Comparison between immune responses and resistance induced in BALB/c mice vaccinated with RB51 and Rev. 1 vaccines and challenged with Brucella melitensis bv. 3

BALB/c mice were immunized with live rough Brucella abortus RB51 or smooth Brucella melitensis Rev. 1 vaccines and challenged with a B. melitensis field strain. Protection was assessed by a variety of serological tests and recovery of vaccinal and challenge strains by culture. Mice vaccinated with RB51 gave negative results in the conventional serological tests prior to challenge, namely; standard tube agglutination test (SAT), Rose Bengal plate test (RBPT), buffered acidified plate antigen test (BAPAT), and mercaptoethanol test (MET). Sero-conversion took place to a whole-cell bacterial buffered RB51 antigen after vaccination and persisted for 7 weeks post-vaccination. Mice challenged with B. melitensis were assessed for bacterial load and immune response for 12 weeks after challenge. Protection units were showed that Rev. 1 vaccine was superior to RB51 vaccine in protection of mice against B. melitensis. However, RB51 vaccine has the advantage that it would not elicit antibodies to standard serological tests based on the LPS O antigen. RB51 vaccine could therefore be used for control of B. melitensis infection and avoid confusion in the use of standard sero-diagnostic tests.     

Anti-chlamydial immunity in ewes conferred by vaccination with a combination of three live chlamydia, brucella and salmonella vaccines

Live attenuated vaccines against Chlamydia psittaci var ovis, Brucella melitensis and Salmonella abortus ovis have previously been shown to be compatible in mice by subcutaneous administration. Immunity against challenge with virulent chlamydia was, however, slightly decreased in associations including the B melitensis Rev 1 vaccine. The chlamydia strain 1B vaccine was administered to four- to five-month-old female lambs, either alone or in combination with the B melitensis Rev1 and the S abortus ovis Rv6 vaccines. Clinical, serological and bacteriological observations demonstrated the compatibility of the three vaccines. Control, singly and triply vaccinated ewes were challenged with a virulent strain of chlamydia during their second pregnancy, 15 months after vaccination. Five of the 12 control ewes lambed normally and 10 of them were infected, as shown by the excretion of the challenge chlamydia in genital secretions. Sixteen of the 17 ewes in the triple vaccine group lambed normally and none was infected. All 12 in the single vaccine group lambed normally and three of the 12 were infected. In spite of this unusually poor protection by the single vaccine, antichlamydial immunity was clearly not decreased by the association with the two other vaccines.     

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.     

Protection against Brucellosis in Goats,Five Years after Vaccination with Reduced-Dose Brucella melitensis Rev 1 Vaccine

The protection conferred by the reduced-dose Rev 1 Brucella melitensis vaccine in goats that had been immunized 5 years previously was evaluated. Sixteen goats vaccinated 5 years before with Rev 1 (1 x 10(5) cfu) and 5 non-vaccinated goats were challenged with B. melitensis 16M (4 x 10(5) cfu) using the conjunctival route. After giving birth or aborting, the goats were sacrificed and tissue samples were taken for bacteriological study. The challenge strain was recovered in 12%, of the animals from the vaccinated group, and in (80% of the control group. It is concluded, therefore that the use of reduced-dose Rev 1 protects goats vaccinated in endemic areas for at least 5 years after immunization.     

Pathogenicity and protective activity in pregnant goats of a Brucella melitensis Deltaomp25 deletion mutant

The Brucella melitensis mutant BM 25, which lacks the major 25 kDa outer membrane protein Omp25, has previously been found to be attenuated in the murine brucellosis model. In the present study, the capacity of the Deltaomp25 mutant to colonise and cause abortions in the caprine host was evaluated. The vaccine potential of BM 25 was also investigated in goats. Inoculation of nine pregnant goats in late gestation with the B. melitensis mutant resulted in 0/9 abortions, while the virulent parental strain, B. melitensis 16M, induced 6/6 dams to abort (P<0.001, n=6). BM 25 also colonised fewer adults (P<0.05, n=6) and kids (P<0.01, n=6) than strain 16M. The Deltaomp25 mutant was found capable of transient in vivo colonisation of non-pregnant goats for two weeks post-infection. Owing to the ability of BM 25 to colonise both non-pregnant and pregnant adults without inducing abortions, a vaccine efficacy study was performed. Vaccination of goats prior to breeding with either BM 25 or the current caprine vaccine B. melitensis strain Rev. 1 resulted in 100 per cent protection against abortion following challenge in late gestation with virulent strain 16M (P<0.05, n=7). However, unlike strain Rev. 1, BM 25 does not appear to cause abortions in late gestation based on this study with a small number of animals. The B. melitensis Deltaomp25 mutant, BM 25, may be a safe and efficacious alternative to strain Rev. 1 when dealing with goat herds of mixed age and pregnancy status.     

