Abortus Strain Research Articles

Proteomics of Brucella: Technologies and Their Applications for Basic Research and Medical Microbiology

Brucellosis is a global zoonosis caused by Gram-negative, facultative intracellular bacteria of the genus Brucella (B.). Proteomics has been used to investigate a few B. melitensis and B. abortus strains, but data for other species and biovars are limited. Hence, a comprehensive analysis of proteomes will significantly contribute to understanding the enigmatic biology of brucellae. For direct identification and typing of Brucella, matrix-assisted laser desorption ionization—time of flight mass spectrometry (MALDI—TOF MS) has become a reliable tool for routine diagnosis due to its ease of handling, price and sensitivity highlighting the potential of proteome-based techniques. Proteome analysis will also help to overcome the historic but still notorious Brucella obstacles of infection medicine, the lack of safe and protective vaccines and sensitive serologic diagnostic tools by identifying the most efficient protein antigens. This perspective summarizes past and recent developments in Brucella proteomics with a focus on species identification and serodiagnosis. Future applications of proteomics in these fields are discussed.

Different Impacts of MucR Binding to the babR and virB Promoters on Gene Expression in Brucella abortus 2308.

The protein MucR from Brucella abortus has been described as a transcriptional regulator of many virulence genes. It is a member of the Ros/MucR family comprising proteins that control the expression of genes important for the successful interaction of α-proteobacteria with their eukaryotic hosts. Despite clear evidence of the role of MucR in repressing virulence genes, no study has been carried out so far demonstrating the direct interaction of this protein with the promoter of its target gene babR encoding a LuxR-like regulator repressing virB genes. In this study, we show for the first time the ability of MucR to bind the promoter of babR in electrophoretic mobility shift assays demonstrating a direct role of MucR in repressing this gene. Furthermore, we demonstrate that MucR can bind the virB gene promoter. Analyses by RT-qPCR showed no significant differences in the expression level of virB genes in Brucella abortus CC092 lacking MucR compared to the wild-type Brucella abortus strain, indicating that MucR binding to the virB promoter has little impact on virB gene expression in B. abortus 2308. The MucR modality to bind the two promoters analyzed supports our previous hypothesis that this is a histone-like protein never found before in Brucella.

A LIVE ATTENUATED Brucella VACCINE POTENTIATES MACROPHAGE ALARMINS

Abstract Wild-type (wt) brucellae subvert host immune detection leading to their replication in the endoplasmic reticulum (ER) resulting in their persistence. Protection against Brucella infection requires a strong Th1-type immunity, characterized by the production of IFN-γ. Mucosal vaccination with a live attenuated B. abortus double mutant expressing lacZ (znBAZ) provides protection against wt B. abortus challenge via the stimulation of activated T cells. Such finding implicates rendering macrophage activation. To test the hypothesis that znBAZ diminishes subversion to enable host innate immune detection, intracellular survival of wt B. abortus, B. abortus strain 19 (S19) vaccine, and znBAZ in bone marrow-derived macrophages (BMDMs) was measured. The percentage of brucellae in LAMP1+ phagolysosomes was significantly greater in znBAZ-infected BMDMs than S19 or wt B. abortus, and znBAZ induced the alarmins, s100a8, Saa1, and Saa3. These alarmins are responsible for stimulating strong inflammatory responses by interacting with pattern recognition receptors (PRRs). Consequently, IL-1β, IL-6, IL-12, and TNFα were significantly elevated following in vitro znBAZ infection of macrophages. CD80, CD86, CD40, and MHCII expression by znBAZ-infected BMDMs was significantly increased. Analysis of lung macrophages two days after pulmonary infection reveals that znBAZ significantly increases the expression of CD86 and MHCII on interstitial and alveolar macrophages, and increased intracellular IL-1β and IL-6 by interstitial macrophages. These findings show that znBAZ enhances macrophage activation, which in turn leads to the induction of potent T cell immunity. Work is supported by NIH AI123244 & AI125516.

