-Brucellosis control and eradication requires serological tests and vaccines. Effective classical vaccines (S19 in cattle and Rev 1 in small ruminants), however, induce antibodies to the O-polysaccharide of the lipopolysaccharide which may be difficult to distinguish from those resulting from infection and may thus complicate diagnosis. Rough attenuated mutants lack the O-polysaccharide and would solve this problem if eliciting protective immunity; the empirically obtained rough mutants 45/20 and RB51 have been used as vaccines. Strain 45/20 is reportedly unstable and it is not presently used. RB51 is increasingly used instead of S19 in some countries but it is rifampicin resistant and its effectiveness is controversial. Some controlled experiments have found good or absolute protection in adult cattle vaccinated orally (full dose) or subcutaneously (reduced dose) and in one field experiment, RB51 was reported to afford absolute protection to calves and to perform better than S19. Controlled experiments in calves, however, have shown reduced doses of RB51 to be ineffective, full doses only partially effective, and RB51 less effective than S19 against severe challenges. Moreover, other observations suggest that RB51 is ineffective when prevalence is high. RB51 is not useful in sheep and evidence in goats is preliminary and contradictory. Rough mutants obtained by molecular biology methods on the knowledge of the genetics and structure of Brucella lipopolysaccharide may offer alternatives. The B. abortus manB core (rfbK) mutant seems promising in cattle, and analyses in mice suggest that mutations affecting only the O-polysaccharide result in better vaccines than those affecting both core and O-polysaccharide. Possible uses of rough vaccines also include boosting immunity by revaccination but solid evidence on its effectiveness, safety and practicality is not available.

BackgroundThe role of wildlife as a brucellosis reservoir for humans and domestic livestock remains to be properly established. The aim of this work was to determine the aetiology, apparent prevalence, spatial distribution and risk factors for brucellosis transmission in several Iberian wild ungulates.MethodsA multi-species indirect immunosorbent assay (iELISA) using Brucella S-LPS antigen was developed. In several regions having brucellosis in livestock, individual serum samples were taken between 1999 and 2009 from 2,579 wild bovids, 6,448 wild cervids and4,454 Eurasian wild boar (Sus scrofa), and tested to assess brucellosis apparent prevalence. Strains isolated from wild boar were characterized to identify the presence of markers shared with the strains isolated from domestic pigs.ResultsMean apparent prevalence below 0.5% was identified in chamois (Rupicapra pyrenaica), Iberian wild goat (Capra pyrenaica), and red deer (Cervus elaphus). Roe deer (Capreolus capreolus), fallow deer (Dama dama), mouflon (Ovis aries) and Barbary sheep (Ammotragus lervia) tested were seronegative. Only one red deer and one Iberian wild goat resulted positive in culture, isolating B. abortus biovar 1 and B. melitensis biovar 1, respectively. Apparent prevalence in wild boar ranged from 25% to 46% in the different regions studied, with the highest figures detected in South-Central Spain. The probability of wild boar being positive in the iELISA was also affected by age, age-by-sex interaction, sampling month, and the density of outdoor domestic pigs. A total of 104 bacterial isolates were obtained from wild boar, being all identified as B. suis biovar 2. DNA polymorphisms were similar to those found in domestic pigs.ConclusionsIn conclusion, brucellosis in wild boar is widespread in the Iberian Peninsula, thus representing an important threat for domestic pigs. By contrast, wild ruminants were not identified as a significant brucellosis reservoir for livestock.

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...

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Prevention of Salmonella contamination of poultry products requires detailed knowledge of the most important risk factors associated with its presence in the production system. The aim of this study was to identify the risk factors for Salmonella contamination in 44 broiler and 51 layer farms and determine the biofilm development capacity of the strains isolated. Then, glutaraldehyde (50% vol/vol), formaldehyde (37% vol/vol), and hydroxide peroxide (35% vol/vol) were applied to evaluate their capacity to remove Salmonella, biofilm and nonbiofilm strains, isolated from each risk factor in an artificial contamination test in field conditions. Samples of feces, dust, surfaces, meconium, delivery-box liners, water tank, water dispensers, litter, vectors (rodents, flies, and beetles), and surfaces of the slaughter trucks were taken throughout the rearing period. All samples were analyzed in accordance with ISO 6579:2002 (Annex D). To evaluate biofilm development, a screening method based on the fluorescence of Salmonella colonies on calcofluor agar plates was used. In the artificial contamination test, the chemical solutions were prepared at a concentration of 1.0% and applied at exact times (1, 15, or 60 min). Our results showed that the most important risk factors for Salmonella contamination were dust, surfaces, and feces. Moreover, day-old broiler chicks reached the farm highly contaminated, and wild carriers played an important role in the recirculation of Salmonella in laying hen houses. The serotype most commonly isolated from each risk factor was Salmonella enteritidis, and irrespective of the origin of different serotypes, around 50% were able to produce biofilm. Finally, the use of glutaraldehyde, formaldehyde, and peroxygen at a concentration of 1.0% in field conditions are inadequate for Salmonella elimination irrespective of the serotype, the biofilm development capacity, and the disinfectant contact time.

