Genomic and phenotypic analysis of invasive Streptococcus suis isolated in Spain reveals genetic diversification and associated virulence traits.

Streptococcus suis is a zoonotic pathogen that causes a major health problem in the pig production industry worldwide. Spain is one of the largest pig producers in the world. This work aimed to investigate the genetic and phenotypic features of invasive S. suis isolates recovered in Spain. A panel of 156 clinical isolates recovered from 13 Autonomous Communities, representing the major pig producers, were analysed. MLST and serotyping analysis revealed that most isolates (61.6%) were assigned to ST1 (26.3%), ST123 (18.6%), ST29 (9.6%), and ST3 (7.1%). Interestingly, 34 new STs were identified, indicating the emergence of novel genetic lineages. Serotypes 9 (27.6%) and 1 (21.8%) prevailed, followed by serotypes 7 (12.8%) and 2 (12.2%). Analysis of 13 virulence-associated genes showed significant associations between ST, serotype, virulence patterns, and clinical features, evidencing particular virulence traits associated with genetic clusters. The pangenome was generated, and the core genome was distributed in 7 Bayesian groups where each group included a variable set of over- and under-represented genes of different categories. The study provides comprehensive data and knowledge to improve the design of new vaccines, antimicrobial treatments, and bacterial typing approaches.

Invasive Streptococcus suis isolated in Spain contain a highly promiscuous and dynamic resistome.

Introduction: Streptococcus suis is a major pathogen for swine and human. Here we aimed to know the rates of antimicrobial resistance (AMR) in invasive S. suis isolates recovered along Spain between 2016 - 2021 and elucidate their genetic origin. Methods: Antibiotic susceptibility testing was performed for 116 isolates of different genetic backgrounds and geographic origins against 18 antibiotics of 9 families. The association between AMR and genotypes and the origin of the isolates were statistically analyzed using Pearson´s chi-square test and the likelihood ratio. The antimicrobial resistant genes were identified by whole genome sequencing analysis and PCR screenings. Results: High AMR rates (>80%) were detected for tetracyclines, spectinomycin, lincosamides, and marbofloxacin, medium (20-40%) for sulphonamides/trimethoprim, tiamulin, penicillin G, and enrofloxacin, and low (< 20%) for florfenicol, and four additional ß-lactams. The occurrence of multidrug resistance was observed in 90% of isolates. For certain antibiotics (penicillin G, enrofloxacin, marbofloxacin, tilmicosin, and erythromycin), AMR was significantly associated with particular sequence types (STs), geographic regions, age of pigs, and time course. Whole genome sequencing comparisons and PCR screenings identified 23 AMR genes, of which 19 were previously reported in S. suis (aph(3')-IIIa, sat4, aadE, spw, aac(6')-Ie-aph(2'')-Ia, fexA, optrA, erm(B), mef(A/E), mrs(D), mph(C), lnu(B), lsa(E), vga(F), tet(M), tet(O), tet(O/W/32/O), tet(W)), and 4 were novel (aph(2'')-IIIa, apmA, erm(47), tet(T)). These AMR genes explained the AMR to spectinomycin, macrolides, lincosamides, tiamulin, and tetracyclines. Several genes were located on mobile genetic elements which showed a variable organization and composition. As AMR gene homologs were identified in many human and animal pathogens, the resistome of S. suis has a different phylogenetic origin. Moreover, AMR to penicillin G, fluoroquinolones, and trimethoprim related to mutations in genes coding for target enzymes (pbp1a, pbp2b, pbp2x, mraY, gyrA, parC, and dhfr). Bioinformatic analysis estimated traits of recombination on target genes, also indicative of gene transfer events. Conclusions: Our work evidences that S. suis is a major contributor to AMR dissemination across veterinary and human pathogens. Therefore, control of AMR in S. suis should be considered from a One Health approach in regions with high pig production to properly tackle the issue of antimicrobial drug resistance.

Disease control tools to secure animal and public health in a densely populated world.

