Simon D. Brew

A review of Brucella sp. infection of sea mammals with particular emphasis on isolates from Scotland

Brucellae recovered from sea mammals were first reported in 1994. In the years since both culture and serological analysis have demonstrated that the infection occurs in a wide range of species of marine mammals inhabiting a vast amount of the worlds oceans. Molecular studies have demonstrated that the isolates differ from those found amongst terrestrial animals and also distinguish between strains which have seals and cetaceans as their preferred hosts. At the phenotypic level seal and cetacean strains can also be differed with respect to their CO(2) requirement, primary growth on Farrells medium and metabolic activity on galactose. Two new species B. cetaceae and B. pinnipediae have been proposed as a result. This paper provides a review of Brucella in sea mammals and updates findings from the study of sea mammals from around the coast of Scotland.

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)).

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.

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.

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.

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.

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.