First isolation and characterization of Brucella suis from yak.

Brucella spp., facultative intracellular pathogens that can persistently colonize animal host cells and cause zoonosis, affect public health and safety. A Brucella strain was isolated from yak in Qinghai Province. To detect whether this isolate could cause an outbreak of brucellosis and to reveal its genetic characteristics, several typing and whole-genome sequencing methods were applied to identify its species and genetic characteristics. Phylogenetic analysis based on MLVA and whole-genome sequencing revealed the genetic characteristics of the isolated strain. The results showed that the isolated strain is a B. suis biovar 1 smooth strain, and this isolate was named B. suis QH05. The results of comparative genomics and MLVA showed that B. suis QH05 is not a vaccine strain. Comparison with other B. suis strains isolated from humans and animals indicated that B. suis QH05 may be linked to specific animal and human sources. In conclusion, B. suis QH05 does not belong to the Brucella epidemic species in China, and as the first isolation of B. suis from yak, this strain expands the host range of B. suis.

Brucella suis Infection in Dog Fed Raw Meat, the Netherlands.

A Brucella suis biovar 1 infection was diagnosed in a dog without typical exposure risks, but the dog had been fed a raw meat-based diet (hare carcasses imported from Argentina). Track and trace investigations revealed that the most likely source of infection was the dog's raw meat diet.

MLVA-16 typing of Brucella suis biovar 2 strains circulating in Europe.

Swine brucellosis due to Brucella suis biovar 2 is an emerging disease in Europe, associated with increase of extensive swine farms and high density of infected wild boars. Since knowledge of predominant circulating strains is a prerequisite for any epidemiological study, accurate molecular typing procedures were applied to a collection of 176 B. suis isolates. By using suis-ladder multiplex PCR and PCR-RFLP analysis of omp2a, omp2b and omp31 genes, five haplotypes were identified among 160 biovar 2 isolates, with haplotypes 2d and 2e restricted to Portugal and Spain and haplotypes 2a, 2b and 2c widespread in Europe (except Portugal). MLVA based on 16 genetic markers (MLVA-16) revealed 126 genotypes, with 101 singletons, and grouped biovar 2 isolates in two clusters according to their geographic origins and haplotypes, defining the Iberian (Portugal and Spain) and the Central-European clonal lineages. In order to get insights on the evolutionary associations between B. suis lineages and their host species, an extended analysis was performed using a subset of 11 markers and publicly available data for 350 additional strains. This MLVA-11 analysis revealed a high genetic divergence amongst the 526 B. suis strains based on their hosts and highlighted the close relationship between strains from swine, wild boars and hares. Beyond corroborating the existence of Iberian and Central European biovar 2 clonal lineages and pointing to the evolution of biovar 2 Iberian clonal lineage from Central-European one by an allopatric speciation event, an ongoing colonization of Iberian Peninsula with specific MLVA-11 genotypes is also highlighted.

Brucella suis biovar 2 multi locus sequence type ST16 in wild boars (Sus scrofa) from Abruzzi region, Italy. Introduction from Central-Eastern Europe?

Porcine brucellosis occurs in many countries where pigs are farmed, often representing an underrated problem. B. suis biovar 2 is the most common isolate in Europe, with high prevalence reported in wild boars in which it is generally isolated in the absence of gross lesions. In the last five years, we tested for Brucella spp. 389 lymph nodes of wild boars collected during hunting seasons or during necropsy procedures. In this paper, we describe the first case of isolation of B. suis biovar 2 from a wild boar aborted foetus, and we analyse the genomic relationships with B.suis biovar 2 strains isolated in the past five years in Abruzzi Region, Central Italy. The genetic fingerprint revealed that the isolates under study belong to the MLST ST16 and to the MLVA11 Gt 57, similar to the Central-Eastern European strains. Massive restocking (for hunting purpose) of wild boars from Eastern Europe have been done since 1950 in Italy contributing to the increasing of population size and distribution, as well as to the interbreeding between these foreign breeds and the local population. The contamination of pastures with infected material such as aborted wild boars foetuses can increase the risk of transmission of Brucella among wild and domestic animals. The contact of B. suis with domestic ruminants may also cause serological reactions to brucellosis serological testing, and even unapparent infection, thus hampering the efforts made in the brucellosis eradication campaign.