Clearance of Brucella with formulation rCagA, TN-OMPs & LPS in mice

The development of vaccines against Brucella is necessary. The formulations rCagA/OMP/LPS were evaluated against the clearance of Brucella. All of the mouse groups were immunized three times orally and followed two times by IM. Two weeks after the last immunization mice were challenged with B. abortus strain 544. Blood and spleen samples were collected from mice.Antigen-specific interleukine responses were measured in the spleen of immunized mice before and post-challenge by ELISA. Specific IgG1,2 isotopes were measured from sera before and after challenge. Sandwich ELISA assays were used to determine IFN-γ, IL-4 and IL-10 levels in the supernatants of the splenocyte cells. Statistical analysis of IFN-γ titers indicated a significant difference between all groups with Mock (p = 0.00). However, there was no significant difference between other groups. Furthermore, the analysis of IL-10 titers had similar results. The statistical analysis for IL-4 showed a significant difference between rCagA with LPS-rCagA conjugate and LPS-rCagA+ CpG groups (p = 0.001). Statistical analysis between IFN-γ and IL-4 titers showed a significant difference among all groups (p = 0.00). Statistical analysis between IFN and IL-10 titers showed a significant difference among all groups (p = 0.00). Statistical analysis between IL-10 and IL-4 titers showed no significant difference in any group. Clearance finally was 7 log with OMP/rCagA against B. abortus strain 544. Immunization with the current combination was safe in animal models and was able to induce appropriate responses and also decrease Brucella colonization.

The Role of the Flagellar Protein FlgJ in the Virulence of Brucella abortus.

Brucella abortus is a facultative intracellular pathogen that causes a zoonosis called brucellosis. This disease leads to abortion and infertility in cattle, and diverse complications in humans. B. abortus is a successful intracellular bacterium that has developed the ability to evade the host's immune system and it replicates in professional and non-professional phagocytic cells, persisting in the different tissues, and organs of its hosts. It has been described that Brucella expresses a polar flagellum under certain conditions, but its function is still unknown. In this study we evaluated the role of the FlgJ, a protein, presumably a peptidoglycan hydrolase involved in flagellum formation and in the virulence of B. abortus strain 2308. B. abortus 2308 ΔflgJ mutant and complemented strains were constructed to study the function of the FlgJ protein in the context of the virulence of this pathogen in in vitro and in vivo assays. The results showed that the elimination of the flgJ gene delays the growth rate of B. abortus in culture, reduces its intracellular survival capacity in professional and non-professional phagocytic cells, rendering it unable to escape from the endocytic route and not reaching the endoplasmic reticulum. It also negatively affects their persistence in BALB/c mice. Functionally, the B. abortus 2308 flgJ gene restored motility to an E. coli flgJ mutant gene. Furthermore, it was discovered that the production of FlgJ protein is associated with the bacterial adherence by B. abortus. Therefore, although the specific function of the polar flagellum for Brucella is unknown, the data indicates that the flagellar flgJ gene and its product are required for full virulence of B. abortus 2308, since its deletion significantly reduces the fitness of this pathogen in vitro and in vivo.

Genetic variation of Brucella isolates at strain level in Egypt.

In this study, Multiple Locus Variable Number Tandem Repeat Analysis (MLVA‐16) was performed on 18 Brucella isolates identified bacteriologically and molecularly (AMOS‐PCR) as Brucella abortus (n = 6) and Brucella melitensis (n = 12). This was aimed to study the genetic association among some Egyptian Brucella genotypes isolated during the period from 2002 to 2013 along with the global genotypes database. MLVA‐16 analysis for B. melitensis and B. abortus strains illustrates a total of 11, and 3 genotypes with 10 and 1 singleton genotypes, respectively. B. melitensis strains displayed greater markers diversity by VNTRs analysis of the 16 loci than B. abortus and this was attributed mainly to the diverging in panel 2B markers. B. melitensis genotype M4_Fayoum_Giza (3,5,3,13,1,1,3,3,8,21,8,7,5,9,5,3) was the only predominated genotype circulating between two different governorates. The most common B. abortus genotype, GT A3_Dakahlia (4,5,4,12,2,2,3,3,6,21,8,4,4,3,4,4), was present in three identical isolates. In phylogeny, Egyptian B. abortus bv1 genotypes were closely related to East Asian strain (for the first time), Western Mediterranean and Americas clonal lineages. B. melitensis local genotypes exhibit a genetic relatedness mostly to Western Mediterranean clonal lineage and one strain of Eastern Mediterranean clonal lineage. In conclusion, the geographic location is not the only factor stands behind the high genetic similarity of the Egyptian Brucella genotypes. These low variations may be a result of a stepwise mutational event of the most variable loci from a very limited number of ancestors especially during the transmission through non‐preference hosts. The authors encourage the authorities in charge to establish pre‐movement testing to reduce the risk of brucellosis spread.