Immunogenicity of Major Cell Surface Protein(s) of Brucella melitensis Rev 1

Brucella melitensis Rev 1 organisms were salt-extracted and the cell surface proteins (BCSPs) were found to be mainly 39-42 kDa (group 2 porin proteins) in addition to 31.6, 32.5, 58.5 and 14.7 kDa proteins. DEAE-Sephadex anion-exchange column chromatography of BCSPs yielded fraction 1, which contained one major protein (39.8-42.0 kDa) and a minor protein (31.6 kDa). All these proteins were found to be immunogenic by Western blotting. Fraction 1 along with monophosphoryl lipid A and trehalose dicorynomycolate adjuvants as well as BCSPs alone induced significant (p < or = 0.05) protection in BALB/c mice. Both these immunizing agents produced almost equivalent protection to live B. melitensis Rev 1 vaccine at 15 and 30 days post challenge. Lymphocyte stimulation test as well as delayed-type hypersensitivity reaction revealed that both these preparations induced cell-mediated immune response. These preparations also induced humoral immune response as indicated by indirect ELISA. Neither of the immune responses was significantly less (p < or = 0.05) than that with live B. melitensis Rev 1 vaccine, except that their duration was short.     

Comparison of subcutaneous and conjunctival routes of Rev 1 vaccination for the prophylaxis of Brucella ovis infection in rams

The serological response and protection conferred against Brucella ovis by the Rev 1 vaccine was evaluated in both adult (experiment 1) and young rams (experiment 2) vaccinated either subcutaneously or conjunctivally. In experiment 1 the Rev 1 vaccine protected 55.5 per cent and 100 per cent, respectively, of subcutaneously and conjunctivally vaccinated rams against three consecutive challenges that infected 100 per cent of unvaccinated controls. In experiment 2, Rev 1 protected 100 per cent of rams vaccinated subcutaneously and 70 per cent of those vaccinated conjunctivally against a challenge dose able to infect all the unvaccinated controls. The serological response after vaccination was significantly lower in rams vaccinated conjunctivally than in those vaccinated subcutaneously.     

Evaluation of a new immunocapture test for the diagnosis of ovine brucellosis caused by Brucella melitensis

A new immunocapture technique has been applied to the diagnosis of ovine brucellosis under experimental conditions. The tests were made on a serum bank derived from both young and adult ewes vaccinated conjunctivally with the Rev 1 strain at a dose of 10(8) to 10(9) colony-forming units. Adult ewes were infected experimentally two-and-a-half years after they had been vaccinated and the results were compared with an unvaccinated control group. The condition of each animal in terms of infection with Brucella melitensis was determined by clinical and bacteriological investigations. The development of the immune response was compared by the rose bengal test, the complement fixation test, the Coombs' test and the immunocapture technique for 180 days after the vaccination and for 410 days after the experimental infection, that is, the two following gestations. The results suggest that the new technique is more specific in animals vaccinated conjunctivally, regardless of their age when they were vaccinated. After the experimental infection, significantly (P < 0.05) fewer of the vaccinated sheep which were free of clinical signs and were not excreting B melitensis reacted positively to the test.     

An ELISA with Brucella lipopolysaccharide antigen for the diagnosis of B. melitensis infection in sheep and for the evaluation of serological responses following subcutaneous or conjunctival B. melitensis strain Rev 1 vaccination.

An indirect enzyme-linked immunosorbent assay (ELISA) with unpurified Brucella melitensis smooth lipopolysaccharide (S-LPS) as antigen was evaluated for the serological diagnosis of B. melitensis infection in sheep in comparison with the Rose Bengal (RB), complement fixation (CF), radial immunodiffusion (RID), microplate agglutination (MA) and rivanol agglutination (RIV) tests. Tests RB and CF detected as positive each of the 77 sera from B. melitensis-infected animals tested, the RID (74), MA (76) and the RIV (72) were less sensitive. However, all tests compared were negative when 77 sera from Brucella-free rams were tested. While subcutaneous Rev 1 vaccination induced high response levels in any of the tests, low level responses were obtained upon conjunctival vaccination, particularly in ELISA and RID tests.     

