Brucella Melitensis Rev Research Articles

The Prevalence and Molecular Distinguishing of Brucella melitensis Rev1 Strain among Field Isolates the Brucella from Sheep and Goat Milk through PCR-RFLP Analysis of omp2 Gene Polymorphism

The present study was undertaken to characterize the prevalence and molecular distinguishing of Brucella melitensis field and vaccine Rev1 strains isolated from milk among the sheep and goat population. The study was carried out on flocks of sheep and goats suffering from Brucella infection outbreaks. A total of 171 milk samples were collected from different districts of the West Bank, part of Palestine, and isolated on Brucella agar. 86 cultures were confirmed by standard biochemical methods and screened for Brucella melitensis 16M of IS711 element by a conventional PCR technique. All these samples were also amplified for the omp2 gene to identify differences between field and Rev 1 strains in an infected flock. The results obtained from the PstI restriction enzyme pattern revealed that only 2 (4.5%) samples have specific polymorphism accomplished with the Brucella melitensis Rev1 strain. All other samples were restricted according to the Brucella melitensis 16M polymorphism pattern and originated from nonvaccinated flocks. Despite the adverse side effects of the Brucella melitensis vaccine, only a well-organized whole-flock vaccination and awareness campaign may inhibit the virulence of the field strain and, subsequently, reduce the prevalence of brucellosis infection among animals and humans in Palestine.

Immune response strategies of Brucella melitensis and their antigens in rats

Brucella melitensis is an intracellular bacterium and is the main brucella species that cause abortion and placenta retention in sheep and goats. It has many mechanisms to evade the immune response. The current study aimed to investigate Brucella melitensis strategies for producing immune responses in rats after challenging the bacterium. For this purpose, live and killed Brucella melitensis REV1 strain was given to rats subcutaneously, and immunological markers like TLR2, TLR4, IFN- γ, and anti-brucella antibodies were determined. The results showed that the level of immunological markers like TLR2 and TLR4 did not significantly increase in rat groups inoculated with live Brucella melitensis, while it increased in the rats’ groups vaccinated with the sonicated Brucella melitensis; also, the results showed an increase in the level of IFN-γ and anti-brucella antibody titers in all animal groups. The study concluded that the inoculation with killed bacteria and REV1 could protect the animals against challenging doses, as seen when the groups were inoculated with challenge dose of the bacterium.

Brucella melitensis Wzm/Wzt System: Changes in the Bacterial Envelope Lead to Improved Rev1Δwzm Vaccine Properties.

The lipopolysaccharide (LPS) O-polysaccharide (O-PS) is the main virulence factor in Brucella. After synthesis in the cytoplasmic membrane, O-PS is exported to the periplasm by the Wzm/Wzt system, where it is assembled into a LPS. This translocation also engages a bactoprenol carrier required for further biosynthesis pathways, such as cell wall biogenesis. Targeting O-PS export by blockage holds great potential for vaccine development, but little is known about the biological implications of each Wzm/Wzt moiety. To improve this knowledge and to elucidate its potential application as a vaccine, we constructed and studied wzm/wzt single- and double-deletion mutants, using the attenuated strain Brucella melitensis Rev1 as the parental strain. This allowed us to describe the composition of Brucella peptidoglycan for the first time. We observed that these mutants lack external O-PS yet trigger changes in genetic transcription and in phenotypic properties associated with the outer membrane and cell wall. The three mutants are highly attenuated; unexpectedly, Rev1Δwzm also excels as an immunogenic and effective vaccine against B. melitensis and Brucella ovis in mice, revealing that low persistence is not at odds with efficacy. Rev1Δwzm is attenuated in BeWo trophoblasts, does not infect mouse placentas, and is safe in pregnant ewes. Overall, these attributes and the minimal serological interference induced in sheep make Rev1Δwzm a highly promising vaccine candidate.

Silver nanoparticle-conjugated antibiotics inhibit in vitro growth of Brucella melitensis.