Evaluation of real-time PCR as an alternative for potency testing of Brucella abortus vaccines

The aim of the research was to evaluate real-time PCR (qPCR) as an alternate method for quantitative detection of Brucella abortus strain 544 (S544) in the spleen of mice for potency testing of live B. abortus strain 19 (S19) vaccine. IS711 and eryC gene-based qPCR were optimized for calculating copy number. The copy number was further correlated with live Brucella count in the spleen by standard plate count (SPC) method. The mice were immunized with S19 and challenged with S544 on 30th Day post-immunization. The spleen of mice was collected at 15th, 21st, and 30th days post challenge (DPC) for estimation of S19 and S544 load via SPC as well as qPCR. The noteworthy difference was observed between immunized and unimmunized group by both methods at all time points. The maximum correlation between SPC and qPCR method was observed at 15th DPC in both immunized and unimmunized group. Repeated experiments at 15th DPC gave the parallel significant difference between immunized and unimmunized group by both methods. Thus novel, risk-free qPCR method can be used for the indirect culture-free potency evaluation of S19 vaccine in order to preclude the cultivation of zoonotic Brucella organisms from spleen samples.

The seroprevalence of brucellosis and molecular characterization of Brucella species circulating in the beef cattle herds in Albania.

BackgroundBrucellosis is a ubiquitous zoonotic disease globally. It is endemic among bovines, sheep, and goats in Albania. The national control and eradication programs for brucellosis has been applied on sheep and goat farms as well as large dairy cattle farms, i.e., those with more than ten milking cows. The current study aims at estimating the herd and average individual animal prevalence of brucellosis in the national beef cattle herds, the missing information that was essential to propose the most appropriate control measures for this sub-population. Rose Bengal Test (RBT), Fluorescence Polarization Assay (FPA), and Enzyme-Linked Immunosorbent Assay (ELISA) were used as serological tests and classical bacteriology for isolation. Results were also used to investigate the difference in sensitivity between the assays used.MethodologyIn total, 655 animals from 38 beef cattle herds from six southern districts of Albania were sampled. Sera were tested using RBT, FPA, and ELISA. Fifteen positive cows and a bull from eight high-prevalence positive herds were slaughtered, and particular tissue samples were collected for bacteriology.ResultsThe overall herd seroprevalence in the tested beef cattle population was 55%, while the overall average within-herd prevalence (including only positive herds) was 38.3%, 42.7%, and 45.6% determined by the RBT, ELISA, and FPA, respectively. FPA was used for the first time in the diagnosis of bovine brucellosis in Albania, and its sensitivity was higher than RBT and ELISA. Three B. abortus strains were identified, two from the supra-mammary lymph node of two cows and one from the epididymis of a seropositive bull.ConclusionBrucellosis was highly prevalent in beef cattle in the southern part of Albania, and B. abortus was isolated from this subpopulation. To the best of our knowledge, this is the first statistically based survey of bovine brucellosis in beef herds in Albania. Using the FPA in parallel with other serological tests improved overall diagnostic sensitivity. Test and slaughter policy is not a rational approach for the control of brucellosis in beef cattle in Albania, and vaccination is only applicable, including strict control of the movement of animals.

Simultaneous Immunization with Omp25 and L7/L12 Provides Protection against Brucellosis in Mice.

