Attenuation of a Brucella abortus mutant lacking a major 25 kDa outer membrane protein in cattle

OBJECTIVE: To determine the virulence of a Brucella abortus mutant, BA25, lacking a major 25 kd outer membrane protein (Omp25) in cattle. ANIMALS: 20 mixed-breed heifers in late gestation. PROCEDURE: 10 heifers were inoculated with 1 x 10(7) colony-forming units of the Omp25 mutant via the conjunctival sac, and an equal number were infected with the virulent parental strain B. abortus 2308. The delivery status of the dams was recorded, and colonization was assessed following necropsy. The ability of BA25 to replicate inside bovine phagocytes and chorionic trophoblasts was also evaluated in vitro because of the propensity of virulent brucellae to replicate inside these cells in vivo. RESULTS: The parental strain induced abortions in 5 of 10 inoculated cattle, whereas only 1 of 10 dams exposed to BA25 aborted. Brucella abortus strain 2308 colonized all of the cow-calf pairs and induced Brucella-specific antibodies in 100% of the dams. In contrast, BA25 was isolated by bacteriologic cultural technique from 30% of the calves and 50% of the inoculated dams (n = 10). Of the 10 heifers inoculated with BA25, 4 did not develop Brucella-specific antibodies nor were they colonized by the mutant strain. In bovine macrophages and chorionic trophoblasts, BA25 replicated in significantly lower numbers than the virulent parental strain (n = 3). CONCLUSIONS AND CLINICAL RELEVANCE: The 25 kd outer membrane protein may be an important virulence factor for B. abortus in cattle. The attenuation of the Omp25 mutant in cattle may involve the inability of BA25 to replicate efficiently in bovine phagocytes and chorionic trophoblasts.     

Relationship of fetal age at conjunctival exposure of pregnant heifers and Brucella abortus isolation

Pregnant heifers were exposed by a conjunctival inoculation with 1 X 10(7) colony-forming units of Brucella abortus strain 2308. At parturition, milk and uterine samples from dams plus samples from dead calves were cultured bacteriologically for Brucella. The logistic regression probability of B abortus isolation increased from 0.22 to 0.90, as fetal age at exposure of heifers increased from 60 to 150 gestation days. Strain 2308 was recovered at parturition from 14 (64%) of 22, 17 (71%) of 24, and all 28 (100%) heifers that were at gestation days less than 127, 127 to 157, and greater than 157, respectively, at time of exposure. The number of infected heifers and the number of samples positive for B abortus were significantly increased as fetal age at exposure of heifers increased from gestation days less than 127 to greater than 157 (chi 2 greater than 10, P less than 0.005).     

Efficacy of calfhood vaccination with Brucella abortus strain RB51 in protecting bison against brucellosis

In the studies reported here, protection induced by calfhood vaccination of bison with 1.2-6.1 x 10(10)CFU of Brucella abortus strain RB51 (SRB51) against a virulent strain of B. abortus was evaluated. Non-vaccinated and SRB51-vaccinated bison were intraconjunctivally challenged during midgestation with 3 x 10(7)CFU of virulent B. abortus strain 2308 (S2308). Maternal and fetal tissues were obtained within 24hour after abortion or parturition. Incidence of abortion was greater (P<0.05) in non-vaccinated as compared to SRB51-vaccinated bison (62% and 15%, respectively), with abortions occurring between 5 and 8 weeks after experimental challenge. Calves from bison vaccinated with SRB51 had a reduced (P<0.05) prevalence of fetal infection with S2308 as compared to calves from non-vaccinated bison (19% and 62%, respectively). Although the ability to recover the 2308 challenge strain from maternal tissues did not differ (P>0.05) between nonvaccinates and vaccinates (100% and 78%, respectively), calfhood vaccination with SRB51 reduced (P<0.05) recovery of S2308 from uterine or mammary gland tissues. In bison which did not abort, S2308 was routinely recovered in low numbers from maternal lymphatic tissues; particularly the parotid, bronchial, supramammary, and mandibular lymph nodes. The RB51 vaccine strain was not recovered at any time from maternal or fetal samples obtained at necropsy. Histological lesions associated with Brucella-induced abortions were suppurative placentitis, fetal broncho-interstitial pneumonia, and fetal histiocytic splenitis. The results of our studies suggest that calfhood vaccination of bison with SRB51 is efficacious in protecting against intramammary, intrauterine, and fetal infection following exposure to a virulent strain of B. abortus during pregnancy. As brucellosis is transmitted horizontally through fluids associated with the birth or abortion of an infected fetus, or vertically to the calf through the ingestion of milk containing B. abortus, our data suggest that calfhood vaccination with SRB51 will be beneficial in preventing transmission of brucellosis in bison.     