Animal health is a prerequisite for global health, economic development, food security, food quality, and poverty reduction, while mitigating against climate change and biodiversity loss. We did a qualitative review of 53 infectious diseases in terrestrial animals with data from DISCONTOOLS, a specialist database and prioritisation model focusing on research gaps for improving infectious disease control in animals. Many diseases do not have any appropriate control tools, but the prioritisation model suggests that we should focus international efforts on Nipah virus infection, African swine fever, contagious bovine pleuropneumonia, peste des petits ruminants, sheeppox and goatpox, avian influenza, Rift Valley fever, foot and mouth disease, and bovine tuberculosis, for the greatest impact on the UN's Sustainable Development Goals. Easy to use and accurate diagnostics are available for many animal diseases. However, there is an urgent need for the development of stable and durable diagnostics that can differentiate infected animals from vaccinated animals, to exploit rapid technological advances, and to make diagnostics widely available and affordable. Veterinary vaccines are important for dealing with endemic, new, and emerging diseases. However, fundamental research is needed to improve the convenience of use and duration of immunity, and to establish performant marker vaccines. The largest gap in animal pharmaceuticals is the threat of pathogens developing resistance to available drugs, in particular for bacterial and parasitic (protozoal, helminth, and arthropod) pathogens. We propose and discuss five research priorities for animal health that will help to deliver a sustainable and healthy planet: vaccinology, antimicrobial resistance, climate mitigation and adaptation, digital health, and epidemic preparedness.

Resistance to colistin and production of extended-spectrum ß-lactamases and/or AmpC enzymes in Salmonella isolates collected from healthy pigs in Northwest Spain in two periods: 2008-2009 and 2018.

Salmonellosis is a common subclinical infection in pigs and therefore apparently healthy animals may represent a reservoir of antibiotic-resistant Salmonella for humans. This study estimates and characterizes resistance to two classes of antimicrobials considered of the highest priority within the critically important antimicrobials for humans, i.e. colistin (CR) and 3rd generation cephalosporins (3GC), on a collection of Salmonella isolates from pigs from two periods: between 2008 and 09, when colistin was massively used; and in 2018, after three years under a National Plan against Antibiotic Resistance. Prevalence of CR was low (6 out of 625; 0.96%; 95%CI: 0.44-2.1) in 2008-09 and associated mostly to the mcr-1 gene, which was detected in four S. 4,5,12:i:- isolates. Polymorphisms in the pmrAB genes were detected in a S. 9,12:-:- isolate. No CR was detected in 2018 out of 59 isolates tested. Among 270 Salmonella isolates considered for the assessment of resistance to 3GC in the 2008-2009 sampling, only one Salmonella Bredeney (0.37%; 95%CI: 0.07-2.1) showed resistance to 3GC, which was associated with the blaCMY-2 gene (AmpC producer). In 2018, six isolates out of 59 (10.2%; 95%CI: 4.7-20.5) showed resistance to 3GC, but only two different strains were identified (S. 4,12:i:- and S. Rissen), both confirmed as extended-spectrum ß-lactamases (ESBL) producers. The blaCTX-M-3 and blaTEM-1b genes in S. 4,12:i:- and the blaTEM-1b gene in S. Rissen seemed to be associated with this resistance. Overall, the prevalence of CR in Salmonella appeared to be very low in 2008-2009 despite the considerable use of colistin in pigs at that time, and seemed to remain so in 2018. Resistance to 3GC was even lower in 2008-2009 but somewhat higher in 2018. Resistance was mostly coded by genes associated with mobile genetic elements. Most serotypes involved in these antimicrobial resistances displayed a multidrug resistance pattern and were considered zoonotic.

Assessment of performance of selected serological tests for diagnosing brucellosis in pigs.

Swine brucellosis due to Brucella suis is considered an emerging zoonotic disease whose control is based on serological testing and the subsequent culling of seropositive animals or the full depopulation of affected flocks. Here we assessed the performance of several serological tests (Rose Bengal Test [RBT], indirect ELISA [i-ELISA], blocking ELISA [b-ELISA], and two competitive ELISAs [c-ELISA]) for diagnosing swine brucellosis caused by B. suis biovar 2. Both frequentistic and Bayesian statistical inference were used. A frequentistic analysis, using sera from known gold standard (GS) populations (i.e., from truly infected or brucellosis free animals), resulted in maximum (100%) diagnostic sensitivity (Se) and specificity (Sp) in the RBT, i-ELISA and b-ELISA tests. However, c-ELISAs resulted in lower diagnostic Se (ranging from 68.5% to 92.6%, according to the different cut-offs selected). A Bayesian analysis of tests yielding the best diagnostic performance with GS sera (RBT, i-ELISA and b-ELISA), but using a large collection of field sera, resulted in similar Se among tests but markedly lower (≈ 80%) than that resulting from the frequentistic analysis using the GS serum populations. By contrast, the estimated Sp in the Bayesian analysis was only slightly lower than 100%, thus similar to that obtained frequentistically. Our results show that adequate diagnostic tests for brucellosis in swine are available, but also emphasize the need for more extensive validation studies before applying these tests under field conditions.

