A potential virulence factor: Brucella flagellin FliK does not affect the main biological properties but inhibits the inflammatory response in RAW264.7 cells.

The bacterial flagellum is an elongated filament that protrudes from the cell and is responsible for bacterial motility. It can also be a pathogen-associated molecular pattern (PAMP) that regulates the host immune response and is involved in bacterial pathogenicity. In contrast to motile bacteria, the Brucella flagellum does not serve a motile purpose. Instead, it plays a role in regulating Brucella virulence and the host's immune response, similar to other non-motile bacteria. The flagellin protein, FliK, plays a key role in assembly of the flagellum and also as a potential virulence factor involved in the regulation of bacterial virulence and pathogenicity. In this study, we generated a Brucella suis S2 flik gene deletion strain and its complemented strain and found that deletion of the flik gene has no significant effect on the main biological properties of Brucella, but significantly enhanced the inflammatory response induced by Brucella infection of RAW264.7 macrophages. Further experiments demonstrated that the FliK protein was able to inhibit LPS-induced cellular inflammatory responses by down-regulating the expression of MyD88 and NF-κB, and by decreasing p65 phosphorylation in the NF-κB pathway; it also inhibited the expression of NLRP3 and caspase-1 in the NLRP3 inflammasome pathway. In conclusion, our study suggests that Brucella FliK may act as a virulence factor involved in the regulation of Brucella pathogenicity and modulation of the host immune response.

Immunoinformatic-guided designing of multi-epitope vaccine construct against Brucella Suis 1300.

Brucella suis mediates the transmission of brucellosis in humans and animals and a significant facultative zoonotic pathogen found in livestock. It has the capacity to survive and multiply in a phagocytic environment and to acquire resistance under hostile conditions thus becoming a threat globally. Antibiotic resistance is posing a substantial public health threat, hence there is an unmet and urgent clinical need for immune-based non-antibiotic methods to treat brucellosis. Hence, we aimed to explore the whole proteome of Brucella suis to predict antigenic proteins as a vaccine target and designed a novel chimeric vaccine (multi-epitope vaccine) through subtractive genomics-based reverse vaccinology approaches. The applied subsequent hierarchical shortlisting resulted in the identification of Multidrug efflux Resistance-nodulation-division (RND) transporter outer membrane subunit (gene BepC) that may act as a potential vaccine target. T-cell and B-cell epitopes have been predicted from target proteins using a number of immunoinformatic methods. Six MHC I, ten MHC II, and four B-cell epitopes were used to create a 324-amino-acid MEV construct, which was coupled with appropriate linkers and adjuvant. To boost the immunological response to the vaccine, the vaccine was combined with the TLR4 agonist HBHA protein. The MEV structure predicted was found to be highly antigenic, non-toxic, non-allergenic, flexible, stable, and soluble. To confirm the interactions with the receptors, a molecular docking simulation of the MEV was done using the human TLR4 (toll-like receptor 4) and HLAs. The stability and binding of the MEV-docked complexes with TLR4 were assessed using molecular dynamics (MD) simulation. Finally, MEV was reverse translated, its cDNA structure was evaluated, and then, in silico cloning into an E. coli expression host was conducted to promote maximum vaccine protein production with appropriate post-translational modifications. These comprehensive computer calculations backed up the efficacy of the suggested MEV in protecting against B. suis infections. However, more experimental validations are needed to adequately assess the vaccine candidate's potential. HIGHLIGHTS: • Subtractive genomic analysis and reverse vaccinology for the prioritization of novel vaccine target • Examination of chimeric vaccine in terms of allergenicity, antigenicity, MHC I, II binding efficacy, and structural-based studies • Molecular docking simulation method to rank based vaccine candidate and understand their binding modes.

Screening of potential vaccine candidates against pathogenic Brucella spp. using compositive reverse vaccinology.

