Abortus S19 Research Articles

Brucella abortus S19 rfbD mutant is highly attenuated, DIVA enable and confers protection against virulent challenge in mice

Brucella abortus S19 is an important tool for controlling bovine brucellosis across the globe. However, vaccination with S19 suffers critical shortcomings such as, presence of residual virulence, induction of abortion and sero-diagnostic interference. In this study, rfbD gene deleted mutant S19 was developed. The mutant strain designated S19ΔR displayed rough LPS phenotype, which was confirmed by acriflavine dye-agglutination and LPS-SDS-PAGE analysis. The virulence was amply reduced as suggested by increased sensitivity to complement killing; reduction in splenic-bacterial load and the recovery time RT50 as validated in mice model. Anti-brucella humoral response was significantly lower as compared to S19 immunization. The minimal induction of Brucella specific IgG1, IgG2a & IgG2b, and IgG3 resulted in no apparent reactivity to RBPT antigen. S19ΔR showed protective index of 1.90 against virulent challenge. S19ΔR being highly attenuated and DIVA compatible may facilitate a platform for developing a safer bovine adulthood vaccine.

Transcriptome analysis of Brucella abortus S19∆per immunized mouse spleen revealed activation of MHC-I and MHC-II pathways.

The mouse (Mus musculus) has been extensively used for studying brucellosis, regarding pathogenesis, immunity and the evaluation of vaccines and therapeutics. In this work, RNA-seq was applied to explore the immunological potential of a live Brucella abortus S19∆per, a perosamine synthetase gene mutant of B. abortus S19. Comparison of transcriptome data was carried out for identifying differentially expressed genes among PBS (control) and B. abortus S19∆per immunized mice at 15 days post-immunization. Functional analysis revealed 545 significant differentially expressed genes related to mouse immunity. Specific activation of MHC-I and MHC-II antigen-processing pathways were identified as the highly enriched pathways based on Kyoto Encyclopedia of Genes and Genomes annotation. Other major immune response pathways regulated within the host were NF-kappa B signalling, chemokine signalling, T-cell receptor pathway, apoptosis, TNF signalling and nucleotide-binding oligomerization domain-like receptor signalling. These data provided new insights into the molecular mechanisms of B. abortus S19∆per-induced immune response in mice spleen that might facilitate the development of a highly immunogenic vaccine against brucellosis.

A combined subunit vaccine comprising BP26, Omp25 and L7/L12 against brucellosis.

The current vaccines against brucellosis, namely Brucella abortus strains 19 and RB51, prevent infection in animals but pose potential risks like virulence and attenuation reversal. In this milieu, although subunit vaccination using a single potent immunogen of B. abortus, e.g. BP26 or Omp25 or L7/L12 etc., appears as a safer alternative, nonetheless it confers inadequate protection against the zoonosis compared to attenuated vaccines. Hence, we have investigated the prophylactic potential of a combined subunit vaccine (CSV) comprising the BP26, Omp25 and L7/L12 antigens of B. abortus, in mice model. Sera obtained from CSV immunized mice groups showed heightened IgG titers against all the three components and exhibited specificity upon immunoblotting, reiterating their authenticity. Further, the IgG1/IgG2a ratio obtained against each antigen revealed a predominant Th2 immune response in CSV immunized mice group. However, on assessing the levels of Th1-dependent (IFN-γ and TNF-α) and Th2-dependent (IL-4 and IL-10) cytokines in different formulations, prominent IFN-γ levels were elicited in CSV immunized mice. Further, upon infection with virulent B. abortus 544, the combined subunit vaccinated mice displayed superior degree of protection (Log10 reduction) than the individual vaccines; however, B. abortus S19 showed the highest protection. Altogether, this study suggests that co-immunization of three B. abortus immunogens as a CSV complements and triggers a mixed Th1/Th2 immune response leading to superior degree of protection against pathogenic B. abortus 544 infection.

New Brucella abortus S19 Mutant to Improve Distinction Between Infected and Vaccinated Animals.

