Brucella cetiinfection in dolphins from the Western Mediterranean sea
© Isidoro-Ayza et al.; licensee BioMed Central Ltd. 2014
Received: 2 March 2014
Accepted: 28 August 2014
Published: 17 September 2014
Brucella ceti infections have been increasingly reported in cetaceans. Brucellosis in these animals is associated with meningoencephalitis, abortion, discospondylitis’, subcutaneous abscesses, endometritis and other pathological conditions B. ceti infections have been frequently described in dolphins from both, the Atlantic and Pacific Oceans. In the Mediterranean Sea, only two reports have been made: one from the Italian Tyrrhenian Sea and the other from the Adriatic Sea.
We describe the clinical and pathological features of three cases of B. ceti infections in three dolphins stranded in the Mediterranean Catalonian coast. One striped dolphin had neurobrucellosis, showing lethargy, incoordination and lateral swimming due to meningoencephalitis, A B. ceti infected bottlenose dolphin had discospondylitis, and another striped dolphin did not show clinical signs or lesions related to Brucella infection. A detailed characterization of the three B. ceti isolates was performed by bacteriological, molecular, protein and fatty acid analyses.
All the B. ceti strains originating from Mediterranean dolphins cluster together in a distinct phylogenetic clade, close to that formed by B. ceti isolates from dolphins inhabiting the Atlantic Ocean. Our study confirms the severity of pathological signs in stranded dolphins and the relevance of B. ceti as a pathogen in the Mediterranean Sea.
KeywordsBrucella ceti Cetacean Neurobrucellosis Discospondylitis Mediterranean sea
After the first descriptions of Brucella infections in dolphins and seals and the definition of Brucella ceti and Brucella pinnipedialis as two new species within the genus -, there has been an increasing recognition of brucellosis in marine mammals (see  and  for recent reviews). Brucella strains from marine mammal origin have been isolated from humans . Antibodies against Brucella have been detected in 28 out of 42 cetacean species investigated, and B. ceti has been isolated from 10 of these species . B. ceti infection in cetaceans is associated to meningoencephalomyelitis ,,-, abortion ,, discospondylitis, subcutaneous abscesses, endometritis, and a wide range of other pathological conditions ,,. However, with the exception of the striped dolphin (Stenella coeruleoalba),-,- the proportion of other cetacean species showing clinicopathological signs associated with brucellosis is low, suggesting that most of these infected animals overcome clinical disease, eventually remaining as Brucella carriers and shedders.
Presumptive Brucella infections in Western Mediterranean Sea dolphins was first established by serology in two striped dolphins and one bottlenose dolphin (Tursiops truncatus) stranded on the Mediterranean Catalonian coast . Recently, Brucella strains were isolated from striped dolphins in the Tyrrhenian and Adriatic Seas ,. Here, we describe the clinical and pathological features of three cases of brucellosis in dolphins stranded on the Mediterranean Catalonian coast, and provide detailed information on the phenotypic and molecular characterization of these three B. ceti isolates.
Dolphin stranding and serological, pathological and bacteriological examinations
Biological data of Brucella ceti infected Mediterranean dolphins
Place, coordinates and date of stranding
Pathological diagnoses (Macro/Micro)
Tissues sampled for bacteriological examination
B. cetistrain isolated from
Non-suppurative encephalitis by CeMV. RT-PCR and IHC for CeMV both positive only in CNS
Encephalon, spleen, diaphragmatic and preescapular lymph nodes, lung
Male, 1.94 m, 74.5 Kg
Found alive Sept 11th-2009
Mycotic pyogranulomatous-necrotizing meningoencephalomyelitis
Encephalon, CSF (swab from lateral ventricle), vertebral abscess (swab), spleen.
