Indirect enzyme-linked immunosorbent assay for detection of Brucella melitensis-specific antibodies in goat milk.

Brucella melitensis is the cause of brucellosis in sheep and goats, which often results in abortion. Few cases of B. melitensis infection in goats have occurred in the United States over the last 25 years. However, vigilance must be maintained, as it is for the bovine milk industry, to ensure that brucellosis is not introduced into the U.S. goat population. The objective of this study was to develop a sensitive and specific indirect enzyme-linked immunosorbent assay (iELISA) for the detection of B. melitensis-specific antibodies in goat milk. Brucella salt-extractable protein extract was employed as an antigen, and a horseradish peroxidase-labeled polyclonal anti-goat antibody was used as an anti-species conjugate. Thirteen of 13 (100%) individual infected goat milk samples tested positive and 134 of 134 (100%) uninfected bulk milk samples tested negative by the developed iELISA. Three positive milk samples with high, medium, and low absorbance values were used to simulate one positive animal in an otherwise negative herd. By this estimation, one high-titer animal could be detected in a herd of >1,600 animals. Detection estimates for medium- and low-titer animals were one positive animal per herd of <200 and 50 animals, respectively. Based on this estimation, it is recommended that herds be sampled in groups of 50 animals or less for bulk milk testing. The iELISA developed for this study was found to be sensitive and specific and shows potential for use as a bulk milk test for the detection of B. melitensis-specific antibodies in goat milk.

Purification and characterization of the Pasteurella haemolytica 35 kilodalton periplasmic iron-regulated protein.

Pasteurella haemolytica serovar A1 is the causative agent of acute fibrinohemorrhagic pneumonia also known as shipping fever. Many pathogens, including P. haemolytica, survive in their respective hosts through the up-regulation of an iron acquisition system. In this study we identified, purified and characterized a 35-kDa periplasmic iron-regulated protein. The N-terminal sequence of the iron-regulated protein ANEVNVYSYRQP YLIEPMLK was identical to the deduced amino acid sequence of the ferric binding protein, FbpA, of P. haemolytica. Growth of P. haemolytica in a synthetic medium (RPMI-1640), without iron and supplemented with 50 gM 2,2' dipyridyl, facilitated the expression, isolation and purification of the native P. haemolytica FbpA. The protein was purified to homogeneity by using ammonium sulfate precipitation followed by CM-Sepharose ion exchange chromatography. SDS-PAGE showed a single band with a molecular weight of 35,369. Isoelectric focusing showed multiple bands with pIs of 5.5, 5.6, 5.8, and one major band with pI of 6.4. The molecular weight obtained by electrospray mass spectrometry was 35,822. Equilibrium velocity ultracentrifugation established that the protein existed as a monomer under native conditions with an apparent molecular weight of 33,795. Analysis of secondary structure of FbpA by circular dichroism showed 42.1% alpha helical structure. This protein is the second periplasmic iron-regulated protein described for P. haemolytica.

Composition and protein fractions of different meat by-products used for petfood compared with mechanically separated chicken (MSC).

Pork by-products (lung lobes, kidneys), chicken viscera (head, feet and viscera) and mechanically separated chicken (MSC) were evaluated for proximate composition, protein distribution and connective tissue. Proximate composition varied among meat by-products and MSC. Pork by-products contained the most crude protein (p<0.05). Low levels of high ionic strength soluble (HIS) proteins were obtained from meat by-products. Pork lungs and chicken viscera contained the greatest amounts of insoluble (IN) proteins (p<0.05). Total collagen values were positively correlated to IN proteins, intramuscular collagen (IMC) and elastin. Types I and III collagen could not be detected by SDS-PAGE for the different meat by-products though collagen solubility appeared to be significant. These results suggest functional property differences between specific by-products are likely when used in petfood product formulations.

Conservation of expression and N-terminal sequences of the Pasteurella haemolytica 31-kilodalton and Pasteurella trehalosi 29-kilodalton periplasmic iron-regulated proteins.