Epidemiological investigation of the first human brucellosis case in Spain due to Brucella suis biovar 1 strain 1330.

INTRODUCTION: No cases of human brucellosis caused by Brucella suis has been reported in Spain. METHODS: This study involved interviews with the case and his co-workers, inspection of their workplace, checking infection control measures, and typing the Brucella strain isolated in the blood culture. RESULTS: Brucella suis biovar 1 strain 1330 was isolated from a patient who worked in a waste treatment plant. Food borne transmission, contact with animals, and risk jobs were ruled out. An accidental inoculation with a contaminated needle from a research laboratory waste container was identified as the most probable mode of transmission. CONCLUSION: There should be controls to ensure that waste containers are sealed.

Whole-genome mapping reveals a large chromosomal inversion on Iberian Brucella suis biovar 2 strains.

Optical mapping is a technology able to quickly generate high resolution ordered whole-genome restriction maps of bacteria, being a proven approach to search for diversity among bacterial isolates. In this work, optical whole-genome maps were used to compare closely-related Brucella suis biovar 2 strains. This biovar is the unique isolated in domestic pigs and wild boars in Portugal and Spain and most of the strains share specific molecular characteristics establishing an Iberian clonal lineage that can be differentiated from another lineage mainly isolated in several Central European countries. We performed the BamHI whole-genome optical maps of five B. suis biovar 2 field strains, isolated from wild boars in Portugal and Spain (three from the Iberian lineage and two from the Central European one) as well as of the reference strain B. suis biovar 2 ATCC 23445 (Central European lineage, Denmark). Each strain showed a distinct, highly individual configuration of 228-231 BamHI fragments. Nevertheless, a low divergence was globally observed in chromosome II (1.6%) relatively to chromosome I (2.4%). Optical mapping also disclosed genomic events associated with B. suis strains in chromosome I, namely one indel (3.5kb) and one large inversion (944kb). By using targeted-PCR in a set of 176 B. suis strains, including all biovars and haplotypes, the indel was found to be specific of the reference strain ATCC 23445 and the large inversion was shown to be an exclusive genomic marker of the Iberian clonal lineage of biovar 2.

Brucella suis bacteremia misidentified as Ochrobactrum anthropi by the VITEK 2 system.

Ochrobactrum and Brucella are genetically related genera of the family Brucellaceae, sharing 98.8% rRNA similarity. Because of their phenotypic similarity, Ochrobactrum can be miscoded as Brucella by automated identification systems. The misidentification on blood cultures (BCs) of B. suis as O. anthropi by the VITEK 2 system is herein described. A 67-year-old male with a prosthetic mitral valve and fever was admitted with bacteremia due to a Gram-negative coccobacillus identified as O. anthropi by VITEK 2. The patient's fever persisted along with positive blood cultures despite specific antimicrobial treatment. Due to this adverse outcome, the patient was interrogated again and admitted having domestic swine. Serological tests were positive for acute brucellosis. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) of BC strains identified B. suis biovar 1. Timely identification of Brucella is essential for providing proper treatment to the patient and for advising safe handling of laboratory cultures in biological safety cabinets to prevent laboratory-acquired infection. Countries where brucellosis is endemic must be aware of this possibility.

A genome-wide SNP-based phylogenetic analysis distinguishes different biovars of Brucella suis.

Brucellosis is an important zoonotic disease caused by Brucella spp. Brucella suis is the etiological agent of porcine brucellosis. B. suis is the most genetically diverged species within the genus Brucella. We present the first large-scale B. suis phylogenetic analysis based on an alignment-free k-mer approach of gathering polymorphic sites from whole genome sequences. Genome-wide core-SNP based phylogenetic tree clearly differentiated and discriminated the B. suis biovars and the vaccine strain into different clades. A total of 16,756 SNPs were identified from the genome sequences of 54 B. suis strains. Also, biovar-specific SNPs were identified. The vaccine strain B. suis S2-30 is extensively used in China, which was discriminated from all biovars with the accumulation of the highest number of SNPs. We have also identified the SNPs between B. suis vaccine strain S2-30 and its closest homolog, B. suis biovar 513UK. The highest number of mutations (22) was observed in the phosphomannomutase (pmm) gene essential for the synthesis of O-antigen. Also, mutations were identified in several virulent genes including genes coding for type IV secretion system and the effector proteins, which could be responsible for the attenuated virulence of B. suis S2-30.

