Combined multilocus short-sequence-repeat and mycobacterial interspersed repetitive unit-variable-number tandem-repeat typing of Mycobacterium avium subsp. paratuberculosis isolates.

Short-sequence-repeat (SSR) sequencing was applied to 127 Mycobacterium avium subsp. paratuberculosis isolates typed by mycobacterial interspersed repetitive unit-variable-number tandem repeats (MIRU-VNTR) and IS900 restriction fragment length polymorphism (RFLP). Combined MIRU-VNTR and SSR typing followed by secondary IS900 RFLP typing is an improved approach to high-resolution genotyping of this pathogen.

Lipopentapeptide induces a strong host humoral response and distinguishes Mycobacterium avium subsp. paratuberculosis from M. avium subsp. avium.

BACKGROUND: Many non-tuberculous mycobacteria synthesize abundant glycopeptidolipids (GPLs). These surface-located GPLs are involved in pathogenicity by interfering with the host immune system. In Mycobacterium avium subsp. avium (Mav), GPLs consist of a lipopeptide core composed of a tetrapeptide O-linked to mono- and oligo-saccharides. The biosynthesis pathway of the simplest GPLs is now relatively well understood and involves probably more than fifteen genes. Whereas it is very obvious that most, if not all, of the Mav isolates produce GPLs, the picture is not as clear for M. avium subsp. paratuberculosis (Map), the etiologic agent of Johne's disease in cattle, and several conflicting data have been produced. METHODS: Biochemical analysis of a large set of characterized Map isolates showed that all Map strains tested produce a lipopentapeptide (L5P) instead of GPLs. To provide a genomic basis for the synthesis of this compound, the recently published genome sequence of Map was explored using in silico methods. Even though Map produces a lipopeptide rather than GPL, its genome contains nevertheless a locus highly similar to the GPL biosynthetic pathway of Mav. We showed that the module composition of the non-ribosomal protein synthase (Nrp) of Map, the enzyme involved in the synthesis of the peptidyl moiety, is dramatically different from that of other GPL producers such as M. smegmatis (Ms) and Mav and is in agreement with the amino acid content of the L5P. We also showed that the peptidyl moiety of the L5P is a target for a strong specific humoral response in Map infected animals. CONCLUSIONS: These genomic and biochemical differences may help to unambiguously distinguish Map from Mav and also from M. bovis, to reclassify related strains of the Map species and to allow the convenient and specific diagnosis of paratuberculosis.

New variable-number tandem-repeat markers for typing Mycobacterium avium subsp. paratuberculosis and M. avium strains: comparison with IS900 and IS1245 restriction fragment length polymorphism typing.

Mycobacterium avium subsp. paratuberculosis, the etiological agent of paratuberculosis, affects a wide range of domestic ruminants and has been suggested to be involved in Crohn's disease in humans. Most available methods for identifying and differentiating strains of this difficult species are technically demanding and have limited discriminatory power. Here, we report the identification of novel PCR-based typing markers consisting of variable-number tandem repeats (VNTRs) of genetic elements called mycobacterial interspersed repetitive units (MIRUs). Eight markers were applied to 183 M. avium subsp. paratuberculosis isolates from bovine, caprine, ovine, cervine, leporine, and human origins from 10 different countries and to 82 human isolates of the closely related species M. avium from France. Among the M. avium subsp. paratuberculosis isolates, 21 patterns were found by MIRU-VNTR typing, with a discriminatory index of 0.751. The predominant R01 IS900 restriction fragment length polymorphism type, comprising 131 isolates, was divided into 15 MIRU-VNTR types. Among the 82 M. avium isolates, the eight MIRU-VNTR loci distinguished 30 types, none of which was shared by M. avium subsp. paratuberculosis isolates, resulting in a discriminatory index of 0.889. Our results suggest that MIRU-VNTR typing is a fast typing method that, in combination with other methods, might prove to be optimal for PCR-based molecular epidemiological studies of M. avium/M. avium subsp. paratuberculosis pathogens. In addition, presumably identical M. avium subsp. paratuberculosis 316F vaccine strains originating from the Weybridge laboratory and from different commercial batches from Mérial actually differed by one or both typing methods. These results indicate a substantial degree of genetic drift among different vaccine preparations, which has important implications for prophylactic approaches.

Oxidative metabolic profiles of Brucella strains isolated from marine mammals: contribution to their species classification.

Since the 1990s, Brucella strains not matching the characteristics of any of the six conventional species have been isolated worldwide from marine mammals. In this study, 31 Brucella strains isolated from various marine mammals were examined for their oxidative metabolic pattern on 12 amino-acid and carbohydrate substrates. Three main oxidative profiles different from those of the Brucella terrestrial mammal strains were identified for the marine mammal strains: one gathering strains isolated from pinnipeds and two gathering strains from cetaceans. Thus, both oxidative metabolism results and previous molecular studies are in agreement with the proposal of two new Brucella species, Brucella pinnipediae and Brucella cetaceae, to classify the Brucella strains isolated from marine mammals, and are also in accordance with a classification of species of the Brucella genus based on host preference.