This study examined the conservation of expression of a 31-kDa iron-regulated protein by serotypes of Pasteurella haemolytica and Pasteurella trehalosi associated with pasteurellosis of cattle and sheep. A polyclonal antibody prepared against the purified 31-kDa periplasmic iron-regulated protein from P. haemolytica serotype A1 showed that all P. haemolytica serotypes expressed similar 31-kDa proteins with identical N-terminal sequences, whereas P. trehalosi serotypes expressed immunologically different 29-kDa proteins with a different N-terminal sequence. Antibody to the 31-kDa iron-regulated protein was a useful tool to distinguish similarities and differences of the iron-regulated proteins of P. haemolytica and P. trehalosi.

Purification and characterization of a 31-kilodalton iron-regulated periplasmic protein from Pasteurella haemolytica A1.

A prominent iron-regulated periplasmic protein was purified from Pasteurella haemolytica grown in an iron-deficient chemically defined medium. The protein was purified by anion exchange chromatography and appeared as a single band by SDS-PAGE with a molecular weight of 32,000. A yield of five mg was obtained from 91 mg of protein extract. The iron-regulated protein existed as a monomer in the native state with an average molecular weight of 29,877 as determined by analytical ultracentrifugation. The protein had a molecular weight of 30,880 as determined by matrix-assisted laser desorption mass spectrometry, hence the protein is referred to as the 31 kDa protein. Isoelectric focusing showed four bands with pIs of 7.15, 6.8, 6.6, and 5.9. The secondary structure of the protein was determined by circular dichroism and contained 16% alpha-helical structure. The N-terminal sequence, EPFKVVTTFTVIQDIAQNVAGDKAT, showed a 95% identity with the 31 kDa iron-binding protein from Haemophilus influenzae. Isolation and characterization of iron-regulated proteins are of particular interest because of their potential roles in iron assimilation and microbial virulence.

Variation of Brucella abortus 2308 infection in BALB/c mice induced by prior vaccination with salt-extractable periplasmic proteins from Brucella abortus 19.

The study compared the immune and protective responses induced in BALB/c mice vaccinated with six salt-extractable periplasmic protein fractions (Brucella cell surface proteins [BCSP]) of Brucella abortus 19 and later challenge exposed with B. abortus 2308. BCSP70 was precipitated with ammonium sulfate at 70% saturation, and BCSP100 was precipitated with ammonium sulfate at 100% saturation by use of supernatant fluid of BCSP70 that had been precipitated with 70% ammonium sulfate. Four subfractions were separated from BCSP100 by anion-exchange high-performance liquid chromatography (HPLC). Monophosphoryl lipid A (MPL) from Salmonella typhimurium Re mutant strain was used as a potential immune response modifier in some vaccines. Reduced or increased numbers of CFU and increased spleen size in the principal groups of mice relative to that of the nonvaccinated control group were considered protectiveness or virulence (survival) criteria. Results indicated that vaccines prepared from BCSP70 and BCSP100 were moderately protective and immunogenic. The subfractions designated BCSP100-A through BCSP100-D purified by anion-exchange HPLC were not protective when MPL was not used as an immune response modifier. However, two subfractions were associated with significant (P < 0.05) increases in CFU per spleen and splenomegaly in vaccinated mice compared with those in nonvaccinated challenge-exposed mice. MPL enhanced protection or was neutral when used with BCSP70, BCSP100, BCSP100-C, and BCSP100-D. Serologic results of an enzyme-linked immunosorbent assay indicated that MPL modulated the immunoglobulin G responses induced by BCSP70, BCSP100, and subfraction BCSP100-B vaccines only. The overall results suggest that certain proteinaceous periplasmic fractions might serve as virulence or survival factors in B. abortus infections.

Structural characterization of the active site of Brucella abortus Cu-Zn superoxide dismutase: a 15N and 1H NMR investigation.

