Brucella suis

While serological tests are essential in surveillance and control programs of animal diseases, to date none of the common serological tests approved in the EU (complement fixation test or Rose-Bengal test) has been shown to be reliable in routine individual diagnosis of porcine brucellosis, and some more recent tests like ELISA have not been fully evaluated yet. In the absence of a gold standard, this study allowed the estimation of sensitivities and specificities of these tests with a Bayesian approach using Markov Chain Monte Carlo algorithms.

Background: The 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. Methods: A 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. Results: Mean 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. Conclusions: In 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.

Background: The 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. Methods: A 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. Results: Mean 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. Conclusions: In 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 suis is a dangerous biological warfare agent already used for military purposes. This bacteria cause brucellosis, a zoonosis highly infective and difficult to fight. An important selective target for chemotherapy against this disease is nucleoside hydrolase (NH), an enzyme still not found in mammals. We present here the first three-dimensional structure of B. suis NH (BsNH) and propose this enzyme as a molecular target to the drug design in the fight against brucellosis. In addition, we performed molecular docking studies, aiming to analyze the three-dimensional positioning of nine known inhibitors of Chritidia fasciculata NH (CfNH) in the active sites of BsNH and CfNH. We also analyzed the main interactions of some of these compounds inside the active site of BsNH and the relevant factors to biological activity. These results, together with further molecular dynamics (MD) simulations, pointed out to the most promising compound as lead for the design of potential inhibitors of BsNH. Most of the docking and MD results corroborated to each other and the docking results also suggested a good correlation with experimental data.

Sequencing of bp26, the gene encoding the Brucella sp. immunogenic BP26 periplasmic protein, was performed in the reference strains of Brucella abortus, B. suis, and B. ovis. The three bp26 sequences were almost identical to that published for B. melitensis 16M bp26, and only minor nucleotide substitutions, without modifying the amino acid sequence, were observed between species. The bp26 genes of the seven B. abortus biovar reference strains and B. abortus S19 and RB51 vaccine strains were also sequenced. Again, only minor differences were found. Surprisingly, the bp26 nucleotide sequence for B. abortus S19 was almost identical to that found for B. melitensis 16M and differed from the sequence described previously by others O. L. Rossetti, A. I. Arese, M. L. Boschiroli, and S. L. Cravero, J. Clin. Microbiol. 34:165-169, 1996) for the same B. abortus strain. The epitope mapping of BP26, performed by using a panel of monoclonal antibodies and recombinant DNA techniques, allowed the identification of an immunodominant region of the protein interesting for the diagnosis of B. melitensis and B. ovis infection in sheep. A recombinant fusion protein containing this region of BP26 reacted indeed, in Western blotting, as the entire recombinant BP26 against sera from B. melitensis-or B. ovis-infected sheep while it avoided false-positive reactions observed with sera from Brucella-free sheep when using the entire recombinant BP26. Thus, use of this recombinant fusion protein instead the entire recombinant BP26 could improve the specific serological diagnosis of B. melitensis or B. ovis infection in sheep.

A new selective medium, named LNIV-M, has been developed for isolation of Brucella suis. In this work, we evaluated the growth of B. suis reference and field strains from domestic pigs in different basal media and the susceptibility to different antibiotics contained in the currently used Farrell’s and modified Thayer-Martin media. We also determined the efficacy of LNIV-M and its diagnostic performance for isolating B. suis from wild boar tissue samples. A total of 1649 samples from 918 hunter-harvested wild boars were cultured in LNIV-M, Farrell’s and modified Thayer-Martin media. One hundred and thirty-nine (8.4%) samples from 63 (6.9%) animals resulted in a positive culture. LNIV-M detected 93.6% and 62.6% of positive animals and samples, respectively, while Farrell’s and modified Thayer-Martin media detected, respectively, 92.1% and 79.4% of positive animals and 58.3% and 59.7% of samples. These results confirm the adequate diagnostic performance of LNIV-M in the isolation of B. suis.