Currently used Brucella vaccines, Brucella abortus strain 19 and RB51, comprises of live attenuated Brucella strains and prevent infection in animals. However, these vaccines pose potential risks to recipient animals such as attenuation reversal and virulence in susceptible hosts on administration. In this context, recombinant subunit vaccines emerge as a safe and competent alternative in combating the disease. In this study, we formulated a divalent recombinant vaccine consisting of Omp25 and L7/L12 of B. abortus and evaluated vaccine potential individually as well as in combination. Sera obtained from divalent vaccine (Omp25+L7/L12) immunized mice group exhibited enhanced IgG titers against both components and indicated specificity upon immunoblotting reiterating its authenticity. Further, the IgG1/IgG2a ratio obtained against each antigen predicted a predominant Th2 immune response in the Omp25+L7/L12 immunized mice group. Upon infection with virulent B. abortus 544, Omp25+L7/L12 infected mice exhibited superior Log10 protection compared to individual vaccines. Consequently, this study recommends that simultaneous immunization of Omp25 and L7/L12 as a divalent vaccine complements and triggers a Th2 mediated immune response in mice competent of providing protection against brucellosis.

Use of recombinant Brucella outer membrane proteins 19, 25, and 31 for serodiagnosis of bovine brucellosis.

Background and Aim:Brucellosis remains one of the most common zoonoses. The current anti-brucellosis measures are largely deemed ineffective due to a lack of specificity of conventional serological tests. This study evaluated the use of Brucella outer membrane protein (Omp)19 for serodiagnostic testing.Materials and Methods:The antigenicity of recombinant Brucella Omp19, Omp25, and Omp31 was examined in serum samples from mice and rabbits immunized with Omp19 or Brucella abortus 19 whole cell (WC) and 12 and 152 cows experimentally or naturally infected with brucellosis, respectively. Serum samples were collected from 151 cows that were vaccinated with B. abortus 19 and 12 unvaccinated heifers that were maintained on a brucellosis-free farm.Results:Immunization with Omp19 resulted in antibody production in mice after a single injection without the use of adjuvant. Serum antibodies obtained from rabbits immunized with inactivated B. abortus strain 19 WC targeted Omps by enzyme-linked immunosorbent assay (ELISA) and Western blot. Antibodies targeting Omp19 were identified in all B. abortus strain 544 experimentally infected cows at day 14 post-inoculation (p.i.); Omp25 was detected by ELISA at day 28 p.i., while an ELISA targeting Omp31 was negative for 25% of cows at this time point. Omp19 and Omp25 were readily detected by sera from cows from a new epizootic focus. Antibodies recognizing Omps were also detected in >50% of the animals maintained in a brucellosis-free herd at 10 months after vaccination.Conclusion:Brucella Omp19 in combination with Omp25 and Omp31 may be utilized as target antigens in an ELISA designed for serological testing of unvaccinated cattle.

Experimental substantiation of the use of a small dose of the vaccine to prevent the specific brucellosis of cattle

The expansion of zones with the cattle without anti-brucellosis immunity in healthy areas increases the risk of cattle infection from neighboring infected regions and states. There is an urgent problem of developing the anti-brucellosis immunity in the adult cattle. The initial full-dose vaccination with the vaccine from strain B. abortus 82 is fraught with post-vaccination abortion. The study is aimed at the exploration of the immune response and immunity in cows vaccinated against brucellosis according to different schemes with small doses of vaccines from B. abortus strains 19 and 82. The results of the experiment showed that the small-dose revaccination with a vaccine from strain 82 is the most promising, because without any harm to the formed immunity, the problem associated with the reactogenicity and agglutinogenicity of the vaccine can be solved. In addition, a ten-time reduction in the vaccine dose can reduce vaccine production volumes and decrease financial expenses of the farmers.

The Influence of Brucella Abortus Strain RB51 Vaccine Which is Given to the Mice (Mus musculus) and Infected by Local Isolat Brucella Suis for the Figures of Hepatic Fibrosis of the Mice (Mus musculus)

The Influence of Brucella Abortus Strain RB51 Vaccine Which is Given to the Mice (Mus musculus) and Infected by Local Isolat Brucella Suis for the Figures of Hepatic Fibrosis of the Mice (Mus musculus)

COMPARISON OF HUMORAL IMMUNE RESPONSES BETWEEN HEAT-INACTIVATED BRUCELLA ABORTUS BIOVAR 3 AND STRAIN RB51 VACCINES IN INDIGENOUS CATTLE OF BANGLADESH

