Biomedical subjects

L B Tabatabai

Publications and source records attributed to L B Tabatabai.

At least 37 records · Page 2Linked to original sources

PubMed1990

A protein isolated from Brucella abortus is a Cu-Zn superoxide dismutase.

Brucella abortus contains a protein that elicits an antigenic response in cattle previously exposed to the organism. The amino acid sequence of the recombinant form of this antigenic protein was determined by gas-phase sequencing of the pyridylethylated protein and its peptides obtained by digestion with cyanogen bromide (CNBr), clostripain, and Staphylococcus aureus V8 protease. The Brucella protein demonstrated 53.6% identity with the Cu-Zn superoxide dismutase (SOD) from Photobacterium leiognathi. Residues essential for metal coordination and enzymatic activity and cysteines required for the formation of the intrasubunit disulfide bridge of Cu-Zn SOD were conserved in the Brucella protein. also exhibited SOD activity that was inhibited by cyanide, which is characteristic of a Cu-Zn SOD. Brucella abortus Cu-Zn SOD is the second prokaryotic Cu-Zn SOD to be sequenced, and the fifth found in prokaryotes. The high degree of conservation between Photobacterium and Brucella Cu-Zn SOD supports the hypothesis of a separately evolved prokaryotic and eukaryotic Cu-Zn SOD gene.

Amino Acid Sequence↗

PubMed1990

Oral immunization of mice with attenuated Salmonella typhimurium containing a recombinant plasmid which codes for production of a 31-kilodalton protein of Brucella abortus.

Salmonella typhimurium chi 4064, an attenuated delta cya delta crp mutant of S. typhimurium SR-11, was used as a carrier for the plasmid pBA31-R7. This plasmid codes for the expression of a 31-kilodalton (kDa) protein from Brucella abortus (BCSP31). Recombinant S. typhimurium chi 4064(pBA31-R7) expressed BCSP31 in vitro as shown by Western blot (immunoblot) analysis. The plasmid was stable in vitro and in vivo and did not affect the ability of the mutant to invade and colonize the small intestine, mesenteric lymph nodes, liver, or spleen of BALB/cByJ mice. Animals orally immunized with S. typhimurium chi 4064(pBA31-R7) developed serum and intestinal antibody responses to the B. abortus 31-kDa protein and to salmonella endotoxin as measured by enzyme-linked immunosorbent assay. Mice orally immunized with S. typhimurium chi 4064pBA31-R7 did not develop a delayed-type hypersensitivity following a footpad injection with recombinant BCSP31. Antigen-specific blastogenic data also support these in vivo results. All data indicate that this route of antigen delivery is effective for stimulating antibody-mediated immunity but that the B. abortus 31-kDa protein is a poor immunogen for inducing a cell-mediated immune response in BALB/cByJ mice.

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

Immunogenicity of Brucella-extracted and recombinant protein vaccines in CD-1 and BALB/c mice.

A study was conducted to determine whether subcomponent proteins (previously identified as BCSP20, BCSP31, and BCSP45, and the corresponding recombinant proteins rBCSP20, rBCSP31, and rBCSP45) that were recovered from the cell surface of Brucella abortus strain 19 were immunogenic and protective for mice when compared with Brucella cell surface protein (BCSP) and with a proteinase K-treated lipopolysaccharide (PKLPS) extracted from B abortus strain 2308. Protection was evaluated after challenge exposure with a virulent culture of B abortus strain 2308, using CD-1 or BALB/c mice or both inoculated with vaccines of various combinations and concentrations, with and without PKLPS or BCSP. Protection was assessed by enumeration of splenic colony-forming units, reduced mean splenic weight relative to controls, and the relative serologic responses (immune response) in an ELISA. The general results indicate that BCSP, PKLPS, BCSP20, and BCSP31 are immunogenic or protective or both. Protectiveness was not observed for each of the recombinant proteins; however, results from the combined recombinant protein vaccine study suggest the immunogenicity of the recombinant proteins. The apparent immune-inducing properties of BCSP20 and BCSP31 are thought to be attributable to the presence of an immunogenic and protective BCSP fraction (possibly lipopolysaccharide) still associated. Serologic results support our conclusion that each of the recombinant protein vaccines did not induce a protective response comparable to that of BCSP or PKLPS, even when the subcomponents were combined. Although the results suggest that the subcomponents of BCSP apparently induced partial protection, they are thought to be only a part of the antigens contained in BCSP that influence the serologic response.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

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

Identification of a virulence factor for Brucella abortus infection in BALB/c mice.

