Alastair P. MacMillan

Characterisation of the genetic diversity of Brucella by multilocus sequencing

BackgroundBrucella species include economically important zoonotic pathogens that can infect a wide range of animals. There are currently six classically recognised species of Brucella although, as yet unnamed, isolates from various marine mammal species have been reported. In order to investigate genetic relationships within the group and identify potential diagnostic markers we have sequenced multiple genetic loci from a large sample of Brucella isolates representing the known diversity of the genus.ResultsNine discrete genomic loci corresponding to 4,396 bp of sequence were examined from 160 Brucella isolates. By assigning each distinct allele at a locus an arbitrary numerical designation the population was found to represent 27 distinct sequence types (STs). Diversity at each locus ranged from 1.03–2.45% while overall genetic diversity equated to 1.5%. Most loci examined represent housekeeping gene loci and, in all but one case, the ratio of non-synonymous to synonymous change was substantially <1. Analysis of linkage equilibrium between loci indicated a strongly clonal overall population structure. Concatenated sequence data were used to construct an unrooted neighbour-joining tree representing the relationships between STs. This shows that four previously characterized classical Brucella species, B. abortus, B. melitensis, B. ovis and B. neotomae correspond to well-separated clusters. With the exception of biovar 5, B. suis isolates cluster together, although they form a more diverse group than other classical species with a number of distinct STs corresponding to the remaining four biovars. B. canis isolates are located on the same branch very closely related to, but distinguishable from, B. suis biovar 3 and 4 isolates. Marine mammal isolates represent a distinct, though rather weakly supported, cluster within which individual STs display one of three clear host preferences.ConclusionThe sequence database provides a powerful dataset for addressing ongoing controversies in Brucella taxonomy and a tool for unambiguously placing atypical, phenotypically discordant or newly emerging Brucella isolates. Furthermore, by using the phylogenetic backbone described here, robust and rationally selected markers for use in diagnostic assay development can be identified.

Identification and Characterization of Variable-Number Tandem-Repeat Markers for Typing of Brucella spp.

ABSTRACT Members of the genus Brucella infect many domesticated and wild animals and cause serious zoonotic infection in humans. The availability of discriminatory molecular typing tools to inform and assist conventional epidemiological approaches would be invaluable in controlling these infections, but efforts have been hampered by the genetic homogeneity of the genus. We report here on a molecular subtyping system based on 21 variable-number tandem-repeat (VNTR) loci consisting of 13 previously unreported loci and 8 loci previously reported elsewhere. This approach was applied to a collection of 121 Brucella isolates obtained worldwide and representing all six classically recognized Brucella species. The size of repeats selected for inclusion varied from 5 to 40 bp giving VNTR loci with a range of diversities. The number of alleles detected ranged from 2 to 21, and Simpsons diversity index values ranged from 0.31 to 0.92. This assay divides the 121 isolates into 119 genotypes, and clustering analysis results in groups that, with minor exceptions, correspond to conventional species designations. Reflecting this, the use of six loci in isolation was shown to be sufficient to determine species designation. On the basis of the more variable loci, the assay could also discriminate isolates originating from restricted geographical sources, indicating its potential as an epidemiological tool. Stability studies carried out in vivo and in vitro showed that VNTR profiles were sufficiently stable such that recovered strains could readily be identified as the input strain. The method described here shows great potential for further development and application to both epidemiological tracing of Brucella transmissions and in determining relationships between isolates worldwide.