Background and Aim:Brucellosis is a contagious livestock disease with a significant economic impact. This study aimed to compare the efficacy of antibiotics used alone or in combination with silver nanoparticles (AgNPs) against Brucella melitensis Rev 1 in vitro.Materials and Methods:AgNps conjugated with ciprofloxacin was synthesized and thoroughly characterized by ultraviolet visible spectrophotometry (UV-vis). The antimicrobial effect of ciprofloxacin alone and ciprofloxacin conjugated with AgNPs against B. melitensis Rev 1 was determined by minimum inhibitory concentration (MIC) and the erythrocyte hemolytic assay determined the capability of conjugation to cause hemolysis in human erythrocyte.Results:The UV-vis spectra of both silver-drug nanoconjugates showed a characteristic surface plasmon resonance band at 420 nm. The MIC assays showed that AgNPs conjugation to antibiotics enhanced the antibacterial potential of the selected antibiotics against B. melitensis Rev 1 relative to non-conjugated antibiotics. The results show that low concentrations of AgNPs can kill B. melitensis Rev 1. The MICs of ciprofloxacin and ciprofloxacin–AgNPs were 0.75 and 0.05 μM, respectively.Conclusion:The conjugation of ciprofloxacin with AgNPs enhanced the antibacterial effects against B. melitensis Rev 1. In addition, this conjugation appears to inhibit the capability of this bacterium to adapt to the presence of antibiotics, thereby inhibiting bacterial resistance. Further studies are required to examine its potential as an in vivo treatment.

Human brucellosis in the southern West Bank, 2015–17: a seroprevalence and molecular characterisation study

BackgroundBrucellosis is a global zoonotic disease ranked by WHO as being among the top seven neglected zoonoses. After implementing a livestock vaccination strategy in 1998, the incidence rate of human brucellosis in Palestine dropped significantly. However, in 2015 the disease re-emerged with alarming incidence rates similar to those in the period before 1998. The aim of this study was to characterise the strains associated with this episode using genetic and genomic tools. MethodsAll patients were referred to the department of preventive medicine from several public or private clinics in the southern West Bank due to undulant fever. After a careful analysis of a patients’ history to evaluate exposure to Brucella, blood samples from patients suspected of brucellosis were collected. Samples positive for the Rose Bengal test with an antibody titre of 1:80 IU/mL or more by Brucella standard agglutination test, and not under antibiotic treatment, were further cultured using the BACTEC 9240 blood culture system. DNA was extracted from the positive culture samples and analysed using three biomarkers to validate the presence of the Brucella genus, establish the species, and identify the Brucella melitensis Rev 1 vaccine strain. Nine randomly chosen isolates were sequenced using targeted next generation sequencing technology to screen for the rpoB gene mutations. Multiple-locus variable number of tandem repeats analysis (MLVA) with 16 variable number of tandem repeats markers was used to genotype the isolates and BioNumerics version 7.6 software was used to analyse the data using the categorical coefficient and unweighted pair group method using the arithmetic averages algorithm. FindingsBlood samples were obtained from a total of 1324 patients during 2015–17. 704 (53·2%) of 1324 serum samples showed agglutination in the Rose Bengal test and 559 (79·4%) of the 704 were considered seropositive based on a standard agglutination test titre of 1:80 IU/mL or more. Only 130 (23·3%) of the 559 seropositive samples were from patients not under antibiotic treatment. Upon culturing, only 72 (12·9%) of the seropositive samples showed growth signals and were positive by PCR for the Brucella genus marker, and all isolates were B melitensis. None of the samples were associated with the B melitensis Rev 1 vaccine strain. The results of sequencing the rpoB gene by next generation sequencing showed four different single nucleotide variations that were not located within the rifampicin resistance-determining region. Using MLVA-16 analysis and the MLVA bank benchmark, the studied isolates were divided into 22 unique genotypes. A hierarchical clustering analysis for the 72 isolates and 18 B melitensis global isolates representing four major regions in the Middle East, Europe, Africa, and America, clustered the 73 isolates in the Middle East clade, providing a similarity cutoff value of more than 97% for genotype and more than 75% for clade. InterpretationThis study shows that the brucellosis episode of 2015–17 was due to B melitensis local isolates and was not associated with the Rev 1 vaccine stain. The results show that MLVA-16 is a useful tool for the public health surveillance of human brucellosis. The present study analysed samples from the Hebron governorate only. It is recommended to include more human and animal samples from different areas in Palestine to understand the molecular epidemiology of brucellosis in Palestine and to make more effective decisions regarding control and treatment programmes. FundingPalestine Polytechnic University.

