C. M. Marín

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.

Efficacy of several serological tests and antigens for diagnosis of bovine brucellosis in the presence of false-positive serological results due to Yersinia enterocolitica O:9

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 α-(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.

Characterization of Brucella abortus O-polysaccharide and core lipopolysaccharide mutants and demonstration that a complete core is required for rough vaccines to be efficient against Brucella abortus and Brucella ovis in the mouse model.

ABSTRACT Brucella abortus rough lipopolysaccharide (LPS) mutants were obtained by transposon insertion into two wbk genes (wbkA [putative glycosyltransferase; formerly rfbU] and per [perosamine synthetase]), into manB (pmm [phosphomannomutase; formerly rfbK]), and into an unassigned gene. Consistent with gene-predicted roles, electrophoretic analysis, 2-keto-3-manno-d-octulosonate measurements, and immunoblots with monoclonal antibodies to O-polysaccharide, outer and inner core epitopes showed no O-polysaccharide expression and no LPS core defects in the wbk mutants. The rough LPS of manB mutant lacked the outer core epitope and the gene was designated manBcore to distinguish it from the wbk manBO-Ag. The fourth gene (provisionally designated wa**) coded for a putative glycosyltransferase involved in inner core synthesis, but the mutant kept the outer core epitope. Differences in phage and polymyxin sensitivity, exposure or expression of outer membrane protein, core and lipid A epitopes, and lipid A acylation demonstrated that small changes in LPS core caused significant differences in B. abortus outer membrane topology. In mice, the mutants showed different degrees of attenuation and induced antibodies to rough LPS and outer membrane proteins. Core-defective mutants and strain RB51 were ineffective vaccines against B. abortus in mice. The mutants per and wbkA induced protection but less than the standard smooth vaccine S19, and controls suggested that anti O-polysaccharide antibodies accounted largely for the difference. Whereas no core-defective mutant was effective against B. ovis, S19, RB51, and the wbkA and per mutants afforded similar levels of protection. These results suggest that rough Brucella vaccines should carry a complete core for maximal effectiveness.

Neurobrucellosis in Stranded Dolphins, Costa Rica

Ten striped dolphins, Stenella coeruleoalba, stranded along the Costa Rican Pacific coast, had meningoencephalitis and antibodies against Brucella spp. Brucella ceti was isolated from cerebrospinal fluid of 6 dolphins and 1 fetus. S. coeruleoalba constitutes a highly susceptible host and a potential reservoir for B. ceti transmission.

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.

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.

Experimental Brucella ovis infection in pregnant ewes

Forty yearling Brucella-free ewes were inoculated with Brucella ovis by the conjunctival route in mid or late first pregnancy. Only a few ewes excreted B ovis during pregnancy and gave birth to stillborn lambs, but most of them excreted the organism at lambing or during lactation. One of the 11 lambs which were born alive but died before they were weaned was found to be infected postmortem. In contrast, none of the 46 surviving lambs which were reared in isolation until adulthood, was found to be infected. At weaning, the 40 ewes were mated again with five Brucella-free rams. Although many of the ewes excreted B ovis, none of the rams was found to be infected when necropsied after mating. Most of the ewes that became pregnant, all having excreted B ovis during their first pregnancy, cleared the infection during the second pregnancy. However, three remained persistently infected and excreted B ovis in their milk throughout the second lactation. None of the lambs born to these three ewes was found to be infected when necropsied at weaning.

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

Efficacy of bp26 and bp26/omp31 B. melitensis Rev.1 deletion mutants against Brucella ovis in rams

Brucella melitensis Rev.1 is the most effective vaccine against B. ovis infection in sheep but induces antibodies interfering with B. melitensis diagnosis. Brucella BP26 and Omp31 proteins are differential diagnostic antigens. Single or double bp26 and omp31 Rev.1 deletion mutants have been proven effective against B. melitensis in sheep. Here, the CGV26 (deleted in bp26 gene) and CGV2631 (deleted in both bp26 and omp31 genes) mutants have been tested for efficacy against B. ovis in rams. Either inoculated subcutaneously or conjunctivally, both mutants conferred significant protection against B. ovis. The protection induced by CGV26 was similar to that of Rev.1 but significantly higher than that conferred by CGV2631. In conclusion, the CGV26 mutant, in association with the adequate diagnostic strategy, could be a useful alternative to Rev.1 for sheep vaccination against B. ovis infections in those countries performing simultaneously B. melitensis and B. ovis eradication campaigns.