Background: Live attenuated Brucella abortus strains 19 and RB51 vaccines have been used as a key method for the control and eradication of brucellosis in cattle worldwide for decades. Due to certain limitations of these live vaccines, research has been undertaken for the development of an ideal more effective and safer vaccine for animals and human brucellosis. Objective: The main objective of this study was to compare the humoral immune responses (HIR) between the heat-inactivated Brucella abortus biovar 3 and attenuated live RB51 vaccines in native cattle of Bangladesh. Materials and Methods: The methods of isolation, identification, preparation of inoculum dose (10 × 1010 cfu/5 ml) and heat inactivation of B. abortus biovar 3 was followed as described earlier. Each of the three B. abortus sero-negative native cows was inoculated with heat-inactivated B. abortus vaccine @ 5.0 ml (10 × 1010 cfu /5 ml)/ cow SC single injection. Similarly, each of five native calves of 6 to 9 months old was inoculated with live attenuated RB51 vaccine (CZ Veterinaria, SA, Spain) @ 2.0 ml (10-34×109) SC as single dose. The sera of cows were collected at 0, 7, 14, 21, 28, 40, 60 and 90 days post vaccination, whereas the sera of the calves were collected at 0, 7, 14, 21, 28, 60, 90, 120, 150 and 180 days post-vaccination. All the collected sera of both the groups were tested to evaluate antibody titer by RBT followed by ELISA with commercial tests kits. Results: The HIR of the cows inoculated with heat-inactivated vaccine showed antibody (Ab) titer started to rise significantly (p < 0.05) from the 14 days (OD 0.2116 ± 0.0397, Ab titer 1:120) and reached a peak level at 28 days (OD 0.319 ± 0.172, Ab titer 1:800) and then started to decline significantly (p < 0.05) from 40 days (OD 0.234 ± 0.0415, Ab titer 1:35) to 60 days (OD 0.094 ± 0.0075, Ab titer 0). The mean Ab titer in calves inoculated with RB51 vaccine showed that Ab titer started to appears insignificantly (p ˃ 0.05) from day 7 (OD 0.094 ± 0.01603) and reached peak level at day 60 days (OD 0.592 ± 0.398), changes are very significant from day 0 (p < 0.05), after 60 days Ab level start to decrease and reach at lowest level at day 150 (OD 0.112 ± 0.0188), Ab level found similar to day 0 (OD 0.0826 ± 0.00517) at 180 days (OD 0.0822 ± 0.00249). Conclusions: The S19 and RB51 are the approved B. abortus vaccine strains have been widely and successfully used with some limitations to prevent bovine brucellosis worldwide. In addition to live attenuated and inactivated vaccines, recombinant genes, proteins, vectors, DNA and recombinant mutant vaccines have also been evaluated for the prevention of brucellosis but further research would be required to develop an ideal vaccine for both the humans and animals.

Multilocus variable number tandem repeat analysis (MLVA)-typing of Brucella abortus isolates of India reveals limited genetic diversity.

Multilocus variable number tandem repeat analysis (MLVA) technique has wide applications in studying phylogenies and short-term epidemiology of pathogens. The technique has been extensively used worldwide in molecular epidemiology of Brucella genus. Only one study on this aspect is reported from India despite its economic and public health significance in country. The present study isolated B. abortus from domesticated bovines of Jammu region of Jammu and Kashmir state, India, and applied MLVA for 16 loci (MLVA-16). MLVA results were compared with the results of a previous study and with MLVA data of Indian isolates present in http://microbesgenotyping.i2bc.paris-saclay.fr/database. In the study, 136 samples from bovines (cattle and buffaloes) of 47 farms of Jammu region were processed for isolation. Eleven isolates of B. abortus biovar 1 from 6 farms were obtained. In MLVA-16 analysis, although the isolates were classified in a single cluster, 5 genotypes were obtained with a specific genotype being prevalent on each farm. The study identifies that MLVA-16 is capable to differentiate B. abortus strains in an area having high genetic similarity among isolates. On comparing the results with previous study and database, the isolates were found to have high genetic similarity indicating that the genetic diversity of B. abortus in India is very limited. It probably indicates that India is contaminated recently with B. abortus. To test this hypothesis, analysis of whole genome sequencing data of diverse collection of Indian B. abortus strains is required.