Improvement of the attenuated mutant strain of Brucella melitensis Rev1 as a potential vaccine candidate

Vaccination is the best approach to prevent brucellosis. The existing vaccines such as Rev1 are associated with disadvantages such as abortion in pregnant animals. They also induce antibodies to the O-polysaccharide which may complicate diagnosis. Therefore, it is necessary to generate an appropriate vaccine. The aim of this study is the improvement of the available mutant strain of Brucella melitensis Rev1, in which the prosamine synthetize coding sequence from the O-PS region has been replaced with kanR coding sequence, via the replacement of kanR with GFP reporter encoding sequence by using homologous recombination. The syntactic deletion cassette, containing the GFP encoding sequence flanked with the upstream and downstream sequences of kanR encoding sequence from Rev1 mutant strain, was cloned into pBluescriptIISK(-) vector by SacI and KpnI, and the suicide vector pBluescriptSK-∆kanR/gfp was electroporated into mutant strain. The constructs were confirmed by using PCR, restriction mapping and DNA sequencing. The loss of kanR function in transfected cells was confirmed by using the GFP phenotyping with flow cytometry and fluorescence microscopy, and following the phenotypic characterization of the mutant strain on selective media and the genomic DNA analysis with specific primers. This kanR-free Rev1 mutant strain lack the O-polysaccharide and have a reduced ability to enter and survive in the host cell. Therefore, it could be evaluated as a potential vaccine candidate if eliciting protective immunity.

Farm practices and risk factors which influence the high prevalence of brucellosis in small ruminant flocks in Northeast Portugal.

The region of Tras-os-Montes e Alto Douro in northeast Portugal displayed the highest prevalence of brucellosis in small ruminants of the country. A vaccination programme of the whole population with Brucella melitensis Rev-1 was carried out from 2001 to 2004 in an attempt to reduce prevalence levels and protect public health. Although individual prevalence decreased from 5.6% in 2001 to 0.4% in 2007, several flocks continued to present individual prevalence ≥ 5.0%. Given the multifactorial characteristics of brucellosis, the current study evaluated farming practices and risk factors in flocks with an individual prevalence over 5% by multifactorial correspondence analysis. Results showed that a lack of recognition of the symptoms of brucellosis and lack of Rev-1 vaccination were the main factors contributing to the high individual prevalence of brucellosis in flocks. Other factors such as the consumption of raw milk, presence of dog commingling with animals and use of communal pastures also contributed to the persistence of the disease. Family farms with low economical profit, minimal training/education of farmers, and a scarcity of veterinary support may explain the persistence of factors contributing to the high prevalence of brucellosis. The results of this study highlight several risk factors and farming practices that might have contributed to the maintenance of a high prevalence of brucellosis in flocks with high brucellosis prevalence. These results could be used to adopt new approaches to improve the efficiency of brucellosis eradication programs.

Production of Brucella lumazine Synthase Recombinant Protein to Design a Subunit Vaccine against Undulant Fever.

Brucella bacterium causes Brucellosis, an infectious disease spreading from animals to human. Brucella lumazine synthase (BLS) is a highly immunogenic protein with adjuvant properties, which has been introduced as an effective protein carrier for vaccine development. This protein also plays a significant role in inducing immune system. This study aimed to clone, express, and purify the BLS gene from Brucella melitensis Rev1. The BLS gene was amplified by particular primers with the restriction enzyme sites as a linker and it was inserted into pTZ57R/T vector. Subsequently, it was ligated into pET32(a)+ expression vector. Recombinant expression vector containing coding sequence of BLS was transformed into E. coli BL21 (DE3) host gene expression and stimulated by 0.1mM IPTG. The results of sequencing showed that there were not any mutations in BLS encoding sequence. The expression results were set by sequencing and endorsed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses and western blotting that showed 35 kDa protein band appropriately.

