BackgroundThe 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.Methodology/Principal FindingsTo 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.Conclusions/SignificanceThe 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.

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.Brucellosis is caused by a facultative intracellular bacterium of the genus Brucella, and it is one of the most frequent bacterial zoonoses in low-income countries, where the control programs have not succeeded in eradicating this neglected zoonosis. The disease is a major cause of direct economic losses and an impediment to trade and exportation. The genus Brucella consists of six recognized species, designated on the basis of differences in pathogenicity and host preference: B. melitensis (goats and sheep), B. abortus (cattle and bison), B. suis (infecting primarily swine but also hares, rodents, and reindeer), B. ovis (sheep), B. canis (dogs), and B. neotomae (wood rats) (7). The discovery of Brucella in a wide variety of marine mammals has led to the proposal of two new species: B. ceti (cetaceans) and B. pinnipedialis (pinnipeds) (8). Some of these species include several biovars, which are currently distinguished from each other by an analysis of approximately 25 phenotypic characteristics, including requirement for CO 2 , H 2 S production, sensitivity to dyes and phages, and other metabolic properties (1). However, all these tests are time-consuming, require skilled technicians, and are not straightforward, and some reagents are not commercially available. In addition, handling of this microorganism represents a high risk for laboratory personnel, since most Brucella strains are highly pathogenic for humans. Accurate diagnostic and typing procedures are critical for the success of the eradication and control of the disease, and therefore the identification of the different species is of great epidemiological importance. In order to overcome most of these difficulties, PCR-based assays have been employed for molecular typing of Brucella species. However, one of the challenges of using DNA-based techniques for differentiating the various Brucella species and strains is their high degree of genetic homology (16). This article describes the evaluation of a new multiplex PCR assay (10), named Bruce-ladder, in seven different European laboratories. The PCR protocol was standardized previously (10), and the same protocol was used in all laboratories (see the supplemental material). The selection of the DNA sequences to design the PCR primers was based on species-specific or strain-specific genetic differences (Table 1). Each laboratory used its own Brucella strain collection, typed by standard bacteriological procedures (1). A total of 625 Brucella strains were used (see the complete list in the supplemental material). The collection included the reference strains of all biovars of B. abortus, B. melitensis, B. suis, and B. ovis, B. canis, B. neotomae, the B. abortus S19, B. ...

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 manB core to distinguish it from the wbk manB O-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.Brucellosis is a zoonotic disease that causes heavy economic losses and human suffering. Under most conditions, vaccination and serological identification and culling of infected animals are the only practical means to achieve its eradication, but the best vaccines available (Brucella abortus S19 for cattle and B. melitensis Rev1 for sheep and goats) may induce abortions when used in pregnant animals and are virulent for humans. Moreover, like field strains, they carry a cell surface smoothtype lipopolysaccharide (S-LPS) whose immunodominant section (the N-formylperosamine O-polysaccharide) induces an antibody response that may be difficult to distinguish from that resulting from a true infection (25,48). This complicates serodiagnosis because the tests currently used detect antibodies to the O-polysaccharide.To overcome these problems, several strategies are possible. The early observation that rough (R) B. abortus strains are attenuated and do not agglutinate with antibody elicited by S bacteria (63) soon led to the concept of Brucella R vaccines and, more than 50 years ago, the spontaneous R mutant B. abortus 45/20 was studied for this purpose. However, strain 45/20 was unstable, and its use was abandoned (1, 48). The same strategy was followed to devel...

Ten striped dolphins, Stenella coeruleoalba, stranded along the Costa Rican Pacifi c coast, had meningoencephalitis and antibodies against Brucella spp. Brucella ceti was isolated from cerebrospinal fl uid of 6 dolphins and 1 fetus. S. coeruleoalba constitutes a highly susceptible host and a potential reservoir for B. ceti transmission. Brucellosis is a zoonotic disease of terrestrial and marine mammals. During the past 3 decades, contacts between cetaceans and humans have increased worldwide (1), augmenting the risk for transmission of pathogenic Brucella spp. from these animals to people (2). Indeed, Brucella marine strains are capable of infecting humans and livestock (3,4). The StudyFrom August 2004 through April 2007, 10 live striped dolphins, Stenella coeruleoalba (3 female adults, 2 female juveniles, 1 female calf, 4 juvenile males), were found stranded in populated areas at the Pacifi c shoreline of the Puntarenas Province of Costa Rica. All animals had swimming problems compatible with neurologic disorders and died within 48 hours of being found. Corpses were kept on ice and transported to the Pathology Unit, Veterinary School, National University, Costa Rica, for sampling; necropsy; and histopathologic, immunohistochemical, and serologic studies. With exception of 1 dwarf sperm whale, Kogia sima, these 10 dolphins were the only cetaceans we were able to examine during this 32-month period.Because marine Brucella spp. have been reported to cause intracerebral infections (3), we decided to perform immunohistochemical and serologic tests. For these tests, rabbit immunoglobulin (Ig) G anti-B. abortus lipopolysaccharide (LPS) was produced and isolated as described elsewhere (5). Antibodies against dolphin Steno bredanensis IgG were produced in rabbits, purifi ed according to described protocols (6). Both rabbit antibodies were linked to fl uorescein isothiocyanate and peroxidase and were assayed by using immunofl uorescent and Western blot techniques, respectively (5,7). Rose Bengal agglutination test, immunofl uorescent assays, and competitive ELISA were designed and used as described (8,9).Blood was collected from the live dolphins in situ, serum was obtained, and physical and chemical examinations were performed, followed after death by necropsies and gross pathologic and histopathologic studies. Tissues were fi xed in formalin, embedded in paraffi n wax, sectioned, and stained with hematoxylin and eosin (10). Organs and tissues of 5 adult females and 1 juvenile male were analyzed for bacteria (11); however, no samples for bacteriologic studies were available from the other dolphins that were stranded before July 2006. Identifi cation of the bacterial isolates was performed according to standard protocols (11,12). Fresh tissue impressions or pellets from supernatants of macerated tissues were fi xed with cold 3% paraformaldehyde for 15 min on ice and subjected to immunofl uorescence for detection of Brucella spp. (9). Genotyping of Brucella isolates was performed by PCR, using 5′-GGCTGATCTCGCAAAGAT-3′ an...

Fifteen different Rose Bengal antigens showed large differences with respect to pH, cell concentration and agglutination with the international standard anti-Brucella abortus serum, demonstrating the lack of international standardisation. Their sensitivity and specificity, compared with that of the complement fixation test, were evaluated for the diagnosis of B melitensis infection in culture-positive sheep, brucella-free ewes, and sheep and goats belonging to field flocks under different epidemiological conditions. All the Rose Bengal antigens and the complement fixation test had 100 per cent specificity when testing brucella-free sheep or animals belonging to flocks in unvaccinated brucellosis-free areas, but there were large differences in sensitivity between the Rose Bengal antigens with sera from culture-positive sheep or from animals belonging to infected flocks. When using the most sensitive antigen, no difference was observed in Rose Bengal sensitivity between animals infected with either biovar 1 or biovar 3 of B melitensis. The relationship between the sensitivity of the Rose Bengal antigens and cell concentration was unclear, but their sensitivity was related to the standardisation of the antigens with the international standard serum. The complement fixation test was less sensitive than the Rose Bengal test when testing culture-positive sheep. When testing sera from animals belonging to infected flocks with antigens standardised according to European Union rules, no great differences were observed in the sensitivities of the two tests. However, great differences in sensitivity between the Rose Bengal antigens were observed with sera from animals belonging to flocks with low levels of prevalence.(ABSTRACT TRUNCATED AT 250 WORDS)