Background: Using Brucella abortus Strain 19 (S19) to control bovine brucellosis is restricted due to induce antibodies to the O-side chain of the smooth lipopolysaccharide (LPS) which may be difficult to differentiate vaccinated and infected animals. Furthermore, it is virulent for humans and can induce abortion to cattle.Objectives: The aim of this study was to employ gene knockout B. abortus S19 for the first time to eliminate diagnostic defects and obtain the attenuated mutant strain.Material and Methods: The wbkA gene, which is one of the LPS O-chain coding genes, was knocked out in vaccinal Brucella abortus S19. The proliferative response and immunoglobulin M production were analyzed in wbkA deletion strain-infected BALB/c mice.Results: The loss of wbkA gene function resulted in induction of the splenocyte proliferative response in mice infected by the mutant S19 strain compare to those induced by parental S19 and RB51 strains. Moreover, wbkA mutant did not induce any IgM antibody response using the enzyme-linked immunosorbent assay.Conclusions: As a result, the new mutant S19 strain had deficiency in its LPS O-chain structure, besides cannot induce IgM response then, reduce mistakes to discriminate between vaccinated and infected animal, and also can be considered as a new vaccine candidate.

Vaccine safety studies of Brucella abortus S19 and S19ΔvjbR in pregnant swine

Brucellosis in swine is caused by Brucella suis, a bacterial infection of nearly worldwide distribution. Brucella suis is also transmissible to humans, dogs and cattle and is considered a reemerging disease of public health concern. To date, there is no effective vaccine for swine. This prompted us to investigate the potential use of the commercially available vaccine for cattle or the live attenuated vaccine candidate S19ΔvjbR. As the first step, we sought to study the safety of the vaccine candidates when administered in pregnant sows, since one of the major drawbacks associated with vaccination using Live Attenuated Vaccines (LAV) is the induction of abortions when administered in pregnant animals. Fifteen pregnant gilts at mid-gestation were divided into four groups and subsequently vaccinated subcutaneously using different formulations containing 2.0 ± 0.508 × 109 CFU of either S19 or S19ΔvjbR. Vaccination in pregnant animals with the vaccine candidates did not induce abortion, stillbirths or a reduction in litter size. Multiple tissues in the gilts and piglets were examined at the time of delivery to assess bacterial colonization and histopathological changes. There was no evidence of vaccine persistence in the gilts or bacterial colonization in the fetuses. Altogether, these data suggest that both vaccine candidates are safe for use in pregnant swine. Analysis of the humoral responses, specifically anti-Brucella IgG levels measured in serum, demonstrated a robust response induced by either vaccine, but of shorter duration (4–6 weeks post-inoculation) compared to that observed in cattle or experimentally infected mice. Such a transient humoral response may prove to be beneficial in cases where the vaccine is used in eradication campaigns and in the differentiation of vaccinated from infected animals. This study provides evidence to support future efficacy studies of both vaccine candidates in swine.

A Case of Identity Confirmation of Brucella abortus S99 by Phage Typing and PCR Methods.

Brucellosis is a zoonotic infection that is associated with fever in humans and abortion in animals. The agent of this disease is a facultative intracellular gram-negative coccobacillus called Brucella. There are six classic species, including B. abortus, B. melitensis, B. suis, B. canis, B. neotomae, and B. ovis. In recent years, four new species have been reported, including Brucella ceti, B. microti, B. pinnipedialis, and B. inopinata. Human disease causes hygienic and economic losses, including inactivity of workforces in the community and high cost of treatment. The disease also causes catastrophic losses in the livestock industry. There is no effective vaccine against human brucellosis. Hence, attempts to prevent human infection with Brucella are focused on preventative measures, including control of infection in livestock, which lead to a reduction in its incidence in humans. The common methods for diagnosis of this disease are serologic methods including Rose Bengal, Wright -2 ME and the ring test. B. abortus strain S99 is used to produce these diagnostic antigens. The production of these antigens requires the presence of a well-characterized seed with full identity. The aim of this work was confirmation of the identity of B. abortus S99 by phage typing, AMOS and multiplex PCR techniques. Therefore, it is essential to carry out the identification of the strains used as seed for the production of the brucellosis diagnostic antigens. In this project, B. abortus strain 99 was supplied by the bacterial collection of the Brucellosis Department of Razi Vaccine and Serum Research Institute. Then, the main aim of the present study was the confirmation of the seed identity by doing the tests through the standard phage typing method, AMOS PCR and multiplex PCR (Brucladder) methods. Results were in support of the identity of the studied strain, and the molecular methods could also be used as the sensitive approaches for validation of antigenic seed.