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.Brucellosis is a worldwide zoonosis affecting mainly lowincome countries (23). Besides two new species (Brucella ceti and B. pinnipedialis) isolated recently from marine mammals, the genus Brucella includes six classical species named according to their host preference (18). Among them, B. melitensis, B. abortus, B. suis, and B. ovis (which preferentially infect sheep and goats, cattle, pigs, and sheep, respectively) are the most important from a socioeconomic standpoint, since in addition to decreasing productivity in animals, the first three species are the main ones responsible for brucellosis in human beings (19). In infected animals, brucellae can be isolated from vaginal discharges, placental and fetal tissues, milk, and semen of live animals and, after necropsy, from the lymph nodes, spleen, liver, mammary glands, epididymides, and male sexual glands. Due to its specificity, the isolation and identification of Brucella in these animal fluids and tissues are the only incontestable demonstration of brucellosis in a given animal or flock. Since primary Brucella isolation requires 4 to 7 days of incubation, the presence in the above field samples of overgrowing fungi as well as commensal and environmental bacteria explains the frequent contamination of culture plates and the reduced sensitivity of bacteriological diagnosis. ...

Rapid and specific identification of Brucella suis at the biovar level is necessary because some of the biovars that infect animals are pathogenic for humans. None of the molecular typing methods described so far are able to discriminate B. suis biovars in a single test and differentiation of B. suis from Brucella canis by molecular approaches can be difficult. This article describes a new multiplex PCR assay, Suis-ladder, for fast and accurate identification of B. suis at the biovar level and the differentiation of B. suis, B. canis and Brucella microti. An advancement of the original Bruce-ladder PCR protocol which allows the correct discrimination of all known Brucella species is also described.

The gene coding for the major outer membrane protein Omp31 was sequenced in five Brucella species and their biovars. Although the omp31 genes appeared to be highly conserved in the genus Brucella, nine nucleotide substitutions were detected in the gene of Brucella ovis compared to that of Brucella melitensis. As shown by differential binding properties of monoclonal antibodies (MAbs) to the two Brucella species, these nucleotide substitutions result in different antigenic properties of Omp31. The antigenic differences were also evidenced when sera from B. ovis-infected rams were tested by Western blotting with the recombinant B. melitensis or B. ovis Omp31 proteins. Twelve available sera reacted with recombinant B. ovis Omp31, but only four of them reacted with recombinant B. melitensis Omp31. These results validate previous evidence for the potential of Omp31 as a diagnostic antigen for B. ovis infection in rams and demonstrate that B. ovis Omp31, instead of B. melitensis Omp31, should be used to evaluate this point. The antigenic differences between the B. melitensis and B. ovis Omp31 proteins should also be taken into account when Omp31 is evaluated as a candidate for the development of subcellular vaccines against B. ovis infection. No reactivity against recombinant B. melitensis Omp31 was detected, by Western blotting, with sera from B. melitensis-infected sheep. Accordingly, Omp31 does not seem to be a good diagnostic antigen for B. melitensis infections in sheep. Two immunodominant regions were identified on the B. ovis Omp31 protein by using recombinant DNA techniques and specific MAbs. Sera from B. ovis-infected rams that reacted with the recombinant protein were tested by Western blotting against one of these immunodominant regions shown to be exposed at the bacterial surface. Only 4 of the 12 sera reacted, but with strong intensity.As a result of DNA-DNA hybridization studies, the genus Brucella has been proposed as monospecific (31,32). However, the classical six-species organization of the genus is still maintained, as it is in accordance with the pathogenicity and host preference characteristics of each species. Moreover, DNA markers distinguishing the Brucella species and some of their biovars have been found (33).Ovine brucellosis is mainly caused by Brucella melitensis (also responsible for caprine brucellosis) and Brucella ovis, the latter being the most frequent cause of contagious ram epididymitis. Infection by B. ovis reduces fertility in rams, and abortions in ewes have also been reported, constituting an important limiting factor for the development of sheep production in many countries.Diagnosis of B. ovis infection is mainly performed with serological methods, of which the complement fixation test (CFT) is most extensively used (3). However, the CFT has several disadvantages, including its complexity, lack of sensitivity, prozone phenomena, and incompatibility with hemolyzed or anticomplementary sera (25,26,28). In order to find a serological test to substitute for the CFT, several enzym...