Mucosal immunization with polymeric antigen BLSOmp31 using alternative delivery systems against Brucella ovis in rams

Subcellular vaccines against ovine contagious epididymitis due Brucella ovis can solve some shortcomings associated with the use of Brucella melitensis Rev 1. We have demonstrated that the parenteral immunization with polymeric antigen BLSOmp31 emulsified in oil adjuvant conferred significant protection against B. ovis in rams. In our previous studies, we have characterized chitosan microspheres (ChMs) and a thermoresponsive and mucoadhesive in situ gel (Poloxamer 407-Ch) as two novel formulation strategies for the delivery of BLSOmp31 in nasal as well as conjunctival mucosa. In the present work, we evaluated the immunogenicity and protection conferred by the intranasal and conjunctival immunization with these two mucosal delivery systems against B. ovis in rams. BLSOmp31-ChM administered by intranasal route and BLSOmp31-P407-Ch applied by intranasal or conjunctival routes induced systemic, local and preputial IgG and IgA antibody response. Neither formulation showed interference in the serological diagnosis. Thus, mucosal immunization using either formulation induced significant specific cellular immune responses (in vitro and in vivo) and it prevented the excretion of B. ovis in semen. Although these vaccines did not prevent infection in immunized rams, colonization reduction of infected organs and bacterial distribution differed significantly between vaccinated and unvaccinated rams.

Serological response of dromedary camels vaccinated with Brucella abortus RB51 and Brucella melitensis REV 1 Vaccines

Serological response of dromedary camels vaccinated with <i>Brucella abortus</i> RB51 and <i>Brucella melitensis</i> REV 1 Vaccines

Ontology-Based Meta-Analysis of Animal and Human Adverse Events Associated With Licensed Brucellosis Vaccines.

Brucella abortus strain 19 (S19), Brucella melitensis Rev 1 (Rev1), and B. abortus strain RB51 (RB51) are the three licensed animal brucellosis vaccines, and they have been most commonly and successfully used in prevent brucellosis in animals. However, many adverse events (AEs) have been associated with these three vaccines after their administering to animals or being accidentally exposed to humans. In this study, 27 peer-reviewed publications containing animal and human AE reports associated with these three brucellosis vaccines were manually annotated from the PubMed database. Our meta-analysis identified 20 animal AEs and 46 human AEs associated with the three vaccines. Based on the Ontology of Adverse Events (OAE) hierarchical classification, these animal AEs were enriched in the immune and reproductive systems that might eventually result in the occurrence of abortion or infertility. The human AEs were concentrated in the behavioral and neurological conditions, and these AEs showed flu-like symptoms that are consistent with human brucellosis. Furthermore, an analysis of variance (ANOVA) statistics analysis with linear model fits was used to determine the major variables that might affect the occurrence of abortion AE in animals. The ANOVA results indicated that three variables (P-value < 0.05) are significantly associated with the occurrence of abortion AE: animal species, vaccination dose, and vaccination route. The other two variables (i.e., vaccine type and animal age at vaccination) did not significantly (P-value > 0.05) associated with the occurrence of abortion AE. Overall, this study represents the first ontology-based meta-analysis of adverse events associated with animal vaccines. The results of such a study led to the better understanding of brucellosis vaccine AEs, facilitating rational design of more secure and effective vaccines.

