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2.  Chromatographic purification and characterization of antigens A and D from Mycobacterium paratuberculosis and their use in enzyme-linked immunosorbent assays for diagnosis of paratuberculosis in sheep. 
Journal of Clinical Microbiology  1991;29(8):1659-1664.
The protein antigens A and D were purified from culture filtrates and sonic extracts of laboratory strains of Mycobacterium paratuberculosis by salt precipitation and chromatography. The characterization of antigen A is shown here, and both antigens were evaluated along with lipoarabinomannan antigen in indirect enzyme-linked immunosorbent assays (ELISA) for the serodiagnosis of ovine paratuberculosis. After anion-exchange (DEAE-5PW) and hydrophobic (phenyl-5PW) chromatography using high-performance liquid chromatography, antigen A showed a prominant band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 31 kDa with small amounts of low-molecular-mass proteins but with no evidence of antigen D. A single precipitin arc was evident with purified antigen A in crossed immunoelectrophoresis. The determination of the N-terminal amino acid sequence showed a high degree of homology between the 31-kDa component of antigen A and antigens of the BCG85 complex of Mycobacterium bovis BCG, a total of 24 of 26 residues being identical to those of BCG85C. A prominant SDS-PAGE band at 400 kDa and a single crossed-immunoelectrophoresis arc was also evident for antigen D after gel filtration (Sephacryl S-200), anion-exchange (DEAE-Sephacel), and concanavalin A-Sepharose affinity chromatography. By ELISA, purified antigen A detected antibody in the sera of 18 of 22 paratuberculosis-infected sheep (82% sensitivity), whereas the purified antigen D detected antibody in all 22 infected animals (100% sensitivity). Combined ELISA results showed increased specificity with some loss in sensitivity.
PMCID: PMC270180  PMID: 1761688
3.  A comparison of standard serological tests for the diagnosis of bovine brucellosis in Canada. 
Six agglutination and two complement fixation tests were compared with respect to specificity, sensitivity and relative sensitivity for the serodiagnosis of bovine brucellosis. Based on 1051 sera from brucellosis free herds, the specificity of the tests was 98.9% for the buffered plate antigen test (BPAT), 99.2% and 99.3% for the standard tube and plate agglutination tests (STAT and SPAT), respectively, and 99.8% for the 2-mercaptoethanol test (2MET). On this small sample, the rose bengal plate test (RBPT), card test (CARD) and the complement fixation test (CFT) correctly classed all sera as negative. On a sample of 167 culture positive cattle, the sensitivities of the tests were CFT: 79.0%, BPAT: 75.4, RBPT: 74.9%, CARD: 74.3%, SPAT: 73.1%, STAT: 68.9%, and 2MET: 59.9%. All tests combined detected only 82% of these infected cattle. Analysis of the relative sensitivity of the six agglutination tests gave the following ranking: BPAT greater than RBPT greater than CARD greater than SPAT greater than STAT. The 2MET ranked between the BPAT and RBPT or between the RBPT and CARD depending on the analysis used. The use of the BPAT as a screening test is recommended provided that a test of high specificity and sensitivity such as the CFT is used to confirm screening test reactions.
PMCID: PMC1236197  PMID: 4075239
4.  An investigation of the etiology of Brucella abortus singleton reactors. 
Single animals in a herd that react serologically to Brucella abortus for no apparent reason are a problem. A number of such reactors from Ontario and Quebec were gathered for extensive clinical, serological, pathological and bacteriological examination in an attempt to investigate the etiology of these single serological reactions. While a variety of pathological changes were found, there was no apparent correlation to the serological, clinical or bacteriological findings.
PMCID: PMC1320091  PMID: 6778597
5.  The Ras-specific exchange factors mouse Sos1 (mSos1) and mSos2 are regulated differently: mSos2 contains ubiquitination signals absent in mSos1. 
Molecular and Cellular Biology  1997;17(12):7132-7138.
We have compared aspects of the mouse sos1 (msos1) and msos2 genes, which encode widely expressed, closely related Ras-specific exchange factors. Although an msos1 plasmid did not induce phenotypic changes in NIH 3T3 cells, addition of a 15-codon myristoylation signal to its 5' end enabled the resulting plasmid, myr-sos1, to induce approximately one-half as many foci of transformed cells as a v-H-ras control. By contrast, an isogenic myr-sos2 plasmid, which was made by fusing the first 102 codons from myr-sos1 at homologous sequences to an intact msos2 cDNA, did not induce focal transformation directly, although it could form foci in cooperation with c-H-ras. Pulse-chase experiments indicated that the half-life of Sos¿1 in NIH 3T3 cells was greater than 18 h, while that of Sos2 was less than 3 h. While in vitro-translated Sos1 was stable in a rabbit reticulocyte lysate, Sos2 was degraded in the lysate, as were each of two reciprocal chimeric Sos1-Sos2 proteins, albeit at a slower rate. In the lysate, Sos2 and the two chimeric proteins could be stabilized by ATPgammaS. Unlike Sos1, Sos2 was specifically immunoprecipitated by antiubiquitin antibodies. In a myristoylated version, the chimeric gene encoding Sos2 at its C terminus made a stable protein in NIH 3T3 cells and induced focal transformation almost as efficiently as myr-msos1, while the myristoylated protein encoded by the other chimera was unstable and defective in the transformation assay. We conclude that mSos2 is much less stable than mSos1 and is degraded by a ubiquitin-dependent process. A second mSos2 degradation signal, mapped to the C terminus in the reticulocyte lysate, does not seem to function under the growth conditions of the NIH 3T3 cells.
