[show abstract][hide abstract] ABSTRACT: 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.
Veterinary Immunology and Immunopathology 03/2012; 146(2):150-8. · 1.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: The World Organisation for Animal Health (OIE) requested an International Standard anti-Brucella melitensis Serum (ISaBmS) to standardise diagnostic tests and reagents for sheep and goats. The agreed criteria were the highest dilution (in negative serum) of the standard which must give a positive result and the lowest dilution (in negative serum) which must simultaneously give a negative result. The two dilutions for each assay were, respectively: indirect enzyme-linked immunosorbent assay (iELISA) 1/64 and 1/750, competitive ELISA (cELISA) 1/8 and 1/300, fluorescent polarisation assay (FPA) 1/16 and 1/200, Rose Bengal test (RBT) 1/16 and 1/200. The OIE International Standard Serum (OIEISS) will remain the primary standard for the RBT; the ISaBmS is an additional standard. It was impossible to set criteria for the complement fixation test, therefore the OIEISS will remain the primary standard. The ISaBmS can be used to standardise iELISA, cELISA and FPA to diagnose sheep and goat brucellosis. This standard should facilitate harmonisation of tests used for brucellosis surveillance and international trade in these species.
Revue scientifique et technique (International Office of Epizootics) 12/2011; 30(3):809-19. · 0.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: Bacteriological diagnosis of brucellosis is performed by culturing animal samples directly on both Farrell medium (FM) and modified Thayer-Martin medium (mTM). However, despite inhibiting most contaminating microorganisms, FM also inhibits the growth of Brucella ovis and some B. melitensis and B. abortus strains. In contrast, mTM is adequate for growth of all Brucella species but only partially inhibitory for contaminants. Moreover, the performance of both culture media for isolating B. suis has never been established properly. We first determined the performance of both media for B. suis isolation, proving that FM significantly inhibits B. suis growth. We also determined the susceptibility of B. suis to the antibiotics contained in both selective media, proving that nalidixic acid and bacitracin are highly inhibitory, thus explaining the reduced performance of FM for B. suis isolation. Based on these results, a new selective medium (CITA) containing vancomycin, colistin, nystatin, nitrofurantoin, and amphotericin B was tested for isolation of the main Brucella species, including B. suis. CITA's performance was evaluated using reference contaminant strains but also field samples taken from brucella-infected animals or animals suspected of infection. CITA inhibited most contaminant microorganisms but allowed the growth of all Brucella species, to levels similar to those for both the control medium without antibiotics and mTM. Moreover, CITA medium was more sensitive than both mTM and FM for isolating all Brucella species from field samples. Altogether, these results demonstrate the adequate performance of CITA medium for the primary isolation of the main Brucella species, including B. suis.
Journal of clinical microbiology 01/2011; 49(4):1458-63. · 4.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Genetics and Genomics 11/2010; 37(11):725-36. · 2.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
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.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
Veterinary Immunology and Immunopathology 12/2008; 127(3-4):295-303. · 1.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: An evaluation of a multiplex PCR assay (Bruce-ladder) was performed in seven laboratories using 625 Brucella strains from different animal and geographical origins. This robust test can differentiate in a single step all of the classical Brucella species, including those found in marine mammals and the S19, RB51, and Rev.1 vaccine strains.
Journal of clinical microbiology 09/2008; 46(10):3484-7. · 4.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
PLoS ONE 01/2008; 3(7):e2760. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Clinical Microbiology 01/2008; 45(12):4070-2. · 4.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: The objective of the present study was to compare the efficacy of gentamicin given alone or combined with doxycycline with that of standard combination therapies in BALB/c mice experimentally infected with the Brucella melitensis vaccine strain Rev 1.
A standard broth microdilution method was applied to determine the susceptibility of strain Rev 1 to the clinically most relevant aminoglycosides. Eight groups of BALB/c mice were inoculated intraperitoneally (ip) with 1 x 10(6) cfu/mouse of strain Rev 1. While one group remained untreated, the other seven groups were treated 10 days later once a day for 14 days with (i) doxycycline given orally at 2 mg/day; (ii) streptomycin given ip at 0.4 mg/day; (iii) gentamicin given ip at 0.4 mg/day; (iv) rifampicin given orally at 0.5 mg/day; (v) doxycycline plus streptomycin; (vi) doxycycline plus gentamicin; and (vii) doxycycline plus rifampicin. The number of cfu per spleen and clearance of Rev 1 were assessed 34 days after inoculation.
With the exception of streptomycin, strain Rev 1 was susceptible to all aminoglycosides tested. As expected, the combination doxycycline/streptomycin was ineffective against Rev 1 infection. In contrast, the combinations doxycycline/gentamicin and doxycycline/rifampicin were effective in the clearance of Rev 1 infection, but only the former improved significantly the therapeutic efficacy as compared with that of the antibiotics given alone.
Gentamicin may be used along with doxycycline when the classical combination is considered the first choice in the treatment of patients with brucellosis due to B. melitensis vaccine strain Rev 1.
Journal of Antimicrobial Chemotherapy 10/2006; 58(3):622-6. · 5.34 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
The Canadian veterinary journal. La revue veterinaire canadienne 11/2005; 46(10):913-6. · 0.77 Impact Factor
[show abstract][hide abstract] ABSTRACT: Yersinia enterocolitica O:9 bears a smooth lipopolysaccharide (S-LPS) of Brucella sp. O-chain A+C/Y epitopic structure and is a cause of false-positive serological reactions (FPSR) in standard tests for cattle brucellosis. Brucella S-LPS, cross-reacting S-LPSs representing several O-chain epitope combinations, Brucella core lipid A epitopes (rough LPS), Brucella abortus S-LPS-derived polysaccharide, native hapten polysaccharide, rough LPS group 3 outer membrane protein complexes, recombinant BP26, and cytosolic proteins were tested in enzyme-linked immunosorbent assays (ELISA) and precipitation tests to detect cattle brucellosis (sensitivity) and to differentiate it from FPSR (specificity). No single serological test and antigen combination showed 100% sensitivity and specificity simultaneously. Immunoprecipitation tests with native hapten polysaccharide, counterimmunoelectrophoresis with cytosolic proteins, and a chaotropic ELISA with Brucella S-LPS were 100% specific but less sensitive than the Rose Bengal test, complement fixation, and indirect ELISA with Brucella S-LPSs and native hapten or S-LPS-derived polysaccharides. A competitive ELISA with Brucella S-LPS and M84 C/Y-specific monoclonal antibody was not 100% specific and was less sensitive than other tests. ELISA with Brucella suis bv. 2 S-LPS (deficient in C epitopes), Escherichia hermannii S-LPSs [lacking the contiguous alpha-(1-2)-linked perosamine residues characteristic of Y. enterocolitica S-LPS], BP26 recombinant protein, and Brucella cytosolic fractions did not provide adequate sensitivity/specificity ratios. Although no serological test and antigen combination fully resolved the diagnosis of bovine brucellosis in the presence of FPSR, some are simple and practical alternatives to the brucellin skin test currently recommended for differential diagnosis.