布鲁氏菌弱毒疫苗粘膜免疫及检测方法的研究

为研究布鲁氏菌弱毒疫苗粘膜免疫及其检测方法,本实验采用粘膜点眼途径对健康母羊接种布鲁氏杆菌猪2号疫苗(S2)、牛19号疫苗(A19)和羊强毒株(M16),筛选布鲁氏杆菌病鉴别检测方法。将12月龄～14月龄母羊60只随机分为3组,以常规疫苗推荐剂量进行半量粘膜点眼接种。采集血液、淋巴、脏器进行布鲁氏菌病血清学检测和细菌学分离以及PCR检测。结果表明:布鲁氏菌弱毒疫苗抗体水平持续6个月,其中血清学的试管凝集试验、半胱氨酸凝集试验与补体结合试验的阳性符合率达到100%。细菌分离期为6个月,乳腺、乳腺淋巴、髂淋巴分离率较高;而强毒株M16的抗体水平和细菌分离持续12个月以上。结果显示以常规血清学和细菌学检测方法在点眼免疫布鲁氏菌S2、A19苗6个月后可以进行野毒感染和疫苗免疫畜的鉴别诊断。     

The characteristics of a variant strain of Brucella melitensis Rev 1

Circumstantial evidence is presented for the occurrence of a variant of a vaccine strain of B. melitensis Rev 1, designated "FSA" (foreign South African). FSA resembles Rev 1 in its reactions to penicillin and streptomycin but reacts closer to a field strain of B. melitensis as regards dye (thionine and basic fuchsin) sensitivity and colony size. Although colonies of Rev 1 were consistently smaller than other B. melitensis strains, their size was 0,75 mm as opposed to the 1-2 mm reported in the literature, while B. melitensis 16M colonies were 1,25-1,5 mm as opposed to the 3-4 mm previously reported. Rev 1 was found to be urease positive, unless a test of low sensitivity was applied.     

绒山羊布氏杆菌病、衣原体和弓形虫病的血清学检测

采用布氏杆菌虎红平板凝集试验、弓形虫和衣原体间接血凝试验对360份绒山羊血清进行布氏杆菌病、弓形虫病和衣原体病的血清学调查。结果在绒山羊的血清样品中检出布氏杆菌病阳性血清8份,布氏杆菌病的血清阳性率为2．22％;检出弓形虫15份,弓形虫病血清阳性率为4．17％;检出衣原体23份,衣原体病血清阳性率为6．39％。     

The 16MΔvjbR as an ideal live attenuated vaccine candidate for differentiation between Brucella vaccination and infection

Brucellosis brings great economic burdens for developing countries. Live attenuated vaccines are the most efficient means for prevention and control of animal Brucellosis. However, the difficulties of differentiating of infection from vaccine immunization, which is essential for eradication programs, limit their applications. Therefore, the development of a vaccine that could differentiate infection from immunization will overcome the limitations and get extensive application. VjbR is a quorum sensing regulator involving in Brucella's intracellular survival. The vjbR∷Tn5 mutants have been proven effective against wild type strain challenge, implying its possibility of use in vaccine candidate development. To further evaluate this candidate gene, in the present study, the antigenicity of purified recombinant VjbR protein was analyzed. Antibodies to Brucella melitensis VjbR could be detected in sera from patients and animals with brucellosis but not in control ones, implying the potential use of this protein as a diagnostic antigen. Then a vjbR mutant of B. melitensis 16M was constructed by replacing the vjbR with kanamycin gene. The mutant showed reduced survival in macrophage and mice. Vaccination of BALB/c mice with 16MΔvjbR conferred significant protective immunity against B. melitensis strain 16M challenges, being equivalent to which induced by the license vaccine Rev.1. The vjbR deletion mutant elicited an anti-Brucella-specific immunoglobulin G response and induced the secretion of gamma interferon and interleukin-10. The most importance is that, the use of vjbR mutants as vaccines in association with diagnostic tests based on the VjbR antigen would allow the serological differentiation between infected and vaccinated animals. These results suggest that 16MΔvjbR is an ideal live attenuated vaccine candidate against B. melitensis and deserves further evaluation for vaccine development.     