Brucella spp. are Gram-negative, facultative intracellular bacteria that cause brucellosis in humans and various animals. The threat of brucellosis has increased, yet currently available live attenuated vaccines still have drawbacks. Therefore, subunit vaccines, produced using protein antigens and having the advantage of being safe, cost-effective and efficacious, are urgently needed. In this study, we used core proteome analysis and a compositive RV methodology to screen potential broad-spectrum antigens against 213 pathogenic strains of Brucella spp. with worldwide geographic distribution. Candidate proteins were scored according to six biological features: subcellular localization, antigen similarity, antigenicity, mature epitope density, virulence, and adhesion probability. In the RV analysis, a total 32 candidate antigens were picked out. Of these, three proteins were selected for assessment of immunogenicity and preliminary protection in a mouse model: outer membrane protein Omp19 (used as a positive control), type IV secretion system (T4SS) protein VirB8, and type I secretion system (T1SS) protein HlyD. These three antigens with a high degree of conservation could induce specific humoral and cellular immune responses. Omp19, VirB8 and HlyD could substantially reduce the organ bacterial load of B. abortus S19 in mice and provide varying degrees of protection. In this study, we demonstrated the effectiveness of this unique strategy for the screening of potential broad-spectrum antigens against Brucella. Further evaluation is needed to identify the levels of protection conferred by the vaccine antigens against wild-type pathogenic Brucella species challenge.

Development of attenuated live vaccine candidates against swine brucellosis in a non-zoonotic B. suis biovar 2 background.

Brucella is a genus of gram-negative bacteria that cause brucellosis. B. abortus and B. melitensis infect domestic ruminants while B. suis (biovars 1-3) infect swine, and all these bacteria but B. suis biovar 2 are zoonotic. Live attenuated B. abortus S19 and B. melitensis Rev1 are effective vaccines in domestic ruminants, though both can infect humans. However, there is no swine brucellosis vaccine. Here, we investigated the potential use as vaccines of B. suis biovar 2 rough (R) lipopolysaccharide (LPS) mutants totally lacking O-chain (Bs2ΔwbkF) or only producing internal O-chain precursors (Bs2Δwzm) and mutants with a smooth (S) LPS defective in the core lateral branch (Bs2ΔwadB and Bs2ΔwadD). We also investigated mutants in the pyruvate phosphate dikinase (Bs2ΔppdK) and phosphoenolpyruvate carboxykinase (Bs2ΔpckA) genes encoding enzymes bridging phosphoenolpyruvate and the tricarboxylic acid cycle. When tested in the OIE mouse model at the recommended R or S vaccine doses (108 and 105 CFU, respectively), CFU/spleen of all LPS mutants were reduced with respect to the wild type and decreased faster for the R than for the S mutants. At those doses, protection against B. suis was similar for Bs2ΔwbkF, Bs2Δwzm, Bs2ΔwadB and the Rev1 control (105 CFU). As described before for B. abortus, B. suis biovar 2 carried a disabled pckA so that a double mutant Bs2ΔppdKΔpckA had the same metabolic phenotype as Bs2ΔppdK and ppdK mutation was enough to generate attenuation. At 105 CFU, Bs2ΔppdK also conferred the same protection as Rev1. As compared to other B. suis vaccine candidates described before, the mutants described here simultaneously carry irreversible deletions easy to identify as vaccine markers, lack antibiotic-resistance markers and were obtained in a non-zoonotic background. Since R vaccines should not elicit antibodies to the S-LPS and wzm mutants carry immunogenic O-chain precursors and did not improve Bs2ΔwbkF, the latter seems a better R vaccine candidate than Bs2Δwzm. However, taking into account that all R vaccines interfere in ELISA and other widely used assays, whether Bs2ΔwbkF is advantageous over Bs2ΔwadB or Bs2ΔppdK requires experiments in the natural host.

Development of an auxotrophic, live-attenuated Brucella suis vaccine strain capable of expressing multimeric GnRH.

Feral swine cost around $1.5 billion each year in agricultural, environmental, and personal property damages. They are also the most widespread carriers of the zoonotic disease brucellosis, which threatens both livestock bio-security and public health. Currently, there is no approved vaccine against brucellosis in pigs. This is a preliminary report on the development of a live-attenuated B. suis vaccine that could be employed to deliver heterologous antigens to control swine populations. An attenuated vaccine strain provided significant protection against B. suis challenge in mice. Leucine auxotrophy in the vaccine strain allowed the over-expression of heterologous antigens without the use of antibiotic resistant markers. Vaccinated mice showed the development of antibodies against expressed antigen. Further evaluation is required to assess its ability to cause infertility using the mouse model prior to further testing for use as a tool for feral swine population and disease control.

