Immune responses of bison and efficacy after booster vaccination with Brucella abortus strain RB51.

Thirty-one bison heifers were randomly assigned to receive saline or a single vaccination with 10(10) CFU of Brucella abortus strain RB51. Some vaccinated bison were randomly selected for booster vaccination with RB51 at 11 months after the initial vaccination. Mean antibody responses to RB51 were greater (P < 0.05) in vaccinated bison after initial and booster vaccination than in nonvaccinated bison. The proliferative responses by peripheral blood mononuclear cells (PBMC) from the vaccinated bison were greater (P < 0.05) than those in the nonvaccinated bison at 16 and 24 weeks after the initial vaccination but not after the booster vaccination. The relative gene expression of gamma interferon (IFN-γ) was increased (P < 0.05) in the RB51-vaccinated bison at 8, 16, and 24 weeks after the initial vaccination and at 8 weeks after the booster vaccination. The vaccinated bison had greater (P < 0.05) in vitro production of IFN-γ at all sampling times, greater interleukin-1ß (IL-1ß) production in various samplings after the initial and booster vaccinations, and greater IL-6 production at one sampling time after the booster vaccination. Between 170 and 180 days of gestation, the bison were intraconjunctivally challenged with approximately 1 × 10(7) CFU of B. abortus strain 2308. The incidences of abortion and infection were greater (P < 0.05) in the nonvaccinated bison after experimental challenge than in the bison receiving either vaccination treatment. Booster-vaccinated, but not single-vaccinated bison, had a reduced (P < 0.05) incidence of infection in fetal tissues and maternal tissues compared to that in the controls. Compared to the nonvaccinated bison, both vaccination treatments lowered the colonization (measured as the CFU/g of tissue) of Brucella organisms in all tissues, except in retropharyngeal and supramammary lymph nodes. Our study suggests that RB51 booster vaccination is an effective vaccination strategy for enhancing herd immunity against brucellosis in bison.

Immune responses and protection against experimental Brucella suis biovar 1 challenge in nonvaccinated or B. abortus strain RB51-vaccinated cattle.

Twenty Hereford heifers approximately 9 months of age were vaccinated with saline (control) or 2 × 10(10) CFU of the Brucella abortus strain RB51 (RB51) vaccine. Immunologic responses after inoculation demonstrated significantly greater (P < 0.05) antibody and proliferative responses to RB51 antigens in cattle vaccinated with RB51 than in the controls. Pregnant cattle received a conjunctival challenge at approximately 6 months of gestation with 10(7) CFU of B. suis bv. 1 strains isolated from naturally infected cattle. The fluorescence polarization assay and the buffered acid plate agglutination test had the highest sensitivities in detecting B. suis-infected cattle between 2 and 12 weeks after experimental infection. Serologic responses and lymphocyte proliferative responses to B. suis antigens did not differ between control and RB51 vaccinees after experimental infection. No abortions occurred in cattle in either treatment group after challenge, although there appeared to be an increased incidence of retained placenta after parturition in both the control and the RB51 vaccination treatment groups. Our data suggest that the mammary gland is a preferred site for B. suis localization in cattle. Vaccination with RB51 did not reduce B. suis infection rates in maternal or fetal tissues. In conclusion, although B. suis is unlikely to cause abortions and fetal losses in cattle, our data suggest that RB51 vaccination will not protect cattle against B. suis infection after exposure.

Seroconversion and abortion in cattle experimentally infected with Brucella sp. isolated from a Pacific harbor seal (Phoca vitulina richardsi).

Previously unrecognized Brucella species have been isolated from a number of marine mammals, including harbor seals (Phoca vitulina richardsi) in the Puget Sound area of the state of Washington. Because of the presence of dairy herds in proximity to the harbor seal populations, a study was conducted to determine the effects of the harbor seal Brucella isolate in experimentally inoculated cattle. Six pregnant cattle were exposed by intravenous injection (n = 3) or intraconjunctival inoculation (n = 3). Two pregnant cows were intravenously injected with saline and served as controls. All of the cows receiving the Brucella seroconverted on 1 or more tests commonly used for the detection of Brucella abortus infection. Two of the cattle receiving the intravenous inoculation aborted, and brucellae were demonstrated in the fetuses and dams immediately following abortion. The remaining 4 Brucella-inoculated animals and their fetuses were culture negative for the organism at 14 weeks postinoculation. Results of this study indicate the marine mammal Brucella is capable of producing seroconversion and abortion in cattle but is less pathogenic in that species than B. abortus.

Prevalence of antibodies against Mycobacterium avium subsp paratuberculosis among beef cow-calf herds.