Evaluation of a rough mutant of Brucella melitensis in pregnant goats

Brucella melitensis strain VTRM1, a rough derivative of B melitensis strain 16M, is able to colonise the lymph nodes of goats, does not induce abortion in pregnant goats when used at doses leading to abortions with virulent strain 16M, and does not induce anti-O chain antibodies. However, strain VTRM1 as a single dose vaccine induces only partial protection against both infection and abortion following challenge.     

Deletion of the GI-2 integrase and the wbkA flanking transposase improves the stability of Brucella melitensis Rev 1 vaccine

Brucella melitensis Rev 1 is the best vaccine available for the prophylaxis of small ruminant brucellosis and, indirectly, for reducing human brucellosis. However, Rev 1 shows anomalously high rates of spontaneous dissociation from smooth (S) to rough (R) bacteria, the latter being inefficacious as vaccines. This S-R instability results from the loss of the O-polysaccharide. To overcome this problem, we investigated whether some recently described mechanisms promoting mutations in O-polysaccharide genes were involved in Rev 1 S-R dissociation. We found that a proportion of Rev 1 R mutants result from genome rearrangements affecting the wbo O-polysaccharide loci of genomic island GI-2 and the wbkA O-polysaccharide glycosyltransferase gene of the wbk region. Accordingly, we mutated the GI-2 int gene and the wbk IS transposase involved in those arrangements, and found that these Rev 1 mutants maintained the S phenotype and showed lower dissociation levels. Combining these two mutations resulted in a strain (Rev 2) displaying a 95% decrease in dissociation with respect to parental Rev 1 under conditions promoting dissociation. Rev 2 did not differ from Rev 1 in the characteristics used in Rev 1 typing (growth rate, colonial size, reactivity with O-polysaccharide antibodies, phage, dye and antibiotic susceptibility). Moreover, Rev 2 and Rev 1 showed similar attenuation and afforded similar protection in the mouse model of brucellosis vaccines. We conclude that mutations targeting genes and DNA sequences involved in spontaneous O-polysaccharide loss enhance the stability of a critical vaccine phenotype and complement the empirical stabilization precautions taken during S Brucella vaccine production.     

Study of interleukin-10 and interleukin-12 productions in response to lipopolysaccharides extracted from two different Brucella strains

The aim of the present study is to investigate the cytokines induction by smooth lipopolysaccharides (S-LPS) extracted from Brucella melitensis (Rev1 vaccine strain) and Brucella abortus (a field isolate). These lipopolysaccharides were used to induce inflammatory cytokines production in peripheral blood cell culture of healthy individuals. Secretion of IL-10 and IL-12 (p70) were measured by means of specific Elisa. In addition, intracellular expression of IL-12 was assessed in CD14+ cells by flow cytometry. It was shown that Brucella LPS is a potent inducer of IL-10. However interferon gamma (IFN-gamma) priming was able to significantly decrease the production of IL-10. Flow cytometry studies showed that LPS alone was not able to induce intracellular IL-12 expression in CD14+ cells. Nevertheless, IFN-gamma priming significantly increased the percentage of CD14+ IL-12+ cells. In conclusion, it was demonstrated that the Brucella LPS could be a potent inducer of IL-10 and induction of IL-12 production needs the most favorable conditions.     

A review of the use of B. melitensis Rev 1 vaccine in adult sheep and goats

The live Brucella melitensis Rev 1 strain is considered the best vaccine available for the prophylaxis of brucellosis in small ruminants. The classically recommended exclusive vaccination of young replacement animals has failed to control brucellosis in some developed countries and is frequently inapplicable in the developing world. Accordingly, whole-flock vaccination is the only feasible alternative to control B. melitensis infection in small ruminants under the extensive management conditions characteristic of these countries. This review describes the practical problems encountered and the experience acquired over the past decade (particularly in Spain) using the Rev 1 based control strategy. The vaccination of pregnant animals with full standard doses of Rev 1 administered subcutaneously is followed by abortion in most vaccinated animals. Reducing the dose of vaccine has been suggested as a method of avoiding this problem and, accordingly, a reduced-dose vaccination strategy has been widely used and has been reported as a safe and effective method of controlling small ruminant brucellosis. However, we reviewed field and experimental results supporting the fact that as a result of the induction of abortion in pregnant animals and the low degree of immunity conferred, reduced doses of Rev 1 should not be recommended as an alternative to the full standard doses.

