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.

Highly Sensitive Bacteriophage-Based Detection of Brucella abortus in Mixed Culture and Spiked Blood.

For decades, bacteriophages (phages) have been used for Brucella species identification in the diagnosis and epidemiology of brucellosis. Traditional Brucella phage typing is a multi-day procedure including the isolation of a pure culture, a step that can take up to three weeks. In this study, we focused on the use of brucellaphages for sensitive detection of the pathogen in clinical and other complex samples, and developed an indirect method of Brucella detection using real-time quantitative PCR monitoring of brucellaphage DNA amplification via replication on live Brucella cells. This assay allowed the detection of single bacteria (down to 1 colony-forming unit per milliliter) within 72 h without DNA extraction and purification steps. The technique was equally efficient with Brucella abortus pure culture and with mixed cultures of B. abortus and α-proteobacterial near neighbors that can be misidentified as Brucella spp., Ochrobactrum anthropi and Afipia felis. The addition of a simple short sample preparation step enabled the indirect phage-based detection of B. abortus in spiked blood, with the same high sensitivity. This indirect phage-based detection assay enables the rapid and sensitive detection of live B. abortus in mixed cultures and in blood samples, and can potentially be applied for detection in other clinical samples and other complex sample types.

Brucella abortus RB51 in milk of vaccinated adult cattle.

The aim of this study was to evaluate the shedding of Brucella abortus in the milk of cows vaccinated with a full dose of RB51 during lactation. Eighteen cows, nine previously vaccinated with S19 as calves and nine non-vaccinated, were immunized subcutaneously with 1.3×10(10)CFU of B. abortus RB51, 30-60days after parturition. Milk samples from all animals were collected daily until day 7, and at weekly interval for the next 9 weeks after vaccination. To evaluate the shedding of B. abortus, milk samples were submitted for culture and PCR. No B. abortus was isolated from any sample tested. Only one sample, collected on first day after vaccination from a cow previously vaccinated, was faintly positive in the PCR. In conclusion, the public health hazard associated with milk consumption from cows vaccinated with RB51 in post-partum is very low, despite vaccination with the full dose and regardless of previous S19 vaccination.

VirJ Is a Brucella Virulence Factor Involved in the Secretion of Type IV Secreted Substrates.

The VirB secretion apparatus in Brucella belongs to the type IV secretion systems present in many pathogenic bacteria and is absolutely necessary for the efficient evasion of the Brucella-containing vacuole from the phagocytic route in professional phagocytes. This system is responsible for the secretion of a plethora of effector proteins that alter the biology of the host cell and promote the intracellular replication process. Although many VirB substrates have been identified in Brucella, we still know very little about the secretion mechanism that mediates their translocation across the two membranes and the periplasmic space. In this manuscript, we describe the identification of a gene, virJ, that codes for a protein with periplasmic localization that is involved in the intracellular replication process and virulence in mice. Our analysis revealed that this protein is necessary for the secretion of at least two VirB substrates that have a periplasmic intermediate and that it directly interacts with them. We additionally show that VirJ also associates with the apparatus per se and that its absence affects the assembly of the complex. We hypothesize that VirJ is part of a secretion platform composed of the translocon and several secretion substrates and that it probably coordinates the proper assembly of this macromolecular complex.

Epidemiology of brucellosis among cattle in Korea from 2001 to 2011.

In the present study, the outbreak patterns of bovine brucellosis in Korea from 2000 to 2011 were analyzed to understand the epidemiological evolution of this disease in the country. A total of 85,521 brucella reactor animals were identified during 14,215 outbreaks over the 12-year study period. The number of bovine brucellosis cases increased after 2003 and peaked in 2006 before decreasing thereafter. The majority of the bovine brucellosis cases were Korean native cattle, Han Woo. The numbers of human brucellosis cases and cattle outbreaks increased and decreased in the same pattern. The correlation coefficient for human and bovine cases per year was 0.96 (95% confidence interval = 0.86 ˜ 0.99; p < 10⁻³). The epidemiological characteristics of bovine brucellosis appeared to be affected by the intensity of eradication programs that mainly involved a test- and-slaughter policy. Findings from the present study were based on freely available statistics from web pages maintained by government agencies. This unlimited access to information demonstrates the usefulness of government statistics for continually monitoring the health of animal populations.