The first report of Brucella suis biovar 1 isolation in human in Turkey.

BACKGROUND: Brucella melitensis and B. abortus are the species generally isolated from human samples in Turkey. Several studies have also demonstrated the presence of antibodies against B. canis. CASE REPORT AND STUDY: Brucella spp. was isolated from blood culture from a 35-year-old male with clinical signs and symptoms of acute meningitis, including fever lasting for 1 week. Multiplex PCR demonstrated B. suis, and biochemical features indicated biovar 1. CONCLUSIONS: This report is the first emphasizing that B. suis should be considered among the causes of brucellosis in Turkey.

Brucella suis Vaccine Strain 2 Induces Endoplasmic Reticulum Stress that Affects Intracellular Replication in Goat Trophoblast Cells In vitro.

Brucella has been reported to impair placental trophoblasts, a cellular target where Brucella efficiently replicates in association with the endoplasmic reticulum (ER), and ultimately trigger abortion in pregnant animals. However, the precise effects of Brucella on trophoblast cells remain unclear. Here, we describe the infection and replication of Brucella suis vaccine strain 2 (B.suis.S2) in goat trophoblast cells (GTCs) and the cellular and molecular responses induced in vitro. Our studies demonstrated that B.suis.S2 was able to infect and proliferate to high titers, hamper the proliferation of GTCs and induce apoptosis due to ER stress. Tunicamycin (Tm), a pharmacological chaperone that strongly mounts ER stress-induced apoptosis, inhibited B.suis.S2 replication in GTCs. In addition, 4 phenyl butyric acid (4-PBA), a pharmacological chaperone that alleviates ER stress-induced apoptosis, significantly enhanced B.suis.S2 replication in GTCs. The Unfolded Protein Response (UPR) chaperone molecule GRP78 also promoted B.suis.S2 proliferation in GTCs by inhibiting ER stress-induced apoptosis. We also discovered that the IRE1 pathway, but not the PERK or ATF6 pathway, was activated in the process. However, decreasing the expression of phosphoIRE1α and IRE1α proteins with Irestatin 9389 (IRE1 antagonist) in GTCs did not affect the proliferation of B.suis.S2. Although GTC implantation was not affected upon B.suis.S2 infection, progesterone secretion was suppressed, and prolactin and estrogen secretion increased; these effects were accompanied by changes in the expression of genes encoding key steroidogenic enzymes. This study systematically explored the mechanisms of abortion in Brucella infection from the viewpoint of pathogen invasion, ER stress and reproductive endocrinology. Our findings may provide new insight for understanding the mechanisms involved in goat abortions caused by Brucella infection.

First report of Brucella suis biovar 2 in a semi free-range pig farm, Italy.

This communication describes the isolation of Brucella suis (B. suis) biovar 2 in semi­free­range pigs located in the province of Rome, Italy. Sera of 28 pigs from a herd with reproductive problems were tested for brucellosis. Twenty-five sera (89%) were found positive to Rose Bengal Test (RBT), while 22 (79%) were positive to Complement Fixation Test (CFT). Two positive pigs were slaughtered, organs were collected and tested for the presence of bacteria. Brucella spp. was isolated from the spleens and the abdominal lymph nodes of the 2 subjects. The isolates were identified as B. suis biovar 2 by biochemical and Polymerase Chain Reaction (PCR) tests. The frequent infringement in the fences of the premises and the birth of striped piglets provided evidence that sows mated with wild boar, the major reservoir of B. suis biovar 2. Conversely, the isolation of B. suis biovar 2 from spleens and lymphnodes of seropositive slaughtered animals only, as well as the constant negative results from all vaginal swabs and the abortion materials tested, raise doubts on the implication of B. suis biovar 2 in the infertility of the holding.