Fast and accurate identification of Brucella suis at the biovar level is an important issue for public health laboratories because some of the biovars that infect suidae (boars and pigs) are pathogenic for humans while others are not. Since classical biovar typing methods are often time-consuming, hard to standardize and require high-level biosafety containment, methodological improvements are desirable. This article describes new single nucleotide polymorphism (SNP) signatures for the rapid identification and biovar characterization of B. suis. These SNPs were included together with previously described ones in real-time PCR assays applicable to low-biosafety conditions. Allelic profiles unique for each B. suis biovar were defined and the most relevant signatures were determined on a collection of 137 field strains of worldwide origin characterized previously. Biovars assigned with both present and classical methods were globally consistent except for some biovar 3 field strains which matched the allelic profile of biovar 1.

Background The 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. Methods A 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. Results Mean 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), fall...

The risk of transmission of pathogens from free-ranging wild boars (Sus scrofa scrofa) to outdoor domestic pigs (S. scrofa domesticus) is of increasing concern in many European countries. We assess this risk, using Switzerland as an example. We estimated ...

In prokaryotes, the ureases are multi-subunit, nickel-containing enzymes that catalyze the hydrolysis of urea to carbon dioxide and ammonia. The Brucella genomes contain two urease operons designated as ure1 and ure2. We investigated the role of the two Brucella suis urease operons on the infection, intracellular persistence, growth, and resistance to low-pH killing.

Brucella spp., like other pathogens, must cope with the environment of diverse host niches during the infection process. In doing this, pathogens evolved different type of transport systems to help them survive and disseminate within the host. Members of the TolC family have been shown to be involved in the export of chemically diverse molecules ranging from large protein toxins to small toxic compounds. The role of proteins from the TolC family in Brucella and other ␣-2-proteobacteria has been explored little. The gene encoding the unique member of the TolC family from Brucella suis (BepC) was cloned and expressed in an Escherichia coli mutant disrupted in the gene encoding TolC, which has the peculiarity of being involved in diverse transport functions. BepC fully complemented the resistance to drugs such as chloramphenicol and acriflavine but was incapable of restoring hemolysin secretion in the tolC mutant of E. coli. An insertional mutation in the bepC gene strongly affected the resistance phenotype of B. suis to bile salts and toxic chemicals such as ethidium bromide and rhodamine and significantly decreased the resistance to antibiotics such as erythromycin, ampicillin, tetracycline, and norfloxacin. Moreover, the B. suis bepC mutant was attenuated in the mouse model of infection. Taken together, these results suggest that BepC-dependent efflux processes of toxic compounds contribute to B. suis survival inside the host.

This paper reports an analysis of the functional interactions between type IV secretion systems (T4SS) that are part of the conjugative machinery for horizontal DNA transfer (cT4SS), and T4SS involved in bacterial pathogenicity (pT4SS). The authors' previous work showed that a conjugative coupling protein (T4CP) interacts with the VirB10-type component of the T4SS in order to recruit the protein-DNA complex to the transporter for conjugative DNA transfer. This study now shows by two-hybrid analysis that conjugative T4CPs also interact with the VirB10 element of the pT4SS of Agrobacterium tumefaciens (At), Bartonella tribocorum (Bt) and Brucella suis (Bs). Moreover, the VirB10 component of a cT4SS (protein TrwE of plasmid R388) could be partially substituted by that of a pT4SS (protein TrwE of Bt) for conjugation. This result opens the way for the construction of hybrid T4SS that deliver DNA into animal cells. Interestingly, in the presence of part of the Bs T4SS the R388 T4SS protein levels were decreased and R388 conjugation was strongly inhibited. Complementation assays between the Trw systems of R388 and Bt showed that only individual components from the so-called 'core complex' could be exchanged, supporting the concept that this core is the common scaffold for the transport apparatus while the other 'peripheral components' are largely system-specific.

Brucella suis has been recognized as the major etiological agent of human brucellosis in areas free from Brucella melitensis infection. However, with changes in swine management, the occurrence of swine brucellosis has decreased as has the human incidence of B. suis infection. A swine brucellosis outbreak within a herd from Jaboticabal (São Paulo, Brazil) was detected in July 2006. The herd comprised approximately 300 sows and 1,500 finishing animals. Many sows within this herd experienced abortions, while others exhibited vaginal discharge; three sows suffered posterior paralysis. Among 271 sows, 254 (93.7%) tested positive for brucellosis by complement fixation, and among 62 randomly bled finishing animals, 17 (27.4%) also tested positive. The B. suis biovar 1 was cultured from 14 aborted fetuses and six sows. Brucella was identified using routine methods. Fourteen farm workers were tested using agglutination tests, with three workers showing evidence of Brucella antibody titers. A 39-year-old woman, who worked with maternal pigs and had direct contact with aborted fetuses, presented an agglutinating titer of 480 IU/mL and displayed clinical signs of infection. Our findings suggest that despite a reduction of swine brucellosis throughout Brazil, B. suis infection still occurs, thereby posing a zoonotic risk.