Prokaryotic Cu-Zn superoxide dismutases (SODs) are rare and poorly characterized compared to their eukaryotic counterparts. To better characterize the structure of the prokaryotic enzyme, an NMR investigation of Brucella abortus Cu-Zn SOD in the reduced form was undertaken. The enzyme studied was a recombinant form, expressed in Escherichia coli. The enzyme initially lacked a full complement of Cu and Zn ion. After demetallation and remetallation with a stoichiometric amount of Cu and Zn ion, the specific activity of the recombinant B. abortus Cu-Zn SOD was comparable to the specific activity of the bovine enzyme. The 15N and 1H resonances of seven active site histidine imidazole rings were assigned using two-dimensional NMR methods. A self-consistent set of nuclear Overhauser effects between imidazole ring protons was observed, which was in agreement with the predictions of a model based on the X-ray crystallographic structure of the oxidized bovine enzyme (Tainer, J.A., Getzoff, E. D., Beem, K. M., Richardson, J.S., & Richardson, D.C. (1982) J. Mol. Biol. 160, 181-217). These observations strongly suggest that the structure of the active site of the prokaryotic enzyme is similar to that of the eukaryotic enzyme. Differences in the observed and predicted nuclear Overhauser effects could be ascribed to differences in the oxidation state of the Cu ion (Cu(I) in the reduced B. abortus enzyme and Cu(II) in the oxidized bovine enzyme), as much as they could to the different origins of the enzymes. The NMR data were also compared to a similar 1H NMR study of the human enzyme (Bertini, I., Capozzi, F., Luchinat, C., Piccioli, M., & Viezzoli, M. S. (1991) Eur. J. Biochem. 197, 691-697). The pattern of nuclear Overhauser effects and the chemical shifts of corresponding resonances were very similar in 1H NMR spectra of the human and B. abortus enzymes. Significant differences in the chemical shifts or exchange behavior of a few resonances indicated differences in the environments of several histidines in the active sites of reduced B. abortus and human Cu-Zn SODs. This is consistent with the presence of a number of insertions and deletions in the loop regions that make up the active site as indicated by amino acid sequence alignment studies. The tautomeric and protonation states of the active site histidines were also determined in this study, and the results were in agreement with previous studies. The resonances of nitrogen atoms coordinated to metal ions were found to fall between those of protonated and unprotonated nitrogens on histidine imidazoles.

Fetuin is a substrate for Pasteurella haemolytica O-sialoglycoprotease.

A soluble bovine glycoprotein, fetuin, was used as an alternative substrate to identify O-sialoglycoprotease activity in culture supernatant protein fractions of Pasteurella haemolytica. An aliquot of a 24-hour incubation mixture containing fetuin and O-sialoglycoprotease was denatured and examined after gradient sodium dodecyl polyacrylamide gel electrophoresis. The Coomassie-Brilliant-Blue-stained gel was examined for the disappearance of the fetuin band at an apparent molecular mass of 64 KDa. Four major hydrolysis products were identified: an N-terminal fragment of 45 kDa, a 20 kDa fragment at Val50, and two C-terminal fragments at Val273 and His287.

Alteration of protective and serologic responses in BALB/c mice vaccinated with chemically modified versus nonmodified proteins of Brucella abortus 19.

A study was conducted to determine whether the covalent chemical modification of Brucella abortus 19 salt-extractable proteins (BCSP) and BCSP derivatives would modulate the immune responses in BALB/c mice. Salt-extractable proteins BCSP 0-70 and BCSP 70-100 were modified with acetoacetic anhydride, and recombinant proteins rBCSP20 (20 kDa), rBCSP31 (31 kDa), and rBCSP45 (45 kDa) were modified with succinic and dodecanoyl anhydrides. Four weeks after mice were vaccinated with the different preparations, principal and control mice were challenge exposed with a virulent culture of B. abortus 2308, and mice were necropsied 2 weeks later. Serum samples were obtained immediately before mice were challenge exposed and at necropsy. Sera were tested for specific immunoglobulin M (IgM) and G (IgG) antibodies by using an enzyme-linked immunosorbent assay. Acylation decreased the immune responses (increased IgG antibodies and reduced spleen CFU and splenomegaly) induced by both BCSP 0-70 and BCSP 70-100. Modification of the recombinant proteins by dodecanoyl and succinic anhydrides had no effect on the protection induced; however, the IgG serologic responses to the homologous and heterologous proteins were altered. Monophosphoryl lipid A markedly enhanced the immunogenicity of BCSP 0-70.