Evaluation of immune responses in mice and sheep inoculated with a live attenuated Brucella melitensis REV1 vaccine produced in bioreactor

The Brucella melitensis REV1 vaccine is the most widely employed vaccine for prophylaxis against brucellosis in sheep and goats. The objective of vaccination is disease control in herds or preventing infection in farms. In this study, we produced REV1 vaccine with a protocol, based on the use of liquid medium in a bioreactor, that resulted efficient, safe, relatively fast, and cost-effective. The live attenuated vaccine produced was tested in mice and sheep to investigate its immunogenicity and efficacy. Seventy-two female BALB/c mice were obtained and subdivided in 2 groups, one was stimulated with 1 × 106 colony-forming units (CFUs) of B. melitensis while the other with physiological solution alone and acting as control group. Furthermore, 25 sheep were subdivided into 5 groups: four were inoculated with a B. melitensis dose, ranging from 0.6 × 109 and 3.2 × 109 CFUs and the other was the control group. In addition, a serological diagnosis was performed for sheep by rapid serum agglutination and the complement-fixation test. Immunocompetent cells from both experiment were collected at different times post vaccination and immunostained to evaluate innate and adaptive-immune responses. In mice flow cytometry was used to detect macrophages, T lymphocytes, dendritic cells, memory cells, naïve cells, natural killer cells, major histocompatibility complex type II, B lymphocytes, regulatory T lymphocytes, T helper lymphocytes, cytotoxic T lymphocytes and recently activated CD4+ and CD8+ lymphocytes. In sheep, macrophages, T helper cells, cytotoxic T lymphocytes, regulatory T lymphocytes, dendritic cells, memory cells and naïve lymphocytes, by the same method, were analyzed. The results showed, both in mice and sheep, that the live, attenuated REV1 vaccine stimulated all immunocompetent cells tested, with a balanced innate and adaptive response. In the sheep experiment, the administered vaccine dose was very important because, at the lower doses, immunological tolerance tended to disappear, while, at the highest dose, the immunological tolerance remained active for a long period. In our experimental conditions, the optimal vaccine dose for sheep was 3.2 × 109 CFUs, although a good immune response was found using a dose of 1.6 × 109 CFUs. The vaccine produced in this study could be extensively employed in developing countries to control the brucellosis in sheep and goats.

Diagnostic potential of Brucella melitensis Rev1 native Omp28 precursor in human brucellosis.

Serologic tests for brucellosis aim to detect antibodies produced against membranous lipopolysaccharide of bacteria. Diagnostic use of this method is limited due to false positiveness. This study evaluates an alternative antigen to lipopolysaccharides (LPS), outer membrane 28-precursor-protein, of Brucella melitensis Rev1 for its diagnostic value. Omp28 precursor of B. melitensis Rev1 was cloned, expressed, and purified. 6-His and sumo epitope tags were used to tag the protein at N-termini. Omp28 gene was amplified based on the ORF sequence and cloned into a pETSUMO vector. The recombinant construct was propagated in Escherichia coli One Shot® Mach1™ cells then transformed into E. coli BL21(D3) cells for protein expression. The purified protein was studied in an indirect ELISA for diagnosis of brucellosis. Sera samples from 60 patients were screened by ELISA and the results were compared to Rose Bengal plate test. Recombinant antigen-based iELISA has given a successful outcome with the sensitivity, specificity, positive predictive value, and negative predictive value of 87.8%, 96.2%, 96.6%, and 78.78%, respectively. In conclusion, recombinant production and purification of the immunodominant Omp28 precursor protein has been achieved successfully in a one-step process with efficient yield and can be used for diagnosis of brucellosis in humans.

Using real-time polymerase chain reaction as an alternative rapid method for enumeration of colony count in live Brucella vaccines.

Aim::Brucellosis is a major bacterial zoonosis of global importance affecting a range of animal species and man worldwide. It has economic, public health, and bio-risk importance. Control and prevention of animal brucellosis mainly depend on accurate diagnostic tools and implementation of effective and safe animal vaccination program. There are three types of animal Brucella live vaccines - Brucella melitensis Rev-1 vaccine, Brucella abortus S19, and B. abortus RB51. Evaluation of these vaccines depends mainly on enumeration of Brucella viable count. At present, used colony count method is time consuming, costly and requires especial skills. Hence, the aim of this study is to use and standardize real-time polymerase chain reaction (RT-PCR) as an alternative, quantitative, sensitive, and rapid method to detect the colony count of Brucella in live Brucella vaccine.Materials and Methods::Four batches of different live Brucella vaccines were evaluated using of conventional bacterial count and RT-quantitative PCR (RT-qPCR) using BSCP31 gene specific primers and probe. Standard curve was generated from DNA template extracted from 10-fold serial dilution of living B. abortus RB51 vaccine to evaluate the sensitivity of RT-qPCR.Results::Results revealed that three batches of living Brucella vaccines were acceptable for Brucella colony count when traditional bacterial enumeration method was used. Results of RT-qPCR were identical to that of conventional bacterial count.Conclusions::Results concluded that RT-qPCR was relatively sensitive compared to traditional bacterial colony count of these vaccines.

