P. M. Muñoz

Evaluation of a Multiplex PCR Assay (Bruce-ladder) for Molecular Typing of All Brucella Species, Including the Vaccine Strains

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.

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

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. CITAs 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.

Immunopathological responses and kinetics of Brucella melitensis Rev 1 infection after subcutaneous or conjunctival vaccination in rams

The innocuousness of the Brucella melitensis Rev 1 live attenuated vaccine strain has never been fully assessed in rams. The immunopathological responses and the kinetics and distribution of the infection induced by this strain were determined after subcutaneous or conjunctival vaccination in both young (3-4 months old) and adult (12 months old) rams. At regular intervals after vaccination the animals were bled for serological studies, and slaughtered for both pathological and bacteriological examinations. The serological response after conjunctival inoculation was of lower intensity and duration than that induced subcutaneously, being the differences more evident in young rams. No genital lesions were produced and genital organs and accessory sexual glands were never found infected, being Rev 1 infection restricted to lymph nodes and spleen. Immunostained Rev 1 bacteria were located intracellularly in plasmablasts, dendritic follicular cells and macrophages in the target lymph nodes, in which cellular hyperplasia was the dominant pathological response. Subcutaneous vaccination induced a generalized infection by 2 weeks after vaccination, being then restricted to the prescapular target lymph node. Infection after conjunctival vaccination was less generalized, being restricted essentially to the cranial lymph nodes. Rev 1 infection was fully cleared by 3 months after vaccination in all animals. These results confirm the innocuousness of B. melitensis Rev 1 vaccine in rams.

Isolation of Brucella species from a live-stranded striped dolphin (Stenella coeruleoalba) in Spain.

BRUCELLOSIS affects human beings and a wide range of animal species, including marine mammals. The disease in marine mammals was first recognised in 1994, when organisms resembling Brucella species were isolated from free-ranging seals and cetaceans (Ewalt and others 1994, Ross and others 1994). Infections of cetaceans, seals and otters (Foster and others 1996, Van Bressem and others 2001) are now well documented, providing evidence that disease due to Brucella species is highly prevalent in most seas around the world. By contrast, few studies have established a direct relationship between Brucella species infection in marine mammals and pathological changes, including placentitis, abortion, blubber abscesses and chronic meningoencephalitis (Miller and others 1999, Patterson and others 2000, Gonzalez and others 2002). This short communication describes the first known case in Spain of Brucella species infection in a striped dolphin (Stenella coeruleoalba). An adult, female striped dolphin, 166 cm in length, was live-stranded on June 23, 2004, in the Cantabric Sea (Cuchia beach, Cantabria). Records of stranded animals in Cantabria during the past century indicate that 22 per cent of stranding were dolphins of this species, which is abundant in this region. The animal was still alive and apparently not disorientated when the technicians arrived to attempt to return it to deep water. The beach was well protected, and there were no sea currents forcing the stranding. Despite several attempts, the animal refused to move to open waters and always returned to the sand, and eventually died during transportation. The dolphin had a normal external appearance, showing only excoriations, several minor (2 to 3 cm) cuts and one severe ulceration on the muzzle, containing several Cyamus balaenopteridae parasites. The only gross lesions observed were hyperaemia and several white, firm nodules, approximately 2 mm in diameter, scattered throughout the lungs. The digestive tract was empty and the animal showed clear signs of starvation. A sample of serum was tested by the standard rose Bengal agglutination test for brucellosis (Alton and others 1988), and gave a positive result. Samples of lungs, brainstem, cerebellum, uterus, spleen, and cranial and mammary lymph nodes were taken for bacteriological or pathological studies. Portions of the lungs, brainstem and cerebellum were fixed in formalin, embedded in paraffin, sectioned and stained with haematoxylin and eosin by routine procedures for pathological examination. Bronchiolar microcalcifications and small aggregates of leucocytes were evident in the peribronchiolar connective tissue. Fibromuscular hyperplasia of alveolar septa and areas of atelectasis were observed in the lung parenchyma. Several eosinophilic granulomas, containing neutrophils, eosinophils and a few macrophages and associated with parasites (probably Pseudalius inflexus lungworms), were also observed. Most of these lesions are the consequence of infestations with parasites, and have been reported previously in seals infected with Brucella species (Garner and others 1997). Although no specific studies were performed in the present case, Brucella species organisms have previously been identified within the uterus of Parafilaroides species lungworms, and a potential role for these parasites in transmitting brucellosis cannot be ruled out (Garner and others 1997). The main lesions in the central nervous system were multifocal haemorrhage, multifocal or diffuse microgliosis, and perivascular cuffing with lymphocytes and plasmocytes. Perivascular necrotic foci with macrophages, gitter cells and lymphocytes were also present (Fig 1a). Most of the lesions appeared to be angiocentric and were essentially located in Agglutination Growth in Growth in Requirement for with serum thionin fuchsin Strain carbon dioxide anti-A anti-M 10 μg 20 μg 10 μg 20 μg

The extradomain A of fibronectin (EDA) combined with poly(I:C) enhances the immune response to HIV-1 p24 protein and the protection against recombinant Listeria monocytogenes-Gag infection in the mouse model.