Mycobacterium smegmatis produces an HBHA homologue which is not involved in epithelial adherence.

Mycobacterium tuberculosis produces heparin-binding hemagglutinin (TB-HBHA), an adhesin involved in binding to non-professional phagocytes and in extrapulmonary dissemination. TB-HBHA binds sulphated glycoconjugates through its C-terminal lysine-rich domain and can be purified by heparin-Sepharose chromatography. Homologues of HBHA are found in other pathogenic mycobacteria, but previous investigations failed to demonstrate them in non-pathogenic Mycobacterium smegmatis. We identified a gene encoding a HBHA-like protein, named MS-HBHA, from the complete M. smegmatis genome. The deduced MS-HBHA amino acid sequence revealed 68% identity with that of TB-HBHA and contains lysine-rich repeats in its C-terminal domain. However, in contrast to TB-HBHA, the lysine-rich domain of MS-HBHA is preceded by a stretch of acidic residues. This difference likely explains the low affinity for heparin displayed by MS-HBHA compared to TB-HBHA. Isolation by heparin-Sepharose chromatography procedure and mass spectrometry analysis indicated that MS-HBHA, similar to TB-HBHA contains several methylated lysine residues in its C-terminal domain. Although MS-HBHA is associated with M. smegmatis cell wall fractions, it does not seem to play a role in epithelial adherence and its function remains unknown. We therefore conclude that TB-HBHA may have evolved as an adhesin in pathogenic mycobacteria from a homolog that serves a different function in a saprophytic mycobacterium.

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.

Immunological responses and protective efficacy against Brucella melitensis induced by bp26 and omp31 B. melitensis Rev.1 deletion mutants in sheep.

The commonly used live attenuated vaccine in ovine brucellosis prophylaxis is Brucella melitensis Rev.1. This vaccine is known to induce antibody responses in vaccinated animals indistinguishable by the current conventional serological tests from those observed in challenged animals. Brucella BP26 and Omp31 proteins have shown an interesting potential as diagnostic antigens for ovine brucellosis. Accordingly, the bp26 gene and both bp26 and omp31 genes have been deleted from the vaccine strain Rev.1. Immunogenicity and vaccine efficacy of the parental Rev.1 strain and of both mutants in protecting sheep against B. melitensis strain H38 challenge was evaluated by clinical and bacteriological examination of ewes. They were conjunctivally or subcutaneously vaccinated when 4 months old and then challenged with B. melitensis H38 at the middle of the first pregnancy following vaccination. Deletion of bp26 and omp31 genes did not significantly affect the well recognised capacity of Rev.1 to protect sheep against B. melitensis challenge. However, the protection conferred by the CGV2631 mutant was significantly lower than that conferred by the CGV26 mutant or the Rev.1 strain. Vaccinated and challenged animals were detected positive in classical serological tests and in the IFN-gamma assay. A BP26-based ELISA was investigated to discriminate between ewes vaccinated by the mutants and ewes challenged with B. melitensis H38. The cut-off which was chosen in order to have 100% specificity resulted in a moderate sensitivity for the detection of challenged ewes. The use in the field of one of the mutants as vaccine against a B. melitensis infection, combined with classic diagnostic tests and a BP26 ELISA, could thus give an improvement in the differentiation between vaccinated and infected animals and contribute to the objective of eradication of brucellosis in small ruminants.

Development of a multiplex PCR assay for polymorphism analysis of Brucella suis biovars causing brucellosis in swine.

Swine brucellosis is caused by the biovars 1, 2 and 3 of Brucella suis the identification of which up to now relies on microbiological tests lacking adequate specificity together with time consuming and expensive molecular procedures. Based on sequence variation of the omp2b gene, we have developed a four primer set multiplex PCR assay that was tested for polymorphism analysis of B. suis biovars causing brucellosis in swine. The assay exploits the single nucleotide polymorphisms found in omp2b gene of B. suis reference biovars which are conserved in 43 B. suis field isolates from different geographic origins and hosts. Three specific amplification patterns (S1, S2 and S3) were obtained for reference strains of B. suis biovars 1, 2 and 3, respectively. However, some B. suis field isolates identified as biovars 2 or 3 according AMOS-PCR, PCR-RFLP of omp31 and omp2 genes and classical bacteriological methods, resulted also in S1 patterns, limiting the typing usefulness of the method.

Evaluation and selection of tandem repeat loci for a Brucella MLVA typing assay.

