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B L Deyoe

Publications and source records attributed to B L Deyoe.

At least 19 recordsLinked to original sources
PubMed1991

Differentiation by western blotting of immune responses of cattle vaccinated with Brucella abortus strain 19 or infected experimentally or naturally with virulent Brucella abortus.

Brucella abortus strain 19 salt-extractable proteins fractionated by differential ammonium sulfate precipitation were used in a western blotting method to detect bovine immunoglobulin G antibodies to B. abortus. Sera from infected cattle and from cattle vaccinated with strain 19 and subsequently exposed to virulent B. abortus bound to a common group of antigens ranging in molecular weights from 31,000 to 45,000 daltons. Immunoglobulin G antibodies in sera from the latter group in addition also bound to antigens with molecular weights of 66,000 to 71,000 daltons. Some sera from cattle vaccinated when sexually mature reacted similar to those from infected cattle, while immunoglobulin G antibodies in sera from Brucella-free cattle and vaccinated calves did not bind to either group of antigens. In general, fractionation of the proteins by ammonium sulfate precipitation offered no advantage for detecting differences between groups of sera. Ammonium sulfate fraction 0 to 35% reacted with a larger number of sera from a naturally infected group than fraction 0 to 70%. Both fractions reacted equally well with sera from the other groups of cattle, while fractions 35 to 70% and 70 to 100% reacted poorly in this technique. The attractive feature of the blot is that sera from calfhood-vaccinated cattle did not react.

Animals↗
PubMed1991

Effect of endocytic and metabolic inhibitors on the internalization and intracellular growth of Brucella abortus in Vero cells.

Uptake, transfer to rough endoplasmic reticulum, and intracellular growth of Brucella abortus were studied in Vero cells treated with endocytic and metabolic inhibitors. Infection of Vero cells was suppressed when inhibitors of energy metabolism (iodoacetate, dinitrophenol), receptor-mediated endocytosis (monodansylcadaverine, amantadine, methylamine), or endosomal acidification (chloroquine, ammonium chloride, monensin) were added to the inoculum. Inhibition was not observed when these drugs were added after the inoculation period. Infection of Vero cells by B abortus was inhibited by dibutyryl-cyclic adenosine monophosphate and Vibrio cholerae enterotoxin, but was stimulated by dibutyryl-cyclic guanosine monophosphate and escherichia coli heat-stable enterotoxin a. Uptake of B abortus by Vero cells was not prevented by colchicine, but was abolished by cytochalasin B. Uptake of heat-killed B abortus and noninvasive E coli was similar to that of viable brucellae. Intracellular growth of B abortus was not affected by cycloheximide. Results indicate that: B abortus may be internalized by a receptor-mediated phagocytic process; transfer of B abortus from phagosomes to rough endoplasmic reticulum may require endosomal acidification; and replication of B abortus within the rough endoplasmic reticulum may not depend on protein synthesis by the host cell.

Amantadine↗
PubMed1991

Effect of milk stasis on Brucella abortus infection of the mammary gland in goats.

To compare the effects of milk stasis and milk flow on Brucella abortus infection of the mammary gland under the same systemic conditions, primiparous goats (n = 5) were inoculated IV with B abortus on the day of parturition, and suckling by their neonates was restricted to one mammary gland. Goats were euthanatized and necropsied at 3 weeks after inoculation, and milk, mammary glands, and supramammary lymph nodes were evaluated by bacteriologic, histologic, and immunoenzymatic staining techniques. Nonnursed mammary glands had high titers of brucellae in milk, moderate interstitial mastitis, and brucellar antigen in macrophages located primarily in alveolar and ductal lumina. Brucellae often filled the macrophage cytoplasm. In contrast, nursed mammary glands had fewer brucellae in milk, minimal inflammatory changes, and no detectable brucellar antigen in histologic sections. Hyperplastic changes were only seen in supramammary lymph nodes draining nonnursed mammary glands; these contained more brucellae than lymph nodes draining nursed mammary glands. These studies show that milk stasis may be the sole cause of increased susceptibility of nonnursed mammary glands to B abortus infection.