Cattle serologically positive for Brucella abortus have antibodies to B. abortus Cu-Zn superoxide dismutase.

In this study, we demonstrated by a Cu-Zn superoxide dismutase-specific enzyme-linked immunoassay that cattle that are serologically positive for Brucella abortus have serum immunoglobulin G antibodies to B. abortus Cu-Zn superoxide dismutase. The specificity of the antibody reactivity was confirmed by Western blot (immunoblot) analysis with B. abortus salt-extractable proteins containing native Cu-Zn superoxide dismutase and with recombinant B. abortus Cu-Zn superoxide dismutase. The results represent a first step in the direction of the development of a multiprotein diagnostic reagent for bovine brucellosis.

Immune responses to superoxide dismutase and synthetic peptides of superoxide dismutase in cattle vaccinated with Brucella abortus strain 19 or RB51.

Antibody and lymph node cell-mediated immune responses to recombinant Brucella abortus strain 19 Cu-Zn superoxide dismutase (rSOD) and to three synthetic strain 19 Cu-Zn SOD peptides were measured during 2 to 12 weeks following vaccination of cattle with B. abortus strain 19 or RB51. Cattle vaccinated with strain 19 or RB51 did not produce antibody to rSOD and to the SOD peptides. Lymph node cells from cattle vaccinated with strain 19, but not with strain RB51, proliferated when incubated with either rSOD or one of the three tested SOD peptides (GGDNYSDKPEPLGG). These results suggest that neither the strain 19 nor the strain RB51 vaccine induces antibody production to SOD and only the strain 19 vaccine induces lymph node cell-mediated immune responses to SOD.

Modulation of immune responses in Balb/c mice vaccinated with Brucella abortus Cu-Zn superoxide dismutase synthetic peptide vaccine.

Three peptides, peptide 1 (GGDNYSDKPEPLGG), peptide 2 (LAEIKQRSLMVHGG) and peptide 3 (GGAPGEKDGKIVPAG), were synthesized based on the amino acid sequence of Brucella abortus Cu-Zn superoxide dismutase. These peptides were selected on the basis of their predicted hydrophilicity, flexibility and antigenicity profiles. The three peptides, singly or in combination, with or without the adjuvant monophosphoryl lipid A were administered to Balb/c mice as vaccines for brucellosis. The protective and immune responses induced by the peptide vaccines after challenge exposure to virulent B. abortus strain 2308 were compared to those obtained with salt-extractable proteins (BCSP) vaccine prepared from B. abortus strain 19, recombinant B. abortus Cu-Zn superoxide dismutase (rSOD) vaccine and non-vaccinated mice. Mice vaccinated with 30 micrograms of peptide 3 plus 50 micrograms monophosphoryl lipid A afforded two logs of protection (reduction in log10 colony-forming units compared with control mice) and one log of protection when given without monophosphoryl lipid A, whereas 5 micrograms of the salt-extractable proteins afforded three logs of protection. The rSOD and peptides 1 and 2 given with or without monophosphoryl lipid A afforded no protection. Superoxide dismutase-specific IgG antibody was present in postchallenge sera only if BCSP was present in the vaccine. Peptide-specific IgG antibodies were present in postchallenge sera of mice, and antibody concentrations were generally enhanced when monophosphoryl lipid A was included in the vaccine. The overall results with the peptide vaccines suggest that peptide 3 probably contains a specific sequence preferentially recognized by the cellular immune system leading to modulation of immune response mechanisms responsible for decreasing splenic infection.

Acidic residues are involved in substrate recognition by two soluble protein tyrosine phosphatases, PTP-5 and rrbPTP-1.