Patho-Physiological Response of LPS Defective Brucella abortus S19∆per in Experimentally Infected Mice

Patho-Physiological Response of LPS Defective Brucella abortus S19∆per in Experimentally Infected Mice

Differential expression of cytokines in PBMC of Bos indicus and Bos taurus × Bos indicus cattle due to Brucella abortus S19 antigen

Brucellosis is the most dreadful disease among bovines, although breed differences have been observed in prevalence of disease, worldwide. In present study, antibody response and relative expression of proinflammatory cytokines was compared in Bos indicus (zebu) and Bos taurus × Bos indicus (crossbred) cattle vaccinated by live attenuated Brucella abortus S19 antigen. Six female calves (4–6 months age) of both groups were vaccinated with B.abortus S19 strain. Blood samples were collected before vaccination (0d) and 7th (7d), 14th (14d) and 28th (28d) days after vaccination. Indirect ELISA showed high (p < .05) anti-Brucella antibody level after vaccination; with no significant difference between the groups. During Real-time expression, IFNγ, TNFα, IL6 and IL10 genes initially showed down regulation followed by upregulation in both the groups; however, the trend was much prominent in crossbreds. The expressions of IFNγ, TNFα and IL6, proinflammatory molecules important for initial containment of the Brucella were significantly (p < .01) higher in crossbred. The study showed that the Sahiwal cattle were less responsive to B.abortus S19 antigen than crossbreds, indicating its lower sensitivity to the Brucella, comparatively. In contrary, higher expression of the proinflammatory molecules in crossbreds could be important for containment of the organism during initial stage of infection.

Development of an indirect ELISA based on whole cell Brucella abortus S99 lysates for detection of IgM anti-Brucella antibodies in human serum.

Brucellosis is the most common zoonotic diseases worldwide. The aim of this study was to develop and evaluate the diagnostic performance of an indirect-ELISA (I-ELISA) method based on whole cell Brucella abortus S99 lysates for detection of IgM anti-Brucella antibodies in a human serum. The study was conducted in two species-rich endemic areas of Iran (Tehran and Lorestan provinces). Serum samples of 102 patients and 150 healthy individuals were tested by the new kit and the commercial Vircell kit for the presence of anti-Brucella IgM antibodies. The disease status was confirmed by Wright agglutination test. The difference in the mean optical densities (OD) recorded by the new and the Vircell kits for patients and healthy individuals were tested using Two-tailed Student t-test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the new kit were informed using Receiver operating curve analysis. The results were used to guide the choice of cutoff. Agreements in ODs recorded by the new and the Vircell kit was visually inspected using Bland-Altman plot. The new I-ELISA showed excellent diagnostic performance (sensitivity and PPV = 95.7%, specificity and NPV = 97.8%) for the diagnosis of brucellosis. The cut-off area for the antibody index (AI) was determined to be 8-10, where AIs less than 8 and greater than 10 were considered Brucella-negative and -positive, respectively. AIs of 8-10 show equivocal results, requiring re-testing. The Vircell kit showed low (36.8%) sensitivity and perfect (100%) specificity on the same samples. The Bland-Altman plot showed low agreement between both tests in recording the OD values for the same individuals. The new I-ELISA based on whole cell Brucella abortus S99 showed a good performance for the detection of Brucella spp. Lack of agreement between the new and the Vircell kit suggest that the performance of ELISA kits might be dependent on the geographical area under study. Hence, validation of the new and the Vircell kits is recommended prior to their implementation in other geographical areas.