Protection levels in vaccinated heifers with experimental vaccines Brucella abortus M1-luc and INTA 2

Brucella abortus M1-luc is a mutant strain derived from S19 vaccine strain in which most of bp26 sequence has been replaced by the luciferase coding gene. Strain I2 is a double mutant derived from M1-luc in which most of omp19 has been deleted without introduction of any genetic markers. In BALB/c mice, M1-luc presented equivalent performance to S19 regarding persistence, splenomegaly and protection against challenge. Interestingly, I2 was more attenuated than S19, with no reduction of protection against challenge. In order to evaluate the potential for vaccine use of these strains in the natural host, four groups of 15 heifers, 6-month old, were either non-vaccinated or vaccinated with S19, M1-luc or I2. To at reached 17-month old, heifers were synchronized with two doses of PGF2alpha and received natural service during 60 days with two bulls. Pregnant heifers were challenged at approximately six gestation months with virulent B. abortus S2308. Blood samples post-challenge of heifers were collected for serologic test as well as specimens of aborted fetuses and premature calves for bacterial isolation and histopathological analyses. Protection levels against abortion were 78.6% for S19, 81.8% for M1-luc and 45.5% for I2, compared to the 25% that did not abort from the non-vaccinated group. These results indicate that in bovines BP26 had no influence in protective capacity of S19, correlating with the results obtained in mice. However, contrarily to what was previously observed in mice, lack of expression of Omp19 rendered in less protection capacity of S19 in the natural host.     

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)     

Re-examination of the role of the Brucella melitensis HtrA stress response protease in virulence in pregnant goats.

Based on previously reported studies describing the experimental infection of pregnant goats with B. melitensis strain RWP5, we proposed that the HtrA protease plays an important role in the virulence of B. melitensis in its natural ruminant host. Subsequent studies, however, have shown that RWP5 is actually an htrA cycL double mutant. In order to definitively evaluate the role of the B. melitensis htrA in virulence, we constructed an authentic htrA mutant and examined this strain in pregnant goats. The findings of these studies indicate that the contribution of the htrA gene product to the virulence of B. melitensis in its natural host is not as great as was previously proposed.     

Survival of smooth, rough and transposon mutant strains of Brucella abortus in bovine mammary macrophages

Transposon mutants offer a unique way to evaluate the role of lipopolysaccharide (LPS) by producing a theoretical single-gene difference between the original strain and the transposon mutant strain. Comparative survival of Brucella abortus smooth strain 2308, rough RB51, smooth strain 19, and two transposon mutant strains (rough strain 2308::Tn5 Lac Z [m106] and rough strain 19::Tn5 Lac Z [m3], was tested in restrictive bovine mammary macrophages that were able to effectively reduce the percentage of intracellular bacterial survival and permissive bovine mammary macrophages that were unable to control the intracellular replication of B. abortus. The theoretical single-gene difference between strain 19 and strain 19::Tn5 lac Z [m3] and between smooth virulent strain 2308 and rough transposon mutant 2308::Tn5 lacZ [m106] is likely related to differences in LPS content or structure. Significant (P less than 0.05) reduction in the survival of rough strain 19::Tn5 Lac Z [m3] with no significant reduction in the rough transposon mutant strain 2308::Tn5 lacZ [m106] indicated that at least one factor other than LPS contributes to the intracellular survival of B. abortus in bovine macrophages.     