The mechanisms for substrate recognition by two cytoplasmic protein tyrosine phosphatases, PTP-5 and rrbPTP-1, were investigated. Phosphorylation sites on tyrosine-phosphorylated casein, a model PTP substrate, were characterized. Two peptides based on casein phosphorylation sites and one peptide based on the tyrosine phosphorylation site of reduced, carboxamidomethylated and maleylated (RCM) lysozyme were tested as PTP substrates. The three peptides were dephosphorylated by PTP-5 and rrbPTP-1 at rates comparable to those of the corresponding sites on the intact proteins. This indicates that peptides based on the two model PTP substrates, casein and RCM-lysozyme, contained all or most of the structural information necessary for PTP-5 and rrbPTP-1 substrate recognition. Structural elements required for substrate recognition by PTP-5 and rrbPTP-1 were also investigated. Km values for dephosphorylation of three simple aromatic phosphate esters (phosphotyrosine, p-nitrophenyl phosphate, and phenyl phosphate) by rrbPTP-1 were about 5000-fold higher than those obtained for the peptide and protein substrates. This indicates that recognition of protein and peptide substrates involves structural elements in addition to the phosphate group and the aromatic tyrosine ring of phosphotyrosine. Analysis of the effects of truncations and Ala for polar substitutions on the reactivity with PTP-5 and rrbPTP-1 of peptides based on casein, RCM-lysozyme, and angiotensin II indicated that Asp or Glu within the first five residues on the N-terminal side of phosphotyrosine increased peptide reactivity with both PTP's. Asn residues were unable or only weakly able to substitute for Asp residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Monophosphoryl lipid A-induced immune enhancement of Brucella abortus salt-extractable protein and lipopolysaccharide vaccines in BALB/c mice.

A study was conducted to determine the effect of monophosphoryl lipid A (MPL) and trehalose dimycolate (TDM) as adjuvants on the protective responses in BALB/c mice vaccinated with Brucella abortus salt-extractable protein (BCSP) or proteinase-K-treated B abortus lipopolysaccharide (PKLPS). Mice were vaccinated with different doses of BCSP or PKLPS given alone or in combination with MPL or TDM. Mice were challenge-exposed 4 weeks later with virulent B abortus strain 2308. Two weeks after challenge exposure, the number of B abortus colony-forming units (CFU) per spleen, spleen weights, and spleen cell interleukin 1 production were measured. Serum IgG and IgM concentrations specific for vaccinal immunogens were measured before and after challenge exposure with B abortus. Spleen weights and mean B abortus CFU per vaccine group were significantly lower in BCSP- and PKLPS-vaccinated mice, compared with those of nonvaccinated control mice. Monophosphoryl lipid A enhanced the suppression of splenic infection when given with the BCSP vaccine, but not when given with the PKLPS vaccine. Trehalose dimycolate had no effect on mean CFU when given with BCSP, but incorporation of TDM resulted in a significant increase in mean CFU when given with PKLPS. Spleen weights in BCSP- or PKLPS-vaccinated mice were not different when these vaccines were combined with MPL or TDM. Because of the wide variation in the results, we could not conclude that vaccination with BCSP or PKLPS alone, or in combination with MPL altered spleen cell interleukin-1 production in B abortus-infected mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of gamma interferon and indomethacin in preventing Brucella abortus infections in mice.

Increased resistance to infection with Brucella abortus 2308 resulted when recombinant murine gamma interferon (rMuIFN-gamma) was given to mice both before and during infection but not when given only before infection. Mice given rMuIFN-gamma had enhanced peritoneal and splenic macrophage bactericidal activity against B. abortus. Treatment of mice with rMuIFN-gamma plus indomethacin did not further enhance resistance to infection or macrophage bactericidal activity compared with that after treatment of mice with rMuIFN-gamma alone.