OBJECTIVE: To estimate the prevalence of Mycobacterium avium subsp paratuberculosis infection among cows on beef operations in the United States. DESIGN: Cross-sectional seroprevalence study. Sample Population-A convenience sample of 380 herds in 21 states. PROCEDURES: Serum samples were obtained from 10,371 cows and tested for antibodies to M avium subsp paratuberculosis with a commercial ELISA. Producers were interviewed to collect data on herd management practices. RESULTS: 30 (7.9%) herds had 1 or more animals for which results of the ELISA were positive; 40 (0.4%) of the individual cow samples yielded positive results. None of the herd management practices studied were found to be associated with whether any animals in the herd would be positive for antibodies to M avium subsp paratuberculosis. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the prevalence of antibodies to M avium subsp paratuberculosis among beef cows in the United States is low. Herds with seropositive animals were widely distributed geographically.

Sensitivity and specificity of the complement fixation test for detection of cattle persistently infected with Anaplasma marginale.

The complement fixation (CF) test commonly is used to identify cattle infected with Anaplasma marginale prior to interstate or international movement. Estimates of the accuracy of the CF test in detecting animals persistently infected with A. marginale vary widely. In this study, the sensitivity and specificity of the CF test for detection of carrier animals was determined using serum from 232 cattle previously defined as A. marginale positive or negative by nested polymerase chain reaction methods and hybridization. Considering results from 2 independent laboratories and interpreting a 1:5 suspect reaction as positive, the best estimate of CF test sensitivity was 20%, with a specificity of 98%. Using a 1:10 cutoff, sensitivity decreased to 14% and specificity increased to 99%. Results of this study indicate that the CF test is ineffective for identifying cattle persistently infected with A. marginale and thus is inadequate for anaplasmosis regulatory and surveillance programs.

Detection of equine antibodies to babesia caballi by recombinant B. caballi rhoptry-associated protein 1 in a competitive-inhibition enzyme-linked immunosorbent assay.

A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was developed for detection of equine antibodies specific for Babesia caballi. The assay used recombinant B. caballi rhoptry-associated protein 1 (RAP-1) and monoclonal antibody (MAb) 79/17.18.5, which is reactive with a peptide epitope of a native 60-kDa B. caballi antigen. The gene encoding the recombinant antigen was sequenced, and database analysis revealed that the gene product is a rhoptry-associated protein. Cloning and expression of a truncated copy of the gene demonstrated that MAb 79/17.18.5 reacts with the C-terminal repeat region of the protein. The cELISA was used to evaluate 302 equine serum samples previously tested for antibodies to B. caballi by a standardized complement fixation test (CFT). The results of cELISA and CFT were 73% concordant. Seventy-two of the 77 serum samples with discordant results were CFT negative and cELISA positive. Further evaluation of the serum samples with discordant results by indirect immunofluorescence assay (IFA) demonstrated that at a serum dilution of 1:200, 48 of the CFT-negative and cELISA-positive serum samples contained antibodies reactive with B. caballi RAP-1. Four of five CFT-positive and cELISA-negative serum samples contained antibodies reactive with B. caballi when they were tested by IFA. These data indicate that following infection with B. caballi, horses consistently produce antibody to the RAP-1 epitope defined by MAb 79/17.18.5, and when used in the cELISA format, recombinant RAP-1 is a useful antigen for the serologic detection of anti-B. caballi antibodies.

Evaluation of North American antibody detection tests for diagnosis of brucellosis in goats.

The sensitivities and specificities of 17 antibody detection tests for brucellosis in goats were estimated. Tests evaluated included the U.S. Department of Agriculture (USDA) card test with 8% cell concentration (8%Card), USDA rapid automated presumptive test (RAP), Mexican rose bengal plate tests with 8 and 3% cell concentrations (8%RB and 3%RB), French rose bengal plate test with 4.5% cell concentration (4.5%RB), USDA standard plate test (SPT), USDA buffered acidified plate agglutination test (BAPA), USDA and Mexican rivanol tests (URIV and MRIV), USDA standard tube tests with Brucella abortus and Brucella melitensis antigens (SATA and SATM), serum enzyme-linked immunosorbent assay (ELISA), USDA cold-fixation complement fixation tests with B. abortus and B. melitensis antigens (CFA and CFM), USDA and Mexican milk ring tests (UBRT and MBRT), and a milk ELISA. Test sensitivity was evaluated by using two groups of 10 goats experimentally infected with B. melitensis or B. abortus and monitored for 24 weeks. Specificity was evaluated by using 200 brucellosis-free nonvaccinated goats from 10 California herds. The 3%RB was considered a good screening test because of high sensitivity at week 24 postinfection (90%), ease of performance, and low cost. The cold-fixation CFA and CFM had 100% specificity in the field study and were considered appropriate confirmatory tests. The milk ELISA was significantly more sensitive (P < 0.05) than the UBRT and significantly more specific (P < 0.05) than the MBRT. The milk ELISA also had the advantage of objectivity and ease of interpretation.

