Claire Dawson

Marine mammal Brucella genotype associated with zoonotic infection.

Marine Mammal Brucella Genotype Associated with Zoonotic Infection

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.

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.

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.

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], [

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

ABSTRACT 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.

Isolation of Brucella species from a bottlenosed dolphin (Tursiops truncatus)

Brucella was first recovered from marine mammals in 1994 (Ross and others 1994). Since then, there have been numerous further accounts of serological evidence and isolations of Brucella species originating from marine mammals inhabiting many of the world’s oceans. This short communication describes the first confirmed case of Brucella species infection in a wild bottlenosed dolphin (Tursiops truncatus) in the UK. In December 2004, a male bottlenosed dolphin was found dead on a beach at Gwithian Hayle, Cornwall. It was taken to the Veterinary Laboratories Agency (VLA) – Truro for postmortem examination. The animal, considered to be in moderate condition with moderate autolysis, was 258 cm long and weighed 160 kg. There were numerous deep rake or bite marks present over the entire body (Fig 1), possibly caused by other bottlenosed dolphins. There had been some scavenger damage to the left eye and left pectoral flipper. Several longstanding cestode (Phyllobothrium delphini) cysts were present in the blubber around the vent; abscesses associated with these cysts were similar to the abscess from which Brucella species was later isolated. Postmortem examination revealed numerous nematodes in the bronchi and bronchioles, which were associated with a parasitic pneumonia. All three sections of the stomach were empty, and chyle was not present in the mesentery, indicating that the animal had not fed recently. The rake or bite marks were not associated with any additional pathology and the cause of death was not established. The exact age of the animal had yet to be determined. Samples of heart blood, lung, liver, kidney and intestine were prepared for routine bacteriological culture on blood agar and MacConkey’s agar. A mixed growth of Edwardsiella tarda and a non-haemolytic Staphylococcus species was recovered from the heart blood, lungs and intestine. Analysis of pericardial fluid by the rose bengal plate test revealed antibodies to Brucella species. Tissue samples of lung, liver, kidney, spleen, testes and brainstem, together with samples of pericardial and abdominal fluid, were therefore submitted to VLA – Weybridge to examine for the presence of Brucella species. Nematode worms recovered from the cardiac section of the stomach and from the lung were also subjected to examination by culture. Brucella was isolated from a purulent abscess within blubber taken from around the vent, and a fine bacterial growth was produced after seven days of incubation in enrichment broth and two days after subculture on Farrell’s media (Farrell 1974). The isolate was initially confirmed as Brucella species by classical biotyping methods (Alton and others 1988). It did not require additional carbon dioxide for growth and produced lysis by phages BK2 and Wb (Table 1). Amplification by PCR of an IS711 element downstream of the bp26 gene (Cloeckaert and others 2000) confirmed that the isolate possessed this unique feature specific to marine mammal strains of Brucella species. Molecular characterisation of the outer membrane protein of the strain using a selection of restriction enzymes (Cloeckaert and others 2001) revealed the type to be N-(K), as is found in common dolphins (Delphinus delphis) and striped dolphins (Stenella coeruleoalba). A competitive ELISA (cELISA) and two indirect ELISAs (iELISAs) were used to screen the pericardial and abdominal fluids for the presence of Brucella antibodies. The cELISA (MacMillan 1990) uses a lipopolysaccharide (LPS) Brucella melitensis antigen and a monoclonal antibody conjugate. For the two iELISAs, B melitensis 16M antigen was used for the first, as described for the cELISA, and for the other a Brucella abortus LPS antigen was used. The iELISAs require an antiglobulin conjugate with specificity for the immunoglobulin isotypes of the species under test; however, protein A has been shown to bind to the immunoglobulin G of a range of marine mammals (Eliasson and others 1989, Sikkema 1989). Positive/negative thresholds for these assays were set with some uncertainty, but were based on those used for testing a wide range of terrestrial mammals from Britain for brucellosis (Table 2). Samples of both pericardial and abdominal fluids were found to be positive for Brucella antibodies by all three assays. A previous report described the characterisation of Brucella species isolated from an aborted fetus of a captive FIG 1: Rake or bite marks on the body of a bottlenosed dolphin (Tursiops truncatus) found dead on a beach in Cornwall

Infection with Brucella ceti and high levels of polychlorinated biphenyls in bottlenose dolphins ( Tursiops truncatus ) stranded in south-west England

Eight bottlenose dolphins (Tursiops truncatus) that stranded in Cornwall, south-west England, between June 2004 and December 2007 were examined using standardised postmortem examination and bacteriological methods. Evidence of Brucella species infection was found in four of these dolphins on culture. In addition, of the eight dolphins, four were positive and two were weakly positive for antibodies to Brucella species on serological analyses of pericardial and other fluids using a competitive ELISA and two indirect ELISAs. High or very high levels of the sum of 25 individual chlorobiphenyl congeners (∑25CBs) were also determined in blubber samples from two of the dolphins (45.5 and 446.6 mg/kg lipid weight).

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.