A review of three decades of use of the cattle brucellosis rough vaccine Brucella abortus RB51: myths and facts.

Cattle brucellosis is a severe zoonosis of worldwide distribution caused by Brucella abortus and B. melitensis. In some countries with appropriate infrastructure, animal tagging and movement control, eradication was possible through efficient diagnosis and vaccination with B. abortus S19, usually combined with test-and-slaughter (T/S). Although S19 elicits anti-smooth lipopolysaccharide antibodies that may interfere in the differentiation of infected and vaccinated animals (DIVA), this issue is minimized using appropriate S19 vaccination protocols and irrelevant when high-prevalence makes mass vaccination necessary or when eradication requisites are not met. However, S19 has been broadly replaced by vaccine RB51 (a rifampin-resistant rough mutant) as it is widely accepted that is DIVA, safe and as protective as S19. These RB51 properties are critically reviewed here using the evidence accumulated in the last 35 years. Controlled experiments and field evidence shows that RB51 interferes in immunosorbent assays (iELISA, cELISA and others) and in complement fixation, issues accentuated by revaccinating animals previously immunized with RB51 or S19. Moreover, contacts with virulent brucellae elicit anti-smooth lipopolysaccharide antibodies in RB51 vaccinated animals. Thus, accepting that RB51 is truly DIVA results in extended diagnostic confusions and, when combined with T/S, unnecessary over-culling. Studies supporting the safety of RB51 are flawed and, on the contrary, there is solid evidence that RB51 is excreted in milk and abortifacient in pregnant animals, thus being released in abortions and vaginal fluids. These problems are accentuated by the RB51 virulence in humans, lack diagnostic serological tests detecting these infections and RB51 rifampicin resistance. In controlled experiments, protection by RB51 compares unfavorably with S19 and lasts less than four years with no evidence that RB51-revaccination bolsters immunity, and field studies reporting its usefulness are flawed. There is no evidence that RB51 protects cattle against B. melitensis, infection common when raised together with small ruminants. Finally, data acumulated during cattle brucellosis eradication in Spain shows that S19-T/S is far more efficacious than RB51-T/S, which does not differ from T/S alone. We conclude that the assumption that RB51 is DIVA, safe, and efficaceous results from the uncritical repetition of imperfectly examined evidence, and advise against its use.

Patterns of ambulatory dispersal in Tetranychus urticae can be associated with host plant specialization.

Dispersal can be an essential factor affecting the biological control of pests. Tetranychus urticae Koch (Acari: Tetranychidae) is a cosmopolitan and polyphagous species that may reach the pest status in many cropping systems including clementine orchards, where it may be found both in the trees and the associated flora. In a previous study, we demonstrated that the use of a ground cover of Festuca arundinacea Schreber (Poaceae) offered a better regulation of T. urticae populations than traditional alternatives (bare soil, multifloral wild cover). Therefore, we decided to study the ambulatory dispersal of mites crawling up and down tree trunks in a clementine mandarin orchard grown in association with a F. arundinacea cover for one season. The highest ambulatory migration rate was upward from the cover to the canopy. Multivariate regressions showed that the dynamics of T. urticae populations in the trees was strongly related to that of Phytoseiidae mites, their main natural predators. Surprisingly, canopy populations were not related to those on the ground cover or to those dispersing from it. When T. urticae individuals collected from the ground cover, the tree trunk, and the canopy were subjected to molecular analyses, the optimal number of genetic clusters (demes) was two. One clustergrouped individuals dispersed from the ground cover (e.g. collected on tree trunks) and 27.5% of individuals collected in the ground cover. The second cluster grouped all the individuals collected from trees and 72.5% of those collected in the cover. Interestingly, none of the individuals collected from the tree canopies was grouped with the first deme. This result may be taken as indicative that grass-adapted T. urticae individuals are unable to satisfactorily colonize and establish on the trees and provides evidence that host adaptation can hamper dispersal and establishment of the ground cover deme on trees, contributing to a better natural regulation of this pest species in citrus.

Studies on a suitable antibiotic therapy for treating swine brucellosis.

