Lorraine L. Perrett

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.

Brucella papionis sp. nov., isolated from baboons (Papio spp.)

Two Gram-negative, non-motile, non-spore-forming coccoid bacteria (strains F8/08-60(T) and F8/08-61) isolated from clinical specimens obtained from baboons (Papio spp.) that had delivered stillborn offspring were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA gene sequence similarities, both strains, which possessed identical sequences, were assigned to the genus Brucella. This placement was confirmed by extended multilocus sequence analysis (MLSA), where both strains possessed identical sequences, and whole-genome sequencing of a representative isolate. All of the above analyses suggested that the two strains represent a novel lineage within the genus Brucella. The strains also possessed a unique profile when subjected to the phenotyping approach classically used to separate species of the genus Brucella, reacting only with Brucella A monospecific antiserum, being sensitive to the dyes thionin and fuchsin, being lysed by bacteriophage Wb, Bk2 and Fi phage at routine test dilution (RTD) but only partially sensitive to bacteriophage Tb, and with no requirement for CO2 and no production of H2S but strong urease activity. Biochemical profiling revealed a pattern of enzyme activity and metabolic capabilities distinct from existing species of the genus Brucella. Molecular analysis of the omp2 locus genes showed that both strains had a novel combination of two highly similar omp2b gene copies. The two strains shared a unique fingerprint profile of the multiple-copy Brucella-specific element IS711. Like MLSA, a multilocus variable number of tandem repeat analysis (MLVA) showed that the isolates clustered together very closely, but represent a distinct group within the genus Brucella. Isolates F8/08-60(T) and F8/08-61 could be distinguished clearly from all known species of the genus Brucella and their biovars by both phenotypic and molecular properties. Therefore, by applying the species concept for the genus Brucella suggested by the ICSP Subcommittee on the Taxonomy of Brucella, they represent a novel species within the genus Brucella, for which the name Brucella papionis sp. nov. is proposed, with the type strain F8/08-60(T) ( = NCTC 13660(T) = CIRMBP 0958(T)).

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

ISOLATION AND CHARACTERIZATION OF BRUCELLA FROM THE LUNGWORMS OF A HARBOR PORPOISE (PHOCOENA PHOCOENA)

Adult female nematodes identified as Pseudalius inflexus were collected from the lungs of a juvenile male harbor porpoise (Phocoena phocoena) found dead on a beach in Cornwall, UK. Classic and molecular typing methods, immunologic and electron microscopy immunolabeling techniques, provided evidence of Brucella sp. infection within the uterine tissue of nematodes of this marine mammal. This finding presents further evidence to suggest parasites should be considered as a potential means of transfer of bacterial infection in marine mammals and highlights the zoonotic implications for humans exposed to marine mammals through occupation or leisure.

A new homogeneous assay for high throughput serological diagnosis of brucellosis in ruminants.

The control and eradication of brucellosis is highly desirable but heavily resource intensive as high throughput serological testing is required. The aim of this study was to meet the needs of high throughput screening laboratories involved in this process through the development of a new assay. An existing cELISA used for the serodiagnosis of brucellosis in ruminants was converted to an AlphaLISA homogenous proximity based assay. This assay requires no separation steps and can be performed in low volume microtitre format. The Brucella AlphaLISA was validated on a panel of bovine, ovine and caprine sera from infected and uninfected animals. The diagnostic sensitivities (>96%) and specificities (>98%) obtained compared well to those from cELISA, iELISA and FPA performed on the same samples. The AlphaLISA met the testing criteria set for ELISAs as defined by the OIEELISA standards and had an analytical sensitivity similar to that of the parent cELISA. The method was also used on a small panel of serum samples from cattle that were experimentally infected with Yersinia enterocolitica O:9. Some false positive reactions were obtained as was also the case with results from FPA, iELISA, cELISA, CFT and SAT. Despite this, the methodological advantages of the AlphaLISA mean that this assay is well suited to high throughput serodiagnosis. This report is the first description of the use of AlphaLISA to detect pathogen specific antibodies. Furthermore, the relative ease with which the cELISA was converted to this platform indicates that this technology is ready to meet the high throughput testing requirements for the diagnosis of many other diseases.

Meningoencephalitis associated with Brucella species in a live-stranded striped dolphin (Stenella coeruleoalba) in south-west England

THE first recovery of a Brucella species from marine mammals was reported in Scotland in 1994 ([Ross and others 1994][1]). Since then, there have been many reports of both serological evidence and isolations of Brucella species from many marine mammal species around the world ([Dawson 2005][2], [

An evaluation of the capability of existing and novel serodiagnostic methods for porcine brucellosis to reduce false positive serological reactions.

Porcine brucellosis is a zoonotic disease of truly global significance because even in countries without the disease the occurrence of false positive serological reactions (FPSRs) creates significant problems. Statutory diagnostic testing is required in many disease free countries or regions and is often a prerequisite for the movement of live animals. Currently this testing is dependent almost entirely on serological assays and these may result in a significant number of FPSRs. The aim of this study was to examine existing and novel serodiagnostic assays to evaluate their diagnostic sensitivity and resilience to FPSRs. The existing assays evaluated were the RBT, smooth lipopolysaccharide (sLPS) indirect (i) ELISA, sLPS competitive (c) ELISA, and the FPA. The novel assays evaluated were the sLPS TR-FRET assay, a rough (r) LPS iELISA, a recombinant protein BP26 iELISA and a cytoplasmic protein extract (Brucellergene™) iELISA. Four populations of sera were evaluated: those from Brucella suis infected swine (n=34), randomly selected samples from non-infected swine (n=161), sera from non-infected swine within herds exhibiting FPSRs (n=132) and sera from swine experimentally infected with Yersinia enterocolitica O:9 (n=4). The results show that all the assays dependent on the sLPS O-polysaccharide (OPS) for their sensitivity (the RBT, sLPS ELISAs, FPA and the sLPS TR-FRET) had significantly reduced diagnostic specificity when applied to the FPSR population, the RBT being most affected. Of the two rapid homogeneous assays, the TR-FRET was diagnostically superior to the FPA in this study. Neither of the protein based iELISAs demonstrated sufficient diagnostic sensitivity to resolve the FPSRs. The rLPS iELISA showed no cross reaction with the FPSRs and had diagnostic sensitivity similar to that of the OPS based assays.