PMCID: PMC232570  PMID: 9372945
6.  Affinity purification of bovine antibodies to Brucella abortus Lipopolysaccharide. 
Journal of Clinical Microbiology  1983;17(2):323-326.
Alkali-treated lipopolysaccharide from Brucella abortus S1119.3 coupled to agarose beads by cyanogen bromide activation resulted in an immunoadsorbent with which a large amount of B. abortus-specific antibodies could be purified. The method described gave alkali-treated lipopolysaccharide binding efficiencies of up to 98%. There was little loss of alkali-treated lipopolysaccharide from the column after several pH shifts, allowing the immunoadsorbent to be regenerated and used repeatedly.
PMCID: PMC272629  PMID: 6403576
7.  Synovial immunoglobulin and antibody in vaccinated and nonvaccinated calves challenged with Mycoplasma bovis. 
Intravenous injection of Mycoplasma bovis produced in calves arthritis with synovial infiltration of lymphocytes, macrophages and neutrophils. Necrosis was observed focally around blood vessels. Joint spaces contained fibrinopurulent exudate. Parenterally vaccinated calves had a markedly reduced frequency of arthritic joints. Immunoglobulin classes and specific antibody in joint fluids were quantitatively less than in sera but significantly greater in arthritic than in normal joints. The possible mechanisms of induction of joint fluid antibody are discussed.
PMCID: PMC1320127  PMID: 7272847
9.  Bovine reaginic antibody III. Cross-reaction of antihuman IgE and antibovine reaginic immunoglobulin antisera with sera from several species of mammals. 
Using antisera specific for the heavy chain of human IgE and bovine reaginic immunoglobulin, the degree of cross-reaction amongst sera from pig, rat, rabbit, guinea pig, goat, cow, horse, dog, cat and human was tested. Antihuman IgE antiserum gave strong reactions with pig, rabbit, cow, goat and human sera (100% to 15.1%) and weak reactions with rat, guinea pig, horse, dog and cat sera (10.1% to 3.22%). Antibovine reagin antiserum produced a considerable amount of cross-reaction with sera from pig, rat, rabbit, goat, horse and human (43.6% to 20.1%) with limited reactions with guinea pig, dog and cat sera (13.9% to 9.3%).
PMCID: PMC1277601  PMID: 907910
10.  Bovine IgG2a antibodies to Haemophilus somnus and allotype expression. 
Bovine IgG2a has been implicated in protection against pyogenic infections, including those caused by Haemophilus somnus. To further investigate the role of IgG2a in defense against H. somnus, IgG1 and IgG2a antibodies were purified from antiserum against an immunodominant 40 kDa outer membrane protein (p40) of H. somnus, which was previously shown to passively protect calves against H. somnus pneumonia. The passive protective capacity of anti-p40 IgG1 or IgG2a was evaluated in vivo in calves. Purified anti-p40 IgG1 or IgG2a was incubated with H. somnus for 15 min before intrabronchial inoculation of calves. Bacteria incubated with anti-p40 IgG1 or IgG2a were inoculated into one caudal lung lobe and bacteria incubated with IgG1 or IgG2a from the respective preimmunization serum were inoculated into the contralateral lobe. The volumes of pneumonia in the right and left lungs were determined 24 h later. The difference in volume of pneumonia with H. somnus preincubated in IgG1 pre- and postimmunization anti p40 was less (16 cm3, P = 0.298) than the difference in volume of pneumonia with H. somnus preincubated in IgG2a pre- and postimmunization anti p40 (30 cm3, P = 0.146). Although the differences in lesion size between pre- and postimmunization serum were not statistically significant, the trend suggests IgG2a may be more protective than IgG1. To examine this further, the peptide specificity of these IgG1 and IgG2a antibodies to p40 was examined. After limited proteolysis of p40, IgG2a antibodies reacted with 2 peptides not recognized by IgG1 antibodies. Other peptides were recognized by both isotypes. Since these studies suggested that IgG2a may be important in protection against infection, we then investigated some aspects of the role of the 2 IgG2a allotypes, A1 and A2. In retrospective studies of age differences in expression of IgG2a allotypes, no heterozygotes were detected in calves of 60 d old or less, and fewer heterozygotes were detected in calves 61-120 d old than in cattle older than 270 d (P < 0.01). In a subsequent prospective study of the time course of allotype expression, Holstein calves shown to be heterozygotes expressed the IgG2aA1 allotype early but the IgG2aA2 allotype was not usually detected until 3 to 4 mo of age. Thus, both the retrospective and the prospective studies showed age related differences in expression of the IgG2aA1 and A2 allotypes. This could have implication in protection.
