Brucella melitensis in France: Persistence in Wildlife and Probable Spillover from Alpine Ibex to Domestic Animals

Bovine brucellosis is a major zoonosis, mainly caused by Brucella abortus, more rarely by Brucella melitensis. France has been bovine brucellosis officially-free since 2005 with no cases reported in domestic/wild ruminants since 2003. In 2012, bovine and autochthonous human cases due to B. melitensis biovar 3 (Bmel3) occurred in the French Alps. Epidemiological investigations implemented in wild and domestic ruminants evidenced a high seroprevalence (>45%) in Alpine ibex (Capra ibex); no cases were disclosed in other domestic or wild ruminants, except for one isolated case in a chamois (Rupicapra rupicapra). These results raised the question of a possible persistence/emergence of Brucella in wildlife. The purpose of this study was to assess genetic relationships among the Bmel3 strains historically isolated in humans, domestic and wild ruminants in Southeastern France, over two decades, by the MLVA-panel2B assay, and to propose a possible explanation for the origin of the recent bovine and human infections. Indeed, this genotyping strategy proved to be efficient for this microepidemiological investigation using an interpretation cut-off established for a fine-scale setting. The isolates, from the 2012 domestic/human outbreak harbored an identical genotype, confirming a recent and direct contamination from cattle to human. Interestingly, they clustered not only with isolates from wildlife in 2012, but also with local historical domestic isolates, in particular with the 1999 last bovine case in the same massif. Altogether, our results suggest that the recent bovine outbreak could have originated from the Alpine ibex population. This is the first report of a B. melitensis spillover from wildlife to domestic ruminants and the sustainability of the infection in Alpine ibex. However, this wild population, reintroduced in the 1970s in an almost closed massif, might be considered as a semi-domestic free-ranging herd. Anthropogenic factors could therefore account with the high observed intra-species prevalence.     

Brucellosis in captive Rocky Mountain bighorn sheep (Ovis canadensis) caused by Brucella abortus biovar 4

Nine (four female, five male) captive adult Rocky Mountain bighorn sheep (Ovis canadensis) contracted brucellosis caused by Brucella abortus biovar 4 as a result of natural exposure to an aborted elk (Cervus elaphus) fetus. Clinical signs of infection were orchitis and epididymitis in males and lymphadenitis and placentitis with abortion in females. Gross pathologic findings included enlargement of the testes or epididymides, or both, and yellow caseous abscesses and pyogranulomas of the same. Brucella abortus biovar 4 was cultured in all bighorn sheep from a variety of tissues, including testes/epididymides, mammary gland, and lymph nodes. All bighorn sheep tested were positive on a variety of standard Brucella serologic tests. This is the first report of brucellosis caused by B. abortus in Rocky Mountain bighorn sheep. It also provides evidence that bighorn sheep develop many of the manifestations ascribed to this disease and that infection can occur from natural exposure to an aborted fetus from another species. Wildlife managers responsible for bighorn sheep populations sympatric with Brucella-infected elk or bison (Bison bison) should be cognizant of the possibility of this disease in bighorn sheep.     

Brucella: a Mr "Hide" converted into Dr Jekyll

Dr David Bruce (1855-1931) first identified the causative agent of brucellosis as a small Gram-negative alpha-Proteobacterium, which was later on called Brucella melitensis in his honor by Meyer and Shaw. Nowadays, four strains exhibit pathogenicity in humans with B. melitensis being the least host specific and also the most infectious for humans. The other strains are Brucella suis and Brucella abortus and more recently human cases being infected with Brucella cetaceae have been reported. Why such a reemerging disease is so difficult to fight, evidence shows that the pathogenic bacterium has developed strategies to hide from immune recognition.     

Brucella suis infections in collared peccaries in Venezuela

A bacteriologic and serologic study was conducted on two ranches in the states of Apure and Guarico, Venezuela for brucellosis in collared peccaries (Tayassu tajacu). One hundred thirty-nine peccaries were necropsied and tissues were cultured. Forty-three isolations of Brucella suis biovar 1, were made from lymph nodes and spleens of 25 males and 18 females. Antibody to Brucella sp. was detected in sera from 122 animals by the rapid plate agglutination, standard tube agglutination, 2-mer-captoethanol, rivanol, complement fixation and card tests. Young animals had infection and reactor rates nearly as high as the older animals indicating most were infected at a relative early age. Results suggest that this species may transmit brucellosis when living with domestic animals. This is the first report of B. suis biovar 1 from collared peccaries in Venezuela.     