Background: Due to the parallel increase of the number of free-ranging wild boar and domestic pigs reared outdoor, the risk that they interact has become higher. Contacts with wild boar can be the origin of disease outbreaks in pigs, as it has been documented for brucellosis in some European countries. This study aimed at quantifying the occurrence of contacts between wild boar and outdoor domestic pigs in Switzerland, and identifying risk factors for these contacts. Furthermore, exposed pigs were tested for pathogen spill-over, taking Brucella suis as an example because B. suis is widespread in Swiss wild boar while domestic pigs are officially free of brucellosis.

A female wild boar, aged approximately two years, was found dead by local veterinary services in Pianola di Roio in L'Aquila Province situated in the Abruzzo Region of central Italy. The carcass was submitted to the Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise 'G. Caporale' in Teramo for necropsy. Brucella suis biovar 2 was isolated from submandibular lymph nodes. This is the first report of isolation of B. suis in the Abruzzo Region. Several authors agreed in the past on the hypothesis that B. suis biovar 2 had been introduced into Italy through the importation of hares from European countries in which the infection is endemic in wild populations. This lead the Italian authorities to reinforce existing controls for hares imported for restocking purposes. However, no provisions for brucellosis control are currently in place (or have been in place in the past) for wild boar movements either at the national or the European level. The isolation of B. ...

A b-carbonic anhydrase (CA, EC 4.2.1.1) from the bacterial pathogen Brucella suis, bsCA II, has been cloned, purified, and characterized kinetically. bsCA II showed high catalytic activity for the hydration of CO 2 to bicarbonate, with a k cat of 1.1 Â 10 6 , and k cat /K m of 8.9 Â 10 7 M À1 s À1. A panel of sulfonamides and sulfamates have been investigated for inhibition of this enzyme. All types of activities, from the low nanomolar to the micromolar, have been detected for these derivatives, which showed inhibition constants in the range of 7.3 nM-8.56 lM. The best bsCA II inhibitors were some glycosylated sulfanilamides, aliphatic sulfamates, and halogenated sulfanilamides, with inhibition constants of 7.3-87 nM. Some of these dual inhibitors of bsCA I and II, also inhibited bacterial growth in vitro, in liquid cultures. These promising data on live bacteria allow us to propose bacterial b-CA inhibition as an approach for obtaining anti-infective agents with a new mechanism of action compared to classical antibiotics.

The risk of transmission of pathogens from free-ranging wild boars (Sus scrofa scrofa) to outdoor domestic pigs (S. scrofa domesticus) is of increasing concern in many European countries. We assess this risk, using Switzerland as an example. We estimated 1) the prevalence of important pathogens in wild boars and 2) the risk of interactions between wild boars and outdoor pigs. First, we tested 252 wild boars from selected areas between 2008 and 2010 for infection with Brucella spp. Bacterial prevalence was estimated to 28.8% (confidence interval [CI] 23.0-34.0) when using bacterial culture (B. suis Biovar 2) and real-time polymerase chain reaction. Antibody prevalence was 35.8% (CI 30.0-42.0), which was significantly higher than in previous studies in Switzerland. We also tested 233 wild boars for porcine reproductive and respiratory syndrome virus (PRRSV). Antibody prevalence was 0.43% (CI 0.01-2.4) for EU-PRRSV and real-time reverse transcription polymerase chain reaction results were negative. These findings suggest that B. suis is increasingly widespread in wild boars and PRRSV is currently not of concern. Second, we documented the spatial overlap between free-ranging wild boars and outdoor piggeries by mapping data on their respective occurrence. Wild boars are most widespread in the mountain range along the western and northern Swiss borders, while most piggeries are located in central lowlands. A risk of interaction is mainly expected at the junction between these two bioregions. This risk may increase if wild boars expand eastward and southward beyond anthropogenic barriers believed to limit their range. Therefore, we evaluated the potential of expansion of the wild boar population. Population trends suggest a continuous increase of wild boars for the past 15 yr. Surveillance of selected wildlife passages using cameras on highways and main roads indicates that these barriers are permeable (average of up to 13 wild boar crossings per 100 days). Thus an increase of wild boar range should be considered. There may be a risk of B. suis spillover from wild boars in Switzerland, which could increase in the future. Data on the occurrence of interactions between pigs and wild boars are needed to assess this risk.