BACKGROUND: The classification of Brucella into species and biovars relies on phenotypic characteristics and sometimes raises difficulties in the interpretation of the results due to an absence of standardization of the typing reagents. In addition, the resolution of this biotyping is moderate and requires the manipulation of the living agent. More efficient DNA-based methods are needed, and this work explores the suitability of multiple locus variable number tandem repeats analysis (MLVA) for both typing and species identification. RESULTS: Eighty tandem repeat loci predicted to be polymorphic by genome sequence analysis of three available Brucella genome sequences were tested for polymorphism by genotyping 21 Brucella strains (18 reference strains representing the six 'classical' species and all biovars as well as 3 marine mammal strains currently recognized as members of two new species). The MLVA data efficiently cluster the strains as expected according to their species and biovar. For practical use, a subset of 15 loci preserving this clustering was selected and applied to the typing of 236 isolates. Using this MLVA-15 assay, the clusters generated correspond to the classical biotyping scheme of Brucella spp. The 15 markers have been divided into two groups, one comprising 8 user-friendly minisatellite markers with a good species identification capability (panel 1) and another complementary group of 7 microsatellite markers with higher discriminatory power (panel 2). CONCLUSION: The MLVA-15 assay can be applied to large collections of Brucella strains with automated or manual procedures, and can be proposed as a complement, or even a substitute, of classical biotyping methods. This is facilitated by the fact that MLVA is based on non-infectious material (DNA) whereas the biotyping procedure itself requires the manipulation of the living agent. The data produced can be queried on a dedicated MLVA web service site.

Development and evaluation as vaccines in mice of Brucella melitensis Rev.1 single and double deletion mutants of the bp26 and omp31 genes coding for antigens of diagnostic significance in ovine brucellosis.

The live attenuated Brucella melitensis Rev.1 strain is considered the best vaccine available for the prophylaxis of brucellosis in sheep caused by either B. melitensis or Brucella ovis. However, its application stimulates antibody responses in vaccinated animals indistinguishable by the current conventional serological tests from those observed in infected animals. The periplasmic protein BP26 and the outer membrane protein (OMP) Omp31 are immunodominant antigens in the serological responses of B. melitensis and B. ovis infected sheep, respectively. Accordingly, vaccine strain Rev.1 single and double deletion mutants of the bp26 and omp31 genes were developed, based on the principle that the use of such mutants as vaccines in association with diagnostic tests based on BP26 and Omp31 antigens would allow the serological differentiation between infected and vaccinated animals. The deletion mutants obtained were indistinguishable from the parental Rev.1 strain by conventional bacteriological and typing tests. The expression of their major surface antigens, as determined by reactivity with specific monoclonal antibodies (MAbs), remained unaffected, i.e. smooth-lipopolysaccharide (S-LPS) and OMPs besides in the expression of the antigens whose respective genes were deleted. The bp26 and omp31 deletions did not modify the kinetics of splenic infection nor the residual virulence of Rev.1 in the BALB/c mouse model. Vaccination of BALB/c mice with the deletion mutants conferred significant protective immunity against B. melitensis strain H38 or B. ovis strain PA challenges, to the same extent as that induced by parental Rev.1 strain. Thus, these Rev.1 bp26 or omp31 deletion mutants are promising vaccine candidates against B. melitensis and B. ovis infections and will be further evaluated in sheep.

Epitope mapping of the Brucella melitensis BP26 immunogenic protein: usefulness for diagnosis of sheep brucellosis.

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.

Classification of Brucella strains isolated from marine mammals by infrequent restriction site-PCR and development of specific PCR identification tests.

Brucella strains have been isolated since the 1990s from a wide variety of marine mammals and represent potential zoonotic pathogens. They have distinctive phenotypic and molecular characteristics from the terrestrial mammal Brucella species, and two new species names have been previously proposed based on DNA polymorphism at the omp2 locus and their preferential host, i.e. Brucella cetaceae for cetacean isolates and Brucella pinnipediae for pinniped isolates. The results presented in this study on characterization of these strains by infrequent restriction site-PCR (IRS-PCR), taking into account the higher number of IS711 elements in their genome compared to terrestrial mammal Brucella species, supports this classification. The nucleotide sequences of specific DNA fragments detected by IRS-PCR were determined and used to develop PCR identification tests for either B. cetaceae or B. pinnipediae.

Lipopolysaccharide heterogeneity in Brucella strains isolated from marine mammals.

