bacterium was proposed as a mechanism for maintenance of the asymptomatic cervicovaginal carrier state in the presence of antibody.

Agglutination Tests↗