Spontaneous excision of the O-polysaccharide wbkA glycosyltranferase gene is a cause of dissociation of smooth to rough Brucella colonies.

The brucellae are Gram-negative pathogens that cause brucellosis, a zoonosis of worldwide importance. The genus Brucella includes smooth and rough species that differ in that they carry smooth and rough lipopolysaccharides, respectively. Brucella abortus, B. melitensis, and B. suis are typical smooth species. However, these smooth brucellae dissociate into rough mutants devoid of the lipopolysaccharide O-polysaccharide, a major antigen and a virulence determinant encoded in regions wbo (included in genomic island-2) and wbk. We demonstrate here the occurrence of spontaneous recombination events in those three Brucella species leading to the deletion of a 5.5-kb fragment carrying the wbkA glycosyltranferase gene and to the appearance of rough mutants. Analysis of the recombination intermediates suggested homologous recombination between the ISBm1 insertion sequences flanking wbkA as the mechanism generating the deletion. Excision of wbkA was reduced but not abrogated in a recA-deficient mutant, showing the existence of both RecA-dependent and -independent processes. Although the involvement of the ISBm1 copies flanking wbkA suggested a transpositional event, the predicted transpositional joint could not be detected. This absence of detectable transposition was consistent with the presence of polymorphism in the inverted repeats of one of the ISBm1 copies. The spontaneous excision of wbkA represents a novel dissociation mechanism of smooth brucellae that adds to the previously described excision of genomic island-2. This ISBm1-mediated wbkA excision and the different %GC levels of the excised fragment and of other wbk genes suggest that the Brucella wbk locus is the result of at least two horizontal acquisition events.

New Bruce-ladder multiplex PCR assay for the biovar typing of Brucella suis and the discrimination of Brucella suis and Brucella canis.

Rapid and specific identification of Brucella suis at the biovar level is necessary because some of the biovars that infect animals are pathogenic for humans. None of the molecular typing methods described so far are able to discriminate B. suis biovars in a single test and differentiation of B. suis from Brucella canis by molecular approaches can be difficult. This article describes a new multiplex PCR assay, Suis-ladder, for fast and accurate identification of B. suis at the biovar level and the differentiation of B. suis, B. canis and Brucella microti. An advancement of the original Bruce-ladder PCR protocol which allows the correct discrimination of all known Brucella species is also described.

Gene expression changes in spleens of the wildlife reservoir species, Eurasian wild boar (Sus scrofa), naturally infected with Brucella suis biovar 2.

Brucella suis is responsible for swine brucellosis worldwide. Of the five different B. suis biovars (bv.), bv. 2 appears restricted to Europe where it is frequently isolated from wild boar and hares, can infect pigs and can cause human brucellosis. In this study, the differential gene expression profile was characterized in spleens of Eurasian wild boar naturally infected with B. suis bv. 2. Of the 20,201 genes analyzed in the microarray, 633 and 1,373 were significantly (fold change > 1.8; P < 0.01) upregulated and downregulated, respectively, in infected wild boar. The analysis was focused on genes that were over represented after conditional test for biological process gene ontology. Upregulated genes suggested that B. suis bv. 2 infection induced cell maturation, migration and/or proliferation in infected animals. The genes downregulated in infected wild boar impaired the activity of several important cellular metabolic pathways such as metabolism, cytoskeleton organization and biogenesis, immune response and lysosomal function and vesicle-mediated transport. In addition, the response to stress, sperm fertility, muscle development and apoptosis seemed to be also impaired in infected animals. These results suggested that B. suis bv. 2 may use strategies similar to other smooth brucellae to facilitate intracellular multiplication and the development of chronic infections. To our knowledge, this is the first report of the analysis of gene expression profile in hosts infected with B. suis bv. 2, which is important to understand the molecular mechanisms at the host-pathogen interface in the main reservoir species with possible implications in the zoonotic cycle of the pathogen.

Spatial distribution and risk factors of Brucellosis in Iberian wild ungulates.