Animals↗
PubMed1990

Penetration and intracellular growth of Brucella abortus in nonphagocytic cells in vitro.

In pregnant ruminants, Brucella abortus localizes and replicates within the rough endoplasmic reticulum of trophoblastic epithelial cells. In this study, Vero cells were exposed to B. abortus to investigate its internalization and intracellular growth in nonphagocytic cells. A new double-fluorescence staining procedure to discriminate between extracellular and intracellular bacteria was developed. Studies with the double-fluorescence staining procedure and quantitative bacteriologic culture of disrupted host cells showed that various B. abortus strains replicated within Vero cells, including smooth virulent (strains 2308S and 544), smooth attenuated (strain 19), and rough (strains 45/20 and 2308R) strains. Rough brucellae were more adherent and entered a greater number of Vero cells. Intracellular replication occurred in a larger percentage of cells with smooth virulent (2308S and 544) strains than with smooth attenuated (19) or rough (45/20 and 2308R) strains. Differences in adhesiveness and invasiveness were correlated to hydrophobicity of the organism, as measured by hydrocarbon adherence. Ultrastructurally, intracellular smooth (2308S) and rough (45/20) brucellae were consistently found within cisternae of the rough endoplasmic reticulum and nuclear envelope. The results suggest that transfer to the rough endoplasmic reticulum is the limiting step in the infection of nonphagocytic cells by B. abortus.

Animals↗
PubMed1990

Cloning, expression, and occurrence of the Brucella Cu-Zn superoxide dismutase.

Recently, the complete amino acid sequence of a protein expressed in Escherichia coli from cloned Brucella abortus DNA was reported. On the basis of amino acid homology, this protein was identified as a copper-zinc superoxide dismutase (Cu-Zn SOD) (B. L. Beck, L. B. Tabatabai, and J. E. Mayfield, Biochemistry 29:372-376, 1990). We demonstrate in this paper that the sequenced protein is the same as the previously studied salt-extractable protein BCSP20. The plasmid-encoded protein expressed from recombinant E. coli is identical to the Brucella-derived BCSP20 in molecular mass, N-terminal amino acid sequence, and cross-reactivity with homologous and heterologous rabbit sera against either the recombinant gene product or the Brucella-derived protein. A survey of the expression of the Cu-Zn SOD protein in Brucella biovars representing all species was done by Western blotting (immunoblotting) using antisera raised against the recombinant E. coli-derived protein. With the exception of B. neotomae and B. suis biovar 2, the Cu-Zn SOD protein was detectable in all Brucella species and biovars tested, including eight biovars of B. abortus.

Amino Acid Sequence↗
PubMed1990

Entry and intracellular localization of Brucella spp. in Vero cells: fluorescence and electron microscopy.

Vero cells were inoculated with the six species of Brucella (B. abortus, B. melitensis, B. suis, B. neotomae, B. canis, and B. ovis) and examined by fluorescence and electron microscopy. All Brucella spp. were internalized by Vero cells. In all cells except those inoculated with B. canis, the numbers of intracellular brucellae increased with time after inoculation. Intracellular brucellae were first seen within phagosomes and phagolysosomes. Subsequent localization within cisternae of the rough endoplasmic reticulum was seen with all species of Brucella, except B. canis, which was restricted to phagolysosomes. Although rough brucellae were more adherent and entered a greater number of Vero cells, intracellular replication occurred in a larger percentage of cells with smooth rather than with rough brucellae. These results suggest that phagocytosed Brucella spp. are transferred 1) to cisternae of the rough endoplasmic reticulum, where unrestricted bacterial replication takes place; or 2) to phagolysosomes in which Brucella spp. fail to replicate. The various strains of Brucella spp. differ in their ability to induce their own transfer to the rough endoplasmic reticulum.

Animals↗
PubMed1989

Detection of conglutinin in bovine serum by complement-dependent agglutination of Escherichia coli.