Molecular characterization and differential expression analysis of interleukin 1ß from Ovis aries.

The interleukin-1 family is an important component of the innate immune system and plays an important role in regulating immune responses on the invasion of intracellular parasites in the acquired immune system. Interleukin 1ß (IL-1ß) is one of the members of the IL-1 family that predominantly activates downstream signaling pathways to play immunological functions of stimulating T and B lymphocyte activation and promoting the various syntheses of inflammatory substances in conjunction with other cytokines. Here, a full-length IL-1ß cDNA (OaIL-1ß) of sheep (Ovis aries) was cloned using rapid amplification of cDNA ends (RACE), which consists of 1494 bp and contains a 5'-UTR region with a length of 83 bp, a complete ORF of 801 bp in length, and a 3'-UTR region with a length of 642 bp. Recombinant protein OaIL-1ß was expressed and purified, and the monoclonal antibody against IL-1ß of sheep is prepared. Western blotting results showed that the sheep IL-1ß protein was detected in the heart, liver, lung, kidney, stomach, intestine, muscle, lymph nodes and leukocytes with the highest expression in the muscle and the lowest expression in the lung. Different bacteria treating sheep white blood cells induced differential expression of OaIL-1ß. Compared with the normal sheep, OaIL-1ß in the buffy coat was differentially expressed in the Brucella melitensis-challenged group and the B. suis S2 strain-inoculated group. However, whether IL-1ß may be considered as a molecular biomarker for differing Brucella-infected animals from brucellosis-vaccinated animals or not need to be further studied.

Chronic Brucella Infection Induces Selective and Persistent Interferon Gamma-Dependent Alterations of Marginal Zone Macrophages in the Spleen.

The spleen is known as an important filter for blood-borne pathogens that are trapped by specialized macrophages in the marginal zone (MZ): the CD209+ MZ macrophages (MZMs) and the CD169+ marginal metallophilic macrophages (MMMs). Acute systemic infection strongly impacts MZ populations and the location of T and B lymphocytes. This phenomenon has been linked to reduced chemokine secretion by stromal cells. Brucella spp. are the causative agent of brucellosis, a widespread zoonotic disease. Here, we used Brucella melitensis infection as a model to investigate the impact of chronic stealth infection on splenic MZ macrophage populations. During the late phase of Brucella infection, we observed a loss of both MZMs and MMMs, with a durable disappearance of MZMs, leading to a reduction of the ability of the spleen to take up soluble antigens, beads, and unrelated bacteria. This effect appears to be selective as every other lymphoid and myeloid population analyzed increased during infection, which was also observed following Brucella abortus and Brucella suis infection. Comparison of wild-type and deficient mice suggested that MZ macrophage population loss is dependent on interferon gamma (IFN-γ) receptor but independent of T cells or tumor necrosis factor alpha receptor 1 (TNF-αR1) signaling pathways and is not correlated to an alteration of CCL19, CCL21, and CXCL13 chemokine mRNA expression. Our results suggest that MZ macrophage populations are particularly sensitive to persistent low-level IFN-γ-mediated inflammation and that Brucella infection could reduce the ability of the spleen to perform certain MZM- and MMM-dependent tasks, such as antigen delivery to lymphocytes and control of systemic infection.

Activation of bovine neutrophils by Brucella spp.

Brucellosis is a globally important zoonotic infectious disease caused by gram negative bacteria of the genus Brucella. While many species of Brucella exist, Brucella melitensis, Brucella abortus, and Brucella suis are the most common pathogens of humans and livestock. The virulence of Brucella is largely influenced by its ability to evade host factors, including phagocytic killing mechanisms, which are critical for the host response to infection. The aim of this study was to characterize the bovine neutrophil response to virulent Brucella spp. Here, we found that virulent strains of smooth B. abortus, B. melitensis, B. suis, and virulent, rough, strains of Brucella canis possess similar abilities to resist killing by resting, or IFN-γ-activated, bovine neutrophils. Bovine neutrophils responded to infection with a time-dependent oxidative burst that varied little between Brucella spp. Inhibition of TAK1, or SYK kinase blunted the oxidative burst of neutrophils in response to Brucella infection. Interestingly, Brucella spp. did not induce robust death of bovine neutrophils. These results indicate that bovine neutrophils respond similarly to virulent Brucella spp. In addition, virulent Brucella spp., including naturally rough strains of B. canis, have a conserved ability to resist killing by bovine neutrophils.