BACKGROUND: The role of wildlife as a brucellosis reservoir for humans and domestic livestock remains to be properly established. The aim of this work was to determine the aetiology, apparent prevalence, spatial distribution and risk factors for brucellosis transmission in several Iberian wild ungulates. METHODS: A multi-species indirect immunosorbent assay (iELISA) using Brucella S-LPS antigen was developed. In several regions having brucellosis in livestock, individual serum samples were taken between 1999 and 2009 from 2,579 wild bovids, 6,448 wild cervids and4,454 Eurasian wild boar (Sus scrofa), and tested to assess brucellosis apparent prevalence. Strains isolated from wild boar were characterized to identify the presence of markers shared with the strains isolated from domestic pigs. RESULTS: Mean apparent prevalence below 0.5% was identified in chamois (Rupicapra pyrenaica), Iberian wild goat (Capra pyrenaica), and red deer (Cervus elaphus). Roe deer (Capreolus capreolus), fallow deer (Dama dama), mouflon (Ovis aries) and Barbary sheep (Ammotragus lervia) tested were seronegative. Only one red deer and one Iberian wild goat resulted positive in culture, isolating B. abortus biovar 1 and B. melitensis biovar 1, respectively. Apparent prevalence in wild boar ranged from 25% to 46% in the different regions studied, with the highest figures detected in South-Central Spain. The probability of wild boar being positive in the iELISA was also affected by age, age-by-sex interaction, sampling month, and the density of outdoor domestic pigs. A total of 104 bacterial isolates were obtained from wild boar, being all identified as B. suis biovar 2. DNA polymorphisms were similar to those found in domestic pigs. CONCLUSIONS: In conclusion, brucellosis in wild boar is widespread in the Iberian Peninsula, thus representing an important threat for domestic pigs. By contrast, wild ruminants were not identified as a significant brucellosis reservoir for livestock.

Evaluation of particulate acellular vaccines against Brucella ovis infection in rams.

The attenuated Brucella melitensis Rev 1 vaccine, used against brucellosis infection, interferes with serological diagnosis tests, may induce abortions in pregnant animals, and may infect humans. In order to overcome these drawbacks, we developed acellular vaccines based on a Brucella ovis antigenic complex (HS) containing outer membrane proteins and R-LPS entrapped in poly(anhydride) conventional and mannosylated nanoparticles (NP-HS and MAN-NP-HS) or in poly(epsilon-caprolactone) microparticles (HS-PEC) as antigen delivery systems and immunoadjuvants. Brucellosis free rams were vaccinated subcutaneously with a single dose of particles containing 3mg of HS, and challenged 6 months thereafter. Protection was evaluated by clinical, bacteriological and serological examinations, in comparison with non-vaccinated control rams. HS-PEC vaccine induced protection (7 out of 13 animals were infected) equivalent to that induced by the reference Rev 1 vaccine (8/14). In contrast, animals immunized with NP-HS were not protected, showing similar results to that obtained in the control unvaccinated rams. Furthermore HS-PEC vaccine did not interfere against B. melitensis serodiagnostic tests. In summary, HS-PEC microparticles could be used as a safe and effective vaccine against brucellosis in rams.

Characterization of possible correlates of protective response against Brucella ovis infection in rams immunized with the B. melitensis Rev 1 vaccine.

Vaccination with the live attenuated Brucella melitensis Rev 1 vaccine is used to control ovine brucellosis caused by Brucella ovis in sheep. The objective of this study was to identify possible correlates of protective response to B. ovis infection through the characterization by microarray hybridization and real-time RT-PCR of inflammatory and immune response genes differentially expressed in rams previously immunized with B. melitensis Rev 1 and experimentally challenged with B. ovis. Gene expression profiles were compared before and after challenge with B. ovis between rams protected and those vaccinated but found infected after challenge. The TLR10, Bak and ANXI genes were expressed at higher levels in vaccinated and protected rams. These genes provide possible correlates of protective response to B. ovis infection in rams immunized with the B. melitensis Rev 1 vaccine.

Rough mutants defective in core and O-polysaccharide synthesis and export induce antibodies reacting in an indirect ELISA with smooth lipopolysaccharide and are less effective than Rev 1 vaccine against Brucella melitensis infection of sheep.

Classical brucellosis vaccines induce antibodies to the O-polysaccharide section of the lipopolysaccharide that interfere in serodiagnosis. Brucella rough (R) mutants lack the O-polysaccharide but their usefulness as vaccines is controversial. Here, Brucella melitensis R mutants in all main lipopolysaccharide biosynthetic pathways were evaluated in sheep in comparison with the reference B. melitensis Rev 1 vaccine. In a first experiment, these mutants were tested for ability to induce anti-O-polysaccharide antibodies, persistence and spread through target organs, and innocuousness. Using the data obtained and those of genetic studies, three candidates were selected and tested for efficacy as vaccines against a challenge infecting 100% of unvaccinated ewes. Protection by R vaccines was 54% or less whereas Rev 1 afforded 100% protection. One-third of R mutant vaccinated ewes became positive in an enzyme-linked immunosorbent assay with smooth lipopolysaccharide due to the core epitopes remaining in the mutated lipopolysaccharide. We conclude that R vaccines interfere in lipopolysaccharide immunosorbent assays and are less effective than Rev 1 against B. melitensis infection of sheep.