Background The 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. Methods A 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. Results Mean 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), fall...

Secretion systems are protein complexes essential for bacterial virulence and potential targets for antivirulence drugs. In the intracellular pathogen Brucella suis, a type IV secretion system mediates the translocation of virulence factors into host cells and it is essential for pathogenicity. VirB8 is a core component of the secretion system and dimerization is important for functionality of the protein complex. We set out to study dimerization and possible conformational changes of VirB8 from B. suis (VirB8s) using nuclear magnetic resonance, X-ray crystallography, and differential scanning fluorimetry. We identified changes of the protein induced by a concentration-dependent monomer-to-dimer transition of the periplasmic domain (VirB8sp). We also show that the presence of the detergent CHAPS alters several signals in the heteronuclear single quantum coherence (HSQC) spectra and some of these chemical shift changes correspond to those observed during monomer-dimer transition. X-r...

Background: Conjugative plasmid transfer is the prevalent means for spreading antibiotic resistance genes among bacteria. Results: Surface exposure of transfer protein TraM from the Gram-positive (Gϩ) plasmid pIP501 was confirmed, and its crystal structure was solved. Conclusion: Structural relations to type IV secretion (T4S) proteins provide a novel classification scheme. Significance: The novel classification will help elucidate structure-function relationships in Gϩ T4S systems. Conjugative plasmid transfer is the most important means of spreading antibiotic resistance and virulence genes among bacteria and therefore presents a serious threat to human health. The process requires direct cell-cell contact made possible by a multiprotein complex that spans cellular membranes and serves as a channel for macromolecular secretion. Thus far, well studied conjugative type IV secretion systems (T4SS) are of Gramnegative (G؊) origin. Although many medically relevant pathogens (e.g., enterococci, staphylococci, and streptococci) are Gram-positive (G؉), their conjugation systems have received little attention. This study provides structural information for the transfer protein TraM of the G؉ broad host range Enterococcus conjugative plasmid pIP501. Immunolocalization demonstrated that the protein localizes to the cell wall. We then used opsonophagocytosis as a novel tool to verify that TraM was exposed on the cell surface. In these assays, antibodies generated to TraM recruited macrophages and enabled killing of pIP501 harboring Enteroccocus faecalis cells. The crystal structure of the C-terminal, surface-exposed domain of TraM was determined to 2.5 Å resolution. The structure, molecular dynamics, and cross-linking studies indicated that a TraM trimer acts as the biological unit. Despite the absence of sequence-based similarity, TraM unexpectedly displayed a fold similar to the T4SS VirB8 proteins from Agrobacterium tumefaciens and Brucella suis (G؊) and to the transfer protein TcpC from Clostridium perfringens plasmid pCW3 (G؉). Based on the alignments of secondary structure elements of VirB8-like proteins from mobile genetic elements and chromosomally encoded T4SS from G؉ and G؊ bacteria, we propose a new classification scheme of VirB8-like proteins.

Strain subtyping is an important tool for detection of outbreaks caused by Salmonella enterica serotype Enteritidis. Current subtyping methods, however, yield less than optimal subtype discrimination. In this study, we describe the development and evaluation of a multiple-locus variable-number tandem repeat analysis (MLVA) method for subtyping Salmonella serotype Enteritidis. The discrimination ability and epidemiological concordance of MLVA were compared with those of pulsed-field gel electrophoresis (PFGE) and phage typing. MLVA provided greater discrimination among non-epidemiologically linked isolates than did PFGE or phage typing. Epidemiologic concordance was evaluated by typing 40 isolates from four food-borne disease outbreaks. MLVA, PFGE, and, to a lesser extent, phage typing exhibited consistent subtypes within an outbreak. MLVA was better able to differentiate isolates between the individual outbreaks than either PFGE or phage typing. The reproducibility of MLVA was evalu...