Monitoring of the immune response to B. abortus S19 conjunctival vaccine in cattle

Brucella abortus S19strain is one of the most preferred strains in vaccines against brucellosis in cattle. However, monitoring of the B. abortus S19 vaccine is difficult due to non availability of sustainable immunoreactive antigen and accurate test method. In this study, the humoral and the cellular immune responsesto S19 vaccine in one year old heifers and calves were monitored on post vaccination days (pvd) of 46, 85 and 169. Thus, the levels of Immunoglobulin (Ig)G and IgA isotypes against lipopolysaccharide (LPS) for humoral immunity and interferon gamma (IFNg) against brucellergen for cellular immune response were investigated by home-made ELISAs. In this study, significant IgG positivity was observed on pvd 46 in calves (100%) and heifers (96.6%), but IgA positivity and IFNg levels were not over 50%. Moreover, percentage of positive animals for IFNg (13.3-43.3%) and IgA (0-44%) have shown no significance for monitoring the vaccine throughout the study. Therefore, IgG levels can be used to monitor the efficiency of Brucella abortus S19 conjunctival vaccine in cattle. On the other hand, novel antigen combinations along with brucellergen and LPS for monitoring the immunity would enhance the sensitivity of the test and could be recommended for future investigations

Evaluation of a DNA vaccine encoding Brucella BvrR in BALB/c mice.

Brucellosis is an important neglected zoonotic disease, and the pathogens responsible are Brucellae. In order to evaluate the immunogenicity and protective efficacy of a DNA vaccine encoding Brucella BvrR, the recombinant plasmid pCDNA‑BvrR was constructed by inserting the BvrR gene fragment into a pCDNA3.0 vector. The His6‑tagged BvrR was purified with His‑trap FF crude affinity chromatography and verified with an anti‑histidine monoclonal antibody by western blot analysis. The specific immunoglobulin antigens and their isotypes were detected by indirect ELISA. The recombinant His6‑BvrR protein was expressed and purified by affinity chromatography. The optical density 450 value of immunoglobulin G (IgG) in the pCDNA‑BvrR group was significantly increased compared with the pCDNA3.0 vector or PBS groups (P<0.05), and the pCDNA3.0 vector and PBS groups exhibited no significant difference (P>0.05). BvrR induced specific antibodies with a dominance of IgG2a over IgG1 and the T cell‑proliferative response, in addition to a typical T helper‑1 (Th1)‑dominated immune response in mice. The splenocytes from mice of the pCDNA‑BvrR group demonstrated significant proliferative activity compared with the pCDNA3.0 vector group. The present results indicated that immunization with BvrR induced a specific Th1‑type immune response in mice. Subsequent to challenging with B. abortus S19, it was identified that the DNA vaccine pCDNA‑BvrR induced a significant level of protection in BALB/c mice by evaluating systemic bacterial clearance. These results suggested that BvrR may be a good candidate for a DNA vaccine against brucellosis.

Assessment of fluorescence polarization assay: a candid diagnostic tool in Brucella abortus strain 19 vaccinated areas

Brucellosis caused by the bacteria of the genus Brucella is an important zoonosis and constitutes a serious public health hazard. Brucellosis is diagnosed mainly by the Rose Bengal plate test and indirect ELISA, both of which have poor specificity because false positive serological reactions occur when screening animals that have been vaccinated with B. abortus S19. Fluorescence polarization assay (FPA) was evaluated for screening samples from cattle and buffalo calves with persistent antibody titers induced by vaccination. The standardized FPA exhibited relative sensitivity and specificity of 0.94 and 0.95, respectively, and the area under the curve, kappa and accuracy were 0.98, 0.87 and 0.95, respectively. Comparison of competitive ELISA and FPA revealed that, FPA is more specific than competitive ELISA. The high specificity, sensitivity and 95% accuracy of FPA indicate that, it is suitable for testing vaccinated animals because it can distinguish between infected from vaccinated animals.