Relationship of days in gestation at exposure and development of brucellosis in strain 19-vaccinated heifers

Heifers injected with 10(8) (n = 40), 10(9) (n = 39), or 10(10) (n = 39) colony-forming units of Brucella abortus strain 19 were conjunctivally exposed to 10(7) colony-forming units of strain 2308 during gestation. At parturition, milk from each quarter of the udder, a piece of placenta, and 2 swab specimens of the uterus from the dam plus a swab specimen of the rectum from each calf were cultured for Brucella. If the calf was dead or died, additional specimens of lung, stomach contents, and a mediastinal lymph node also were cultured. Days in gestation was determined for each heifer, using data from rectal palpation after breeding and crown-rump length and weight of calf at parturition, with the median value used for data analysis. In each vaccine dosage group, the proportion (%) of heifers developing brucellosis increased as days in gestation at exposure increased. Strain 2308 was isolated from 3 (11%) of 26, 16 (25%) of 64, and 18 (64%) of 28 heifers that were grouped as less than 121, 121 to 150, and greater than 150 days in gestation at time of exposure, respectively. Thirty-two (86%) of the 37 infected heifers were less than 260 days in gestation at parturition, and calves were premature. Heifers with premature calves were more likely to be infected, and tissues were more likely to yield multiple isolations of strain 2308, regardless of days in gestation at exposure or of days after exposure to parturition. Days after exposure to premature parturition of infected heifers ranged from 35 to 110.     

Brucella abortus INTA2, a novel strain 19 (Delta)bp26::luc (Delta)bmp18 double mutant lacking drug resistance markers

Brucella abortus INTA2, a novel mutant strain, was constructed by inactivation of two B. abortus S19 genes: bp26 and bmp18, with the objective of obtaining a mutant strain that could be compatible with a diagnostic test and have less residual virulence than strain 19. The double mutant was constructed by replacing a large section of the bp26 coding region with the luciferase (luc) coding gene, resulting in mutant strain B. abortus M1luc, followed by partial deletion of bmp18 coding sequence. Both genes were inactivated by allelic replacement assisted by sacB counter-selection. Luciferase expression was evaluated and confirmed that it is a valid marker in the construction of mutant strains. When B. abortus INTA2 was inoculated in BALB/c mice, significantly fewer colony forming units (CFUs) were recovered from mice spleens during initial phase of infection. No splenomegaly was observed in strain INTA2-immunized mice at any time suggesting that strain INTA2 has lost some residual virulence of the parental strain. Nevertheless, similar protection levels against virulent challenge were observed in mice immunized with strains INTA2 or S19. Although strain INTA2 would still induce O-side antibodies, it does not express BP26. This would allow differentiation of INTA2-vaccinated animals from animals infected with field strains by measuring anti-BP26 antibodies, either by an agglutination test or ELISA using BP26 as antigen. Altogether these results indicate that B. abortus INTA2 might be a promising vaccine strain against brucellosis.     

Biological properties of RB51; a stable rough strain of Brucella abortus

A rifampin-resistant mutant of Brucella abortus, designated RB51, was derived by repeated passage of strain 2308 on Trypticase soy supplemented with 1.5% agar and varying concentrations rifampin or penicillin. The RB51 colonies absorbed crystal violet and RB51 cell suspensions autoagglutinated, indicating a rough type colonial morphology for this strain. No O-chain component was detected in lipopolysaccharide (LPS) extracted from RB51 on SDS-PAGE gels stained with silver. Western blot analysis with the monoclonal antibody BRU 38, which is specific for the perosamine homopolymer O-chain of smooth Brucella LPS, indicated that the LPS of RB51 is highly deficient in O-chain when compared with the parenteral smooth strain 2308 or rough strain 45/20. Biochemically, RB51 resembles parental strain 2308 in its ability to utilize erythritol. Intraperitoneal inoculation of RB51 into mice results in a splenic colonization which is cleared within four weeks post infection. RB51 does not revert to smooth colony morphology upon passage in vivo (mice) or in vitro. Mice infected with RB51 produce antibodies against B. abortus antigens including class 2 and 3 outer membrane proteins but not against the O-chain. Furthermore, rabbits, goats and cattle hyperimmunized with sonicates of RB51 develop antibodies to B. abortus cellular antigens but do not develop antibodies specific for the O-chain. Immunization of mice with 1 x 10(8) viable RB51 organisms confers significant protection against challenge with virulent B. abortus strain 2308.     