上海地区散发布氏杆菌感染的细菌学及分子鉴定

目的 本研究对我院的1例散发布氏杆菌病患者进行细菌学及分子生物学的分析,并在国内首次尝试了用数目可变串联重复单元(VNTR)分子指纹分析法对其进行了基因分型并和国际流行株进行了分子流行病学比较分析。方法 对临床疑似布氏杆菌病病例作血液细菌培养与生化鉴定,进一步作布氏杆菌特异性基因片段的序列分析鉴定以及利用布氏杆菌基因组中的8个位点构建VNTR指纹图谱,参照国际布氏杆菌VNTR数据库,构建布氏杆菌基因系统树。结果 用细菌学方法确定散发疑似布氏杆菌病病例体内分离到的为布氏杆菌,通过基因序列分析进一步得到证实,但不能鉴定到生物种和生物型。对分离株作VNTR指纹分析提示该散发布氏杆菌病为猪2型布氏杆菌感染所致。结论 通过传统细菌培养方法与布氏杆菌VNTR指纹分析可用于我国布氏杆菌病分子流行病学的系统调查。     

Identification of species of Brucella using Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy (FTIR) is a technique that has been used over the years in chemical analysis for the identification of substances and is one that may be applied to the characterisation of microorganisms. The marked tendency of Brucella towards variation in the smooth rough phase, together with the laboriousness and risk involved in the methods used in their identification, make their classification difficult. We studied the type strains of the different species and biovars of Brucella and 11 isolates of human origin of Brucella melitensis, six corresponding to biovar 1, one to biovar 2 and five to biovar 3. The results of linear discriminant analysis performed using the data provide an above 95% likelihood of correct classification, over half of which are in fact above 99% for the vast majority of Brucella strains. Only one case of B. melitensis biovar 1 has been incorrectly classified. The rest of the microorganisms studied (Staphylococcus aureus, Strteptococcus pyogenes, Enterococcus faecalis, Corynebacterium pseudodiphtheriticum, Clostridium perfringens, Escherichia coli, Acinetobacter calcoaceticus and Pseudomonas aeruginosa) have been classified correctly in all cases to a likelihood of over 80%. In the graphic representation of the analysis, a grouping of these can be seen in clusters, which include the different species. One of these comprises B. melitensis, another Brucella abortus, and another wider one is made up of Brucella suis. The Brucella canis, Brucella ovis and Brucella neotomae strains appear separate from the previously described groups.     

Cohabitation of a <Emphasis Type="Italic">Brucella melitensis</Emphasis> infected Alpine ibex (<Emphasis Type="Italic">Capra ibex</Emphasis>) with domestic small ruminants in an enclosure in Gran Paradiso National Park,in Western Italian Alps

After the first report of Brucella melitensis infection from a 7-year-old alpine ibex (Capra ibex) buck living in Gran Paradiso National Park (GPNP), further studies demonstrated the presence of the infection in ibex and chamois. Considering that livestock herds keep on sharing pastures with more than 3,500 ibex and 9,000 chamois in the park, our aim was to demonstrate under controlled conditions the possibility of Brucella infection passing from wild ruminants to livestock. A 7-year-old male alpine ibex with clinical signs of brucellosis and serologically positive was released in a 5,000 m² enclosure together with five goats and two sheep rams. Due to poor condition, ibex was suppressed at day 40, domestic ruminants stayed into the enclosure potentially contaminated by ibex for further 38 days. During this period, we had monitored our animals taking blood from domestic ruminants every 15 days and tested the serum to Rose Bengal agglutination test and Complement Fixation test. Domestic animals tested negative at serology at all sampling time and at isolation, while B. melitensis biovar 3 was isolated from ibex tissues. Our data show that transmission of infection from ibex to livestock is not easy. After 40 days of strict cohabitation and 38 days of permanence in an area where an infected ibex lives, no one of the domestic animals contracted infection. In spite of the limitation of our field trial, we have demonstrated that long direct and indirect contact between alpine ibex and domestic animals will not easily lead to an infection of the latter. Further investigations are needed to confirm our results and evaluate the effective risk of B. melitensis transmission from alpine ibex to livestock.     

