First confirmed reports of the isolation of Brucella ceti from a Risso's dolphin Grampus griseus and a killer whale Orcinus orca.

Brucella ceti has been recovered from a number species of cetaceans worldwide over the last 25 yr. Here we report, for the first time, the recovery of B. ceti from a Risso's dolphin Grampus griseus and a killer whale Orcinus orca. Recovery from an abdominal mass in the dolphin provides further evidence of the systemic pathogenic potential for B. ceti infection in cetaceans. The isolation of B. ceti ST23 (porpoise cluster) from a killer whale from a group known to eat other marine mammals raises the possibility of infection via ingestion. This report takes the number of cetacean species in UK coastal waters from which B. ceti has been isolated to 11 and highlights the value of routine, comprehensive and specific screening for significant pathogens such as Brucella sp. by strandings networks.

Meningoencephalitis in a common minke whale Balaenoptera acutorostrata associated with Brucella pinnipedialis and gamma-herpesvirus infection.

Fatal marine Brucella infections with histologic lesions specific to the central nervous system (CNS), known as neurobrucellosis, have been described in 5 species of odontocete cetaceans in the UK: striped dolphins Stenella coeruleoalba, Atlantic white-sided dolphins Lagenorhynchus acutus, short-beaked common dolphins Delphinus delphis, long-finned pilot whale Globicephala melas and Sowerby's beaked whale Mesoplodon bidens. To date, these CNS lesions have only been associated with Brucella ceti ST26 and not with B. pinnipedialis, which is rarely isolated from cetaceans and, although commonly found in various seal species, has never been associated with any pathology. This paper describes the first report of neurobrucellosis in a common minke whale Balaenoptera acutorostrata which was associated with the isolation of Brucella pinnipedialis ST24 and co-infection with Balaenoptera acutorostrata gamma-herpesvirus 2. This is the first report of neurobrucellosis in any species of mysticete and the first report of Brucella pinnipedialis in association with any pathology in any species of marine mammal, which may be due to co-infection with a herpesvirus, as these are known to be associated with immunosuppression.

Neurobrucellosis due to Brucella ceti ST26 in Three Sowerby's Beaked Whales (Mesoplodon bidens).

Fatal meningoencephalitis due to Brucella ceti infection has been described in striped dolphins (Stenella coeruleoalba), Atlantic white-sided dolphins (Lagenorhynchus acutus), short-beaked common dolphins (Delphinus delphis) and long-finned pilot whales (Globicephala melas), which are all within the family Delphinidae. We report B. ceti-associated neurobrucellosis in three juvenile male Sowerby's beaked whales (Mesoplodon bidens) that all had typical lesions of lymphocytic meningoencephalitis, which increased in severity from rostral to caudal regions of the brain. In two cases there was loss of ependymal cells lining the cerebral ventricular system, with large numbers of lymphocytes in the underlying neuropil. This finding suggests that B. ceti gains access to, and multiplies in, the cerebrospinal fluid, and confirms that this is the sample of choice for bacteriological recovery of the causative organism. These findings expand the increasing range of cetaceans susceptible to neurobrucellosis to members of the family Ziphiidae.

First isolation of Brucella pinnipedialis and detection of Brucella antibodies from bearded seals Erignathus barbatus.

Brucella species infecting marine mammals was first reported in 1994 and in the years since has been documented in various species of pinnipeds and cetaceans. While these reports have included species that inhabit Arctic waters, the few available studies on bearded seals Erignathus barbatus have failed to detect Brucella infection to date. We report the first isolation of Brucella pinnipedialis from a bearded seal. The isolate was recovered from the mesenteric lymph node of a bearded seal that stranded in Scotland and typed as ST24, a sequence type associated typically with pinnipeds. Furthermore, serological studies of free-ranging bearded seals in their native waters detected antibodies to Brucella in seals from the Chukchi Sea (1990-2011; 19%) and Svalbard (1995-2007; 8%), whereas no antibodies were detected in bearded seals from the Bering Sea or Bering Strait or from captive bearded seals.