Serological and molecular investigation for brucellosis in swine in selected districts of Uganda.

Brucellosis is a notifiable zoonotic disease affecting livestock, humans, and wildlife in Uganda. Pigs can be infected with human pathogenic Brucella suis biovars 1 and 3 and can be a significant source of brucellosis for humans. Uganda has a rapidly growing pig population, and the pork consumption per capita is the highest in East Africa. The objective of this work was to determine the seroprevalence of brucellosis in Ugandan pigs. A cross-sectional serosurvey of pigs was conducted in three of the major pig-keeping districts in Uganda (Masaka (n = 381 samples), Mukono (n = 398), and Kamuli (n = 414)). In addition, pigs originating from these districts were sampled in the major pig abattoir in Kampala (n = 472). In total, 1665 serum samples were investigated by serological and molecular tests. Only three putative brucellosis-positive samples were detected serologically using indirect ELISA. These sera were found negative for Brucella antibodies by CFT; however, two had antibodies against Yersinia enterocolitica as determined by SAT. Presence of antibodies against Yersiniae was confirmed by Y. enterocolitica antibody-specific ELISA. The two Yersiniae ELISA-positive samples were brucellosis negative using real-time PCR. We tested additional 142 sera from the 1665 samples with real-time PCR. All tested negative. Under this type of production system, we expect a maximum B. suis prevalence of less than 1 % at 95 % confidence level, and therefore, the risk of acquiring brucellosis from the pigs or their products is negligible. However, pigs may harbor the zoonotic Y. enterocolitica. This is the first study to investigate the occurrence of brucellosis in pigs in Uganda and the first study to report Y. enterocolitica antibodies in swine in Uganda.

Immunogenicity Analysis of a Novel Subunit Vaccine Candidate Molecule-Recombinant L7/L12 Ribosomal Protein of Brucella suis.

Brucella was an intracellular parasite, which could infect special livestock and humans. After infected by Brucella, livestock's reproductive system could be affected and destroyed resulting in huge economic losses. More seriously, it could be contagious from livestock to humans. So far, there is no available vaccine which is safe enough for humans. On this point, subunit vaccine has become the new breakthrough of conquering brucellosis. In this study, Brucella rL7/L12-BLS fusion protein was used as an antigen to immunize rabbits to detect the immunogenicity. The results of antibody level testing assay of rabbit antiserum indicated rL7/L12-BLS fusion protein could elicit rabbits to produce high-level IgG. And gamma interferon (IFN-γ) concentrations in rabbit antiserum were obviously up-regulated in both the rL7/L12 group and rL7/L12-BLS group. Besides, the results of quantitative real-time PCR (qRT-PCR) showed the IFN-γ gene's expression levels of both the rL7/L12 group and rL7/L12-BLS group were obviously up-regulated. All these results suggested Brucella L7/L12 protein was an ideal subunit vaccine candidate and possessed good immunogenicity. And Brucella lumazine synthase (BLS) molecule was a favorable transport vector for antigenic protein.

Bioluminescence Imaging of Colonization and Clearance Dynamics of Brucella Suis Vaccine Strain S2 in Mice and Guinea Pigs.

PURPOSE: The goal of this study was to develop a plasmid-based lux bio-reporter for use to obtain in vivo images of Brucella suis vaccine strain 2 (B.suis S2) infection with high resolution and good definition. PROCEDURES: The pBBR-lux (pBBR1MCS-2-lxCDABE) plasmid that carries the luxCDABE operon was introduced into B. suis S2 by electroporation yielding B. suis S2-lux. The spatial and temporal transit of B. suis S2 in mice and guinea pigs was monitored by bioluminescence imaging. RESULTS: The plasmid pBBR-lux is stable in vivo and does not appear to impact the virulence or growth of bacteria. This sensitive luciferase reporter could represent B. suis S2 survival in real time. B. suis S2 mainly colonized the lungs, liver, spleen, and uterus in mice and guinea pigs as demonstrated by bioluminescence imaging. CONCLUSION: The plasmid-based lux bioreporter strategy can be used to obtain high resolution in vivo images of B. suis S2 infection in mice and guinea pigs.