Neutrophils exert a suppressive effect on Th1 responses to intracellular pathogen Brucella abortus.

Polymorphonuclear neutrophils (PMNs) are the first line of defense against microbial pathogens. In addition to their role in innate immunity, PMNs may also regulate events related to adaptive immunity. To investigate the influence of PMNs in the immune response during chronic bacterial infections, we explored the course of brucellosis in antibody PMN-depleted C57BL/6 mice and in neutropenic mutant Genista mouse model. We demonstrate that at later times of infection, Brucella abortus is killed more efficiently in the absence of PMNs than in their presence. The higher bacterial removal was concomitant to the: i) comparatively reduced spleen swelling; ii) augmented infiltration of epithelioid histiocytes corresponding to macrophages/dendritic cells (DCs); iii) higher recruitment of monocytes and monocyte/DCs phenotype; iv) significant activation of B and T lymphocytes, and v) increased levels of INF-γ and negligible levels of IL4 indicating a balance of Th1 over Th2 response. These results reveal that PMNs have an unexpected influence in dampening the immune response against intracellular Brucella infection and strengthen the notion that PMNs actively participate in regulatory circuits shaping both innate and adaptive immunity.

Can brucellosis influence the course of chronic hepatitis C in dual infection?

Hepatitis C and brucellosis are infectious diseases that occur worldwide, and both are endemic in Egypt. Co-infection with both agents is possible, and this can involve the liver in various ways. In this study, we investigated serum tissue inhibitor metalloproteinase-1 (TIMP-1), viral load, and liver functions in patients co-infected with hepatitis C virus (HCV) before and after brucellosis treatment. Over 3 years, 241 consecutive HCV patients (before interferon therapy was received) with recurrent fever who had occupational contact with animals were tested for brucellosis co-infection by a standard tube agglutination test. In patients with dual infection, viraemia (RT-PCR), TIMP-1 measured by ELISA, and liver functions were assessed and re-evaluated 2 months after brucellosis treatment. The number of patients with HCV/brucellosis co-infection was 32 out of 241 (13.3%). TIMP-1, viraemia, AST, ALT and bilirubin showed significant decrease (improvement) after brucellosis treatment (p < 0.001) but an insignificant difference (p > 0.05) with regard to serum albumin and prothrombin concentration. The study revealed that brucellosis is an important infection in HCV-infected patients and can aggravate the course of disease, suggesting that early treatment and prevention are important.

Prevalencia de bacterias asociadas a la infertilidad infecciosa en bovinos de Montería, Colombia / Prevalence of bacteria associated with infectious infertility in bovines of Monteria, Colombia

Objetivo. Establecer la prevalencia de algunos gérmenes asociados con infertilidadinfecciosa de bovinos del municipio de Montería. Materiales y métodos. Se recolectaron384 muestras sanguíneas e igual número de hisopados cervicales de hembras quecumplieron con alguno de los siguientes criterios de inclusión: más de tres servicios sinconcepción, vaginitis, cervicitis, metritis, endometritis, útero con contenido, momificaciónfetal, natimortos, reabsorciones embrionarias, antecedentes de abortos y/o hembraspositivas a brucelosis. Las muestras cervicales se sembraron en agar Mc conkey, agarsangre, agar brucella y agar campylobacter. A las colonias sospechosas se le realizaronpruebas bioquímicas y la confirmación se llevó a cabo con pruebas serológicas. Para elserodiagnóstico de Brucella, los sueros fueron analizados por Rosa de Bengala y lospositivos fueron confirmados con ELISA indirecta. Resultados. De las 384 muestrasprocesadas, hubo crecimiento en 281 (73,2%), las bacterias prevalentes fueron Escherichiacoli, Bacilos Gram Negativos Oxidantes, Klepsiella spp y Pseudomonas spp, quecorrespondieron a 194 (69.1%) aislamientos y cinco muestras equivalentes al 1.8%resultaron positivas para Campylobacter spp. Las pruebas serológicas para Brucellaarrojaron como resultado 22 muestras positivas por ELISA (6.3%). Conclusiones. Losresultados de este trabajo describen de forma preliminar la posible asociación bacterianaen la infertilidad infecciosa en hembras vacunas, demostrando la presencia de una granvariedad de microorganismos.