Differential expression of inflammatory and immune response genes in rams experimentally infected with a rough virulent strain of Brucella ovis.

Infection of sheep with Brucella ovis results in ovine brucellosis, a disease characterized by infertility in rams, abortion in ewes and increased perinatal mortality in lambs. During the course of the infection both the ovine immune response and host cell gene expression are modified. The objective of this research was to conduct a preliminary characterization of differential gene expression in rams experimentally infected with B. ovis by microarray hybridization and real-time RT-PCR. Of the 600 ruminant inflammatory and immune response genes that were analyzed in the microarray, 20 and 14 genes displayed an expression fold change >1.75 with a P-value <0.05 at 15 and 60 days post-challenge (dpc), respectively. Of these genes, 16 were upregulated and 4 were downregulated in infected rams at 15 dpc. At 60 dpc, 11 and 3 genes were up- and down-regulated in infected rams, respectively. Only four genes, desmoglein, epithelial sodium channel, alpha subunit (ENaC-alpha), interleukin 18 binding protein (IL18BP) and macrophage migration inhibition factor (MIF) were found upregulated in infected rams at both 15 and 60 dpc. The analysis of differentially expressed genes demonstrated activation of inflammatory and innate immune pathways in infected animals. B. ovis infection also resulted in upregulation of genes involved in phagocytosis and downregulation of protective host defense mechanisms, both of which may contribute to the chronicity of B. ovis infection. The gene expression profiles differed between rams with severe and moderate B. ovis infection. This is the first analysis of differential gene expression in rough brucellae and particularly in B. ovis-infected rams. The characterization of the genes and their expression profiles in response to B. ovis infection further contributes to our understanding of the molecular mechanisms of infection and the pathogenesis of brucellosis.

Brucellosis vaccines: assessment of Brucella melitensis lipopolysaccharide rough mutants defective in core and O-polysaccharide synthesis and export.

BACKGROUND: The brucellae are facultative intracellular bacteria that cause brucellosis, one of the major neglected zoonoses. In endemic areas, vaccination is the only effective way to control this disease. Brucella melitensis Rev 1 is a vaccine effective against the brucellosis of sheep and goat caused by B. melitensis, the commonest source of human infection. However, Rev 1 carries a smooth lipopolysaccharide with an O-polysaccharide that elicits antibodies interfering in serodiagnosis, a major problem in eradication campaigns. Because of this, rough Brucella mutants lacking the O-polysaccharide have been proposed as vaccines. METHODOLOGY/PRINCIPAL FINDINGS: To examine the possibilities of rough vaccines, we screened B. melitensis for lipopolysaccharide genes and obtained mutants representing all main rough phenotypes with regard to core oligosaccharide and O-polysaccharide synthesis and export. Using the mouse model, mutants were classified into four attenuation patterns according to their multiplication and persistence in spleens at different doses. In macrophages, mutants belonging to three of these attenuation patterns reached the Brucella characteristic intracellular niche and multiplied intracellularly, suggesting that they could be suitable vaccine candidates. Virulence patterns, intracellular behavior and lipopolysaccharide defects roughly correlated with the degree of protection afforded by the mutants upon intraperitoneal vaccination of mice. However, when vaccination was applied by the subcutaneous route, only two mutants matched the protection obtained with Rev 1 albeit at doses one thousand fold higher than this reference vaccine. These mutants, which were blocked in O-polysaccharide export and accumulated internal O-polysaccharides, stimulated weak anti-smooth lipopolysaccharide antibodies. CONCLUSIONS/SIGNIFICANCE: The results demonstrate that no rough mutant is equal to Rev 1 in laboratory models and question the notion that rough vaccines are suitable for the control of brucellosis in endemic areas.

Comparison of multiple-locus variable-number tandem-repeat analysis with other PCR-based methods for typing Brucella suis isolates.

Multiple-locus variable-number tandem-repeat analysis (MLVA), multiplex PCR, and PCR-restriction fragment length polymorphism analysis were compared for typing Brucella suis isolates. A perfect concordance was obtained among these molecular assays. However, MLVA was the only method to demonstrate brucellosis outbreaks and to confirm that wildlife is a reservoir for zoonotic brucellosis.