Brucella suis strain 2 vaccine is safe and protective against heterologous Brucella spp. infections.

Brucellosis is a wide spread zoonotic disease that causes abortion and infertility in mammals and leads to debilitating, febrile illness in humans. Brucella abortus, Brucella melitensis and Brucella suis are the major pathogenic species to humans. Vaccination with live attenuated B. suis strain 2 (S2) vaccine is an essential and critical component in the control of brucellosis in China. The S2 vaccine is very effective in preventing brucellosis in goats, sheep, cattle and swine. However, there are still debates outside of China whether the S2 vaccine is able to provide protection against heterologous virulent Brucella species. We investigated the residual virulence, immunogenicity and protective efficacy of the S2 vaccine in BALB/c mice by determining bacteria persistence in spleen, serum antibody response, cellular immune response and protection against a heterologous virulent challenge. The S2 vaccine was of low virulence as there were no bacteria recovered in spleen four weeks post vaccination. The vaccinated mice developed Brucella-specific IgG in 2-3 weeks, and a burst production of IFN-γ at one week as well as a two-fold increase in TNF-α production. The S2 vaccine protected mice from a virulent challenge by B. melitensis M28, B. abortus 2308 and B. suis S1330, and the S2 vaccinated mice did not develop any clinical signs or tissue damage. Our study demonstrated that the S2 vaccine is of low virulence, stimulates good humoral and cellular immunity and protects animals against infection by heterologous, virulent Brucella species.

[The fragments of dead macrophages infected by Brucellasuis S2 strain activates the immune response against Brucella infection].

OBJECTIVE: To investigate the role of the dead macrophages infected by Brucella suis S2 strain in the initiation of immune response to Brucella. METHODS: The mouse peritoneal macrophages were infected with Brucella suis S2 strain in vitro. After one hour, the cells were cultured in serum-free RPMI1640 medium for 5 days until all of them were dead because of starvation. The dead cell fragments and the bone marrow-derived dendritic cells (BMDCs) were co-cultured for 24, 48 and 72 hours, and then interleukin 12 (IL-12), tumor necrosis factor α (TNF-α) in the co-cultivation supernatant were detected by ELISA. The mouse macrophages marked by carboxyfluorescein diacetate succinimidyl ester (CFSE) were infected with Brucella suis S2 strain in vitro, and then were cultured without serum in the dark; the dead macrophages fragments and BMDCs labeled with anti-CD11c-PE were co-cultured for 1 hour away from light, and then the changes that BMDCs swallowed the fragments of macrophages were observed by laser scanning confocal microscopy. BALB/c mice were inoculated with the fragments of dead macrophages infected by S2 strain through abdominal cavity. After one week, a second immunization was done. The serum levels of IL-4, IL-2 and IFN-γ were detected with ELISA at 3 days post-secondary immunization. RESULTS: The macrophages fragments infected by S2 strain could be swallowed by DCs. The level of TNF-α in BMDCs swallowing macrophages fragments infected by S2 strain was significantly higher than that in the control group, but the former did not secret IL-12. The levels of IL-2, IL-4 and IFN-γ in the sera from the mice inoculated with the macrophages fragments infected by S2 strain were dramatically higher than those in the control groups. CONCLUSION: The dead macrophages infected by Brucella suis S2 strain can activate DCs to present antigen and induce the anti-Brucella immune response.

Immune responses and protection induced by Brucella suis S2 bacterial ghosts in mice.

With the purpose of generating Brucella suis bacterial ghosts and investigating the immunogenicity of bacterial ghosts as a vaccine candidate, the lysis gene E and temperature-sensitive regulator cassette were cloned into a shuttle plasmid, pBBR1MCS-2, for construction of a recombinant temperature-sensitive shuttle lysis plasmid, pBBR1MCS-E. pBBR1MCS-E was then introduced into attenuated B. suis live vaccine S2 bacteria, and the resultant transformants were used for production of B. suis ghosts (BSGs) by inducing lysis gene E expression. The BSGs were characterized by observing their morphology by transmission electron microscopy. The safety and immunogenicity of BSGs were further evaluated using a murine model, the result suggested that BSG was as safe as formalin-killed B. suis. In mice, BSG demonstrated a similar capacity of inducing pathogen-specific serum IgG antibody response, spleen CD3(+) and CD4(+) T cell responses, induce secretion of gamma interferon and interleukin-4, and protection levels against Brucella melitensis 16M challenge, as the attenuated B. suis live vaccine. These data suggesting that BSG could confer protection against Brucella infection in a mouse model of disease and may be developed as a new vaccine candidate against Brucella infection.

