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.     

Cell-mediated immune responses in cattle adult-vaccinated with Brucella abortus strain 19 and in cattle infected with Brucella abortus field strain.

Cell-mediated immune responses in cattle adult-vaccinated with Brucella abortus strain 19, cattle infected with B abortus field strain, and nonexposed cattle were studied by an in vitro lumphocyte-stimulation test (LST). Lymphocytes were prepared from peripheral bovine blood by the Ficoll-diatrizoate technique, and results were assayed for [3H]thymidine incorporation into DNA by liquid scintillation spectrometry. Serotests and bacteriologic isolation attempts were conducted simultaneously with LST. Lymphocytes from cattle infected with field strains had significantly (P = 0.01) higher specific lymphocyte-stimulation inexposed controls. The LST, the serum standard-tube agglutination test (STT), the Rivanol (RIV) test, and the complement-fixation (CF) test correctly classified cattle from which field strains and strain 19 of B abortus were isolated. The LST was negative in cattle vaccinated with B abortus strain 19 (nonshedding), but the three serotests had many false-positive reactions. The CF test had the least false-positive reaction, followed by the RIV test, and the STT was the least specific. Well before the three serotests became positive, the LST was positive in samples from some cattle during the incubation period of the infection. There was little or no correlation between cell-mediated immune responses (as measured by LST) and serum antibody responses (as measured by STT, RIV test, and CF test) in vaccinated but culture-negative cattle and in some nonvaccinated cattle during the incubation period.     

Enzyme-linked immunosorbent assay for differentiation of the antibody response of cattle naturally infected with Brucella abortus or vaccinated with strain 19

Purified O chain of Brucella abortus was passively attached to polystyrene to differentiate antibody responses of cattle vaccinated with B abortus strain 19 from those of naturally infected cattle. In the indirect assay, using O polysaccharide as antigen, a single serum dilution was used and mouse monoclonal antibody to bovine L chain conjugated with horseradish peroxidase was the detection reagent. Measurable antibody was not found in sera of vaccinated cattle, except for 3 sera from cattle that were persistently infected with strain 19. Sera from 25 cattle infected with pathogenic strains contained antibody on the basis of results of indirect enzyme immunoassay, using smooth lipopolysaccharide or O chain as antigens, or results of competitive enzyme immunoassay, using the O-chain antigen. Results in sera from calves with experimentally induced Yersinia enterocolitica serotype 0:9 infection or inoculated with a low dose of B abortus strain 2308 were comparable with those in sera of cattle that were vaccinated with strain 19. The data correlated with those from competitive enzyme immunoassay, using one serum dilution and horseradish peroxidase-conjugated mouse monoclonal antibody to smooth lipopolysaccharide. On the basis of results of the indirect enzyme immunoassay, all sera (except those samples obtained before inoculation) contained antibody to smooth lipopolysaccharide.     

Isolation of Brucella abortus and Brucella abortus, strain 19, from cattle.

Tissues from 104 cows in herd were examined for brucellae. Brucella abortus, strain 19, was isolated from 22 cows, a field strain of B abortus, biotype 1, was isolated from 9 cows, and both strains were isolated from 2 cows.     

Immunomodulation in cattle immunized with Brucella abortus strain 19

Regulation of the bovine immune response to immunization with Brucella abortus Strain 19 (S19) was investigated through application of a modification of an assay to measure suppressor T lymphocyte activities in humans and through development and characterization of antigen-stimulated T lymphocyte lines in vitro. A total of nine of steers were alloted into two groups: control (n = 4) and S19-immunized (n = 5). Peripheral blood mononuclear cells (PBMC) from each animal were cultured in vitro with mitogens (concanavalin A (Con A) and pokeweed mitogen (PWM], B. abortus antigens (B. abortus soluble antigen (BASA) and whole heat-killed B. abortus cells (HKC)) and media alone periodically from days 4 through 49 of the experiment. Supernates from these cultures were assayed for immunomodulatory activity(s) by addition to indicator cultures stimulated with suboptimal concentrations of Con A. Supernates from PBMC of S19-immunized steers generated with B. abortus antigens significantly (P less than 0.05) suppressed indicator cell responses as compared to those from control steers on days 35 and 49 post-immunization. This suppressive activity from PBMC of immunized cattle with respect to that of control cattle could also be induced through mitogenic stimulation with Con A or PWM. On day 49 of the study, suppressive activity was spontaneously released from the PBMC of immunized cattle. T lymphocyte lines were initiated from two S19-immunized steers at 2 and 9 weeks post-immunization. These T cell lines were characterized with respect to proliferative responses to B. abortus antigens through in vitro assay and surface marker expression through indirect immunofluorescence with a limited panel of monoclonal antibodies. Results from the present study indicated that S19 immunization induces a subpopulation(s) of cells in the PBMC of cattle capable of regulating the in vitro response to B. abortus. This regulatory activity is detectable by in vitro assay as early as 7 weeks post-immunization. Furthermore, the regulatory cell(s) appear to involve BoCD8+ T, lymphocytes which are specific for B. abortus antigens.     