Swine immunity to an attenuated Salmonella typhimurium mutant 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 shown to be avirulent in swine. S. typhimurium chi 4064 was used as a carrier for plasmid pBA31-R7, which codes for the expression of a 31-kDa protein from Brucella abortus (BCSP31). Given orally, S. typhimurium chi 4064(pBA31-R7) colonized the intestine and mesenteric lymph nodes of 5- to 6-week-old crossbred swine. Orally immunized animals developed serum and intestinal antibody responses to the B. abortus 31-kDa protein and to salmonella endotoxin as measured by enzyme-linked immunosorbent assay. Similarly immunized swine did not develop delayed-type hypersensitivity following a subcutaneous injection of recombinant BCSP31. However, swine parenterally immunized with recombinant BCSP31 incorporated in Freund incomplete adjuvant did develop a delayed-type hypersensitivity response to the homologous antigen. The data indicated that oral presentation of antigen to swine in the context of recombinant S. typhimurium effective stimulated mucosal and systemic antibody-mediated immunity but failed to sensitize swine for either an antigen-specific delayed-type hypersensitivity or a blastogenic response to the cloned BCSP31.

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.

Establishment of dose-response relationships in BALB/c mice, using Brucella cell surface protein and lipopolysaccharide.

A study was conducted to determine the immune (increased antibody) and protective (reduced colony-forming units) responses induced in mice by a: (i) single vaccinal inoculation, using various concentrations of Brucella cell surface protein (BCSP) or lipopolysaccharide (LPS); (ii) primary inoculation, using various concentrations of BCSP, followed by a secondary inoculation, using a standard concentration of BCSP; and (iii) primary inoculation, using 1 concentration of BCSP or LPS, followed by a secondary inoculation, using various concentrations of BCSP or LPS. Four weeks after the primary inoculation, mice were challenge exposed with approximately 1 x 10(4) colony-forming units of Brucella abortus strain 2308 and all mice were euthanatized at 6 weeks. Reduced splenic weights and reduced colony-forming units in the spleens of vaccinated mice, compared with nonvaccinated mice, were the criteria of protection. Increase in serum IgM and IgG was defined as immunity. Both BCSP and LPS induced protective and immune responses that were proportional to the dose given up to an optimal limit. However, concentrations higher than optimal decreased the protective and immune responses. This was true for mice given either 1 or 2 vaccinal inoculations. Enhanced secondary protective responses were seen only when suboptimal doses were used in the primary inoculation. Excessive or optimal doses in the secondary inoculations prevented or obscured the protectiveness and immunity by primary inoculations. The protective effects appeared to be additive when suboptimal doses were used in the primary and secondary inoculations.(ABSTRACT TRUNCATED AT 250 WORDS)

Unresponsiveness of vaccinated BALB/c mice to a second inoculation of lipopolysaccharide from Brucella abortus strain 2308.

A study was conducted to determine whether the protection induced in mice by a primary inoculation of lipopolysaccharide from Brucella abortus would be enhanced by a second inoculation given at different time intervals. Protection was challenged by exposure of the mice to a virulent culture of B. abortus strain 2308. Reduced mean viable count and/or splenic weights were the criteria of protection. There was no significant difference (P greater than 0.05) in the protective responses among mice given a single inoculation. Vaccinated mice were significantly (P less than 0.05) better protected than were nonvaccinated mice. Mice given vaccinal inoculations simultaneous with challenge exposure were less protected (P less than 0.001) than were mice vaccinated prior to challenge, but were better protected (P less than 0.010) than were nonvaccinated mice.

Immunoglobulin G binding activity of Brucella abortus.

A Brucella abortus protein with a molecular weight of 50 kDa has been shown to bind bovine immunoglobulin G from healthy, brucellosis-free animals. The Brucella immunoglobulin G binding molecule appears to be a protein, since it is susceptible to proteolysis. The protein is presumed to be located on the cell surface, since intact cells precipitate bovine immunoglobulin G. Examination of other species of Brucella shows that all Brucella species and strains tested express the protein. B. abortus cells also bound immunoglobulin G from other animal species. These included cat, chicken, dog, guinea pig, horse, human, mouse, rat, sheep, swine, and turkey but not immunoglobulin G from goat or rabbit.