Brucella suis vaccine strain S2-infected immortalized caprine endometrial epithelial cell lines induce non-apoptotic ER-stress.

Brucella, which is regarded as an intracellular pathogen responsible for a zoonotic disease called brucellosis, survives and proliferates within several types of phagocytic and non-phagocytic cells. Brucella infects not only their preferred hosts but also other domestic and wild animal species, inducing abortion and infertility. Therefore, the interaction between uterine cells and Brucella is important for understanding the pathogenesis of this disease. In this study, we describe the Brucella suis vaccine strain S2 (B.suis.S2) infection and replication in the immortalized caprine endometrial epithelial cell line hTERT-EECs and the induced cellular and molecular response modulation in vitro. We found that B.suis S2 was able to infect and replicate to high titers and inhibit the proliferation of EECs and induce non-apoptotic pathways, as determined by B.suis.S2 detection using MTT and acridine orange/ethidium bromide (AO/EB) staining and flow cytometry. We explored the evidence of non-apoptotic pathways using real-time quantitative RT-PCR and by western blot analysis. Finally, we discovered the over-expression of GRP78, ATF4, ATF6, PERK, eIF2α, CHOP, and cytochrome c (Cyt-c) but not IRE1, xbp-1, and caspase-3 in B.suis.S2 (HK)-attacked and B.suis.S2-infected cells, suggesting that the molecular mechanism of ER stress sensor activation by B.suis.S2 is basically concomitant with that by B.suis.S2 (HK) and that ER stress, especially the PERK pathway, plays an important role in the process of B.suis.S2 infecting EEC, which may, in part, explain the role of the uterus in the pathogenesis of B.suis.S2.

Brucella suis infection in domestic pigs in Sardinia (Italy).

During a 4-year (2007-2010) survey, the presence of Brucella suis infection in domestic pigs in Sardinia was investigated. Serum samples were collected from breeding pigs located on 108 commercial farms with documented reproductive problems and analysed using the Rose Bengal (RBT) and complement fixation (CFT) tests for screening and confirmation of Brucella, respectively. Of the 1251 serum samples analysed by RBT, 406 sera, originating from 36 farms, were positive for B. suis. CFT was positive in 292/748 sera analysed, confirming positivity in all 36 pig herds. Pigs with international complement fixation test units per ml (ICFTU/ml) values ⩾160 were slaughtered, and their organs collected for bacteriological examination and testing by polymerase chain reaction (PCR). Brucella spp. strains were isolated in culture from 13/502 organs analysed, and subsequently identified as B. suis biovar 2. PCR detected positivity to Brucella spp. in 19/285 organs analysed. These results confirm the presence and emergence of B. suis infection in domestic pigs in Sardinia.

Clinical and histological features of brucellin skin test responses in Brucella suis biovar 2 infected pigs.

Current serological tests for swine brucellosis detect antibodies to the Brucella O-polysaccharide (O/PS). However, when infections by bacteria carrying cross-reacting O/PS occur, these tests suffer from false positive serological reactions (FPSR), and the skin test with Brucella soluble protein extracts is the best diagnostic alternative to differentiate true Brucella suis infections from FPSR in pigs. Since this test has been seldom used in B. suis infected swine, the clinical and histological features involved have not been described properly. Here, we describe the clinical and histological events in B. suis biovar 2 infected pigs skin tested with a cytosoluble O/PS free protein extract from rough Brucella abortus Tn5::per mutant. A similar extract from rough Ochrobactrum intermedium was also used for comparative purposes. No relevant differences were evidenced between the homologous and heterologous allergens, and the main clinical feature was an elevated area of the skin showing different induration degrees. Moreover, an important vascular reaction with hyperemia and haemorrhage was produced in most infected sows 24-48 h after inoculation, thus facilitating the clinical interpretation of positive reactions. Histologically, combined immediate (type III) and delayed (type IV) hypersensitivity reactions were identified as the most relevant feature of the inflammatory responses produced.