PMCID: PMC1189405  PMID: 9243001
11.  An evaluation of selected screening tests for bovine paratuberculosis. 
The objective of this study was to evaluate the performance of the lipoarabinomannan antigen enzyme-linked immunosorbent assay (LAM-ELISA), carbohydrate antigen complement fixation (CH-CFT), and protein D antigen agar gel immunodiffusion (D-AGID) tests for bovine paratuberculosis, relative to histopathology, and to culture and isolation of Mycobacterium paratuberculosis from tissues and feces. Samples for test evaluation were collected from four sources including blood and tissues from 400 cull cows at three abattoirs in Ontario, blood and feces from a paratuberculosis survey of cattle from 120 dairy farms in Ontario, a serum bank containing samples from cattle from Ontario and Québec, and a bank of sera from cattle from Pennsylvania and the northeastern United States. The data were analyzed using receiver operator characteristic curves, estimates of relative sensitivity and specificity, and kappa statistics of agreement between tests. The LAM-ELISA performed significantly better than both the CH-CFT and the D-AGID tests. The LAM-ELISA was better at predicting fecal shedding status than tissue infection. However, the LAM-ELISA also had limitations. When interpreted as positive or negative (+/-), at a critical optical density of 0.675, its sensitivity and specificity relative to bacteriology were 49% and 87% respectively. Although the serological tests examined in this study provided some information, they did not predict well the infection status of individual animals.
PMCID: PMC1263460  PMID: 1909601
12.  Mycobacterium paratuberculosis antigen D: characterization and evidence that it is a bacterioferritin. 
Journal of Clinical Microbiology  1991;29(8):1652-1658.
By using a combination of agarose and polyacrylamide gel electrophoresis, Mycobacterium paratuberculosis antigen D was resolved from a crude sonicated preparation of the organism and characterized as a component with a molecular mass of approximately 400,000 Da. While this component was composed mainly of protein, with unusually high proportions of glutamic acid and leucine, it was resistant to digestion with a number of proteolytic enzymes. Structural detail revealed by electron microscopy, amino acid sequence data, and the demonstration of a Soret band in its absorption spectrum indicated that antigen D was similar to an Escherichia coli bacterioferritin.
PMCID: PMC270179  PMID: 1761687
13.  Killing of Brucella abortus by bovine serum. 
Infection and Immunity  1988;56(12):3251-3261.
Studies of the serum bactericidal system in bovine brucellosis were undertaken to investigate the role of the humoral immune response in protection of cattle against the facultative intracellular parasite Brucella abortus. Fresh sera from normal control cattle, infected cattle, and cattle immunized with B. abortus cell envelopes were collected before treatment and during the course of immunization or infection. Normal fresh bovine serum or fresh agammaglobulinemic serum from colostrum-deprived calves was effective in killing smooth virulent B. abortus 2308, but rough strains RB51 (a rough mutant of strain 2308) and 45/20 were much more sensitive to serum. The difference in susceptibility to serum was shown to be correlated with differences in lipopolysaccharide chemotype, with the more resistant strain 2308 having O polysaccharide and the more susceptible strains 45/20 and RB51 lacking O side chains. By treatment of fresh serum with MgCl2 and EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] killing was shown to occur via the classical pathway of complement activation. When antibody to B. abortus was present, killing of strain RB51 increased but killing of smooth strain 2308 decreased. The earliest antibody response in serum from infected animals did not interfere with killing. When affinity-purified bovine immunoglobulins specific for B. abortus smooth lipopolysaccharide were added to fresh normal bovine serum, immunoglobulin G1 (IgG1) and IgG2 isotypes blocked killing but IgM and IgA isotypes did not. Thus, it appears that serum from previously unexposed animals or animals early during infection can kill smooth B. abortus, an appropriate defense mechanism before the organism becomes intracellular. At later stages of infection, blocking antibodies predominate.
PMCID: PMC259732  PMID: 3141287
14.  Brucella abortus 1119-3 O-chain polysaccharide to differentiate sera from B. abortus S-19-vaccinated and field-strain-infected cattle by agar gel immunodiffusion. 
Journal of Clinical Microbiology  1988;26(6):1120-1123.
Purified Brucella abortus 1119-3 and Brucella melitensis 16M lipopolysaccharide O-chain polysaccharides were not precipitated in agar gel immunodiffusion by any of 24 sera from vaccinated cattle but were precipitated by 18 of 24 sera from infected cattle. This difference can be used to differentiate sera of cattle vaccinated with B. abortus S-19 from sera of some field-strain-infected cattle.
PMCID: PMC266545  PMID: 3133389

Results 1-14 (14)