The aim of this work was developing effective treatments against Brucella suis biovar 2, responsible for swine brucellosis in Europe. MICs for antibiotics used classically in brucellosis and two new macrolides (tulathromycin and tildipirosin) were determined for 33 B. suis biovar 2 field and B. suis reference strains. MIC90 values ranged from 0.01 to 0.25 µg/mL. The best candidates, given alone or combined, were then evaluated in mice. Ten groups (n = 7) of BALB/c mice were inoculated (1 × 10(5) CFU/mouse) with a virulent B. suis biovar 2 field strain. All groups, excepting untreated control, were treated for 14 days with, respectively, doxycycline, dihydrostreptomycin, tulathromycin (one or two doses), or tildipirosin (one or two doses) given alone, and doxycycline combined with dihydrostreptomycin, tulathromycin, or tildipirosin. Combined tildipirosin treatment was the most effective, then selected for pig studies. Sixteen B. suis biovar 2 naturally infected sows were treated with oxytetracycline (20 mg/kg BW/daily) for 21 days. The half of these received also tildipirosin (4 mg/kg BW) in two doses with a 10-day interval. An extensive bacteriological study conducted ten days after ceasing treatments proved the efficacy of this combined oxytetracycline/tildipirosin treatment.

Brucellosis in terrestrial wildlife.

The epidemiological link between brucellosis in wildlife and brucellosis in livestock and people is widely recognised. When studying brucellosis in wildlife, three questions arise: (i) Is this the result of a spillover from livestock or a sustainable infection in one or more host species of wildlife? (ii) Does wildlife brucellosis represent a reservoir of Brucella strains for livestock? (iii) Is it of zoonotic concern? Despite their different host preferences, B. abortus and B. suis have been isolated from a variety of wildlife species, whereas B. melitensis is rarely reported in wildlife. The pathogenesis of Brucella spp. in wildlife reservoirs is not yet fully defined. The prevalence of brucellosis in some wildlife species is very low and thus the behaviour of individual animals, and interactions between wildlife and livestock, may be the most important drivers for transmission. Since signs of the disease are non-pathognomonic, definitive diagnosis depends on laboratory testing, including indirect tests that can be applied to blood or milk, as well as direct tests (classical bacteriology and methods based on the polymerase chain reaction [PCR]). However, serological tests cannot determine which Brucella species has induced anti-Brucella antibodies in the host. Only the isolation of Brucella spp. (or specific DNA detection by PCR) allows a definitive diagnosis, using classical or molecular techniques to identify and type specific strains. There is as yet no brucellosis vaccine that demonstrates satisfactory safety and efficacy in wildlife. Therefore, controlling brucellosis in wildlife should be based on good management practices. At present, transmission of Brucella spp. from wildlife to humans seems to be linked to the butchering of meat and dressing of infected wild or feral pig carcasses in thedeveloped world, and infected African buffalo in the developing world. In the Arctic, the traditional consumption of raw bone marrow and the internal organs of freshly killed caribou or reindeer is an important risk factor.

Haemophilic arthropathy: basic protocols for clinical examination and imaging.

In haemophilia, screening protocols in the prevention and treatment of common lesions still require unification of criteria. Patients with haemophilia seek medical consultation exclusively for two reasons: because they have requested an appointment for a routine check-up (1-2 times a year in case of severe haemophilia) or because they have developed acute bleeding that requires treatment. The purpose of this paper is to emphasize the importance of an early differential diagnosis of joint damage and to review the techniques that allow an effective evaluation. The World Federation of Haemophilia recommends the 'Primary Prophylaxis' treatment modality, and today, severe haemophilia patients adhering to that factor VIII/IX therapy have significantly reduced common injuries: haematomas, haemarthrosis, synovitis, and haemophilic arthropathy. The basic protocols and minimum data for the control of musculoskeletal health are described. In summary, the primary goal of the haematologist-led multidisciplinary care team treating patients with haemophilia is likely to restore and/or preserve joint and musculoskeletal health, which is essential to promoting quality of life. Appropriate factor replacement regimens are required to prevent bleeding, these should be combined with physical activity and a physiotherapy program, in accordance with the recommendations of the World Health Organization for the general population.

Development of a selective culture medium for primary isolation of the main Brucella species.

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.

The first International Standard anti-Brucella melitensis Serum.

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.

Evaluation of a multiplex PCR assay (Bruce-ladder) for molecular typing of all Brucella species, including the vaccine strains.

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.