Smooth lipopolysaccharides (S-LPSs) from Brucella strains isolated from seals, dolphins, porpoises, an otter and a minke whale were characterized by ELISA using monoclonal antibodies (mAbs) directed against seven previously defined O-polysaccharide (O-PS) epitopes and by Western blot after SDS-PAGE. All strains studied were A-dominant as shown by specific polyclonal sera and this was also confirmed by the mAbs. However, binding patterns in ELISA of mAbs to the specific common (C) epitopes were rather heterogeneous, and for some strains, such as those isolated from striped dolphins, binding of these mAbs was much reduced or negative as had previously been shown for Brucella suis biovar 2 strains. Western blot after SDS-PAGE showed the typical A-dominant strain banding pattern for all marine mammal Brucella isolates, but the average S-LPS size was shorter in many of these compared to reference Brucella abortus strain 544. Thus, S-LPSs of the marine mammal isolates show heterogeneity with regard to their O-PS C epitope content and their average size.

Identification of Brucella melitensis vaccine strain Rev.1 by PCR-RFLP based on a mutation in the rpsL gene.

The live attenuated strain B. melitensis Rev.1 is considered the best vaccine available for the prophylaxis of brucellosis in sheep and goats. The Rev.1 vaccine was obtained in the 1950s by a two-step selection involving firstly streptomycin resistance and dependence and secondly reversion of dependence but keeping streptomycin resistance. Chromosomally acquired streptomycin resistance is frequently due to mutations in the gene encoding the ribosomal protein S12, rpsL. Nucleotide sequencing revealed one mutation in the rpsL gene of vaccine strain Rev.1 compared to that of reference strain 16M leading to an amino acid Pro-to-Leu change at codon position 91 (Pro91Leu). This mutation resulted also in the lack of a NciI restriction site in the gene. PCR-restriction fragment length polymorphism (PCR-RFLP) using NciI applied to a large number of Brucella reference and field strains showed that the mutation detected was specific of vaccine strain Rev.1.

Phenotypic and molecular characterization of a Brucella strain isolated from a minke whale (Balaenoptera acutorostrata).

Isolation of Brucella spp. in marine mammals has been reported during the past several years. A Brucella strain from the spleen and liver of a minke whale (Balaenoptera acutorostrata) was isolated. Conventional typing methods indicated that this isolate was related to the genus Brucella but did not match the profiles of any known Brucella species or biovar. Successful PCR amplification of the Brucella rrs-rrl spacer sequence and of the insertion sequence IS6501 also indicated that the minke whale strain was related to the genus Brucella. In addition, the rrs gene of this strain shared a very high degree of nucleotide identity (>98%) with published Brucella spp. rrs sequences. However, RFLP studies using an IS6501-specific probe showed a unique profile for this strain in comparison with the profiles of the six known Brucella species. Moreover, analysis of the omp2 locus by PCR-RFLP, by Southern hybridization using omp2a- and omp2b-specific probes, and by DNA sequencing showed that the minke whale isolate possesses two copies of the omp2b gene instead of one omp2a and one omp2b gene copy or two copies of the omp2a gene described in the six known Brucella species. Thus, molecular typing methods showed that this isolate is clearly distinct from all other known Brucella species and strains. The specific molecular features of this minke whale Brucella isolate raise questions about the lineage between the Brucella strains isolated from marine mammals and the Brucella species isolated from terrestrial mammals.

DNA polymorphism at the omp-31 locus of Brucella spp.: evidence for a large deletion in Brucella abortus, and other species-specific markers.

The omp-31 gene, encoding a major outer-membrane protein in Brucella melitensis, was PCR-amplified from Brucella strains representing all species and known biovars by using primers selected according to the B. melitensis 16M omp-31 published sequence. Amplification of omp-31 was achieved from DNA of all Brucella species with the exception of Brucella abortus, the only Brucella species where expression of omp-31 was not detected by reactivity with an mAb specific for an epitope located in Omp-31. Southern blot hybridization of plasmid probes, bearing inserts (4.4-17 kb) containing B. melitensis 16M omp-31 and adjacent DNA of different sizes, with HindIII-digested total DNA showed that a large fragment, comprising the entire omp-31 gene and flanking DNA, was actually absent in B. abortus strains. The size of this DNA fragment has been determined to be about 10 kb. Southern blot hybridization with the different plasmid probes identified species-specific markers for B. abortus and B. melitensis. At the biovar level, a specific marker for B. melitensis bv. 1 was also identified. Additionally, PCR-RFLP studies of omp-31 revealed specific markers for Brucella ovis, Brucella canis and Brucella suis bv. 2. Using a combination of omp-31 PCR-RFLP patterns and Southern blot hybridization profiles Brucella species were differentiated with the sole exception of Brucella neotomae which was not differentiated from B. suis bv. 1, 3, 4 and 5. Results presented in this paper demonstrate the potential of omp-31 for differentiating the brucellae and show that B. abortus lacks a large DNA fragment of about 10 kb containing omp-31 and flanking DNA. In such a large deletion, other genes in addition to omp-31 are probably involved. Sequencing of this DNA fragment will help to identify the missing genes in B. abortus which could possibly be involved in the differences of pathogenicity and host preference seen in Brucella species.