Agglutination of Escherichia coli (ECA) by normal bovine serum was shown to be prevented by heating serum to 56 degrees C for 30 min, but restored by normal horse, swine, rabbit or guinea pig sera. Further investigation of the ECA reaction using techniques to distinguish between conglutination and immunoconglutination indicated ECA to be a conglutination reaction. Testing of 264 sera obtained from 22 normal cattle over a period of 5 months did not show individual or seasonal variation in ECA. Changes in ECA and conglutination were detected in sera of periparturient cows. The ECA reaction is a simple technique for detecting conglutinin in bovine serum.

Agglutination↗
PubMed1989

Immunogenicity of Brucella abortus salt-extractable proteins.

The immunogenic properties of salt-extractable proteins and chromatographic fractions thereof from Brucella abortus were evaluated in lemmings (Dicrostonyx rubricatus). The efficacy of the Brucella proteins as immunogens was determined after challenge with virulent B. abortus strain 2308 and was based on protection against clinical signs and gross lesions of brucellosis, as well as on numbers of viable Brucella in the spleen. Vaccination of lemmings with as little as 0.1 microgram of salt-extractable proteins (CSP) suppressed splenic infection, resulting in reduced numbers of viable organisms per spleen of 5-6 logs compared to non-vaccinated controls. Protein fractions separated by column chromatography were generally effective in reducing splenic infection, and contained proteins with molecular weights of 30,000, 20,000 and 12,000. Vaccines containing chemically modified dodecanoyl-CSP offered no additional advantage over unmodified CSP vaccines.

Animals↗
PubMed1989

Pathogenesis of Brucella abortus infection of the mammary gland and supramammary lymph node of the goat.

Goats, both in late pregnancy and soon after parturition, were inoculated intravenously with Brucella abortus, and mammary glands and supramammary lymph nodes were examined by light and electron microscopy at 2 to 55 days post-inoculation. After 7 days, lymphoplasmacytic, histiocytic interstitial mastitis with a lobular and periductal distribution were detected microscopically. Brucellae, identified in tissues with immunoperoxidase staining and antibody-coated colloidal gold stain, were first seen in macrophages and neutrophils throughout mammary parenchyma, but most commonly in mammary alveoli. In subsequent samples, infected phagocytes progressively increased in number, especially in ductal and alveolar lumina, and adjacent parenchyma. B. abortus was in phagosomes and phagolysosomes in macrophages and neutrophils; degenerate and necrotic phagocytes were often filled with brucellae. Extracellular brucellae were associated with ruptured necrotic infected phagocytes. Supramammary lymph nodes draining infected mammary glands were enlarged. Lymphofollicular hyperplasia, medullary plasmacytosis, and sinus histiocytosis were seen microscopically. Brucellae were seen exclusively in macrophages, which were most often located in subcapsular and cortical sinuses. This study suggests that phagocytic leukocytes 1) transport brucellae into mammary glands; 2) provide a site for intracellular replication in mammary secretions; and 3) transport brucellae from mammary glands to supramammary lymph nodes.

Animals↗
PubMed1989

Effect of nursing on Brucella abortus infection of mammary glands of goats.

Eight 1-year-old, goats were inoculated intravenously with Brucella abortus (B. abortus) on the day of parturition and necropsied at 28 days after inoculation. Four nursed their kids and four did not (milk was not removed from the udders). Tissues and fluids were examined by bacterial isolation, light microscopy, and serologic methods. Nonnursing goats had high titers of brucellae (less than or equal to 10(8) organisms/ml) in milk (brucellae were isolated from four of four udders), had marked enlargement of supramammary lymph nodes, and had lymphoplasmacytic and histiocytic interstitial mastitis. Immunoperoxidase staining revealed that brucellae were primarily in macrophages and neutrophils of the mammary alveolar and ductal lumens and in macrophages of the subcapsular sinuses of the supramammary lymph node. In contrast, nursing goats excreted brucellae intermittently at low concentrations (less than 10(3) organisms/ml) in milk; brucellae were isolated at necropsy from one of four udders; supramammary lymph nodes were not enlarged; and mammary lesions were not seen. Brucellae were detected in more tissues other than the udder, and serum anti-Brucella antibody titers were higher in nonnursing goats than in nursing goats. The present study indicates that the failure to nurse or release milk enhances localization and replication of B. abortus in mammary glands of goats after parturition, and that mammary gland infection may result in increased systemic spread and persistence of brucellae in the host.