A Carbonic Anhydrase Pseudogene Sensitizes Select Brucella Lineages to Low CO2 Tension.

Brucella spp. are intracellular pathogens that cause a disease known as brucellosis. Though the genus is highly monomorphic at the genetic level, species have animal host preferences and some defining physiologic characteristics. Of note is the requirement for CO2 supplementation to cultivate particular species, which confounded early efforts to isolate B. abortus from diseased cattle. Differences in the capacity of Brucella species to assimilate CO2 are determined by mutations in the carbonic anhydrase gene, bcaA Ancestral single-nucleotide insertions in bcaA have resulted in frameshifted pseudogenes in B. abortus and B. ovis lineages, which underlie their inability to grow under the low CO2 tension of a standard atmosphere. Incubation of wild-type B. ovis in air selects for mutations that "rescue" a functional bcaA reading frame, which enables growth under low CO2 and enhances the growth rate under high CO2 Accordingly, we show that heterologous expression of functional Escherichia coli carbonic anhydrases enables B. ovis growth in air. Growth of B. ovis is acutely sensitive to a reduction in CO2 tension, while frame-rescued B. ovis mutants are insensitive to CO2 shifts. B. ovis initiates a gene expression program upon CO2 downshift that resembles the stringent response and results in transcriptional activation of its type IV secretion system. Our study provides evidence that loss-of-function insertion mutations in bcaA sensitize the response of B. ovis and B. abortus to reduced CO2 tension relative to that of other Brucella lineages. CO2-dependent starvation and virulence gene expression programs in these species may influence persistence or transmission in natural hosts.IMPORTANCEBrucella spp. are highly related, but they exhibit differences in animal host preference that must be determined by genome sequence differences. B. ovis and the majority of B. abortus strains require high CO2 tension to be cultivated in vitro and harbor conserved insertional mutations in the carbonic anhydrase gene, bcaA, which underlie this trait. Mutants that grow in a standard atmosphere, first reported nearly a century ago, are easily selected in the laboratory. These mutants harbor varied indel polymorphisms in bcaA that restore its consensus reading frame and rescue its function. Loss of bcaA function has evolved independently in the B. ovis and B. abortus lineages and results in a dramatically increased sensitivity to CO2 limitation.

Development of New Modified Simple Polymerase Chain Reaction and Real-time Polymerase Chain Reaction for the Identification of Iranian Brucella abortus Strains.

Brucellosis is primarily a worldwide zoonotic disease caused by Brucella species. The genus Brucella contains highly infectious species that are classified as biological threat agents. In this regard, the identification of Brucella can be a time-consuming and labor-intensive process posing a real risk of laboratory-acquired infection to the laboratory staff. This study aimed to present a novel conventional and real-time polymerase chain reaction (PCR) assay for the identification of Brucella abortus strains. Regarding this, two primers (bru ab2) were designed based on the unique loci encoding autotransporter-associated beta strand repeat-containing protein (ID:YP00113760). A total of 56 Brucella strains (e.g., reference, vaccinal, and field isolates) and Yersinia enterocolitica, as a non-Brucella isolate, were evaluated in conventional and real-time PCR systems. The results of the study indicated that 0.4 ng and 400 FG of genomic DNA of B. abortus strains can be detected by conventional and real-time PCR, respectively. The primers, bru ab2, were suitable for both PCR methods. Both methods were specific for the detection of all strains of the bacterium; however, real-time PCR assay was 1000-fold more sensitive than the conventional PCR method. Therefore, this new detection system could be a suitable selective modified method for the accurate identification of all B. abortus strains.