Antigenic, immunologic and genetic characterization of rough strains B. abortus RB51, B. melitensis B115 and B. melitensis B18

The lipopolysaccharide (LPS) is considered the major virulent factor in Brucella spp. Several genes have been identified involved in the synthesis of the three LPS components: lipid A, core and O-PS. Usually, Brucella strains devoid of O-PS (rough mutants) are less virulent than the wild type and do not induce undesirable interfering antibodies. Such of them proved to be protective against brucellosis in mice. Because of these favorable features, rough strains have been considered potential brucellosis vaccines. In this study, we evaluated the antigenic, immunologic and genetic characteristics of rough strains B. abortus RB51, B. melitensis B115 and B. melitensis B18. RB51 derived from B. abortus 2308 virulent strain and B115 is a natural rough strain in which the O-PS is present in the cytoplasm. B18 is a rough rifampin-resistan mutant isolated in our laboratory. The surface antigenicity of RB51, B115 and B18 was evaluated by testing their ability to bind antibodies induced by rough or smooth Brucella strains. The antibody response induced by each strain was evaluated in rabbits. Twenty-one genes, involved in the LPS-synthesis, were sequenced and compared with the B. melitensis 16M strain. The results indicated that RB51, B115 and B18 have differences in antigenicity, immunologic and genetic properties. Particularly, in B115 a nonsense mutation was detected in wzm gene, which could explain the intracellular localization of O-PS in this strain. Complementation studies to evaluate the precise role of each mutation in affecting Brucella morphology and its virulence, could provide useful information for the assessment of new, attenuated vaccines for brucellosis.     

Identification of immunoreactive proteins of Brucella melitensis by immunoproteomics

Infection with Brucella causes brucellosis, a chronic disease in humans, which induces abortion and sterility in livestock. Among the different Brucella species, Brucella melitensis is considered the most virulent and is the predominant species associated with outbreaks in China. To date, no safe human vaccine is available against Brucella infection. The currently used live vaccines against Brucella in livestock induce antibodies that interfere with the diagnosis of field infection in vaccinated animals, which is harmful to eradication programs. However, there is as yet no complete profile of immunogenic proteins of B. melitensis. Towards the development of a safer, equally efficacious, and field infection-distinguishable vaccine, we used immunoproteomics to identify novel candidate immunogenic proteins from B. melitensis M5. Eighty-eight immunoreactive protein spots from B. melitensis M5 were identified by Western blotting and were assigned to sixty-one proteins by mass spectrometry, including many new immunoreactive proteins such as elongation factor G, F0F1 ATP synthase subunit beta, and OMP1. These provide many candidate immunoreactive proteins for vaccine development.     

Differences in chromosome number and genome rearrangements in the genus Brucella

We have studied the genomic structure and constructed the Spe I, Pac I and I- Ceu I restriction maps of the four biovars of the pathogenic bacterium Brucella suis . B . suis biovar 1 has two chromosomes of 2.1 Mb and 1.15 Mb, similar to those of the other Brucella species: B . melitensis , B . abortus , B . ovis and B . neotomae . Two chromosomes were also observed in the genome of B . suis biovars 2 and 4, but with sizes of 1.85 Mb and 1.35 Mb, whereas only one chromosome with a size of 3.1 Mb was found in B . suis biovar 3. We show that the differences in chromosome size and number can be explained by rearrangements at chromosomal regions containing the three rrn genes. The location and orientation of these genes confirmed that these rearrangements are due to homologous recombination at the rrn loci. This observation allows us to propose a scheme for the evolution of the genus Brucella in which the two chromosome-containing strains can emerge from an hypothetical ancestor with a single chromosome, which is probably similar to that of B . suis biovar 3. As the genus Brucella is certainly monospecific, this is the first time that differences in chromosome number have been observed in strains of the same bacterial species.     

Whole genome sequences of four Brucella strains

Brucella melitensis and Brucella suis are intracellular pathogens of livestock and humans. Here we report four genome sequences, those of the virulent strain B. melitensis M28-12 and vaccine strains B. melitensis M5 and M111 and B. suis S2, which show different virulences and pathogenicities, which will help to design a more effective brucellosis vaccine.     