Evidence of ongoing brucellosis in livestock animals in North West Libya.

Animal brucellosis is thought to be present in small ruminants, cattle, and camels in Libya, particularly in the west coastal strip. Before the system collapsed due to political unrest in 2011, prevalence of the disease did not exceed 0.2% in cattle, 0.1% in camels, 8.3% in sheep, and 14.8% in goats. The aim of this study was to highlight outbreaks of disease that took place during the 18-month period from November 2014 to April 2016. A total of 1612 serum samples, collected opportunistically from 29 herds in 12 different localities in the northwest region of Libya, were investigated for brucellosis. The samples were screened for Brucella antibodies using the Rose Bengal test, and confirmed with either indirect enzyme linked immunosorbent assay in the case of sheep, and/or a serum agglutination test, followed with a complement fixation test, in the case of cattle and camels. Our results showed the highest rates of brucellosis seropositivity in goats (33.4%) and sheep (9.2%). The overall percentage of brucellosis seropositivity was 21%. The high level of brucellosis identified by this study, particularly in small ruminants, strongly suggests re-emergence of the disease in the region. Re-evaluation of intervention measures applied to the control of brucellosis is highly recommended.

Exposure of harbour seals Phoca vitulina to Brucella in declining populations across Scotland.

Since 2000 there has been a major decline in the abundance of Scottish harbour seals Phoca vitulina. The causes of the decline remain uncertain. The aim of this study was to establish the extent to which the seals in the regions of greatest decline have been exposed to Brucella, a bacterial pathogen that causes reproductive failure in terrestrial mammalian hosts. Tissues from dead seals collected between 1992 and 2013 were cultured for Brucella (n = 150). Serum samples collected from live capture-released seals (n = 343) between 1997 and 2012 were tested for Brucella antibodies using the Rose Bengal plate agglutination test (RBT) and a competitive enzyme-linked immunosorbent assay (cELISA). In total, 16% of seals cultured had Brucella isolated from one or more tissues, but there were no pathological signs of infection. The cELISA results were more sensitive than the RBT results, showing that overall 25.4% of seals were seropositive, with the highest seroprevalence in juveniles. As there was no evidence of either a higher seroprevalence or higher circulating antibody levels in seropositive animals in the areas with the greatest declines, it was concluded that Brucella infection is likely not a major contributing factor to recent declines. However, the consistently high proportion of seals exposed to Brucella indicates possible endemicity in these populations, likely due to B. pinnipedialis, which has demonstrated a preference for pinniped hosts. Importantly, given the close proximity between seals, humans and livestock in many areas, there is the potential for cross-species infections.

Characterisation of North American Brucella isolates from marine mammals.

Extension of known ecological niches of Brucella has included the description of two novel species from marine mammals. Brucella pinnipedialis is associated predominantly with seals, while two major Brucella ceti clades, most commonly associated with porpoises or dolphins respectively, have been identified. To date there has been limited characterisation of Brucella isolates obtained from marine mammals outside Northern European waters, including North American waters. To address this gap, and extend knowledge of the global population structure and host associations of these Brucella species, 61 isolates from marine mammals inhabiting North American waters were subject to molecular and phenotypic characterisation enabling comparison with existing European isolates. The majority of isolates represent genotypes previously described in Europe although novel genotypes were identified in both B. ceti clades. Harp seals were found to carry B. pinnipedialis genotypes previously confined to hooded seals among a diverse repertoire of sequence types (STs) associated with this species. For the first time Brucella isolates were characterised from beluga whales and found to represent a number of distinct B. pinnipedialis genotypes. In addition the known host range of ST27 was extended with the identification of this ST from California sea lion samples. Finally the performance of the frequently used diagnostic tool Bruce-ladder, in differentiating B. ceti and B. pinnipedialis, was critically assessed based on improved knowledge of the global population structure of Brucella associated with marine mammals.

Brucella ceti Infection in a Common Minke Whale ( Balaenoptera acutorostrata ) with Associated Pathology.