Identification of Brucella suis from feral swine in selected states in the USA.

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% CL = 29.8-74.3) 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.

Brucella suis biovar 2 isolations from cattle in Poland.

Bovine brucellosis is an infectious disease caused by bacteria of the Brucella genus, primarily by B. abortus, less frequently by B. melitensis, and occasionally by B. suis. In the European Union, brucellosis in cattle has been eradicated in most of the Member States, which are recognized as 'officially free from bovine brucellosis'. Nevertheless, cattle herds continue to be serologically monitored for the potential re-emergence of the disease. The aim of the presented study was to show the results of bacteriological investigations of cattle slaughtered in Poland in years 2002-2011 on account of positive serological reactions for brucellosis. Specimens (sera and tissues) from 176 cows were examined. Sera from the animals were tested using RBT(rose bengal test), SAT (serum agglutination test), CFT (complement fixation test), 2-ME (2-mercaptoethanol test), Coombs (Coombs antiglobulin test) and ELISA (enzyme linked immunosorbant assay). Tissue samples were cultured for Brucella, according to official protocols. All sera were RBT and SAT-positive, 170 of them were CFT- positive, whereas 6 other samples were CFT negative while positive in Coombs and ELISA. In bacteriological examination, B. abortus was not isolated. On the other hand, B. suis biovar 2 was isolated from 5 cows, which had never been reported previously in Poland. Three cows came from the same herd. Conventional, as well as, molecular investigations based on PCR methods, confirmed that the bacteria isolated bacteria belong to the B. suis biovar 2. In Poland, as in many other European countries, wildlife (wild boars and hares) constitutes a huge reservoir of the said biovar. The results of the presented research indicate that B. suis biovar 2 can easily infect cattle, and undoubtedly plays a role in the epidemiology and control of bovine brucellosis.

Human brucellosis at a pig slaughterhouse.

Seventeen workers in a pig slaughterhouse with signs and symptoms compatible with brucellosis were clinically examined at the outpatient service of different health institutions and studied by serological tests during the period 2005-2011. Eleven blood cultures were taken and six Brucella suis strains were isolated, three biovar 1 and three with atypical characteristics. In order to confirm that these cases had no common source, a variable number of tandem repeat (VNTR) analyses were performed on 5 of the 6 strains whose results showed substantial heterogeneity in the genotypes, thereby demonstrating that the immediate origin was not the same. Two hundred adult pigs admitted for slaughter at the plant were sampled by convenience and tested by buffered antigen plate test (BPAT), serum agglutination test (SAT) and 2-mercapto-ethanol test (MET). Seven of 62 males (11%) and 25/138 (18%) females tested positive. The study results contribute information on risk scenarios for packing plant workers and underscore the need to improve plant workers' education on appropriate containment measures and to actively screen animals for swine brucellosis.

Complete genome sequence of Brucella suis field strain BCB025 of sequence type ST22.

Brucella is a genus of relatively conservative pathogenic bacteria. Brucella suis is the most diversified Brucella species. Strains of B. suis belong to different sequence types. Here, we report the genome sequence of B. suis strain BCB025, one isolate of the sequence type 22 epidemic in China.

Genome sequences of three live attenuated vaccine strains of Brucella species and implications for pathogenesis and differential diagnosis.

Live attenuated vaccines play essential roles in the prevention of brucellosis. Here, we report the draft genome sequences of three vaccine strains, Brucella melitensis M5-10, B. suis S2-30, and B. abortus 104M. Primary genome sequence analysis identified mutations, deletions, and insertions which have implications for attenuation and signatures for differential diagnosis.

Genotyping of Brucella species using clade specific SNPs.

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.

Brucellosis due to Brucella suis in a swine herd associated with a human clinical case in the State of São Paulo, Brazil.

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.

Isolation of Brucella suis biovar 2 from a wild boar in the Abruzzo Region of Italy.

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. suis biovar 2 from wild boar in two different and distant regions of Italy may suggest that this infection may have been introduced to the affected areas by wild boar rather than by imported hares. National and European rules managing wildlife brucellosis should be adapted to control the health status of farmed wild boar before movement or release, with the aim of preventing the spread of this pathogen to free territories.