Use of western immunoblot analysis for testing moose serum for Brucella suis biovar 4 specific antibodies.

To determine if 12 moose (Alces alces) from northern Alaska with agglutinating antibodies specific for Brucella spp. had been exposed to either B. suis biovar 4 or B. abortus biovar 1, western immnnoblot serologic analysis was performed. Differential serologic responses to strain specific A and M antigenic variances of the lipopolysaccharide O-polysaccharide sugar allowed strain identification. Prior to examination, test sera were absorbed with killed whole cells from either B. abortus biovar 1, containing predominately A antigen (A+ M-); B. melitensis biovar 1, containing essentially M antigen (A- M+); or B. suis biovar 4, containing both antigenic tyes (A+ M+). The resulting sera were then examined by western immunoblot for recognition of either B. abortus biovar 1, B. melitensis biovar 1, or B. suis biovar 4 cell lysates. The results of this study indicate that these moose were exposed to B. suis biovar 4, a known pathogen of caribou (Rangifer tarandus) from arctic Alaska.     

Purification, refolding and characterization of the trimeric Omp2a outer membrane porin from Brucella melitensis

Brucella melitensis is a gram-negative bacteria known to cause brucellosis and to produce severe infections in humans. Whilst brucella's outer membrane proteins have been extensively studied due to their potential role as antigens or virulence factors, their function is still poorly understood at the structural level, as the 3D structure of Brucella β-barrel membrane proteins are still unknown. In this context, the B. melitensis trimeric Omp2a porin has been overexpressed and refolded in n-dodecyl-β-d-maltopyranoside. We here show that this refolding process is insensitive to urea but is temperature- and ionic strength-dependent. Reassembled species were characterized by fluorescence, size-exclusion chromatography and circular dichroism. A refolding mechanism is proposed, suggesting that Omp2a first refolds under a monomeric form and then self-associates into a trimeric state. This first complete in vitro refolding of a membrane protein from B. melitensis shall eventually lead to functional and 3D structure determination.     

Human Brucellosis in Maghreb: Existence of a Lineage Related to Socio-Historical Connections with Europe

Despite control/eradication programs, brucellosis, major worldwide zoonosis due to the Brucella genus, is endemic in Northern Africa and remains a major public health problem in the Maghreb region (Algeria/Morocco/Tunisia). Brucella melitensis biovar 3 is mostly involved in human infections and infects mainly small ruminants. Human and animal brucellosis occurrence in the Maghreb seems still underestimated and its epidemiological situation remains hazy. This study summarizes official data, regarding Brucella melitensis infections in Algeria, from 1989 to 2012, with the purpose to provide appropriate insights concerning the epidemiological situation of human and small ruminant brucellosis in Maghreb. Algeria and Europe are closely linked for historical and economical reasons. These historical connections raise the question of their possible impact on the genetic variability of Brucella strains circulating in the Maghreb. Other purpose of this study was to assess the genetic diversity among Maghreb B. melitensis biovar 3 strains, and to investigate their possible epidemiological relationship with European strains, especially with French strains. A total of 90 B. melitensis biovar 3 Maghreb strains isolated over a 25 year-period (1989–2014), mainly from humans, were analysed by MLVA-16. The obtained results were compared with genotypes of European B. melitensis biovar 3 strains. Molecular assays showed that Algerian strains were mainly distributed into two distinct clusters, one Algerian cluster related to European sub-cluster. These results led to suggest the existence of a lineage resulting from socio-historical connections between Algeria and Europe that might have evolved distinctly from the Maghreb autochthonous group. This study provides insights regarding the epidemiological situation of human brucellosis in the Maghreb and is the first molecular investigation regarding B. melitensis biovar 3 strains circulating in the Maghreb.     

Comparative proteome analysis of Brucella melitensis vaccine strain Rev 1 and a virulent strain, 16M

The genus Brucella consists of bacterial pathogens that cause brucellosis, a major zoonotic disease characterized by undulant fever and neurological disorders in humans. Among the different Brucella species, Brucella melitensis is considered the most virulent. Despite successful use in animals, the vaccine strains remain infectious for humans. To understand the mechanism of virulence in B. melitensis, the proteome of vaccine strain Rev 1 was analyzed by two-dimensional gel electrophoresis and compared to that of virulent strain 16M. The two strains were grown under identical laboratory conditions. Computer-assisted analysis of the two B. melitensis proteomes revealed proteins expressed in either 16M or Rev 1, as well as up- or down-regulation of proteins specific for each of these strains. These proteins were identified by peptide mass fingerprinting. It was found that certain metabolic pathways may be deregulated in Rev 1. Expression of an immunogenic 31-kDa outer membrane protein, proteins utilized for iron acquisition, and those that play a role in sugar binding, lipid degradation, and amino acid binding was altered in Rev 1.     