Cloning and molecular characterization of Omp31 gene from Brucella melitensis Rev 1 strain

Brucellosis, caused by the genus Brucella bacterium, is a well-known infection among domestic animals. Considering the serious economic and medical consequences of this infection, various preventive efforts have been made through using recombinant vaccines, based on outer membrane protein (OMP) antigens of Brucella species. The objective of the present study was to clone, analyze the sequence, and predict the epitopes of Omp31 gene as a major B. melitensis antigen. The full-length open reading frame (ORF) for this gene was amplified by specific primers and cloned into the pTZ57R/T vector. The gene sequence of B. melitensis Rev 1 strain was submitted to NCBI database. The results of phylogenetic analysis showed that Omp31 is almost similar in different Brucella species. Online prediction software programs were also used to predict B- and T-cell epitopes, secondary and tertiary structures, antigenicity, and enzymatic degradation sites. The bioinformatic tools in the current study were confirmed by the results of three different experimental epitope prediction studies. Bioinformatic analysis identified one T-cell and three B-cell epitopes for Omp31 antigen. Finally, based on the antigenicity and proteosome recognition sites, common B- and T-cell epitopes were predicted for Omp31 (amino acids 191-204). Bioinformatic analysis showed that these regions had proper epitope characterization and could be useful for recombinant vaccine development.

FEATURES OF MASS-SPECTROMETRIC PROTEIN PROFILES OF STRAINS OF BRUCELLOSIS CAUSATIVE AGENT DURING PREPARATION OF CULTURE ON VARIOUS NUTRIENT MEDIA

Carry out comparative analysis using time-of-flight mass-spectrometry with matrix laser desorption/ionization (MALDI-TOF MS) of protein profiles of brucellosis causative agents (Brucella melitensis Rev-1 and Brucella abortus 19BA), cultivated in various nutrient media: Albimi agar, brucellagar and erythrit-agar. Vaccine,strains: Brucella melitensis Rev-1 and Brucella abortus 19BA. Protein profiling in linear mode on Microflex "Bruker Daltonics" MALDI-TOF mass-spectrometer. A number of characteristic features of brucella mass-spectra was detected: in particular, preservation of the total qualitative composition of protein profiles of cultures and significant differences in the intensity of separate peaks depending on the nutrient medium used. Based on the analysis of the data obtained, use of Albimi agar as the nutrient medium for preparation of brucella culture samples for mass-spectrometric analysis was shown to be optimal.

Antibacterial and Antibiofilm Effects of Boron on Different Bacteria

Boron (B) compounds are used in many fields ranging from medicine to industry. In this study, boric acid (BA) and disodium octaborate tetrahydrate (DOT) were evaluated for their antibacterial effects and antibiofilm capacities on selected strains of clinical and type cultures that are of veterinary concern (Staphylococcus aureus ATCC 25923, Aeromonas hydrophila ATCC 19570, Pseudomonas aeruginosa ATCC 27853, Brucella melitensis Rev1 and field isolates of Vibrio anguillarum, Aeromonas hydrophila, Yersinia ruckeri, Pseudomonas aeruginosa, Lactococcus garvieae, and Brucella abortus). Also, the inhibition of biofilm was monitored by scanning electron microscopy. The lowest MIC values of BA and DOT were measured, by broth method using microdilution, from Pseudomonas aeruginosa ATCC 27853, and were 0.385 and 0.644mg/ml, respectively. Staphylococcus aureus was the most resistant to both BA and DOT. Using the microplate method, we observed that the strongest positivities for biofilm production were presented by Pseudomonas aeruginosa ATCC 27853 and also a clinical isolate of Lactococcus garviea. Lower values in the MIC scores for both B compounds were tested by measuring the inhibitory effect on biofilm production. We found that all the bacterial strains inhibited biofilm formation with the exception of the Pseudomonas aeruginosa strains for BA only and an isolate of Lactococcus garviea for DOT only. Such effects by BA and DOT are worth discussing in order to find novel approaches for different functions in medicine and industry using the bacteria tested.