Immunogenic or pathogenic factors of recombinant proteins (rBCSP20, rBCSP-31, and rBCSP45 of Brucella abortus strain 19) for mice were compared with factors of a proteinase K-treated lipopolysaccharide extracted from B abortus strain 2308. Mice were vaccinated with 4 products, using different inoculation schedules and were challenge exposed with a virulent culture of B abortus strain 2308. Blood samples were collected 2 weeks after vaccination and at necropsy and sera were obtained. Spleens were cultured for B abortus at necropsy (3 to 4 weeks after challenge exposure). Mice given proteinase K-treated lipopolysaccharide alone or in conjunction with rBCSP20 or rBCSP45 proteins were protected, but mice given rBCSP31 on the same day as challenge exposure were not. Vaccination with recombinant proteins alone neither provide protection nor significantly (P greater than 0.05) increase the pathogenic effect of the challenge-exposure culture. Seemingly, rBCSP31 might be a virulence factor of B abortus.

Animals↗

PubMed1988

The cloning, expression, and nucleotide sequence of a gene coding for an immunogenic Brucella abortus protein.

Brucella abortus is the causative agent for brucellosis in cattle and man. Development of a single diagnostic test for the differentiation of vaccinated from infected animals and the development of a nonviable 'subunit' vaccine are top priorities of the brucellosis research program in the United States. Preliminary evidence previously showed that a purified 31-kDa protein (thought to be localized at or near the bacterial cell surface) protects against experimental brucellosis in rodents. The gene for this 31-kDa protein has now been cloned in Escherichia coli. The protein is expressed well, apparently from its native promoter, when placed in several different E. coli plasmids. The nucleotide sequence of the flanking and encoding sequences has been determined, and comparison with the N-terminal amino acid (aa) sequence of the mature protein indicates the presence of a putative 28-aa signal sequence. The availability of the 31-kDa protein free of Brucella contaminants now allows rigorous study of the immunological properties of this protein.

Amino Acid Sequence↗

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↗

PubMed1987

Vaccination of cattle with chemically modified and unmodified salt-extractable proteins from Brucella abortus.

Beef heifers were vaccinated on Day 0 with either salt-extractable protein (CSP) or chemically modified CSP (dCSP) from Brucella abortus Strain 19 in Freund's complete adjuvant (FCA). Six weeks later, vaccination was repeated, and heifers received either the homologous or heterologous vaccine. Another group of heifers received only FCA and saline. Vaccinations with CSP or dCSP stimulated marked antibody responses to B. abortus, as detected by standard serologic tests, an enzyme-linked immunosorbent assay, or a quantitative fluorometric immunoassay. Twelve percent of the heifers were seropositive by the CARD test 1 year after vaccination. Vaccination stimulated an increased cell-mediated immune response as measured by lymphocyte blast transformation (LBT) to B. abortus antigens. Fifty-six weeks after the initial vaccination, the heifers were challenged intraconjunctivally with 1.9 X 10(7) colony-forming units of B. abortus strain 2308. Sixty to 83% of the heifers aborted in each group and 70-83% of the heifers were culture positive. There were no significant differences (P greater than 0.05) among groups with respect to the number of abortions or the number of culture-positive heifers. Antibody responses increased rapidly within 4 weeks after challenge. Overall, antibody responses were greater for heifers that aborted than for those that did not abort. These differences were significant (P less than 0.05) only as measured by the fluorometric procedure. The LBT responses appeared to be higher for vaccinates than for the control group, but these differences were not significant (P greater than 0.20). There was a significantly lower (P less than 0.05) LBT response to heat-killed B. abortus in those heifers that aborted compared to those that did not.

Abortion, Veterinary↗

PubMed1987

Characterization of virulent and attenuated strains of pseudorabies virus for thymidine kinase activity, virulence and restriction patterns.

A pseudorabies virus mutant lacking thymidine kinase activity (TK-) was isolated and characterized. The mutant replicated as well in cell culture as the parental TK+ strain, was not temperature sensitive at 38.5 degrees C, and did not revert to TK+. Two pseudorabies virus field isolates and three commercial modified live virus vaccine strains were compared for TK activity and virulence for the mouse; all strains expressed TK: Km values for thymidine of the viral TKs ranged from 2.9 to 3.9 microns; the commercial modified live virus vaccine strains were reduced in virulence for the mouse two to ten-fold. The TK- mutant was avirulent for the mouse. Restriction enzyme analysis of the pseudorabies virus DNA from the strains under study revealed that two of the modified live virus vaccine strains are closely related and that all three modified live virus vaccine strains lack the largest PstI fragment characteristic of the other strains included in the study.