Antibody isotype response in adult cattle vaccinated with Brucella abortus S19

In a chronological study of sera collected from eight adult cattle vaccinated with 3 X 10(-10) cfu of Brucella abortus S19, antibody of each of the four major isotypes was measured by indirect enzyme immunoassay (ELISA) and by direct and modified complement fixation tests (CFT). Six of the cattle gave antibody responses to the vaccine strain that commenced between days 5 and 8 for all the isotypes in the ELISA, peaked by 1 to 4 months and then declined to low levels by 10 months. Direct CFT and modified CFT titers were measurable by 7 or 8 days post-vaccination, and peaked by 1 month; direct CFT titers disappeared by 5 months while the modified CFT titers lingered for 10 months. Two animals gave cyclical direct CFT and modified CFT antibody responses, a cyclical IgG1 response, a low IgG2 and an elevated IgA response. The amplitude of the cycles was uniform over three cycles while the wavelength increased with time. A year post-vaccination, B. abortus S19 was isolated twice from milk from one of the animals (no attempt was made to culture B. abortus from the other). Sera from B. abortus naturally infected cattle were analysed for comparison.     

布鲁菌S2疫苗株免疫牛与牛种、羊种布鲁菌自然感染牛ELISA鉴别诊断方法的建立

为建立区分猪种布鲁菌S2疫苗株接种奶牛与布鲁菌自然感染奶牛,BLAST比对分析羊种、牛种、猪种、犬种、沙林鼠种和绵羊种6种布鲁菌基因序列,发现repA-related基因是猪种布鲁菌与牛种及羊种布鲁菌的差异基因。设计引物PCR扩增获得repA-related基因片段,克隆并原核表达得到了布鲁菌repA-related融合蛋白,以repA-related蛋白建立间接ELISA检测方法。用repA-related蛋白间接ELISA检测猪种S2疫苗株接种动物血清为阳性,检测牛种和羊种布鲁菌自然感染动物血清为阴性。repA-related蛋白间接ELISA能从试管凝聚实验(SAT)及常规ELISA检测阳性的奶牛血清样本中,区分出S2疫苗接种牛与牛种布鲁菌感染牛。     

布鲁菌S2疫苗株免疫牛与牛种、羊种布鲁菌自然感染牛ELISA鉴别诊断方法的建立

为建立区分猪种布鲁菌S2疫苗株接种奶牛与布鲁菌自然感染奶牛,BLAST比对分析羊种、牛种、猪种、犬种、沙林鼠种和绵羊种6种布鲁菌基因序列,发现repA—related基因是猪种布鲁菌与牛种及羊种布鲁菌的差异基因。设计引物PCR扩增获得repA-related基因片段,克隆并原核表达得到了布鲁菌repA—related融合蛋白,以repArelated蛋白建立间接EI．IsA检测方法。用repA—related蛋白间接ELISA检测猪种s2疫苗株接种动物血清为阳性,检测牛种和羊种布鲁菌自然感染动物血清为阴性。repA—related蛋白间接EusA能从试管凝聚实验（SAT）及常规ELIsA检测阳性的奶牛血清样本中,区分出s2疫苗接种牛与牛种布鲁菌感染牛。     

Arthropathy associated with Brucella abortus strain 19 vaccination in cattle. II. Experimental studies

Attempts to reproduce in calves the arthropathy associated with Brucella abortus strain 19 (S19) vaccination by direct intra-articular injection of S19 or virulent Brucella cells were unsuccessful. Intra-articular injection of rabbits and a calf with immune complexes isolated from the synovial fluid of a field case produced clinical and histological signs of arthropathy accompanied by the development of rheumatoid factor.     