Brucella species lacking the major outer membrane protein Omp25 are attenuated in mice and protect against Brucella melitensis and Brucella ovis

To aid in the development of novel efficacious vaccines against brucellosis, Omp25 was examined as a potential candidate. To determine the role of Omp25 in virulence, mutants were created with Brucella abortus (BA25), Brucella melitensis (BM25), and Brucella ovis (BO25) which contain disruptions in the omp25 gene (Deltaomp25 mutants). Western immunoblot analysis and PCR verified that the Omp25 protein was not expressed and that the omp25 gene was disrupted in each strain. BALB/c mice infected with B. abortus BA25 or B. melitensis BM25 showed a significant decrease in mean CFU/spleen at 18 and 4 weeks post-infection, respectively, when compared to the virulent parental strain (P<0.05, n=5). Mice infected with B. ovis BO25 had significantly lower mean CFU/spleen counts from 1 to 8 weeks post-infection, at which point the mutant was cleared from the spleens (P<0.01, n=5). Murine vaccination with either BM25 or the current caprine vaccine B. melitensis strain Rev. 1 resulted in more than a 2log(10) reduction in bacterial load following challenge with virulent B. melitensis (P<0.01, n=5). Vaccination of mice with the B. ovis mutant resulted in clearance of the challenge strain and provided 2.5log(10) greater protection against virulent B. ovis than vaccine strain Rev. 1. Based on these data, the B. melitensis and B. ovis Deltaomp25 mutants are interesting vaccine candidates that are currently under study in our laboratory for their safety and efficacy in small ruminants.     

Differentiation of Brucella abortus and Yersinia enterocolitica serotype 09 infections in cattle: the use of specific lymphocyte transformation and brucellin skin tests

The lymphocyte transformation test (using an in vitro whole-blood lymphocyte stimulation procedure) and the Brucellin skin test were applied to five heifers infected with virulent Brucella abortus strain 544, five cows inoculated with Yersinia enterocolitica serotype 09, and four non-exposed cows. Lymphocytes from Brucella-inoculated animals persistently gave very high blastogenic reactions indicative of active Brucella infection. The test was persistently negative in Yersinia-infected and non-exposed cattle. Four of the five cows infected with Yersinia enterocolitica type 09 and all four control cattle were persistently negative to the delayed hypersensitivity skin reaction with brucellin. All cattle infected with Yersinia enterocolitica type 09 were strongly positive to the Rose Bengal, Serum agglutination, Complement fixation and Antibovine globulin tests using Brucella abortus antigens. One lactating cow infected with Yersinia enterocolitica type 09 was positive to Brucella milk ring test. These results indicate that standard Brucella serological tests are unreliable in differentiating the two infections in cattle and that both the Lymphocyte transformation and brucellin skin tests could be used to differentiate bovine brucellosis from yersiniosis.     

布鲁氏菌vceA基因缺失株的构建及生物学特性研究

试验旨在构建布鲁氏菌Ⅳ型分泌系统vceA基因缺失株,分析其生长特征及其在人胚胎滋养层细胞(HPT-8)中的存活能力。以布鲁氏菌S2308为模板,PCR扩增vceA基因上、下游同源臂,以pUC19K质粒为模板扩增kan基因,然后利用融合PCR技术将3段基因进行融合,再将此片段与pMD19-T载体连接,电转至大肠杆菌DH5α感受态细胞中,构建ΔvceA基因缺失株(S2308ΔvceA),通过卡那抗性对缺失株进行连续筛选,并检测其遗传稳定性。在相同培养条件下,观察亲本株S2308和S2308ΔvceA的生长变化趋势,以侵染复数(MOI)100∶1侵染HPT-8细胞,通过CFU计数检测亲本株S2308和缺失株S2308ΔvceA在细胞内的生存能力。结果显示,试验成功获得片段大小为372、510、1 093 bp的vceA基因上、下游同源臂和kan基因;且成功构建pMD19-T-vceA-kan自杀载体,将其电转入大肠杆菌DH5α感受态细胞,构建基因缺失株,连续传代10次未发现基因回复突变;在体外相同培养条件下,缺失株S2308ΔvceA与亲本株S2308生长趋势相似,均在12 h达到对数生长期,30 h进入平台期;侵染HPT-8细胞12 h时,缺失株S2308ΔvceA在细胞中的数量显著低于亲本株S2308(P<0.05)。综上所述,本试验成功构建并获得了具有良好遗传稳定性的布鲁氏菌vceA基因缺失株,该缺失株在体外培养条件下与亲本株生长趋势相似;但该缺失株在HPT-8细胞内的存活能力显著变弱。     

Prevention of persistent infection in calves by vaccination of dams with noncytopathic type-1 modified-live bovine viral diarrhea virus prior to breeding