Assessment of genetic stability of Brucella melitensis Rev 1 vaccine strain by multiple-locus variable-number tandem repeat analysis.

The assessment of the genetic stability is one of the essential elements to guarantee the biological quality of live anti-bacteria vaccines. Live attenuated Brucella melitensis Rev 1 is the most effective vaccine against brucellosis in small ruminants. Thirty-six B. melitensis Rev 1 vaccine strains isolated from human or animal sources from different geographic regions, from different commercial batches or laboratory collections were typed by the multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) recently described for Brucella spp. Our results demonstrated that B. melitensis Rev 1 group as assayed by MLVA is genetically very homogeneous. We believe that MLVA methodology could be an essential assay to guarantee the quality and stability of live anti-bacterial vaccines being produced worldwide and can be included as in vitro control.

Residual virulence and immunogenicity of CGV26 and CGV2631 B. melitensis Rev. 1 deletion mutant strains in sheep after subcutaneous or conjunctival vaccination.

The CGV26 and CGV2631 strains are novel engineered Brucella melitensis Rev.1 mutant strains deleted for the bp26 gene or for both bp26 and omp31 genes, respectively, coding for proteins of diagnostic significance. The residual virulence and immunogenicity of both mutants were compared to the parental Rev.1 strain in sheep after subcutaneous or conjunctival vaccination. The deletion of the bp26 gene or both bp26 and omp31 genes had no significant effect on the intracellular survival of the Rev.1 strain in ovine macrophage cultures. The kinetics of infection induced by both mutants in sheep was similar to the Rev.1 strain, and inoculation by the subcutaneous route produced wider and more generalized infections than the conjunctival route. All strains were cleared from lymph nodes and organs within 3 months after inoculation. The CGV26 and CGV2631 mutants induced both specific systemic antibody response and lymphoproliferation in sheep. The kinetics of the responses induced by the mutants was quite similar to that of the parental Rev.1 strain, except for the intensity of the lymphoproliferative response, which was attenuated for the CGV2631 mutant. In conclusion, the residual virulence of both CGV26 and CGV2631 mutants in sheep was similar to that of the parental Rev.1 vaccine strain. These mutants induced also significant specific antibody and cell-mediated immunity in sheep and are suitable to be evaluated as potential vaccine candidates against B. melitensis and B. ovis infections in sheep.

Brucella outer membrane complex-loaded microparticles as a vaccine against Brucella ovis in rams.

Due to the important drawbacks of the Brucella melitensis Rev 1 vaccine, a safer vaccine based on an outer membrane complex from Brucella ovis encapsulated in poly-epsilon-caprolactone (PEC) microparticles (MP) was developed and tested in rams. Homogeneous batches of microparticles were prepared by a new double emulsion solvent evaporation method called "Total Recirculation One-Machine System" (TROMS). Such microparticles presented a mean diameter of 2 microm and displayed an antigen loading of about 13 microg HS per mg of microparticles. Subcutaneous vaccination of rams with 800 microg HS (hot saline antigenic extract of B. ovis) in PEC microparticles induced an adequate serological response against B. ovis antigens and conferred similar protection against challenge with B. ovis to that induced by the living attenuated B. melitensis Rev 1 reference vaccine. By contrast, lower doses (80 microg) of HS-PEC evoked reduced serological responses against B. ovis antigens and did not induce significant protection. The revaccination with 800 microg of HS-PEC increased the intensity and duration of the serological response against B. ovis antigens but did not improve the protection conferred by the single vaccination. Sample sera taken from any of the animals immunized with Rev 1 were seropositive in both Rose Bengal and the Complement Fixation tests (RBT, CFT) used for the diagnosis of smooth Brucella infections. By contrast, no positive reactors in both tests were recorded in the animals vaccinated with HS-PEC, being this a target objective of this study. HS-PEC microparticles can be used as a safe vaccine against brucellosis in rams, but further studies using higher doses of antigens are necessary to exploit their full potential for the prophylaxis of brucellosis in sheep.

Specificity dependence between serological tests for diagnosing bovine brucellosis in Brucella-free farms showing false positive serological reactions due to Yersinia enterocolitica O:9.

When brucellosis false positive serological reactions happen in cattle, the serial use of pairs of specificity-correlated serological tests (rose bengal, complement fixation, competitive ELISA) results in specificities lower than expected. In this situation, highly specific tests, such as the indirect ELISA used alone, may be more adequate than serial testing.