SAR interferometry monitoring of subsidence in a detritic basin related to water depletion in the underlying confined carbonate aquifer (Torremolinos, southern Spain).

This research underlines the need to improve water management policies for areas linked to confined karstic aquifers subjected to intensive exploitation, and to develop additional efforts towards monitoring their subsidence evolution. We analyze subsidence related to intensive use of groundwater in a confined karstic aquifer, through the use of the InSAR technique, by the southern coast of Spain (Costa del Sol). Carbonates are overlain by an unconfined detritic aquifer with interlayered high transmissivity rocks, in connection with the Mediterranean Sea, where the water level is rather stable. Despite this, an accumulated deformation in the line-of-sight (LOS) direction greater than -100 mm was observed by means of the ERS-1/2 (1992-2000) and Envisat (2003-2009) satellite SAR sensors. During this period, the Costa del Sol experienced a major population increase due to the expansion of the tourism industry, with the consequent increase in groundwater exploitation. The maximum LOS displacement rates recorded during both time spans are respectively -6 mm/yr and -11 mm/yr, respectively. During the entire period, there was an accumulated descent of the confined water level of 140 m, and several fluctuations of more than 80 m correlating with the subsidence trend observed for the whole area. Main sedimentary depocenters (up to 800 m), revealed by gravity prospecting, partly coincide with areas of subsidence maxima; yet ground deformation is also influenced by other factors, the main ones being the fine-grained facies distribution and rapid urbanization due to high touristic pressure.

[Treatment of the dysfunction of the pelvic floor]. / Tratamiento de la disfunción del suelo pélvico.

The perineum is formed by muscle-aponeurotic elements that are integrated under the control of the nervous system. Their alterations are responsible for urogynecological, coloproctologic and sexual pathologies. In order to obtain a successful treatment, it is obliged not to forget the role that plays the perineum in those pathologies. The treatment of the dysfunction of the pelvic floor groups conservative techniques and procedures like changes in life habits, behavioural therapy, biofeedback, electroestimulation (neuromodulation and peripheral electrical stimulation) and training with muscular exercises of the pelvic floor (perineal rehabilitation). The objective of all of them is to improve or to obtain the urinary continence, the strengthening of its musculature to be able to balance pelvic static, to improve the local vascularization and the anorrectal function besides securing a satisfactory sexuality.

Brucellosis in Sub-Saharan Africa: Current challenges for management, diagnosis and control.

Brucellosis is a highly contagious zoonosis caused by bacteria of the genus Brucella and affecting domestic and wild mammals. In this paper, the bacteriological and serological evidence of brucellosis in Sub-Saharan Africa (SSA) and its epidemiological characteristics are discussed. The tools available for the diagnosis and treatment of human brucellosis and for the diagnosis and control of animal brucellosis and their applicability in the context of SSA are presented and gaps identified. These gaps concern mostly the need for simpler and more affordable antimicrobial treatments against human brucellosis, the development of a B. melitensis vaccine that could circumvent the drawbacks of the currently available Rev 1 vaccine, and the investigation of serological diagnostic tests for camel brucellosis and wildlife. Strategies for the implementation of animal vaccination are also discussed.

Efficacy of antibiotic treatment and test-based culling strategies for eradicating brucellosis in commercial swine herds.

Swine brucellosis caused by Brucella suis biovar 2 is an emerging disease in continental Europe. Without effective vaccines being available, the European Food Safety Authority (EFSA) recommends the full depopulation of infected herds as the only strategy to eradicate B. suis outbreaks. Using data collected from 8 herds suffering natural swine brucellosis outbreaks, we assessed the efficacy of four control strategies: (i) oxytetracycline treatment only, as a default scenario, (ii) oxytetracycline treatment combined with skin testing and removal of positive animals, (iii) oxytetracycline treatment combined with serological testing (Rose Bengal test-RBT-and indirect ELISA -iELISA-) and removal of seropositive animals and (iv) oxytetracycline treatment combined with both serological (RBT/iELISA) and skin testing and removal of positive animals. A Susceptible-Infectious-Removal model was used to estimate the reproduction ratio (R) for each strategy. According to this model, the oxytetracycline treatment alone was not effective enough to eradicate the infection. However, this antibiotic treatment combined with diagnostic testing at 4-monthly intervals plus immediate removal of positive animals showed to be effective to eradicate brucellosis independent of the diagnostic test strategy used in an acceptable time interval (1-2 years), depending on the initial number of infected animals.