A high prevalence of Brucella pinnipedialis serology and bacteriology positive animals has been found in the Northeast Atlantic stock of hooded seal (Cystophora cristata); however no associated gross pathological changes have been identified. Marine mammal brucellae have previously displayed different infection patterns in human and murine macrophages. To investigate if marine mammal Brucella spp. are able to invade and multiply in cells originating from a presumed host species, we infected alveolar macrophages from hooded seal with a B. pinnipedialis hooded seal isolate. Hooded seal alveolar macrophages were also challenged with B. pinnipedialis reference strain (NCTC 12890) from harbor seal (Phoca vitulina), B. ceti reference strain (NCTC 12891) from harbor porpoise (Phocoena phocoena) and a B. ceti Atlantic white-sided dolphin (Lagenorhynchus acutus) isolate (M83/07/1), to evaluate possible species-specific differences. Brucella suis 1330 was included as a positive control. Alveolar macrophages were obtained by post mortem bronchoalveolar lavage of euthanized hooded seals. Phenotyping of cells in the lavage fluid was executed by flow cytometry using the surface markers CD14 and CD18. Cultured lavage cells were identified as alveolar macrophages based on morphology, expression of surface markers and phagocytic ability. Alveolar macrophages were challenged with Brucella spp. in a gentamicin protection assay. Following infection, cell lysates from different time points were plated and evaluated quantitatively for colony forming units. Intracellular presence of B. pinnipedialis hooded seal isolate was verified by immunocytochemistry. Our results show that the marine mammal brucellae were able to enter hooded seal alveolar macrophages; however, they did not multiply intracellularly and were eliminated within 48 hours, to the contrary of B. suis that showed the classical pattern of a pathogenic strain. In conclusion, none of the four marine mammal strains tested were able to establish a persistent infection in primary alveolar macrophages from hooded seal.

While serological tests are essential in surveillance and control programs of animal diseases, to date none of the common serological tests approved in the EU (complement fixation test or Rose-Bengal test) has been shown to be reliable in routine individual diagnosis of porcine brucellosis, and some more recent tests like ELISA have not been fully evaluated yet. In the absence of a gold standard, this study allowed the estimation of sensitivities and specificities of these tests with a Bayesian approach using Markov Chain Monte Carlo algorithms.

Feral pigs are wild animal reservoirs of infectious pathogens transmissible to other species, all of which are transmissible to domestic pigs. The objective of this study was to detect the presence of harmful production-limiting pathogens; Brucella suis, Leptospira species, Lawsonia intracellularis, Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae in a feral pig population within a 10 km radius of two large-scale commercial piggeries in Southern Queensland, Australia. The movement pattern of six pigs within the feral population was also investigated using geographic positioning system collars. All pathogens were present in the feral pig population except for A pleuropneumoniae. The true seroprevalence (TP) from 83 serum samples was 10.5 per cent for B suis, 48.6 per cent for Leptospira species, 100 per cent for L intracellularis and 42.1 per cent for M hyopneumoniae. Of 72 lung samples, 27.6 per cent were positive for M hyopneumoniae. Serum samples from 86 domestic sows within the study region were positive for Leptospira species (TP 2.1 per cent), L intracellularis (TP 100 per cent) and M hyopneumoniae (TP 100 per cent). The majority of feral pig movement was within 5 km of the piggeries, with one approaching to 100 m of the free-range piggery. The presence of pathogens in feral pigs in such close proximity to commercial piggeries could pose a biosecurity risk.