Agglutination Tests↗
PubMed1989

Protection of mice against Brucella abortus infection by inoculation with monoclonal antibodies recognizing Brucella O-antigen.

Monoclonal antibodies recognizing the O-polysaccharide portion of Brucella abortus strain 2308 provided BALB/c mice with passive protection against challenge exposure with the homologous strain. Numbers of colony-forming organisms in the spleen were reduced by IgM and IgG monoclonal antibodies. Active immunization of mice, using B abortus 2308S lipopolysaccharide, resulted in production of IgM antibody at 14 days. Clearance of organisms in the actively immunized mice after challenge exposure at 14 days was nearly identical to that in passively immunized mice. Mice either passively or actively immunized were effectively protected from 0 to 28 days. Bacterial colonization of the spleen was observed to increase in both groups of mice at 56 days and indicated that humoral responses were effective in eliminating the organism in the early stages of infection, but other immune mechanisms were necessary for protection of mice in the later stage of infection with virulent strains of B abortus.

Animals↗
PubMed1989

Chemical and protective properties of Brucella lipopolysaccharide obtained by butanol extraction.

Lipopolysaccharide (LPS) fractions were obtained from smooth cultures of Brucella abortus strains 2308 and S-19 by butanol extraction procedures. The LPS from the initial butanol extraction contained 10 to 15% protein and was reduced to less than 1% protein by treatment with proteinase K. The LPS fractions were identified and characterized on the basis of the chemical analysis, sodium dodecyl sulfate gel electrophoresis, cesium chloride gradients, electron microscopy, and gel immunodiffusion. Results indicated that the butanol procedure is a reliable method in the extraction of LPS from Brucella abortus cells. Proteinase K-treated LPS containing less than 1% protein from strain 2308 was used to vaccinate BALB/cByJ mice. Immune and protective criteria for vaccinated and nonvaccinated mice were increased immunoglobulin (IgG and IgM) titers in sera of prechallenge-exposed mice, reduced colony-forming units/spleen, and splenomegaly in post-challenge-exposed mice. Results indicated that proteinase K-treated LPS was immunogenic as well as protective for mice.

Animals↗
PubMed1989

Duration of strain 2308 infection and immunogenicity of Brucella abortus lipopolysaccharide in five strains of mice.

A study was conducted to compare immunogenicity of a Brucella abortus lipopolysaccharide (LPS) and the duration of infection in 5 strains of mice. Mice of strains CBA/NJ, BALB/c, CD-1, C3H/HeN, and C3H/HeJ were allotted into 2 large groups (vaccinated with proteinase K-treated LPS or nonvaccinated) and 6 subgroups based on the intervals between challenge exposure to B abortus strain 2308 and the week the response data were obtained. Criteria used in comparing responses between the various strains of mice as well as between vaccinated and nonvaccinated mice were splenomegaly, colony-forming units (CFU) from spleens, and antibody titers. Responses were evaluated at 1, 2, 3, 5, 8, and 12 weeks after challenge exposure. Results indicated that all strains of mice became infected and maintained infection throughout the 12-week period, the percentages of mice infected were significantly (P less than 0.05) less in vaccinated mice for the first 5 weeks after challenge exposure, and there were no direct correlations between increased immunoglobulins (IgM and IgG titers) and reduction in CFU. Vaccinated mice of strains BALB/c, CD-1, C3H/HeN, and C3H/HeJ had increased titers when challenge exposed and also had significantly (P less than 0.05) smaller spleens and lower CFU. Vaccinated CBA/NJ mice did not have marked antibody titers. The overall results indicated that vaccination with LPS offers some initial protection against B abortus strain 2308 infection, but this protection disappears gradually and in various degrees in the 5 strains of mice studied.

Animals↗
PubMed1989

Immunologic, histopathologic, and bacteriologic responses of five strains of mice to Brucella abortus strain 2308.