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

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

Diagnostic performance of serological tests for swine brucellosis in the presence of false positive serological reactions.

Swine brucellosis caused by Brucella suis biovar 2 is an emerging disease in Europe. Currently used diagnostic tests for swine brucellosis detect antibodies to the O-polysaccharide (O-PS) of Brucella smooth lipopolysaccharide (S-LPS) but their specificity is compromised by false-positive serological reactions (FPSRs) when bacteria carrying cross-reacting O-PS infect pigs. FPSRs occur throughout Europe, and the only tool available for a specific B. suis diagnosis is the intradermal test with Brucella protein extracts free of O-PS or S-LPS. Using sera of 162 sows naturally infected by B. suis biovar 2, 406 brucellosis-free sows, and 218 pigs of brucellosis-free farms affected by FPSR, we assessed the diagnostic performance of an indirect ELISA with rough LPS (thus devoid of O-PS) and of gel immunodiffusion, counterimmunoelectrophoresis, latex agglutination and indirect ELISA with O-PS free proteins in comparison with several S-LPS tests (Rose Bengal, complement fixation, gel immunodiffusion and indirect ELISA). When adjusted to 100% specificity, the sensitivity of the rough LPS ELISA was very low (30%), and adoption of other cut-offs resulted in poor specificity/sensitivity ratios. Although their specificity was 100%, the sensitivity of protein tests (ELISA, latex agglutination, counterimmunoelectrophoresis, and gel immunodiffusion) was only moderate (45, 58, 61 and 63%, respectively). Among S-LPS tests, gel immunodiffusion was the only test showing acceptable sensitivity/specificity (68 and 100%, respectively). Despite these shortcomings, and when the purpose is to screen out FPSR at herd level, gel immunodiffusion tests may offer a technically simple and practical alternative to intradermal testing.

A fast data acquisition system for neurophysiological signals based on a personal microcomputer.

A general purpose analog-to-digital conversion system and its interface for a low-cost personal computer (Commodore 64) are described. Special emphasis has been put on achieving a high sampling frequency rate (up to 20,000 samples/s) and a high discrimination (12 bits). It has likewise been attempted to increase the number of input channels (up to 8, by means of a multiplexer) and averaging capability since such features may be useful in a great number of neurobiological studies. Commercially available hardware elements have been employed and software has been developed in BASIC and 6510 Machine Code.

Modulation of the cellular immune response after oral or subcutaneous immunization with microparticles containing Brucella ovis antigens.

An antigenic extract (HS) from Brucella ovis was encapsulated in either poly-epsilon-caprolactone (PEC) or poly-lactic-co-glycolic acid 75:25 (PLGA) microparticles containing beta-cyclodextrin and Pluronic F-68 as stabilising agents. The resulting microparticles displayed sub-5 microm sizes. Antigen loading was 5.2 and 3.8 microg/mg for HS-PEC and HS-PLGA microparticles, respectively. Specific HS cytokine profiles were determined after subcutaneous and oral immunisation of BALB/c mice. Gut distribution studies of the formulations after oral administration showed that HS-PEC microparticles interacted more strongly with mucosa and Peyer's patches than HS-PLGA. Accordingly, oral immunisation with HS-PLGA induced a negligible immune response, whereas HS-PEC elicited a Th1 response although of low intensity. Subcutaneous immunisation with HS-PEC induced high IFN-gamma and IL-2 release; in contrast, HS-PLGA particles induced a Th2 profile characterized by significant levels of IL-4. Splenic cells from free-HS immunised mice released IFN-gamma and IL-2 but not IL-4. A less intense Th1 pattern was also found from HS stimulated nai;ve splenic cells. These results suggest that the HS itself possesses Th1 immunopotentiating properties, required to control brucellosis, that can be specifically increased by encapsulation in PEC microparticles. In contrast, PLGA microparticles modulate the response toward a Th2 pathway.

Immunization with Brucella melitensis Rev 1 against Brucella ovis infection of rams.