OBJECTIVE: To determine the ability of a modified-live virus (MLV) bovine viral diarrhea virus (BVDV) type 1 (BVDV1) vaccine administered to heifers prior to breeding to stimulate protective immunity that would block transmission of virulent heterologous BVDV during gestation, thus preventing persistent infection of a fetus. ANIMAL: 40 crossbred Angus heifers that were 15 to 18 months old and seronegative for BVDV and 36 calves born to those heifers. PROCEDURE: Heifers were randomly assigned to control (n = 13) or vaccinated (27) groups. The control group was administered a multivalent vaccine where-in the BVDV component had been omitted. The vaccinated heifers were administered a single dose of vaccine (IM or SC) containing MLV BVDV1 (WRL strain). All vaccinated and control heifers were maintained in pastures and exposed to BVDV-negative bulls 21 days later. Thirty-five heifers were confirmed pregnant and were challenge exposed at 55 to 100 days of gestation by IV administration of virulent BVDV1 (7443 strain). RESULTS: All control heifers were viremic following challenge exposure, and calves born to control heifers were persistently infected with BVDV. Viremia was not detected in the vaccinated heifers, and 92% of calves born to vaccinated heifers were not persistently infected with BVDV. CONCLUSIONS AND CLINICAL RELEVANCE: These results document that vaccination with BVDV1 strain WRL protects fetuses from infection with heterologous virulent BVDV1.     

Bovine T-lymphocyte lines reactive with Brucella abortus

Bovine T-cell lines reactive with Brucella abortus were established by repeated stimulation with B abortus and mitomycin C-treated autologous antigen-presenting cells. Representative results were obtained, using 33 cell lines from 14 cows. Cultures responded to the virulent laboratory strain 2308, the vaccine strain 19, and the rough mutant strain RB51 in thymidine-incorporation assays. The cells in these cultures required antigen-presenting cells for their response to B abortus. Autologous antigen-presenting cells were optimal for most lines tested, although some T-cell lines could respond to B abortus in the presence of some, but not all, allogeneic antigen-presenting cells. The cell lines expressed cell surface markers characteristics of activated bovine T cels. Of the cell lines tested for expression of cluster-determinant (CD) 4 and CD8 cell surface antigens, no cells in any cultures expressed the bovine CD8 equivalent, but all cultures included CD4+ cells in variable amounts. Some cell lines consisted of up to 50% CD2+CD4-CD8- cells. None of the cell lines tested expressed surface immunoglobulin or other bovine B-cell markers. Thus, these long-term cell lines appear to include 2 T-lymphocyte subsets: the helper/inducer subset and a second subset expressing a phenotype similar to major histocompatibility complex-unrestricted cytolytic cells in other species.     

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.     

Responses of cattle to two dosages of Brucella abortus strain RB51: serology, clearance and efficacy

A new brucellosis vaccine, Brucella abortus strain RB51 (SRB51), is currently recommended for use as a calfhood vaccine in the US at dosages between 1 x 10(10)and 3.4 x 10(10)colony-forming units (CFU). The purpose of the study reported here was to compare responses to minimal and maximal recommended SRB51 dosages. Eighteen heifer calves were vaccinated subcutaneously with 1.6 x 10(10)CFU of SRB51, 3.2 x 10(10)CFU of SRB51, or saline (n = 6 per treatment). The vaccine strain was recovered from the superficial cervical lymph node 14 weeks after vaccination in two of six animals that received 1.6 x 10(10)CFU SRB51, but not from any cattle vaccinated with 3.2 x 10(10)CFU SRB51. The higher SRB51 dosage stimulated greater antibody titres. Protection against abortion or infection following B. abortus strain 2308 (S2308) challenge was similar for both SRB51 dosages and greater than resistance of non-vaccinates. The vaccine strain was recovered from one heifer and her fetus at necropsy 1 week prior to estimated parturition. Data from this study suggests that SRB51 induces similar protective immunity across the recommended dosage range. The SRB51 vaccine may persist in some cattle into adulthood but the incidence and significance of this persistence remains unknown.     