Single-tube duplex-PCR for specific detection and differentiation of Brucella abortus S19 vaccine strain from other Brucella spp

A simple single-tube duplex-PCR assay was optimized for rapid and sensitive detection and differentiation of Brucella abortus (B.abortus) S19 vaccine strain from other Brucella spp.(B.abortus 544,B abortus S99,B.melitensis 16M,B.suis).This assay was optimized using two primer pairs that were designed, one targeting genus specific multicopy IS711 and another eryC for the development of the duplex-PCR assay. Specificity of the assay was assessed using DNA templates from various Brucella species (B.abortus, B.melitensis, B.suis) and non Brucella bacteria like Staphylococcus aureus, Proteus vulgaris, Pasteurella multocida, Pseudomonas aeruginosa, Citrobacter freundii, E. coli, Salmonella enteritidis, Campylobacter jejuni and Listeria monocytogenes. The assay was also evaluated on spiked milk samples with known B.abortus cultures. The assay was able to detect up to 2.47 x 103CFU of B. abortus S99 organism in spiked milk sample. The assay was further validated in 53 clinical specimens (aborted foetal stomach content). Results confirm that Single tube duplex-PCR assay was useful for early and rapid detection and differentiation of B. abortus vaccine strain S19 from other Brucella spp in milk as well as in clinical specimen.

Protective and therapeutic efficacy study of divalent fusion protein rL7/L12-Omp25 against B. abortus 544 in presence of IFNγ.

Brucella as intracellular pathogen requires a coordinate interaction between Th1 subset of gamma interferon-secreting CD4 T cells and CD8 T cells for optimal protective immunity. It was previously recognized that L7/L12 as T cell-reactive antigen from the pathogen. On other hand, Omp25 was found as another antigen to provide protection against the Brucella infection by eliciting both Th1 and Th2 type of immune responses in mice. Here, we analyzed the prophylactic and therapeutic efficacy of a divalent fusion protein (rL7/L12-Omp25) comprising these two promising immunogens of Brucella in the presence of murine IFN-gamma in mice against B. abortus 544 challenge. rIFN-gamma with rL7/L12-Omp25 resulted in superior immune response when compared to the animal vaccine strain B. abortus S19. The vaccine candidate caused dominance of IgG1 over IgG2a and upregulated cytokine secretion (IFN-gamma, TNF-α, and IL-10) among immunized mice. Moreover, the antigen in combination with murine IFN-gamma elicited stronger cell-mediated immune response among the immunized animals when compared to standard vaccine (S19). The registered log protection unit among challenged mice with B. abortus 544 pathogen was 2.16, p = 0.0001 when rL7/L12-Omp25 was administered alone and 2.4, p = 0.0001 when it was administered along with rIFN-gamma. However, the molecule upon administration with murine IFN-gamma imparted very minimal or no therapeutic effect against brucellosis. To conclude, our study demonstrates the potential of rL7/L12-Omp25 as an immunogen of prospective and efficient prophylaxis as it is capable of eliciting both cell-mediated and humoral immune responses against brucellosis.

Penggunaan Imunostik sebagai Uji Serologi untuk Deteksi Brucella abortus pada Sapi (APPLICATION IMMUNOSTICK ASSAY FOR SEROLOGICAL TEST BRUCELLA ABORTUS IN BOVINE)

Serological test is one of diagnostic method to detect pathogenicity brucella. Several methods are being improving such as Rose Bengal Test (RBT), Complement Fixation Test (CFT) dan Enzyme-linked Immunosorbent Assay (ELISA). Immunostick has an accuracy equivalent to ELISA and is easy to apply in the field so it is possible to be applied as a rapid test for brucellosis detection. The study aim was to know sensitivity and specificity of immunostick that were used to detecte antibody Brucella abortus using commercial antigens of B. abortus Strain 19 (S19) and B. abortus Strain 99 (S99). The test have compared with ELISA. The tests were conducted in two stages, namely (i) immunostick ability to detect antibodies in seropositive and seronegative serum, and (ii) the immunostick result were compared with ELISA result in serum grup that were be know and unknown status. A total of 250 serums were examined and result indicated that immunostick can be detect B. abortus antibodies in cattle serum with sensitivity 100%. Immunostick specifity were 45,45% for B. abortus S99(1) antigen; 78,79% for B. abortus S99(2) antigen and 51,52% for B. abortus S19 antigen. When the test compared with ELISA, the sensitivity 82,86% and the spesifity were 52,31% for B. abortus S99(1) antigen; 93,54% and 79,71 for B. abortus S99(2) antigen and 82,86% and 58,46% for B. abortus S19 antigen.