There are three major lineages of marine mammal strains of Brucella spp.: Brucella ceti ST23, found predominantly in porpoises; B. ceti ST26, in pelagic delphinids and ziphiids; and Brucella pinnipedialis ST24/25, predominantly in seals. The isolation of Brucella spp. in mysticetes has been described only in common minke whales ( Balaenoptera acutorostrata ) in Norway and Scotland. We report a third case of Brucella infection and isolation in a minke whale associated with a large abscess. In contrast to the two previous reports that involved isolates of B. pinnipedialis ST24 or the porpoise-associated B. ceti complex ST23, this case was associated with the dolphin-associated B. ceti ST26. Thus, minke whales can be infected naturally with members of all the distinct major lineages of Brucella associated with marine mammals. This report is unique in that the B. ceti ST26 did not originate from a pelagic delphinid or a beaked whale.

Extended Multilocus Sequence Analysis to Describe the Global Population Structure of the Genus Brucella: Phylogeography and Relationship to Biovars.

An extended multilocus sequence analysis (MLSA) scheme applicable to the Brucella, an expanding genus that includes zoonotic pathogens that severely impact animal and human health across large parts of the globe, was developed. The scheme, which extends a previously described nine locus scheme by examining sequences at 21 independent genetic loci in order to increase discriminatory power, was applied to a globally and temporally diverse collection of over 500 isolates representing all 12 known Brucella species providing an expanded and detailed understanding of the population genetic structure of the group. Over 100 sequence types (STs) were identified and analysis of data provided insights into both the global evolutionary history of the genus, suggesting that early emerging Brucella abortus lineages might be confined to Africa while some later lineages have spread worldwide, and further evidence of the existence of lineages with restricted host or geographical ranges. The relationship between biovar, long used as a crude epidemiological marker, and genotype was also examined and showed decreasing congruence in the order Brucella suis > B. abortus > Brucella melitensis. Both the previously described nine locus scheme and the extended 21 locus scheme have been made available at http://pubmlst.org/brucella/ to allow the community to interrogate existing data and compare with newly generated data.

First report of Brucella ceti-associated meningoencephalitis in a long-finned pilot whale Globicephala melas.

Fatal Brucella ceti infection with histological lesions specific to the central nervous system has been described in only 3 species of cetaceans: striped dolphins Stenella coeruleoalba, Atlantic white-sided dolphins Lagenorhynchus acutus and short-beaked common dolphins Delphinus delphis. This paper describes the first report of a B. ceti-associated meningoencephalitis in a long-finned pilot whale Globicephala melas, showing the increasing range of species susceptibility. Brucella was recovered in larger numbers from cerebrospinal fluid than from brain tissue and is the sample of choice for isolation.

Isolation of Brucella ceti from a Long-finned Pilot Whale (Globicephala melas) and a Sowerby's Beaked Whale (Mesoploden bidens).

Brucella ceti is an emerging zoonotic pathogen that has been recovered from several species of cetaceans in the world's oceans over the past 20 yr. We report the recovery of B. ceti from a Sowerby's beaked whale (Mesoploden bidens) and a long-finned pilot whale (Globicehala melas). Recovery from the testis of a long-finned pilot whale provides further evidence of potential for B. ceti infection to impact the reproductive success of cetaceans, many of which are threatened species. The addition of another two cetacean species to the growing number from which B. ceti has been recovered also further emphasizes the concern for human infections with this organism.

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)).

Examination of taxonomic uncertainties surrounding Brucella abortus bv. 7 by phenotypic and molecular approaches.