The efficacy of Brucella Melitensis Rev 1 vaccine (Rev 1) for the prophylaxis of Brucella ovis ram epididymitis was evaluated. Twenty-nine 3-month-old rams were vaccinated with 2 X 10(9) Rev 1 and 14 were revaccinated with 5 X 10(8) at 14 months of age. Six rams remained unvaccinated as a control group. All rams were challenged with 5 X 10(8) B. ovis at 21 months of age. Before being slaughtered 8 weeks later, only one vaccinated ram developed epididymitis while four of the six control rams developed testicular alterations. Genital and selected extragenital organs and lymph nodes were removed at slaughter and inoculated on selective media. B. ovis was isolated from 26.6% of the vaccinated rams, 21.4% of the revaccinated rams and 100% of control rams. Portions of epididymis, testes and vesicular glands were also used for pathological studies. More severe lesions were observed in control rams than in vaccinated ones. In conclusion, these results show that vaccination of young lambs, followed or not by revaccination, is a suitable method for the prophylaxis of B. ovis infection of rams.

An ELISA with Brucella lipopolysaccharide antigen for the diagnosis of B. melitensis infection in sheep and for the evaluation of serological responses following subcutaneous or conjunctival B. melitensis strain Rev 1 vaccination.

An indirect enzyme-linked immunosorbent assay (ELISA) with unpurified Brucella melitensis smooth lipopolysaccharide (S-LPS) as antigen was evaluated for the serological diagnosis of B. melitensis infection in sheep in comparison with the Rose Bengal (RB), complement fixation (CF), radial immunodiffusion (RID), microplate agglutination (MA) and rivanol agglutination (RIV) tests. Tests RB and CF detected as positive each of the 77 sera from B. melitensis-infected animals tested, the RID (74), MA (76) and the RIV (72) were less sensitive. However, all tests compared were negative when 77 sera from Brucella-free rams were tested. While subcutaneous Rev 1 vaccination induced high response levels in any of the tests, low level responses were obtained upon conjunctival vaccination, particularly in ELISA and RID tests.

Evaluation of a PCR test for the diagnosis of Brucella ovis infection in semen samples from rams.

The sensitivity and specificity of a PCR assay with primers derived from the insertion sequence IS6501 was compared with that of bacteriological culture and serological tests for the diagnosis of Brucella ovis infection in rams. No amplifications were detected with DNAs from the strains phylogenetically related to Brucella and from the seven bacterial species considered as the main etiologic agents of epididymitis in rams. In addition, the specificity of the PCR was 100% when testing semen samples from Brucella-free rams. The comparison of the semen culture and PCR results from 192 semen samples tested, showed a proportion of agreement of 0.91 between both tests. The PCR-based test described has sensitivity similar to that of semen culture and could be used as a complementary test for the direct diagnosis of Brucella ovis in semen samples of rams.

Evaluation of whole cell and subcellular vaccines against Brucella ovis in rams.

Five antigen preparations from Brucella ovis strain REO 198 were incorporated with the pluronic polymer L-121 and muramyl dipeptide and tested as vaccines against B. ovis infection of rams. The antigenic preparations were: (1) a fraction enriched in outer membrane proteins and rough lipopolysaccharide (hot saline extract, HS); (2) the proteins from HS substantially free of lipopolysaccharide; (3) outer membrane blebs; (4) outer membrane-peptidoglycan complexes extracted with detergent; (5) killed whole cells. The experimental vaccines were compared with two standard vaccines, rough Brucella abortus 45/20 whole killed cells in an oil based adjuvant, and live Brucella melitensis Rev 1. Immunizations with non-living vaccines were performed on two occasions, 18 weeks apart. The rams were challenged with a virulent strain of B. ovis 31 weeks after the second vaccination and slaughtered 15 weeks thereafter. Rates of infection in groups vaccinated with Rev 1 (33%), and HS (40%) were significantly lower (P < 0.005 and P < 0.025, respectively) than that in the non-vaccinated control group (87%). Strain 45/20 was the only other vaccine that conferred a significant level of protection (50%) (P < 0.05). The organ distribution of the infection and the level of colonization of infected organs did not differ significantly between infected animals in the various vaccine groups and those in the unvaccinated control group. No statistically significant relationship was detected between the magnitude of the antibody responses to the HS extract, to outer membrane proteins, or to the rough lipopolysaccharide, and freedom from infection. The results indicate that the HS extract of B. ovis may represent a useful alternative to B. melitensis Rev 1 or B. abortus 45/20 as a vaccine against B. ovis.