Genome Sequence of Brucella melitensis S66, an Isolate of Sequence Type 8, Prevalent in China

Brucella melitensis is the most-represented Brucella species causing human brucellosis in China. Here we report the complete genome sequence of B. melitensis strain S66, a representative strain of sequence type 8 (ST8), which is prevalent in China, making it possible to compare the genome sequences of isolates from different countries.     

Complete genome sequences of Brucella melitensis strains M28 and M5-90, with different virulence backgrounds

Brucella melitensis is a Gram-negative coccobacillus bacteria belonging to the Alphaproteobacteria subclass. It is an important zoonotic pathogen that causes brucellosis, a disease affecting sheep, cattle, and sometimes humans. The B. melitensis strain M5-90, a live attenuated vaccine cultured from the B. melitensis virulent strain M28, has been an effective tool to control brucellosis in goats and sheep in China. Here we report the complete genome sequences of B. melitensis M28 and M5-90, strains with different virulence backgrounds, which will serve as a valuable reference for future studies.     

Brucella melitensis B115-based complement fixation test to detect antibodies induced by Brucella rough strains

Aims:  To assess the efficiency of a Brucella melitensis B115 rough strain, naturally devoid of anticomplementary activity, used as antigen in a complement fixation test (CFT) to detect antibodies induced by Brucella strains with rough phenotype, such as Brucella abortus RB51, Brucella ovis and Brucella canis .
Methods and Results:  Complement fixation testing was performed on sera from RB51-vaccinated cattle and buffaloes, B. ovis -infected sheep and B. canis -infected dogs using B115, RB51 and the hot saline extract (HSE) as antigens. The B115-based CFT proved highly sensitive and specific in detecting rough antibodies and its efficiency was comparable with that of RB51 and HSE-based CFT.
Conclusions:  Brucella melitensis B115 can be successfully used as an antigen in CFT to detect antibodies induced by Brucella rough strains.
Significance and Impact of the Study:  Brucella melitensis B115 antigen may represent an improvement over Brucella rough strains for Brucella antibody detection by CFT, thus enhancing the efficiency of brucellosis surveillance systems. Owing to the absence of anticomplementary activity, it does not require particular growth conditions or modifications and can be accurately standardized. The B115-based CFT may constitute a suitable supplementary test for the diagnosis of human infections owing to rough Brucellae .     

Evaluation of Brucella MLVA typing for human brucellosis

Human brucellosis is still the most common bacterial zoonosis worldwide. Neither well-known molecular tools nor the classical biotyping methods are satisfactory for subtyping of Brucella spp. Loci containing Variable Number of Tandem Repeats (VNTRs) have recently proved their usefulness in typing strains from animal origin despite the high genetic homogeneity within the genus Brucella (DNA-DNA homology >90%). The aim of this study was to evaluate MLVA (Multiple Locus VNTR Analysis) for diagnostic and epidemiological use in human brucellosis. One hundred and twenty-eight B. melitensis isolates of all three biovars were typed using eight minisatellite (panel 1) and eight microsatellite (panel 2) markers. One hundred and ten different genotypes were identified. The MLVA clustering pattern correlated with the geographic origin of the strains. Brucella strains isolated from different patients within the same outbreak or from the same patient before first-line therapy and after relapse showed identical genotypes. Fuchsin sensitive B. melitensis strains were found in closely related clusters giving evidence for an association between VNTRs and some phenotypic characteristics. However, the validity of biovars established by classical microbiological methods could not be confirmed by MLVA clustering. The original data can be queried on the genotyping web page at http://bacterial-genotyping.igmors.u-psud.fr. The MLVA assay is rapid, highly discriminatory, and reproducible within human Brucella isolates. MLVA can significantly contribute to epidemiological trace-back analysis of Brucella infections and may advance surveillance and control of human brucellosis.