Seminal vesiculitis and orchitis caused by Brucella abortus biovar 1 in young bison bulls from South Dakota.

Specimens of blood, lymph nodes, spleens, and genitalia were collected at slaughter from seven 3- and 4-year-old male bison that had recently become seropositive for brucellosis. The animals were from a captive herd of approximately 3,500 bison located in central South Dakota. Brucella abortus biovar 1 was isolated from 2 or more specimens from each of 6 bison. Severe necrotizing and pyogranulomatous orchitis was present in 1 testicle from 1 bull, and 4 animals had mild to marked seminal vesiculitis. Immunohistochemical staining labeled organisms in seminal vesicles and the testicle with orchitis. Ultrastructurally, intact bacilli were present in cytoplasmic vacuoles of some macrophages; other macrophages contained intracytoplasmic aggregates of calcified coccobacilli.

Serologic responses of Brucella abortus strain 19 calfhood-vaccinated cattle following adult vaccination with strain RB51.

This study was designed to determine if Brucella abortus strain RB51, which expresses small amounts of the lipopolysaccharide O side chain, would cause positive responses on brucellosis serologic surveillance tests when given to adult cattle that were vaccinated as calves with B. abortus strain 19. Cattle vaccinated as adults with strain RB51 that had been vaccinated as calves with strain 19 (n = 40) had significantly greater antibody titers (P < 0.05) against strain RB51 at 4 and 8 weeks postvaccination in the dot blot assay than did animals (n = 10) not vaccinated with strain RB51. When evaluated using the card or buffered acid plate agglutination presumptive tests, 7 strain RB51 vaccinates tested positive at either 4 or 8 weeks following vaccination as compared with 4 cattle in the control group that were not vaccinated with strain RB51. One strain RB51 vaccinate was scored as suspect on the standard tube agglutination (STA) test at 8 weeks following vaccination. Remaining samples from strain RB51 vaccinates tested negative on the STA, complement fixation (CF), rivanol, and particle concentration fluorescence immunoassay (PCFIA) confirmatory tests. Samples from 2 control cattle were PCFIA positive at time 0; 1 of these animals was CF positive throughout the study. This study suggests that use of strain RB51 in cattle vaccinated with strain 19 as calves will not cause positive responses on confirmatory tests and will not impair brucellosis serologic surveillance efforts.

Conserved recombinant antigens of Anaplasma marginale and Babesia equi for serologic diagnosis.

The competitive inhibition ELISA (CI-ELISA) format overcomes problems associated with antigen purity since the specificity of the CI-ELISA depends solely on the monoclonal antibody (mAb) used. Therefore, the CI-ELISA format is well suited for use with recombinant antigens. Molecular clones expressing a conserved 19 kDa protein of Anaplasma marginale and a 34 kDa protein of Babesia equi were derived and characterized. The 19 kDa A. marginale protein, conserved in all recognized Anaplasma species, and present in the infected tick salivary gland, was reactive with all bovine immune sera tested. The 34 kDa B. equi protein contains a protein epitope bound by antibody in equine immune sera from 19 countries. Monoclonal antibodies reactive with these proteins were derived and applied with recombinant copies of the 19 kDa A. marginale and 34 kDa B. equi proteins in a CI-ELISA format.

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.

Serologic responses in diagnostic tests for brucellosis in cattle vaccinated with Brucella abortus 19 or RB51.

Serologic responses in the particle concentration fluorescence immunoassay and the card, complement fixation, and tube agglutination tests were measured for 10 weeks after vaccination of cattle with either Brucella abortus 19 or the lipopolysaccharide O-antigen-deficient mutant, strain RB51. The responses of strain 19-vaccinated cattle were positive, whereas those of strain RB51-vaccinated cattle were negative, in all of the tests. These results indicate that cattle vaccinated with strain RB51 fail to produce antibodies that can be detected by conventional serologic tests that are used to diagnose bovine brucellosis.

Reproducibility of a commercial enzyme-linked immunosorbent assay for bovine paratuberculosis among eight laboratories.