The development of effective vaccines against HIV-1 infection constitutes one of the major challenges in viral immunology. One of the protein candidates in vaccination against this virus is p24, since it is a conserved HIV antigen that has cytotoxic and helper T cell epitopes as well as B cell epitopes that may jointly confer enhanced protection against infection when used in immunization-challenge approaches. In this context, the adjuvant effect of EDA (used as EDAp24 fusion protein) and poly(I:C), as agonists of TLR4 and TLR3, respectively, was assessed in p24 immunizations using a recombinant Listeria monocytogenes HIV-1 Gag proteins (Lm-Gag, where p24 is the major antigen) for challenge in mice. Immunization with EDAp24 fusion protein together with poly(I:C) adjuvant induced a specific p24 IFN-γ production (Th1 profile) as well as protection against a Lm-Gag challenge, suggesting an additive or synergistic effect between both adjuvants. The combination of EDA (as a fusion protein with the antigen, which may favor antigen targeting to dendritic cells through TLR4) and poly(I:C) could thus be a good adjuvant candidate to enhance the immune response against HIV-1 proteins and its use may open new ways in vaccine investigations on this virus.

Performance of skin tests with allergens from B. melitensis B115 and rough B. abortus mutants for diagnosing swine brucellosis.

Swine brucellosis by Brucella suis biovar 2 is an emerging disease whose control is based on serological testing and culling. However, current serological tests detect antibodies to the O-polysaccharide (O/PS) moiety of Brucella smooth lipopolysaccharide (S-LPS), and thus lack specificity when infections by Yersinia enterocolitica O:9 and other gram-negative bacteria carrying cross-reacting O/PS occur. The skin test with the protein-rich brucellin extract obtained from rough B. melitensis B115 is assumed to be specific for discriminating these false positive serological reactions (FPSR). However, B115 strain, although unable to synthesize S-LPS, accumulates O/PS internally, which could cause diagnostic problems. Since the brucellin skin test has been seldom used in pigs and FPSR are common in these animals, we assessed its performance using cytosoluble protein extracts obtained from B. abortus rough mutants in manBcore or per genes (critical for O/PS biosynthesis) and B. melitensis B115. The diagnostic sensitivity and specificity were determined in B. suis biovar 2 culture positive and brucellosis free sows, and apparent prevalence in sows of unknown individual bacteriological and serological status belonging to B. suis biovar 2 naturally infected herds. Moreover, the specificity in discriminating brucellosis from FPSR was assessed in brucellosis free boars showing FPSR. The skin test with B. abortus ΔmanBcore and B. melitensis B115 allergens performed similarly, and the former one resulted in 100% specificity when testing animals showing FPSR in indirect ELISA, Rose Bengal and complement fixation serological tests. We conclude that O/PS-free genetically defined mutants represent an appropriate alternative to obtain Brucella protein extracts for diagnosing swine brucellosis.

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.

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.

The extradomain A of fibronectin enhances the efficacy of lipopolysaccharide defective Salmonella bacterins as vaccines in mice

The Extradomain A from fibronectin (EDA) has an immunomodulatory role as fusion protein with viral and tumor antigens, but its effect when administered with bacteria has not been assessed. Here, we investigated the adjuvant effect of EDA in mice immunizations against Salmonella enterica subspecies enterica serovar Enteritidis (Salmonella Enteritidis). Since lipopolysaccharide (LPS) is a major virulence factor and the LPS O-polysaccharide (O-PS) is the immunodominant antigen in serological diagnostic tests, Salmonella mutants lacking O-PS (rough mutants) represent an interesting approach for developing new vaccines and diagnostic tests to differentiate infected and vaccinated animals (DIVA tests). Here, antigenic preparations (hot-saline extracts and formalin-inactivated bacterins) from two Salmonella Enteritidis rough mutants, carrying either intact (SEΔwaaL) or deep-defective (SEΔgal) LPS-Core, were used in combination with EDA. Biotinylated bacterins, in particular SEΔwaaL bacterin, decorated with EDAvidin (EDA and streptavidin fusion protein) improved the protection conferred by hot-saline or bacterins alone and prevented significantly the virulent infection at least to the levels of live attenuated rough mutants. These findings demonstrate the adjuvant effect of EDAvidin when administered with biotinylated bacterins from Salmonella Enteritidis lacking O-PS and the usefulness of BEDA-SEΔwaaL as non-live vaccine in the mouse model.

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.