Validation of FPA and cELISA for the detection of antibodies to Brucella abortus in cattle sera and comparison to SAT, CFT, and iELISA

The fluorescence polarisation assay (FPA) is a recently described test for the serological diagnosis of Brucella infection. It has many methodological advantages over older, more established tests and can be performed in a fraction of the time. To validate the FPA, serum samples from 146 confirmed (by culture) Brucella-infected cattle were tested in conjunction with serum samples from 1947 noninfected cattle. The competitive ELISA (cELISA) was validated using these positive reference samples and 1440 negative samples, while data for the indirect ELISA (iELISA) was generated from 6957 negative samples plus the positive sera. Published diagnostic specificity (DSp) data for the complement fixation test (CFT) and serum agglutination test (SAT) was used in conjunction with the test results on the positive sera to obtain diagnostic specificity plus diagnostic sensitivity (DSn). After selection of a cutoff for the FPA and cELISA, the diagnostic specificity and sensitivity total for each test were compared. The results, with 95% confidence intervals, were: FPA (195.7+/-2.79), iELISA (195.0+/-2.70), cELISA (194.9+/-3.48), CFT (191.7+/-4.45), and SAT (180.4+/-6.33). The data presented supports the use of the FPA in diagnosis of brucellosis and questions the use of the SAT and CFT for either screening or confirmatory testing.

Use of Amplified Fragment Length Polymorphism To Identify and Type Brucella Isolates of Medical and Veterinary Interest

ABSTRACT Amplified fragment length polymorphism (AFLP) is a whole-genome fingerprinting method that relies on the selective PCR amplification of restriction fragments. The potential of this approach for the discrimination of Brucella isolates at the species and intraspecies level was assessed. A number of different combinations of restriction enzymes and selective primers were examined, and one, using EcoRI and MseI with additional selective TC bases on the MseI primer, was selected for full assessment against a panel of Brucella isolates. The technique could readily differentiate Brucella spp. from all Ochrobactrum spp. representing the group of organisms most closely related to Brucella spp. Application of AFLP highlighted the genetic homogeneity of Brucella. In spite of this determination of AFLP profiles of large numbers of isolates of human and animal origin, including Brucella abortus, B. melitensis, B. ovis, B. neotomae, marine mammal isolates (no species name), B. canis, and B. suis, confirmed that all but the latter two species could be separated into distinct clusters based on characteristic and conserved differences in profile. Only B. suis and B. canis isolates clustered together and could not be distinguished by this approach, adding to questions regarding the validity of species assignments in this group. Under the conditions examined in the present study only limited intraspecies genomic differences were detected, and thus this AFLP approach is likely to prove most useful for identification to the species level. However, combination of several of the useful restriction enzyme-primer combinations identified in the present study could substantially add to the discriminatory power of AFLP when applied to Brucella and enhance the value of this approach.

Serological evidence of Brucella species infection in odontocetes from the south Pacific and the Mediterranean

Sera from 58 odontocetes taken in fisheries off Peru in 1993 to 1995 and from 24-cetaceans stranded along the Spanish coast of the Mediterranean in 1997 to 1999 were tested for the presence of Brucella species antibodies in competitive and indirect ELISAS (cELISA and iELISA). Among the animals from Peru, 21 of 27 (77.8 per cent) Lagenorhynchus obscurus, three of six Delphinus capensis, one of two inshore and two of three offshore Tursiops truncatus and five of 20 (25 per cent) Phocoena spinipinnis were positive in the cELISA. Brucella species antibodies were also observed in two of 16 (12.5 per cent) Stenella coeruleoalba and in one of two T truncatus from the Mediterranean. These data provide the first evidence for the presence of cetacean brucellae in the south Pacific Ocean and the Mediterranean Sea.

Phenotypic and molecular characterisation of Brucella isolates from marine mammals

BackgroundBacteria of the genus Brucella are the causative organisms of brucellosis in animals and man. Previous characterisation of Brucella strains originating from marine mammals showed them to be distinct from the terrestrial species and likely to comprise one or more new taxa. Recently two new species comprising Brucella isolates from marine mammals, B. pinnipedialis and B. ceti, were validly published. Here we report on an extensive study of the molecular and phenotypic characteristics of marine mammal Brucella isolates and on how these characteristics relate to the newly described species.ResultsIn this study, 102 isolates of Brucella originating from eleven species of marine mammals were characterised. Results obtained by analysis using the Infrequent Restriction Site (IRS)-Derivative PCR, PCR-RFLP of outer membrane protein genes (omp) and IS711 fingerprint profiles showed good consistency with isolates originating from cetaceans, corresponding to B. ceti, falling into two clusters. These correspond to isolates with either dolphins or porpoises as their preferred host. Isolates originating predominantly from seals, and corresponding to B. pinnipedialis, cluster separately on the basis of IS711 fingerprinting and other molecular approaches and can be further subdivided, with isolates from hooded seals comprising a distinct group. There was little correlation between phenotypic characteristics used in classical Brucella biotyping and these groups.ConclusionMolecular approaches are clearly valuable in the division of marine mammal Brucella into subtypes that correlate with apparent ecological divisions, whereas conventional bioyping is of less value. The data presented here confirm that there are significant subtypes within the newly described marine mammal Brucella species and add to a body of evidence that could lead to the recognition of additional species or sub-species within this group.