Immunomodulation with killed Propionibacterium acnes in guinea pigs simultaneously vaccinated with Brucella abortus strain 19

Immunomodulation with killed Propionibacterium acnes was attempted in guinea pigs simultaneously vaccinated with Brucella abortus strain 19. Two groups, each comprised of 9 guinea pigs, were injected by different routes (s.c. and or i.v.) with 1.4 mg of P. acnes and 5 X 10(8) CFU of B. abortus, S-19, while 3 other groups each received either P. acnes, B. abortus S-19, or saline (s.c.). The antibody titers to B. abortus measured at 6, 10 and 14 weeks after vaccination indicated no significant (P less than 0.01) response in the 2 groups immunopotentiated with P. acnes concurrent with B. abortus S-19 vaccination. The delayed hypersensitivity response to 3 Brucella antigens conducted 8 weeks after immunization did not show a significant difference between the B. abortus S-19 vaccinated group compared with the 2 groups immunopotentiated and vaccinated. However, the proliferative response of lymphocytes to the B. abortus soluble antigen diluted 1:100 indicated significantly enhanced blastogenesis in the (s.c.) immunopotentiated and immunized guinea pigs compared with the B. abortus S-19 vaccinated group. A slightly enhanced response was also observed in the group immunopotentiated (i.v.) and vaccinated (s.c.). The guinea pigs were challenged with B. abortus strain 2308 and necropsied 4 weeks later. The mean splenic CFU of the Brucella in the group immunopotentiated (i.v.) and vaccinated (s.c.) was significantly decreased when compared with the guinea pigs vaccinated with B. abortus S-19 alone. These findings indicated that P. acnes administered simultaneously with B. abortus S-19 vaccine was able to augment the immune response in guinea pigs. Immunomodulation as evidenced by enhanced clearance of B. abortus from the spleens of immunopotentiated animals was presumably brought about by activated macrophages or a T-cell mediated cytolytic mechanism or both.     

Distribution of Brucella abortus in infected cattle

Experimentally and naturally infected cattle were examined bacteriologically to determine the anatomical distribution of specimens yielding Brucella abortus. In 91 experimentally infected pregnant cows, examined 3 to 4.5 months after conjunctival challenge during pregnancy, the most frequently infected specimen was the mammary (syn. supramammary) lymph node. All experimentally infected cows could be identified from cultures of the mammary, mandibular (syn. submaxillary), medial iliac, caudal superficial cervical (syn. prescapular) lymph nodes and uterine caruncles, cotyledons or foetal tissues. Forty-six naturally infected cows were examined and again the most frequently infected specimen was the mammary lymph node. All naturally infected cows could be identified from cultures of the mammary, parotid, mandibular and subiliac (syn. prefemoral) lymph nodes. The distribution of infected specimens was somewhat different in heifers. In 61 naturally infected heifers the most frequently infected specimen was the mandibular lymph node but 8 other specimens would have been required to enable identification of all infected heifers. Specimens from 3 infected bulls were cultured and 11 of the 12 specimens examined were infected in at least one of the bulls. The most frequently infected tissues were the mandibular, caudal superficial cervical, subiliac and scrotal lymph nodes. The results suggest which specimens should be selected for culture, particularly when only a limited amount of effort can be expended.     

Cytokine responses in camels (Camelus bactrianus) vaccinated with Brucella abortus strain 19 vaccine

In the present study, we determined the levels of cytokines produced by camel (Camelus bactrianus) peripheral blood mononuclear cells (PBMCs) in response to live attenuated Brucella abortus (B. abortus) S19 vaccine. Seven camels were vaccinated with commercial B. abortus S19 vaccine, and their cytokine responses were determined using a real-time PCR assay. Cytokine responses to B. abortus S19 were examined at 6 hr, 48 hr and 1, 2 and 3 weeks post-vaccination. Serological tests were performed to further confirm these immune responses. The results revealed that IFN-gamma and IL-6 were upregulated during the first week post-vaccination. Low level expressions of IL-1alpha, IL-1beta, TNFalpha and IL-10 and no expression of IL-2 and IL-4 were observed compared with the control camels. The findings showed that B. abortus stimulates cell-mediated immunity by directly activating camel Th1 cells to secrete IFN-gamma. This quantification of cytokine expression in camels is essential for understanding of Camelidae disease development and protective immune responses. This is the first report of in vivo camel cytokine quantification after vaccination.