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.

A B lymphocyte mitogen is a Brucella abortus virulence factor required for persistent infection.

Microbial pathogens with the ability to establish chronic infections have evolved strategies to actively modulate the host immune response. Brucellosis is a disease caused by a Gram-negative intracellular pathogen that if not treated during the initial phase of the infection becomes chronic as the bacteria persist for the lifespan of the host. How this pathogen and others achieve this action is a largely unanswered question. We report here the identification of a Brucella abortus gene (prpA) directly involved in the immune modulation of the host. PrpA belongs to the proline-racemase family and elicits a B lymphocyte polyclonal activation that depends on the integrity of its proline-racemase catalytic site. Stimulation of splenocytes with PrpA also results in IL-10 secretion. Construction of a B. abortus-prpA mutant allowed us to assess the contribution of PrpA to the infection process. Mice infected with B. abortus induced an early and transient nonresponsive status of splenocytes to both Escherichia coli LPS and ConA. This phenomenon was not observed when mice were infected with a B. abortus-prpA mutant. Moreover, the B. abortus-prpA mutant had a reduced capacity to establish a chronic infection in mice. We propose that an early and transient nonresponsive immune condition of the host mediated by this B cell polyclonal activator is required for establishing a successful chronic infection by Brucella.

Polirradiculopatía brucelósica con afectación axonal / Brucellar polyradiculoneuropathy with axonal involvement

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Model proficiency testing scheme for serological diagnosis of Brucellosis: interlaboratory study.

A model interlaboratory testing scheme was developed by the Italian National Reference Laboratory for Brucellosis. This scheme was planned for both qualitative (Rose Bengal Plate Test; RBPT) and quantitative (Complement Fixation Test; CFT) serological tests and involved a total of 42 laboratories. In the preparation of this scheme, reference was made to general protocols and guidelines and to methods reported in the literature, which were applicable to analytical chemistry laboratories. Six field sera from naturally infected animals, one positive serum at a titer below the European Union (EU) positivity threshold, and 5 sera positive at titers between 20 and 851 International Units of Complement Fixation Test (IUCFT)/mL plus one negative serum were used to produce a panel of test sera. To evaluate laboratory performances in the quantitative test for each tested sample examined, z-scores based on robust summary statistics (the median and normalized interquartile range) were used. To evaluate overall laboratory performance, 2 types of combined z-scores were used: Rescaled Sum of Scores and Sum of Squared Scores. In the case of the qualitative test (RBPT), results were analyzed by a Bayesian approach. A Beta distribution, based on the result of each laboratory, was calculated and used to estimate the probability of each laboratory giving a correct result and its uncertainty.

Brucelosis y gestación / Brucelosis and pregnancy

La brucelosis es una zoonosis cuyo reservorio lo constituyen los animales domésticos, en particular cabras, ovejas, vacas y cerdos. La enfermedad en nuestro medio se adquiere tras la ingestión de productos lácteos no controlados. La mujer embarazada no escapa a esta infección. En la comarca de Jerte la incidencia asciende a unos 300 casos por 105 habitantes, lo que la convierte en la zona de mayor endemia del país. Analizamos 16 casos de brucelosis asociados a la gestación en los dos últimos años, con los siguientes resultados: una gestante fue diagnosticada tras ingresar de urgencias por feto muerto intraútero a las 19 semanas y síndrome febril, resultando la serología y hemocultivos positivos. En otra se advirtió enfermedad aguda (hemocultivos positivos) a las 13 semanas y se instauró tratamiento con 600 mg/día de rifampicina, tras lo que se produjo un aborto espontáneo a las 16 semanas. La tercera paciente con infección activa se diagnosticó a las 24 semanas. Se le administró monoterapia con rifampicina a dosis de 600 mg/día durante 45 días, con lo que se negativizaron los hemocultivos posteriormente y evolucionaron favorablemente la gestación y el recién nacido. Los 13 casos restantes sin bacteriemia (hemocultivos negativos) fueron seguidos muy de cerca con serología y hemocultivos mensuales hasta finalizar la gestación, sin que aparecieran complicaciones ni se aplicara tratamiento médico a ninguna paciente (AU)