Competitive ELISA for bovine brucellosis suitable for testing poor quality samples.

Unutbefor tesin * A COMPETITIVE enzyme-linked immunosorbent assay (CELISA) was evaluated as an alternative confirmatory test to the complement fixation test (CFT) for the diagnosis of brucellosis in cattle. The aim was to show that the cELISA was capable of testing poor quality serum samples which were unsuitable for diagnosis by the CFT. An M dominant epitope lipopolysaccharide (LPS) antigen was extracted from Brucella melitensis strain 16M, by the hot phenol method (Cherwonogrodzky and others 1991). An anti-M epitope monoclonal antibody (mAb) (Greiser-Wilke and others 1985) was conjugated with horseradish peroxidase (HRP) by a method adapted from Nakane and Kawaoi (1974). HRP (20 mg) was dissolved in 5 ml distilled water and 1-2 ml freshly prepared 0*1M sodium periodate was added. After stirring for 20 minutes at room temperature, the activated HRP was dialysed against lmM sodium acetate (pH 4.0) for 15 to 20 hours. The dialysed HRP and 20 mg mAb were adjusted to pH 9-0 before being combined, and then stirred for two hours at room temperature, after which 0 5 ml ascorbic acid (4 mg/ml) was added. After four hours incubation at 4°C, the mixture was dialysed for 15 to 20 hours against several changes of 0-1M phosphate buffered saline. The conjugate was passed through a 100 kD filter, added to equal volumes of glycerol and stored at -20°C. The optimal concentration of antigen and conjugate was determined by titration against standard sera from a known Brucella-free sheep and serum from a goat experimentally infected with B melitensis. The optimum concentrations selected were those that gave the highest ratio between the optical density (OD) of the negative and positive standards in conjunction with an OD of less than 0.100 for the positive standard and greater than 0*700 for the negative standard. The M dominant B melitensis antigen and anti-M mAb were selected as they have been shown to be less affected by false positive serological reactions caused by cross-reacting bacteria, such as Yersinia enterocolitica 0:9, which affects all Brucella serodiagnostic assays (A. P. MacMillan, unpublished observations). Brucella abortus infection is detected by this combination of antigen and conjugate, as antibodies specific for the M epitope are produced. The CELISA was adapted from the method described by MacMillan and others (1990). LPS antigen (100 VI), suspended in carbonate buffer (pH 9.6) aqa predetermined dilution, was added to each well of a 96-well polystyrene polysorp plate (Nunc Life Technologies) and incubated overnight at 40C. Unbound antigen was removed by washing with phosphate buffered saline (PBS) (pH 7.2) containing 0.01 per cent Tween 20 (wash solution). Undiluted test and control sera (20 ill) were dispensed into respective duplicate wells and mixed with 100 VI of conjugate at a predetermined dilution. The reaction took place at room temperature for 30 minutes on a rotary shaker at 160 revs/minute, after which the plates were washed three times with the wash solution. The reaction was developed with 100 il1 of substrate/chromogen solution comprising 30 mg o-phenylenediamine dihydrochloride (OPD) and 300 VIl 3 per cent hydrogen peroxide suspended in 75 ml distilled water. The reaction was stopped after 15 minutes incubation *Numberf sampls whichwere unsuitbl for tsigdeto ecessNive haemolysi andu/oraticomWlplemelntarativt