When tested in a mouse model, differences in residual virulence and immunogenicity have been demonstrated between the different Rev 1 vaccines produced world-wide. These differences could account for the discrepancies in safety results obtained in mass vaccination trials in different countries. The induction of abortions when vaccinating pregnant animals means that there is no entirely safe strategy for Rev 1 vaccination. Conjunctival vaccination is safer than subcutaneous vaccination but is not safe enough to be applied regardless of the pregnancy status of the animals, and should be used only under restricted conditions. For sheep, conjunctival administration of standard doses of Rev 1 during the late lambing season or during lactation is recommended as a whole-flock vaccination strategy.     

粗糙型布鲁菌M111菌壳的制备

将含有裂解酶基因重组温控裂解质粒pBBR1MCS：：PR-PL-E电转化至粗糙型布鲁菌M111中，构建重组布鲁菌M111（pBBRlMCS：：PR-PL-E）。重组菌株在28℃培养，42℃诱导表达裂解酶E，从而制备布鲁菌菌壳。绘制布鲁菌生长曲线及裂解曲线，计算裂解率并用透射电镜观察布鲁菌菌壳的形态。结果显示，成功制备了布鲁菌菌壳，温控裂解质粒pBBRIMCS：：PR-PL-E对布鲁菌的裂解率为100％。透射电镜观察可见细菌内容物部分流出，细菌表面出现不同程度的皱缩，细胞形态发生变化。结果表明，本试验成功制备了粗糙型布鲁菌菌壳，初步研究了其基本特性，为下-步开展布鲁菌菌壳疫苗的研究奠定了基础。     

Immunization with Brucella melitensis Rev 1 against Brucella ovis infection of rams

The efficacy of Brucella Melitensis Rev 1 vaccine (Rev 1) for the prophylaxis of Brucella ovis ram epididymitis was evaluated. Twenty-nine 3-month-old rams were vaccinated with 2 X 10(9) Rev 1 and 14 were revaccinated with 5 X 10(8) at 14 months of age. Six rams remained unvaccinated as a control group. All rams were challenged with 5 X 10(8) B. ovis at 21 months of age. Before being slaughtered 8 weeks later, only one vaccinated ram developed epididymitis while four of the six control rams developed testicular alterations. Genital and selected extragenital organs and lymph nodes were removed at slaughter and inoculated on selective media. B. ovis was isolated from 26.6% of the vaccinated rams, 21.4% of the revaccinated rams and 100% of control rams. Portions of epididymis, testes and vesicular glands were also used for pathological studies. More severe lesions were observed in control rams than in vaccinated ones. In conclusion, these results show that vaccination of young lambs, followed or not by revaccination, is a suitable method for the prophylaxis of B. ovis infection of rams.     

The effect of subunit or modified live bovine herpesvirus-1 vaccines on the efficacy of a recombinant Pasteurella haemolytica vaccine for the prevention of respiratory disease in feedlot calves

The efficacy of a Pasteurella haemolytica vaccine (PhV) administered once to calves within 24 hours of arrival at a feedlot was tested for the ability to prevent morbidity and mortality from all bovine respiratory disease (BRD) and specifically from fibrinous pneumonia mortality. The PhV consisted of two immunizing ingredients: outer membrane proteins extracted from P. haemolytica, plus genetically attenuated leukotoxin produced by recombinant DNA technology. This double blind study was conducted at a large Saskatchewan feedlot using 2,324 high-risk calves purchased at auction markets and kept under typical commercial feedlot conditions. The trial design included four vaccine test groups: 1) PhV and a bovine herpesvirus type-1 (BHV-1) subunit vaccine comprised only of the virus glycoprotein IV (gIV); 2) PhV and a commercial modified live vaccine (MLV) containing BHV-1 and parainfluenza-3 viruses; 3) gIV alone; and 4) MLV alone. Calves were assigned to vaccine groups in a random systematic manner, individually identified, and monitored for 90 days after vaccination. The vaccines were given once, on arrival, to reflect common feedlot practice, although vaccination prior to expected risk would be more appropriate.

The PhV in combination with gIV reduced BRD morbidity by 20% (p < 0.05) compared to gIV alone and 24% (p < 0.05) compared to MLV alone, and reduced BRD mortality by 88% (p < 0.05) and fibrinous pneumonia mortality by 100% (p < 0.05) when compared to either gIV or MLV alone. Vaccination with PhV in combination with MLV significantly reduced the efficacy of the PhV in preventing BRD morbidity, BRD mortality, and fibrinous pneumonia mortality and also reduced the antibody response to P. haemolytica leukotoxin. These results suggest that the MLV interfered with the protective capacity of the PhV.