Duplex PCR for differentiation of the vaccine strain Brucella suis S2 and B. suis biovar 1 from other strains of Brucella spp.

Immunisation with attenuated Brucella spp. vaccines prevents brucellosis, but may also interfere with diagnosis. In this study, a duplex PCR was developed to distinguish Brucella suis vaccine strain S2 from field strains of B. suis biovar 1 and other Brucella spp. The PCR detected 60 fg genomic DNA of B. suis S2 or biovar 1 field strains and was able to distinguish B. suis S2 and wild-type strains of B. suis biovar 1 among 76 field isolates representing all the common species and biovars, as well as four vaccine strains, of Brucella.

A repA-based ELISA for discriminating cattle vaccinated with Brucella suis 2 from those naturally infected with Brucella abortus and Brucella melitensis.

The commonest ways of diagnosing brucellosis in animals include the Rose-Bengal plate agglutination test, the buffered plate agglutination test (BPA), the slide agglutination test, the complement fixation test, and the indirect enzyme linked immunosorbent assay (I-ELISA). However, these methods cannot discriminate the Brucella vaccine strain (Brucella suis strain 2; B. suis S2) from naturally acquired virulent strains. Of the six common Brucella species, Brucella melitensis, Brucella abortus, and B. suis are the commonest species occurring in China. To develop an ELISA assay that can differentiate between cows inoculated with B. suis S2 and naturally infected with B. abortus and B. melitensis, genomic sequences from six Brucella spp. (B. melitensis, B. abortus, B. suis, Brucella canis, Brucella neotomae and Brucella ovis) were compared using Basic Local Alignment Search Tool software. One particular gene, the repA-related gene, was found to be a marker that can differentiate B. suis from B. abortus and B. melitensis. The repA-related gene of B. suis was PCR amplified and subcloned into the pET-32a vector. Expressed repA-related protein was purified and used as an antigen. The repA-based ELISA was optimized and used as specific tests. In the present study, serum from animals inoculated with the B. suis S2 vaccine strain had positive repA-based ELISA results. In contrast, the test-positive reference sera against B. abortus and B. melitensis had negative repA-based ELISA results. The concordance rate between B. abortus antibody-negative (based on the repA-based ELISA) and the Brucella gene-positive (based on the 'Bruce ladder' multiplex PCR) was 100%. Therefore, the findings suggest that the repA-based ELISA is a useful tool for differentiating cows vaccinated with the B. suis S2 and naturally infected with B. abortus and B. melitensis.

MLVA genotyping of Brucella melitensis and Brucella abortus isolates from different animal species and humans and identification of Brucella suis vaccine strain S2 from cattle in China.

In China, brucellosis is an endemic disease and the main sources of brucellosis in animals and humans are infected sheep, cattle and swine. Brucella melitensis (biovars 1 and 3) is the predominant species, associated with sporadic cases and outbreak in humans. Isolates of B. abortus, primarily biovars 1 and 3, and B. suis biovars 1 and 3 are also associated with sporadic human brucellosis. In this study, the genetic profiles of B. melitensis and B. abortus isolates from humans and animals were analyzed and compared by multi-locus variable-number tandem-repeat analysis (MLVA). Among the B. melitensis isolates, the majority (74/82) belonged to MLVA8 genotype 42, clustering in the 'East Mediterranean' group. Two B. melitensis biovar 1 genotype 47 isolates, belonging to the 'Americas' group, were recovered; both were from the Himalayan blue sheep (Pseudois nayaur, a wild animal). The majority of B. abortus isolates (51/70) were biovar 3, genotype 36. Ten B. suis biovar 1 field isolates, including seven outbreak isolates recovered from a cattle farm in Inner Mongolia, were genetically indistinguishable from the vaccine strain S2, based on MLVA cluster analysis. MLVA analysis provided important information for epidemiological trace-back. To the best of our knowledge, this is the first report to associate Brucella cross-infection with the vaccine strain S2 based on molecular comparison of recovered isolates to the vaccine strain. MLVA typing could be an essential assay to improve brucellosis surveillance and control programs.