SEROLOGIC SURVEY FOR BRUCELLA SPP., PHOCID HERPESVIRUS-1, PHOCID HERPESVIRUS-2, AND PHOCINE DISTEMPER VIRUS IN HARBOR SEALS FROM ALASKA, 1976–1999

Harbor seals (Phoca vitulina richardsi) were captured in the coastal regions of Southeast Alaska, Gulf of Alaska, Prince William Sound (PWS), and Kodiak Island during 1976–1999. Blood was collected from 286 seals. Sera were tested for evidence of exposure to Brucella spp., phocid herpesvirus-1 (PhoHV-1), phocid herpesvirus-2 (PhHV-2), and phocine distemper virus (PDV). Antibody prevalence rates were 46% (46/100) for Brucella spp., 93% (225/243) for PhoHV-1, 0% (0/286) for PhHV-2, and 1% (2/160) for PDV. Antibody prevalence for Brucella spp. was directly related to host age. Antibody prevalence for PhoHV-1 was higher in PWS as compared to the other three regions. No evidence of mortality attributable to these four agents was observed during the course of this study. Based on the results of this survey, none of these agents is considered a significant mortality factor in harbor seals from the four regions of coastal Alaska included in the study.

Liposomal delivery of p-ialB and p-omp25 DNA vaccines improves immunogenicity but fails to provide full protection against B. melitensis challenge

BackgroundWe have previously demonstrated protective efficacy against B. melitensis using formulations of naked DNA vaccines encoding genes ialB and omp25. The present study was undertaken to further understand the immune response generated by the protective vaccination regimens and to evaluate cationic liposome adsorption as a delivery method to improve vaccine utility.MethodsThe protective efficacy and immunogenicity of vaccines delivered as four doses of naked DNA, a single dose of naked DNA or a single dose of DNA surface adsorbed to cationic liposomes were compared using the BALB/c murine infection model of B. melitensis. Antigen-specific T cells and antibody responses were compared between the various formulations.ResultsThe four dose vaccination strategy was confirmed to be protective against B. melitensis challenge. The immune response elicited by the various vaccines was found to be dependent upon both the antigen and the delivery strategy, with the IalB antigen favouring CD4+ T cell priming and Omp25 antigen favouring CD8+. Delivery of the p-ialB construct as a lipoplex improved antibody generation in comparison to the equivalent quantity of naked DNA. Delivery of p-omp25 as a lipoplex altered the profile of responsive T cells from CD8+ to CD4+ dominated. Under these conditions neither candidate delivered by single dose naked DNA or lipoplex vaccination methods was able to produce a robust protective effect.ConclusionsDelivery of the p-omp25 and p-ialB DNA vaccine candidates as a lipoplex was able to enhance antibody production and effect CD4+ T cell priming, but was insufficient to promote protection from a single dose of either vaccine. The enhancement of immunogenicity by lipoplex delivery is a promising step toward improving the practicality of these two candidate vaccines, and suggests that this lipoplex formulation may be of value in situations where improvements to CD4+ responses are required. However, in the case of Brucella vaccine development it is suggested that further modifications to the candidate vaccines and delivery strategies will be required in order to deliver sustained protection.

Efficacy of moxifloxacin or gatifloxacin as prophylaxis against experimental murine Brucella melitensis infection

The prophylactic potential of moxifloxacin and gatifloxacin was assessed in comparison with doxycycline, an established therapeutic antibiotic, to limit or control infection by Brucella melitensis in an experimental mouse model, determined by reduced bacterial burden in the spleen. Although moxifloxacin was found to have a small protective effect when administered 6 h following infection, neither moxifloxacin nor gatifloxacin showed significant efficacy in vivo. In comparison, doxycycline provided significant protection when prophylaxis was started at 6 h, 7 days or 14 days following infection. Overall, these results confirm the utility of doxycycline in the prophylaxis of brucellosis and suggest that neither moxifloxacin nor gatifloxacin are likely to be valuable for post-exposure prophylaxis of Brucella infection.