Interlaboratory reproducibility of an absorbed enzyme-linked immunosorbent assay (ELISA) kit for detection of bovine serum antibodies to Mycobacterium paratuberculosis was evaluated. A panel of 30 bovine sera (15 positives and 15 negatives) was tested in triplicate microtiter wells on each of 2 days at 8 different laboratories. One laboratory had invalid results because of positive or negative serum control optical density (OD) readings beyond the acceptable range specified by the kit. The coefficient of variation (CV) for mean OD values was influenced by low ODs on test negative sera at 2 laboratories, thus the CVs on positive sera were considered a more representative measure of kit reproducibility. Between-well CVs averaged 6.7% +/- 2.8% (mean +/- standard deviation), and between-day CVs averaged 14.5% +/- 9.8% among the 7 laboratories with valid assays on the 15 positive sera. The OD values were converted to positive or negative classifications for each assay well, and the results were compared. Among 1,392 assays in 7 laboratories, 98.6% were in agreement. Eleven of 18 discrepant results were due to a sample that consistently gave OD values near the cutoff for a positive test. Exclusion of that serum from the analysis resulted in a 99.8% rate of agreement among laboratories. Results indicated that the absorbed ELISA kit provided reproducible results within and between laboratories.

Antibody to a recombinant merozoite protein epitope identifies horses infected with Babesia equi.

Horses infected with Babesia equi were previously identified by the presence of antibodies reactive with a merozoite surface protein epitope (D. P. Knowles, Jr., L. E. Perryman, L. S. Kappmeyer, and S. G. Hennager. J. Clin. Microbiol. 29:2056-2058, 1991). The antibodies were detected in a competitive inhibition enzyme-linked immunosorbent assay (CI ELISA) by using monoclonal antibody 36/133.97, which defines a protein epitope on the merozoite surface. The gene encoding this B. equi merozoite epitope was cloned and expressed in Escherichia coli. The recombinant merozoite protein, designated equi merozoite antigen 1 (EMA-1), was evaluated in the CI ELISA. Recombinant EMA-1 bound antibody from the sera of B. equi-infected horses from 18 countries. The antibody response to EMA-1 was then measured in horses experimentally infected with B. equi via transmission by the tick vector Boophilus microplus or by intravenous inoculation. Anti-EMA-1 antibody was detected 7 weeks post-tick exposure and remained, without reexposure to B. equi, for the 33 weeks of the evaluation period. The data indicate that recombinant EMA-1 can be used in the CI ELISA to detect horses infected with B. equi.

Bacterial survival, lymph node changes, and immunologic responses of cattle vaccinated with standard and mutant strains of Brucella abortus.

Forty-eight cattle were used in 4 experiments; 6-week-old calves in experiments 1-3 (n = 24) and 10-month-old heifers in experiment 4 (n = 24). In experiments 1-3, 7 groups of 3 calves each were inoculated SC with 5 strains of Brucella abortus: virulent strain 2308 (2 groups), vaccine strain 19 (2 groups), and mutant strains RB51. 19 delta 31K, and 19 delta SOD. Sera and lymph node tissues were examined at 2-week intervals for evidence of infection. At postinoculation (PI) week 12, 2 calves in each group were given dexamethasone for 5 days. Calves were then euthanatized and lymphoid tissue, spleen, liver, and bone marrow were examined for evidence of B abortus. Calves given strain 2308 had large numbers of bacteria in their lymph nodes, marked granulomatous lymphadenitis in the deep cortex, and loss of lymphoid cells in superficial cortical areas. In addition, they had high serum antibody titers at PI week 16. Calves given strain 19, or genetic mutants derived from strain 19, cleared bacteria from lymph nodes more rapidly, had less lymphoid destruction, and developed antibody titers that did not persist for 16 weeks. The RB51 strain (rough) was cleared most rapidly from lymphoid tissues and induced serum antibody responses only to the core of the lipopolysaccharide molecule. Treatment of calves with dexamethasone did not cause B abortus to reappear in tissues of any calves, nor did serum antibody titers increase.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of equine antibody to Babesia equi merozoite proteins by a monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay.

A competitive inhibition enzyme-linked immunosorbent assay (CI ELISA) was developed to detect antibody to Babesia equi. One hundred fifty-four equine serum samples from 19 countries were tested for antibody to B. equi by the complement fixation test and by CI ELISA. The CI ELISA and complement fixation test results agreed in 94% (144) of the serum samples tested. The 10 discrepant serum samples were retested and analyzed for ability to immunoprecipitate in vitro translation products from B. equi merozoite mRNA. Five discrepant results were clearly resolved in favor of the CI ELISA, and the remaining five discrepancies were not definitively resolved.

Rapid identification of dominant Brucella antigens, using a microagglutination test.

A microagglutination test was used to identify Brucella dominant antigens from 400 Brucella and non-Brucella cultures. There was 100% agreement between microagglutination and tube agglutination tests in identifying dominant antigen.