A new biomarker for diagnosis and monitoring of effectiveness of therapy of Brucellosis

Background: Brucellosis is an important zoonotic disease causing huge economic losses to livestock industry and human sufferings worldwide. Current treatment of bovine Brucellosis is ineffective and infected animals become carriers. Brucella abortus Strains S19, producing antibody alone and RB51, inducing cell mediated immunity only, are used as live attenuated vaccines. However, antibody alone is ineffective for the intracellular pathogen and RB51 is Rifampicin resistant. Both strains can infect humans and cause abortions in animals. Phage lysed bacteria are harmless and retain maximum immunogenicity than conventional killing. Methods & Materials: We did immunotherapy of bovine Brucellosis by phage lysates of RB51 (RL) and S19 (SL). A single subcutaneous injection of 2 ml of lysates SL alone or RL alone or cocktail lysate (both RL and SL) was given to brucellosis affected cattle. Antibody titers, interferon gamma production and plasma RNA levels were monitored in the treated and untreated animals at various intervals upto 90 days. Results: The SL induced antibody and RL stimulated CMI. In vitro restimulation of leukocytes from RL immunized cattle induced interferon gamma. A single subcutaneous dose of 2 ml of cocktail lysate (both RL and SL), eliminated live Brucella from Brucellosis affected cattle in 3 months with Brucella specific 223 bp amplicon undetectable in plasma by RT-PCR and blood negative for live Brucella by culture by 90 day postimmunization. Conclusion: We have successfully used plasma RNA specific for live Brucella abortus as a biomarker of Brucella for accurate diagnosis and monitoring of the efficacy of immunotherapy of Brucellosis by phage lysed Brucella abortus S19 and RB51 (lysates).

Trials for bovine diagnosis using molecular biology

Background: Brucellosis is a major bacterial zoonosis of global importance affecting a range of different mammals including cattle, sheep, goats, swine, rodents, marine mammals and man worldwide. In order to control brucellosis, comprehensive vaccination, surveillance and quarantine programs should be implemented. Both of control and prevention procedures are highly depending on accurate diagnostic tools and effective and safe vaccination programs. idea of this ELISA kit depends on the facts that eryC gene is absent in Brucella abortus S19 only but it is present and functional in all other brucella strains and isolates. Methods & Materials: omp31 as a DNA vaccine against Brucellosis were evaluated in Balb/C mice where eryC recombinant protein as a coating antigen for accurte diagnosis of brucellosis and differention between vaccinated and infected cattle were evaluated using IELISA aganist ELISA using hot saline extract and soluble brucella proteins as a coating antigens as a control postive. Results: results of DNA vaccine in Balb/C mice revealed a 60% protection after challange using brucella melitensis 16 M where results of ELISArevealed that in case of sera of naturally infected cattle, sero-positivity was 94.7%, 100%, 100% and 100% with eryC-ELISA, HSE-ELISA, SBP-ELISA and rose Bengal test respectively where in case of sera of S19 vaccinated cattle, all samples were negative when tested with eryC-ELISA where in case of HSE-ELISA, SBP-ELISA and Rose Bengal test, sero-positivity was 92.3%, 84.6% and 100% respectively. All sera of brucella free cattle were negative when tested with Rose Bengal test and different IELISA tests. Conclusion: The Results of this study recommended the use of omp31 DNA vaccine as a safe vaccine for control of bovine brucellosis and use of eryC protein as a good diagnostic antigen to be used in serological tests for diagnosis of bovine brucellosis and differentiation between infected and Brucella abortus S19-vaccinated cattle but more studies are needed to be done on large cattle populations accompanied with bacteriological isolation to detect the sensitivity and specificity of this protein as a diagnostic antigen and also for validate this test.