Identification of novel drug targets for humans and potential vaccine targets for cattle by subtractive genomic analysis of Brucella abortus strain 2308

Brucella abortus is the causative agent of brucellosis, a neglected endemic zoonotic disease. It causes devastating economic losses in low income and developing countries. Clinical symptoms of infected cows include abortion, poor weight, reduced fertility gain and reduction in milk production. Transmission of the zoonotic disease from cattle to human can occur through direct contact with infected cows, their tissues (e.g. placenta or aborted tissues), or their products (e.g. dairy) whereas human-to-human transmission can occur transplacentally or via breastfeeding. Malaise, fatigue, fever, arthritis are some clinical symptom of the disease in humans. Recent studies have revealed that Brucella abortus show resistance to several antibiotics. There are worldwide concerns about rising levels of antibiotic resistance resulting in the treatment failure as well as the reduced usefulness of older broad-spectrum antibiotics. Hence, a rather novel method has been in use to combat resistant pathogens since the last decade. To overcome this challenge, subtractive genomic analysis has been successfully carried out with the whole proteome of Brucella abortus strain 2308 using various bioinformatic tools and servers. Proteins nonhomologous to cattle and human were selected for metabolic analysis. Only three membrane proteins (ABC transporter permease, acriflavine resistance protein B, penicillin-binding protein 2) were found to be potential novel vaccine candidates with cattle as the host whereas one membrane protein (ABC transporter permease) was selected as novel drug target with human as the host. Development of novel vaccines and therapeutics through targeting inhibition of the functions of any of these essential proteins can lead to disruption of pathogen-specific metabolic pathways and thereby to the destruction and the eradication of this pathogen from respective hosts. The results of this study could facilitate the discovery and release of new and effective drugs and help in designing and producing potent vaccines against Brucella abortus strain 2308.

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.

Comparative genomic analysis between newly sequenced Brucella abortus vaccine strain A19 and another Brucella abortus vaccine S19

BackgroundBrucellosis is a bacterial disease caused by Brucella infection. Brucella abortus strain A19 is a spontaneously attenuated vaccine strain that has been used in vaccination of cattle against brucellosis. Until now, the physiological and molecular mechanisms of A19 are still unknown. ResultsIn this paper, the whole-genome sequence of B. abortus A19 was performed using Illumina Hiseq 4000 and PacBio sequencing technology and comparative genomics analysis were carried out with the whole genome sequences of B. abortus strains S19. This analysis indicated that the two vaccine strains have a high degree of similarity in genomic structure. We further analysis of the difference in genomic structure between A19 and S19. And found some differential genes such as eryC, eryD and eryF. Of the other different proteins between A19 and S19, such as outer membrane protein, 2-isopropylmalate synthase, citramalate synthase, GntR family transcriptional regulator and ABC transporters, no clear effects related to bacterial virulence were found, pending further investigation. ConclusionThe data presented here provide a reasonable basis for designing Brucella vaccines that can be used in other strains.

Sustained antigen release polyanhydride-based vaccine platform for immunization against bovine brucellosis

Brucellosis is a bacterial zoonosis and a significant source of economic loss and a major public health concern, worldwide. Bovine brucellosis, as caused primarily by Brucella abortus, is an important cause of reproductive loss in cattle. Vaccination has been the most effective way to reduce disease prevalence contributing to the success of control and eradication programs. Currently, there are no human vaccines available, and despite the success of commercial vaccines for livestock, such as B. abortus strain RB51 (RB51), there is need for development of novel and safer vaccines against brucellosis. In the current study, we report the fabrication of and immune responses to an implantable single dose polyanhydride-based, methanol-killed RB51 antigen containing delivery platform (VPEAR) in cattle. In contrast to animals vaccinated with RB51, we did not observe measurable RB51-specific IFN-γ or IgG responses in the peripheral blood, following initial vaccination with VPEAR. However, following a subsequent booster vaccination with RB51, we observed an anamnestic response in both vaccination treatments (VPEAR and live RB51). The magnitude and kinetics of CD4+ IFN-γ-mediated responses and circulating memory T cell subpopulations were comparable between the two vaccination treatments. Additionally, IgG titers were significantly increased in animals vaccinated with VPEAR as compared to live RB51- vaccinated animals. These data demonstrate that killed antigen may be utilized to generate and sustain memory, IFN-γ-mediated, CD4+ T cell and humoral responses against Brucella in a natural host. To our knowledge, this novel approach to vaccination against intracellular bacteria, such as Brucella, has not been reported before.