A study was conducted to establish baseline data on Brucella abortus infection induced in 5 strains of mice (CBA/NJ, BALB/c, CD-1, C3H/HeN, and C3H/HeJ). The strains were compared on the basis of immunologic, histopathologic, and bacteriologic responses. There were 4 treatment groups for each strain of mice: (1) vaccinated with homologous lipopolysaccharide and challenge exposed to B abortus strain 2308; (2) not vaccinated but challenge exposed; (3) vaccinated and not challenge exposed; and (4) not vaccinated and not challenge exposed. Results indicated that mice can be used for comparative studies on the pathogenesis and immunogenesis of B abortus infections; strains of mice may vary in their responses to Brucella infection, regardless of their vaccination status. Bacteriologic and immunologic responses in mouse strains BALB/c, CD-1, C3H/HeN, and C3H/HeJ, but not those of CBA/NJ, were extrapolative among strains.

Animals↗
PubMed1988

Conservation of antigenicity in a 31-kDa Brucella protein.

A 31-kilodalton (kDa) protein extracted from Brucella abortus was previously cloned into Escherichia coli and expressed at high levels. The E. coli-derived protein can be purified by a simple 2-step procedure entailing detergent extraction followed by ion-exchange chromatography. Subsequent analyses show that the E. coli-derived protein is identical to the Brucella-derived protein in molecular weight and isoelectric point. A partial amino acid sequence of the N-terminus of the protein of E. coli origin matches the predicted sequence, based on DNA sequence data. Using specific antiserum raised against the E. coli-derived protein, 34 strains of Brucella, representing all 6 recognized species, were examined for expression of the 31-kDa protein by Western blotting. This protein was detectable in all, but one Brucella species (B. ovis), including all 8 biovars of B. abortus tested. This degree of conservation supports further study of the 31-kDa protein for potential exploitation as a vaccine or diagnostic component.

Amino Acid Sequence↗
PubMed1988

Bovine ileal dome lymphoepithelial cells: endocytosis and transport of Brucella abortus strain 19.

Ligated ileal loops of calves were inoculated with Brucella abortus and examined at 2, 4, 6, 10, and 24 hours post-inoculation. B. abortus was identified by light and electron microscopy using immunoperoxidase and antibody-coated colloidal gold techniques. B. abortus was detected in vesicles, phagolysosomes, and large vacuoles of lymphoepithelial cells. Numbers of intracellular bacteria decreased with time after inoculation. B. abortus was also seen between and below lymphoepithelial cells and free in the dome interstitium and intestinal lymph vessels. Neutrophils and macrophages in both epithelium and lamina propria contained intact or degraded bacteria within phagosomes, phagolysosomes, and multivesicular bodies. These studies showed that (1) transepithelial migration of B. abortus occurred principally by dome lymphoepithelial cell endocytosis and transport, and (2) B. abortus was degraded by macrophages and neutrophils of the gut-associated lymphoid tissue.

Animals↗
PubMed1988

Pathogenesis of Brucella abortus in chicken embryos.

Chicken embryos inoculated with Brucella abortus at 6, 10, and 12 days of incubation were examined by light and electron microscopy. B. abortus was identified by avidin-biotin immunoperoxidase and immunogold techniques. Death occurred from 2 to 5 days post-inoculation, depending on age of the embryo and route of inoculation. B. abortus was recovered from all infected eggs. Brucellae had spread throughout all tissues and localized preferentially within cells of mesodermal derivation. Organ distribution and degree of bacterial replication varied with age of the embryo at time of inoculation. In 6-day-old embryos, B. abortus localized preferentially in endoderm and mesoderm of yolk sac wall, extra- and intraembryonic serosal epithelia, and glomeruli of the mesonephros. In 10- and 12-day-old embryos, B. abortus spread to all tissues; renal glomeruli, liver, spleen, and heart were most severely infected. Intracellular B. abortus was within the rough endoplasmic reticulum of mesenchymal, mesothelial, yolk endodermal, and hepatic cells. In mononuclear phagocytes, endothelial cells, and granulocytes, bacteria were within membrane-bound vacuoles. Intracellular replication of B. abortus in embryonic tissues, especially yolk endoderm, closely resembled that in experimental infections of trophoblasts.

Animals↗