Major histocompatibility complex (DRB3) geneexpression pattern indicates differences in BrucellaabortusS19 vaccine induced immune responsein Karan Fries and Sahiwal cattle

Brucella abortus S19 strain vaccination is most effectively used as a tool to control the brucellosis in cattle. To understand the genetic basis of differences in immune responsiveness after immunization in cattle of different genotypes, we assessedthe expression of MHC-DRB3 antigen receptor molecule in six each female calves of Karan Fries crossbreds (KF, Bos indicus x Bos taurus) and Sahiwal (Bos indicus) vaccinated with Brucella S19. Serum and peripheral blood mononuclear cells (PBMC) were isolated from blood collected on 0(before vaccination) and 7, 14 and 28 day of vaccination. Antigenic response was assessed for these days in both the groups using Rose Bengal Plate form Test (RBPT). At 0d, the calves of both groups showed no antigen agglutination, confirming the calves free from the infection.The serum of 7d onward started showing the agglutination with more strong response in later stages specifically in KF, indicating increased immune response against Brucella. Therefore, RBPT can be used as earliest screening (7d onward) for Brucella antigenic reactivity in both cattle groups. The expression of DRB3 gene started with slight upregulation after vaccination,in general, however without any significant differences between two different genetic groups upto14d.The significant (p Lass Than 0.01) higher expression (8 times) of DRB3was observed in KF than Sahiwal at 28d. The study indicated that antigenic reactivity and MHC-DRB3 expression elicited by Brucella S19 vaccination was more prominent in KFduring initial days, which may provide an extra advantage to the host for antigen binding, thereby better immune protection at later stage.

Acellular outer membrane vesicles of Brucella abortus strain 19 elicits both humoral and cell mediated immune response in mice

Outer membrane vesicles (OMVs) contain biologically active proteins, lipoolysaccharide (LPS), periplasmic and membrane-bound proteins and are known to perform diverse biological functions. OMVs from Brucella abortus S19 were isolated and characterized by transmission electron microscopy (TEM), SDS-PAGE and immunoreactivity was investigated by western blotting. On TEM, bilayered spherical structures of 50-200 nm were observed. SDS-PAGE of OMVs revealed approximate bands size of 82 kDa, 68 kDa, 38 kDa, 32 kDa, 29 kDa and 18 kDa. Western blot analysis of OMVs revealed a dominant immunoreactive band of 38 kDa that correspond to some major outer membrane proteins. Humoral immune response was measured by indirect ELISA which showed that OMV specific antibodies were detected from 7th day post immunization (DPI) onwards and showed a rising trend up to 35th DPI. Cell mediated immune (CMI) response against OMVs as evidenced by the proliferation of splenocytes have also been observed. Thus OMVs were found to possess immunogenic proteins which had potential to induce both humoral as well as cell mediated immunity. After correlating this immune response with protection it has been concluded that OMV can be used as one of the vaccine candidate against brucellosis.

Site-directed mutagenesis in Brucella abortus S19 by overlap extension PCR-based procedure

Introduction of a site-directed mutation can be effective method to evaluate properties of various genes. Brucellosis is one of the most common zoonotic infectious diseases, which causes great economic losses. Thus, determination of pathogenicity factors in the genus Brucella can lead to the control of this health problem. Due to the importance of site-directed mutations in identification of genomic structure, overlap extension polymerase chain reaction (PCR) has been introduced as an improved technique for the removal and replacement of gene targets. In this study, three DNA fragments were amplified and combined using a two-step PCR with specific primers. The resulting fusion PCR product, obtained without any change in the nucleotide sequence, was cloned in a specific position in the pBluescript II SK (-) plasmid using restriction enzymes. Finally, the construct was transferred into cells of Brucella abortus S19 by electroporation and replaced the target gene (wbkA) in the genome of the bacterium. PCR analysis was performed on kanamycin-resistant colonies to provide genetic evidence that the B. abortus wbkA gene was interrupted by the kanamycin cassette. The results of this study show that the optimized modified technique, splicing by overlap extension PCR, is effective in creating mutations in the bacterial genome and can easily be used in Brucella spp.