Animals↗

PubMed1986

Identification of Brucella abortus in formalin-fixed, paraffin-embedded tissues of cows, goats, and mice with an avidin-biotin-peroxidase complex immunoenzymatic staining technique.

An avidin-biotin-peroxidase complex immunoenzymatic staining technique was evaluated for light microscopic detection of Brucella organisms in formalin-fixed, paraffin-embedded tissues. Tissues from cows, goats, and mice inoculated with B abortus strain 2308 were examined, using rabbit antiserum to Brucella cell surface protein as primary antibody. Stained organisms were identified histologically in tissue sections containing B abortus, as detected by bacteriologic examination of duplicate nonprocessed tissue samples. Bacillus sp, Corynebacterium pyogenes, Escherichia coli, Listeria monocytogenes, Pasteurella haemolytica, P multocida, Salmonella typhimurium, Staphylococcus sp, and Streptococcus sp did not stain with this system, using anti-Brucella cell surface protein primary antibody, thus indicating specificity for Brucella organisms.

Animals↗

PubMed1985

Isolation of components of Brucella abortus responsible for inhibition of function in bovine neutrophils.

The effects of fractions of Brucella abortus strain 2308 on functions of bovine polymorphonuclear neutrophils (PMNs) were examined in vitro. Ingestion of Staphylococcus aureus and reduction of nitroblue tetrazolium dye by bovine PMNs were not inhibited by heat-killed B. abortus. The ability of PMNs to iodinate proteins was significantly inhibited by live or heat-killed B. abortus and supernatant from heat-killed cells but not by washed heat-killed cells. Two inhibitory components isolated from the supernatant by high-performance liquid chromatography were characterized as nucleotide-like substances with molecular weights of less than 1,000. Inhibition of iodination by these components was concentration dependent. These results indicate that one of the mechanisms by which B. abortus may escape intracellular killing by PMNs is through the production of low-molecular-weight components that inhibit the myeloperoxidase-hydrogen peroxide-halide antibacterial system of bovine PMNs.

Animals↗

PubMed1984

Characterization of salt-extractable protein antigens from Brucella abortus by crossed immunoelectrophoresis and isoelectricfocusing.

Salt-extractable protein antigens (CSP) from Brucella abortus strains 19 and 2308 (vaccine and virulent strains, respectively) were analysed by crossed immunoelectrophoresis (CIE) using rabbit antisera to protein antigens and by isoelectricfocusing (IEF) in polyacrylamide gels. The reference immunoelectrophoretic profiles developed for proteins from strain 19 and 2308 of B. abortus contained 20 and 25 immunoprecipitates, respectively. Serum from cows experimentally infected or hyperimmunized with live organisms produced up to 5 immunoprecipitates in CIE with the protein antigens. Absorption of rabbit sera with homologous B. abortus cells reduced, but did not eliminate all of the immunoprecipitates from rabbit sera, suggesting that the majority, but not all of the protein components, are exposed on the surface of the cell. In contrast, antibody to protein antigens in agglutinin-free absorbed serum from infected cattle could still be demonstrated by CIE, even though CIE with protein extracts from whole cells radioiodinated with the cell surface labeling reagent, diazoiodusulfanilic acid, indicated that these antigens may be at or near the surface of the cell. From CIE in heterologous systems we concluded that all proteins present in strain 19 preparations were partially or completely identical to those in strain 2308. The IEF studies paralleled the CIE studies and revealed that the protein profile from strain 2308 was more complex than the profile from strain 19. Major differences between the 2 strains were found in the pH region from 3.9 to 5.0, where strain 2308 exhibited 4 additional protein bands.

Animals↗

PubMed1984

Specific enzyme-linked immunosorbent assay for detection of bovine antibody to Brucella abortus.

Six soluble antigens prepared from Brucella abortus were compared with a salt-extractable protein (CSP) antigen in an enzyme-linked immunosorbent assay for the detection of antibody to B. abortus in cattle sera. Of seven preparations tested, antigens from B. abortus soluble antigen (prepared from an autoclaved cell suspension) and CSP were stable on frozen storage. Enzyme-linked immunosorbent assay with CSP antigen under optimal conditions was from 100- to 700-fold more sensitive than the standard agglutination, card, Rivanol precipitation-plate agglutination, and the complement fixation tests in detecting immunoglobulin G antibody. From a practical point of view, however, using the most stringent criteria for determining an "upper negative" value, the enzyme-linked immunosorbent assay with CSP was at least 12-fold more sensitive than the standard agglutination test and any of the other serological tests. Furthermore, the enzyme-linked immunosorbent assay with CSP was specific for antibody to B. abortus.