Peptide nucleic acids (PNAs) are single-stranded, synthetic nucleic acid analogues containing a pseudopeptide backbone in place of the phosphodiester sugar-phosphate. When PNAs are covalently linked to cell-penetrating peptides (CPPs) they readily penetrate the bacterial cell envelope, inhibit expression of targeted genes and cause growth inhibition both of Gram-positive and Gram-negative bacteria. However, the effectiveness of PNAs against Brucella, a facultative intracellular bacterial pathogen, was unknown. The susceptibility of a virulent Brucella suis strain to a variety of PNAs was assessed in pure culture as well as in murine macrophages. The studies showed that some of the PNAs targeted to Brucella genes involved in DNA (polA, dnaG, gyrA), RNA (rpoB), cell envelope (asd), fatty acid (kdtA, acpP) and protein (tsf) synthesis inhibit the growth of B. suis in culture and in macrophages after 24 h of treatment. PNA treatment inhibited Brucella growth by interfering with gene expression in a sequence-specific and dose-dependent manner at micromolar concentrations. The most effective PNA in broth culture was that targeting polA at ca. 12 M. In contrast, in B. suis-infected macrophages, the most effective PNAs were those targeting asd and dnaG at 30 M; both of these PNAs had little inhibitory effect on Brucella in broth culture. The polA PNA that inhibits wild-type B. suis also inhibits the growth of wild-type Brucella melitensis 16M and Brucella abortus 2308 in culture. This study reveals the potential usefulness of antisense PNA constructs as novel therapeutic agents against intracellular Brucella.

Secondary structure in the target is a property not usually considered in software applications for design of optimal custom oligonucleotide probes. It is frequently assumed that eliminating self-complementarity, or screening for secondary structure in the probe, is sufficient to avoid interference with hybridization by stable secondary structures in the probe binding site. Prediction and thermodynamic analysis of secondary structure formation in a genome-wide set of transcripts from Brucella suis 1330 demonstrates that the properties of the target molecule have the potential to strongly influence the rate and extent of hybridization between transcript and tethered oligonucleotide probe in a microarray experiment. Despite the relatively high hybridization temperatures and 1M monovalent salt imposed in the modeling process to approximate hybridization conditions used in the laboratory, we find that parts of the target molecules are likely to be inaccessible to intermolecular hybridiz...

Background: Brucellosis is a worldwide disease of mammals caused by Alphaproteobacteria in the genus Brucella. The genus is genetically monomorphic, requiring extensive genotyping to differentiate isolates. We utilized two different genotyping strategies to characterize isolates. First, we developed a microarray-based assay based on 1000 single nucleotide polymorphisms (SNPs) that were identified from whole genome comparisons of two B. abortus isolates, one B. melitensis, and one B. suis. We then genotyped a diverse collection of 85 Brucella strains at these SNP loci and generated a phylogenetic tree of relationships. Second, we developed a selective primer-extension assay system using capillary electrophoresis that targeted 17 high value SNPs across 8 major branches of the phylogeny and determined their genotypes in a large collection (n = 340) of diverse isolates. Results: Our 1000 SNP microarray readily distinguished B. abortus, B. melitensis, and B. suis, differentiating B. melitensis and B. suis into two clades each. Brucella abortus was divided into four major clades. Our capillary-based SNP genotyping confirmed all major branches from the microarray assay and assigned all samples to defined lineages. Isolates from these lineages and closely related isolates, among the most commonly encountered lineages worldwide, can now be quickly and easily identified and genetically characterized. Conclusions: We have identified clade-specific SNPs in Brucella that can be used for rapid assignment into major groups below the species level in the three main Brucella species. Our assays represent SNP genotyping approaches that can reliably determine the evolutionary relationships of bacterial isolates without the need for whole genome sequencing of all isolates.

Serologic tests currently available for brucellosis diagnosis detect antibodies to Brucella but do not distinguish between species of Brucella. Although Brucella suis is known to circulate within various feral swine (Sus scrofa) populations, our objective was to determine the primary species of Brucella circulating in feral swine populations in areas of the US with high brucellosis prevalence. We cultured lymph nodes from 183 feral swine. We identified 22 isolates from 21 animals, and all isolates were genotyped as B. suis. Most isolates were B. suis biovar 1, with the exception of two genetically distinct isolates from one feral swine in Hawaii, which were identified as B. suis biovar 3. Serum from each feral swine was also tested by the fluorescence polarization assay when possible, but only 52% (95% CL529.8-74.3) of culture-positive animals were antibody positive. Our results indicate that brucellosis infections in feral swine within the US are typically caused by B. suis. However, improved serologic tests are needed to more accurately determine exposure to Brucella spp. and to monitor disease trends in feral swine populations.