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.

Application of Whole Genome Sequencing and Pan-Family Multi-Locus Sequence Analysis to Characterize Relationships Within the Family Brucellaceae.

The bacterial family Brucellaceae is currently composed of seven genera, including species of the genus Brucella, a number of which are significant veterinary and zoonotic pathogens. The bacteriological identification of pathogenic Brucella spp. may be hindered by their close phenotypic similarity to other members of the Brucellaceae, particularly of the genus Ochrobactrum. Additionally, a number of novel atypical Brucella taxa have recently been identified, which exhibit greater genetic diversity than observed within the previously described species, and which share genomic features with organisms outside of the genus. Furthermore, previous work has indicated that the genus Ochrobactrum is polyphyletic, raising further questions regarding the relationship between the genus Brucella and wider Brucellaceae. We have applied whole genome sequencing (WGS) and pan-family multi-locus sequence analysis (MLSA) approaches to a comprehensive panel of Brucellaceae type strains, in order to characterize relationships within the family. Phylogenies based on WGS core genome alignments were able to resolve phylogenetic relationships of 31 non-Brucella spp. type strains from within the family, alongside type strains of twelve Brucella species. A phylogeny based on concatenated pan-family MLSA data was largely consistent with WGS based analyses. Notably, recently described atypical Brucella isolates were consistently placed in a single clade with existing species, clearly distinct from all members of the genus Ochrobactrum and wider family. Both WGS and MLSA methods closely grouped Brucella spp. with a sub-set of Ochrobactrum species. However, results also confirmed that the genus Ochrobactrum is polyphyletic, with seven species forming a separate grouping. The pan-family MLSA scheme was subsequently applied to a panel of 50 field strains of the family Brucellaceae, isolated from a wide variety of sources. This analysis confirmed the utility of the pan-Brucellaceae MLSA scheme in placing field isolates in relation to recognized type strains. However, a significant number of these isolates did not cluster with currently identified type strains, suggesting the existence of additional taxonomic diversity within some members of the Brucellaceae. The WGS and pan-family MLSA approaches applied here provide valuable tools for resolving the identity and phylogenetic relationships of isolates from an expanding bacterial family containing a number of important pathogens.

Streptococcus caledonicus sp. nov., isolated from sheep.

Five strains of an unidentified Gram-positive, catalase-negative, chain-forming coccus-shaped organism recovered from sheep in Scotland were characterized using phenotypic and molecular taxonomic methods. Based on morphological and biochemical criteria, the strains were tentatively identified as streptococci but they did not appear to correspond to any recognised species of the genus. Comparative 16S rRNA gene sequencing showed the strains were highly related to each other and confirmed their placement in the genus Streptococcus, with a maximum nucleotide identity of around 97 % to extant species. Best matches were with Streptococcus hillyeri followed by Streptococcus porci. Average nucleotide identity and in silico DNA-DNA hybridization values determined from whole-genome sequence were also consistent with the group representing a novel species. Best matches, again seen to S. hillyeri, followed by S. porci and S. plurextorum, were below accepted cut-off values for species delineation. Based on biochemical criteria and molecular genetic evidence, it is proposed that the unknown isolates from sheep be assigned to a new species of the genus Streptococcus as Streptococcus caledonicus sp. nov. The type strain of Streptococcus caledonicus is S784/96/1T=CCUG 73951T=NCTC 14363T.

Forensic microbiology reveals that Neisseria animaloris infections in harbour porpoises follow traumatic injuries by grey seals.

Neisseria animaloris is considered to be a commensal of the canine and feline oral cavities. It is able to cause systemic infections in animals as well as humans, usually after a biting trauma has occurred. We recovered N. animaloris from chronically inflamed bite wounds on pectoral fins and tailstocks, from lungs and other internal organs of eight harbour porpoises. Gross and histopathological evidence suggest that fatal disseminated N. animaloris infections had occurred due to traumatic injury from grey seals. We therefore conclude that these porpoises survived a grey seal predatory attack, with the bite lesions representing the subsequent portal of entry for bacteria to infect the animals causing abscesses in multiple tissues, and eventually death. We demonstrate that forensic microbiology provides a useful tool for linking a perpetrator to its victim. Moreover, N. animaloris should be added to the list of potential zoonotic bacteria following interactions with seals, as the finding of systemic transfer to the lungs and other tissues of the harbour porpoises may suggest a potential to do likewise in humans.

Actinobacillus vicugnae sp. nov., isolated from alpaca (Vicugna pacos).

Ten strains of an Actinobacillus-like organism were isolated from alpaca (Vicugna pacos) in the UK over a period of 5 years, with no known epidemiological linkages. The isolates are distinct, based on both phenotype and genotype, from any previously described Actinobacillus species. Molecular analysis, based on 16S rRNA, rpoB and infB gene sequences, placed the isolates as a novel, early branching, lineage within the currently recognised Actinobacillus sensu stricto. In agreement with the results of the single-gene analysis, average nucleotide identity values, based on whole genome sequences, showed very similar identities to a number of members of the Actinobacillus sensu stricto notably Actinobacillus equuli, Actinobacillus suis and Actinobacillus ureae. At least two phenotypic characteristics differentiate the alpaca isolates from other Actinobacillus sensu stricto species, and from taxa likely falling within this group but awaiting formal species description, with Actinobacillus anseriformium and A. equulisubsp. haemolyticus being the most closely related phenotypically. The alpaca isolates can be differentiated from A. anseriformium by production of ß-galactosidase (ONPG) and acid from raffinose, and from A. equulisubsp. haemolyticus by production of acid from d-sorbitol and failure to produce acid from d-xylose. Isolates were obtained from multiple sites in alpaca including respiratory tract, alimentary tract and internal organs although further evidence is required to understand any pathogenic significance. Based on the results of characterization described here, it is proposed that the isolates constitute a novel species, Actinobacillus vicugnae sp. nov. The type strain is W1618T (LMG30745T NCTC14090T) isolated in the UK in 2012 from oesophageal ulceration in an alpaca (Vicugna pacos).

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.

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.

Evaluation of MALDI-ToF as a method for the identification of bacteria in the veterinary diagnostic laboratory.

Matrix-Assisted Laser Desorption Ionisation-Time of Flight (MALDI-ToF) Mass Spectrometry with Bruker MALDI Biotyper software was evaluated as a method for identifying veterinary bacteria. For 620 isolates (~100 bacterial species), identification by MALDI-ToF and non-16S rDNA methods (mainly phenotypic/biochemical) agreed to species-level (95.3%) and to species/genus-level (100%), but in the absence of 16S rDNA as a gold standard. For a further panel of 107 anaerobes and 234 aerobes (~100 bacteria species) using 16S rDNA results as the gold standard, MALDI-ToF/biochemical tests showed 97.8/96.6% species-level and 99.6/93.5% genus-level agreement for aerobes and 95.3/93.6% species-level and 100/95.3% genus-level agreement for anaerobes compared to the gold standard. Where results were obtained from direct spots, direct spots overlaid with formic acid and extracts, 89.4% of 180 aerobes and 90.1% of 152 anaerobes were identified by MALDI-ToF. MALDI-ToF was shown to be a rapid and reliable method to identify veterinary bacteria.