Brucellosis in heifers weaned from seropositive dams

Fifty-six heifers were weaned from dams that were card-test positive for brucellosis. Forty-four dams were positive by rivanol and complement-fixation tests and Brucella abortus field strain was isolated from 14. Numbers of expected pregnancies following natural breeding and numbers of viable calves produced were not reduced in the heifers. Persistent B abortus infection was documented in 2 of 37 parturient heifers from reactor dams. The frequency of infection was 1 of 10 in strain 19-vaccinated heifers, and 1 of 27 in nonvaccinated heifers. The 2 persistently infected heifers had atypical serologic reaction patterns before normal parturitions.     

Recombinant bovine interleukin 2 enhances immunity and protection induced by Brucella abortus vaccines in cattle

Augmentation of immunization of cattle Brucella abortus S19 or a B. abortus soluble protein extract (SPEBA) vaccine through administration of recombinant bovine IL 2 (rBoIL 2) was evaluated. Seventy-five heifers were divided among 6 groups that were treated with the following: Group 1, no treatment; Group 2, rBoIL 2 (1microg/kg) on day 0; Group 3, SPEBA (2 mg) on day 0 and week 9; Group 4, SPEBA + rBoIL 2 on day 0, SPEBA on week 9; Group 5, S19 (10(7) CFU) on day 0 and week 9; Group 6, S19 + rBoIL 2 on day 0, S19 only on week 9. Approximately, 6 months after vaccination, cattle were bred by natural service, and at mid-gestation pregnant cattle were challenged intraconjunctivally with 9.1 x 10(5) CFU of virulent B. abortus S2308. Pre- and post-challenge antibody responses were measured by an enzyme-linked immunosorbent assay, a particle concentration fluorescence assay, and the card test. Lymphoproliferation (LP) responses to gamma-irradiated B. abortus and SPEBA antigens were measured in peripheral blood mononuclear cells. After vaccination, antibody responses to B. abortus elevated rapidly in SPEBA- and S19-vaccinates with and without rBoIL 2, however, these responses were significantly (P < 0.05) higher in vaccinates which also received rBoIL 2. Antibody levels for all vaccinated groups had returned to those of negative control groups by the challenge date with the exception of the SPEBA/rBoIL 2 group. In general, LP responses were higher in vaccinated or rBoIL 2-treated cattle than for unvaccinated controls. Challenge of 48 pregnant heifers resulted in abortions in 4/9 of Group 1, 0/9 of Group 2, 4/8 of Group 3, 2/9 of Group 4, 1/7 of Group 5, and 0/6 of Group 6 cattle. Treatment with rBoIL 2 alone (Group 2) provided significant (P < 0.05) protection from infection, abortions and induction of sero-positive status compared to untreated (Group 1) cattle. Co-administration of rBoIL 2 with S19 resulted in significant (P < 0.05) augmentation in onset, duration and magnitude of LP responses to B. abortus antigens following challenge. Characterization of the cytokine response of bovine monocyte-derived macrophages by real-time polymerase chain reaction indicated that in vitro stimulation of these cells with rBoIL 2 resulted in a profound up-regulation of genes encoding tumor necrosis factor-alpha, IL 12p40, and interferon-gamma reflecting activation of the cells. Overall, rBoIL 2-treatment was associated with fewer infections, sero-conversions and a significant (P = 0.02) level of protection against abortion as compared to vaccination alone or no treatment.     

布鲁菌在豚鼠和奶牛体内诱导的抗体水平和变态反应强度的相关性

为探索布鲁菌在豚鼠和奶牛体内所引起的抗体水平和变态反应强度的相关性,分别用1×104,3×104CFU牛种布鲁菌强毒2308株各感染12只豚鼠,30 d后测定抗体滴度和变态反应强度。感染后35 d,扑杀豚鼠,取脾脏测定每克脾脏含菌量。用布鲁菌A19疫苗,以5×1010CFU皮下注射布病阴性荷斯坦奶牛50头,分别于免疫后15,30,60 d测定抗体滴度,并于免疫后45 d测定变态反应强度。运用SPSS 17.0-Analyze-Correlate-Bivariate Corre-lations程序分析试验数据,结果显示,不同剂量布鲁菌2308感染豚鼠后30 d所诱导的抗体水平、变态反应强度和克脾脏含菌量三者之间均无相关性。A19疫苗免疫奶牛后60 d的抗体水平与免疫45 d的变态反应强度呈正相关,免疫15 d和30 d的抗体水平和变态反应强度无相关性。豚鼠试验结果表明,抗体水平、变态反应强度与个体对布病的抵抗力均无相关性。