Brucella taxonomy is perpetually being reshuffled, at both the species and intraspecies levels. Biovar 7 of Brucella abortus was suspended from the Approved Lists of Bacterial Names Brucella classification in 1988, because of unpublished evidence that the reference strain 63/75 was a mixture of B. abortus biovars 3 and 5. To formally clarify the situation, all isolates previously identified as B. abortus bv. 7 in the AHVLA and ANSES strain collections were characterized by classical microbiological and multiple molecular approaches. Among the 14 investigated strains, including strain 63/75, only four strains, isolated in Kenya, Turkey, and Mongolia, were pure and showed a phenotypic profile in agreement with the former biovar 7, particularly agglutination with both anti-A/anti-M monospecific sera. These results were strengthened by molecular strategies. Indeed, genus- and species-specific methods allowed confirmation that the four pure strains belonged to the B. abortus species. The combination of most approaches excluded their affiliation with the recognized biovars (biovars 1 to 6 and 9), while some suggested that they were close to biovar 3.These assays were complemented by phylogenetic and/or epidemiological methods, such as multilocus sequence analysis (MLSA) and variable-number tandem repeat (VNTR) analysis. The results of this polyphasic investigation allow us to propose the reintroduction of biovar 7 into the Brucella classification, with at least three representative strains. Interestingly, the Kenyan strain, sharing the same biovar 7 phenotype, was genetically divergent from other three isolates. These discrepancies illustrate the complexity of Brucella taxonomy. This study suggests that worldwide collections could include strains misidentified as B. abortus bv. 7, and it highlights the need to verify their real taxonomic position.

Meningoencephalitis and arthritis associated with Brucella ceti in a short-beaked common dolphin (Delphinus delphis).

Brucella species infection in marine mammal species has been reported to have a global distribution. In 2007, the description of Brucella ceti was published and formally adopted for those isolates originating from cetaceans and pathologic lesions similar to those seen in terrestrial mammals infected with Brucella spp. have been associated with its isolation. Brucella ceti infection specific to the central nervous system has been described in two species of cetacean: striped dolphins (Stenella coeruleoalba) in Europe and Costa Rica and an Atlantic white-sided dolphin (Lagenorhynchus acutus) in the UK. We describe the first report, to our knowledge, of B. ceti-associated meningitis and arthritis in a third species, the short-beaked common dolphin (Delphinus delphis), in an animal that stranded in the UK.

Evaluation of competitive ELISA for detection of antibodies to Brucella infection in domestic animals.

AIM: To evaluate competitive enzyme-linked immunosorbent assay (cELISA) for its suitability as an additional serological test for the diagnosis of animal brucellosis. METHODS: cELISA, which was developed at the Veterinary Laboratories Agency, has been evaluated for its accuracy and suitability as an additional serological test for the diagnosis of animal brucellosis. Samples from naturally and experimentally infected animals and those from Brucella-free flocks and herds were tested. RESULTS: Data obtained since 1991 were analyzed from routine surveillance, animals experimentally infected with Brucella, and stored sera to validate cELISA for the detection of antibodies to Brucella in cows, small ruminants, and pigs. The sensitivity of the test ranged from 92.31% to 100%, in comparison with 77.14% to 100% for the complement fixation test (CFT). Specificities for cELISA, indirect enzyme-linked immunosorbent assay, and CFT were greater than 90%. CONCLUSION: cELISA can be used on a variety of animal species, and an added advantage is its suitability for use on poor-quality samples such as those affected by hemolysis.

Comparative performance of SNP typing and 'Bruce-ladder' in the discrimination of Brucella suis and Brucella canis.

Two novel molecular assays, 'Bruce-ladder' and SNP typing, have recently been described designed to differentiate isolates of the genus Brucella, causative organisms of the significant zoonotic disease brucellosis, at the species level. Differentiation of Brucella canis from Brucella suis by molecular approaches can be difficult and here we compare the performance of 'Bruce-ladder' and SNP typing in correctly identifying B. canis isolates. Both assays proved easy to perform but while 'Bruce-ladder' misidentifies a substantial proportion of B. canis isolates as B. suis, all B. canis isolates were correctly identified by SNP typing.

Phenotypic and molecular characterisation of Brucella isolates from marine mammals.

BACKGROUND: Bacteria 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. RESULTS: In 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. CONCLUSION: Molecular 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.

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.

Characterisation of the genetic diversity of Brucella by multilocus sequencing.

BACKGROUND: Brucella 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. RESULTS: Nine 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. CONCLUSION: The 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.

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 Simpson's 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.