Agglutination Tests↗

PubMed1984

Detection of serum antibody to Brucella abortus in cattle by use of a quantitative fluorometric immunoassay.

A quantitative fluorometric immunoassay (FIAX) was adapted for the detection of serum antibodies to Brucella abortus in cattle. Results are expressed in nanograms of immunoglobulin binding the antigen carrier. The FIAX was compared with the standard tube agglutination, Rivanol precipitation, and complement fixation tests, using 285 serum samples from vaccinated, challenged, or control cattle. Linear regression analysis indicated a significant correlation among all four serological tests; the FIAX test correlated best with the Rivanol test. Ninety sera were from vaccinated and nonvaccinated cattle that were challenged with virulent B. abortus 2308. The sensitivity and specificity of each serological test were determined based on culture results from these cattle. The FIAX was the most sensitive of the four serological tests, detecting 79.2% of the culture-positive animals. The FIAX was the least specific, with 15.4% of the culture-negative animals being classified as positive. Eighty-eight sera were from cattle vaccinated with strain 19 but not challenged. All four serological tests had a statistically significant ability to distinguish sera from control and vaccinates on the basis of mean titers. The mean titer of vaccinates was also significantly different from that of challenged animals. Advantages and disadvantages of the FIAX test for bovine brucellosis are discussed.

Animals↗

PubMed1984

Biochemical and biological properties of soluble protein preparations from Brucella abortus.

Soluble salt-extractable protein antigens (CSP) from Brucella abortus may be of potential value as a vaccine and as a diagnostic reagent for the prevention and diagnosis of bovine brucellosis. These protein antigens, excluding protoplasmic proteins, are those present in the matrix of the outer membrane and in the periplasmic space of gram-negative bacteria. Our research deals mainly with the proteins of the periplasmic space and the nonporin proteins of the outer membrane. We have isolated a group of proteins from intact inactivated cells using mild, nondestructive extraction procedures. The proteins thus obtained range in molecular weight from 10 000 to 51 000 daltons as determined under denaturing conditions and from 10 000 to 124 000 daltons under nondenaturing conditions. The isoelectric pH of these proteins range from 3.5 to 10 with numerous bands in the pH 4.5 to 5.5 region, and a few bands in the pH 6 to 10 region. The low pH region also contains those protein bands which can be labeled with the membrane impermeant reagent, diazotized [125I]-iodosulfanilic acid. The soluble protein preparations are antigenic in guinea pigs, rabbits and cattle, and immunogenic in lemmings, guinea pigs and cattle. Cross-reacting protein antigens are present in protein preparations from B. abortus strains 19 and 2308 when tested with heterologous rabbit sera and with sera from vaccinated and experimentally infected cattle. The soluble protein antigens can be rendered more immunogenic by chemical modification of the primary amino groups with acyl anhydrides, especially with dodecanoyl anhydride. Immunization with dodecanoyl--modified proteins resulted in decreased humoral antibody levels (as measured in guinea pigs and cattle) without loss of protective activity, as measured in lemmings. Furthermore, the soluble proteins have proven to be a sensitive, stable and reliable reagent in the enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Brucella abortus in serum of vaccinated and experimentally infected cattle. Other soluble protein antigen preparations have also been evaluated and compared with strain 19 proteins for immunogenic properties and for use as an ELISA reagent.

Animals↗

PubMed1982

Generation of chemotactic factor for granulocytes and monocytes from serum by fractions of Brucella abortus.

Several fractions isolated from Brucella abortus were examined for their ability to generate chemotactic factor from normal serum. Phenol phase lipopolysaccharides exhibited activity equivalent to that obtained with E. Coli lipopolysaccharide. A carbohydrate-rich aqueous methanol fraction was inhibitory at high concentrations, but a non-dialysable component of this fraction contained a potent stimulator of chemotactic activity. Protein-rich fractions from both strain 19 and strain 2308 were inactive. Preheating the serum at 56 degrees for 30 min prevented generation of chemotactic activity by the various fractions, suggesting a role for serum complement. No chemotactic activity was produced by Brucella fractions in C5-deficient DBA/2J mouse serum.

Animals↗

PubMed1981

Neuraminidase activity of Pasteurella haemolytica isolates.

Neuraminidase activities of Pasteurella haemolytica cell suspensions were measured. Type isolates of the 12 established serotypes and bovine and ovine field isolates were included in the study. Neuraminidase activities ranged from 0 to 0.87 U per mg (dry weight) of P. haemolytica. Activity levels among the isolates studied were serotype associated.

Animals↗