Acute Brucellosis Following Accidental Exposure to Brucella melitensis Rev 1 Vaccine.

Brucellosis is mainly transmitted to humans by direct contact with infected animals, consumption of non-pasteurized dairy products or through inhalation of aerosols. However, the disease may also be transmitted by exposure to Brucella vaccination that is used in veterinary medicine. In the literature, there were a few case reports of persons who developed brucellosis after unintentional inoculation or conjunctival exposure to the live Brucella vaccine. Here, we describe a sheep farmer with acute brucellosis that occurred as a result of unintentional percutaneous exposure to Brucella melitensis Rev 1 animal vaccine while vaccinating lambs.

Impact of B. melitensis Rev-1 vaccination on brucellosis prevalence

TrAis-os-Montes e Alto Douro, in the northeastern region of Portugal, previously exhibited the highest brucellosis prevalence in the entire small ruminant population of Portugal. Consequently, a vaccination program of the whole population with Brucella melitensis Rev-1 was carried out from 2001 to 2004, and further compulsory Rev-1 vaccination of 3- to 6-month-old lambs and kids was carried out between 2005 and 2007. The prevalence of brucellosis decreased by 71.42% in 2004, with a 2-fold decrease occurring from 2005 to 2007. The reduction of brucellosis prevalence was statistically significant (P < 0.001) according to flock size. By species, brucellosis prevalence decreased 14.5-fold in sheep and 5.5-fold in goats in 2004. Regarding flock constitution, it decreased by 77% for pure flocks and 73% in mixed flocks in 2004. Regarding the animals involved in production, brucellosis prevalence decreased during the 7-year period. It was concluded that Rev-1 vaccination of the whole population was effective in decreasing brucellosis prevalence. These results contribute to the scarce information available regarding the effect of Rev-1 vaccination on different characteristics of flocks. They may be used to improve the efficiency of brucellosis eradication programs within livestock management.

Evaluation in mice of Brucella ovis attenuated mutants for use as live vaccines against B. ovis infection.

Brucella ovis causes ram contagious epididymitis, a disease for which a specific vaccine is lacking. Attenuated Brucella melitensis Rev 1, used as vaccine against ovine and caprine brucellosis caused by B. melitensis, is also considered the best vaccine available for the prophylaxis of B. ovis infection, but its use for this purpose has serious drawbacks. In this work, two previously characterized B. ovis attenuated mutants (Δomp25d and Δomp22) were evaluated in mice, in comparison with B. melitensis Rev 1, as vaccines against B. ovis. Similarities, but also significant differences, were found regarding the immune response induced by the three vaccines. Mice vaccinated with the B. ovis mutants developed anti-B. ovis antibodies in serum of the IgG1, IgG2a and IgG2b subclasses and their levels were higher than those observed in Rev 1-vaccinated mice. After an antigen stimulus with B. ovis cells, splenocytes obtained from all vaccinated mice secreted similar levels of TNF-α and IL12(p40) and remarkably high amounts of IFN-γ, a crucial cytokine in protective immunity against other Brucella species. By contrast, IL-1α -an enhancer of T cell responses to antigen- was present at higher levels in mice vaccinated with the B. ovis mutants, while IL-10, an anti-inflammatory cytokine, was significantly more abundant in Rev 1-vaccinated mice. Additionally, the B. ovis mutants showed appropriate persistence, limited splenomegaly and protective efficacy against B. ovis similar to that observed with B. melitensis Rev 1. These characteristics encourage their evaluation in the natural host as homologous vaccines for the specific prophylaxis of B. ovis infection.