Male 3 m
Found dead 7May 23rd-2012
Chronic, severe, focally extensive, suppurative discospondylitis
RT-PCR and IHC for CeMV negative
Non-suppurative meningoencephalitis. RT-PCR and IHC for CeMV negative
Encephalon, spleen, diaphragmatic lymph node, lung
Female, 1.84 m, 54.5 Kg
Found alive June 3rd-2012
Tissue samples of two dolphins (N-275/12 and N-301/12) were collected at the time of necropsy and submitted for bacteriological examination (Table 1). Tissues of the third dolphin (S. coeruleoalba, N-372/09), frozen at −80°C since 2009, were defrosted and submitted also to bacteriological studies, but cerebrospinal fluid (CSF) was not available in this case. Swabs taken at necropsy were each smeared in at least two plates of both Farrell’s  and CITA  culture media. The remaining necropsy samples were homogenized under sterile conditions in the minimum amount possible of sterile buffered saline (PBS pH 6.8) in a Stomacher unit (Seward Medical, Worthing, UK), and 0.5 mL of each tissue homogenate seeded also on at least two plates of each selective culture medium. The plates were checked for growth after 5–8 days of incubation at 37°C both in air and 10% CO2 atmospheres. Brucella colonies were identified by colonial morphology and standard typing procedures ,. One culture was considered as positive when at least one Brucella colony forming unit (CFU) was isolated. The suspected Brucella colonies isolated were further identified and characterized by molecular and chemical methods (see below).
The following strains obtained from the CITA and from PIET/CIET strain collections were used as controls for molecular studies: B. ceti Atlantic dolphin type (B14/94), B. ceti Atlantic porpoise type (B1/94), B. ceti Cantabric Sea isolate from S. coeruleoalba stranded in Northern Spain (C1), B. pinnipedialis seal type (B2/94), Brucella abortus 2308 (biovar 1 virulent reference strain), B. abortus S19 (biovar 1 reference vaccine strain), Brucella melitensis Rev1 (biovar 1 reference vaccine strain), Brucella suis (S2 biovar 1 ), B. canis (CR206-10; Costa Rica isolate), B. neotomae 5 K33 (reference strain), Brucella ovis PA (virulent reference strain) and Brucella microti (CCM4915, reference strain).
Brucella DNA samples from each isolate and control strains were extracted with DNeasy Blood & Tissue kit from QIAGEN®, and stored at −70°C until used. The three Mediterranean dolphin isolates were identified as B. ceti using the multiplex PCR as described elsewhere . DNA samples from these isolates and the marine control strains were also tested by PCR-RFLP of omp2b locus  and by multiplex PCR using the following two pairs of primers: TCA ACT GCG TGA ACA ATG CT (f) / GCG GGC TCT ATC TCA AGG TC (r), and CGT CAA CTC GCT GGC CAA GAG (f) / GCA GGA GAA CCG CAA CCT AA (r). Multiple loci variable number of tandem repeats (MLVA-16) analysis of Brucella species and strains was performed as described previously -. The basic protocol for MLVA-16 was slightly modified to use DreamTaq™ PCR Master Mix (Fermentas®). Amplicon analysis was performed on the ChemiDoc Gel Documentation System XRS, BioRad® using the Quantity One® software, which allowed molecular size determination of amplicons. Brucella control strains were used for validation ,,. The profiles were entered in the database MLVA-NET for the corresponding analysis .
Mass spectrometry analysis of Brucella protein extracts
For MALDI-TOF studies, the three B. ceti isolates and all control strains were grown in trypticase soy agar plates for four days in the presence or in the absence of CO2 (as required) following modifications of previous protocols ,,. For each bacterial strain, three clearly separated colonies were suspended in a 1.5 ml Eppendorf tube containing 1 ml of ultrapure distilled water, and centrifuged at 14,000 rpm for 5 min. Then, the bacterial pellet was thoroughly resuspended in 300 μL of water. After this, 700 μL of absolute ethanol were added, the suspension mixed in a vortex, let rest for five minutes at room temperature and centrifuged at 14,000 rpm for 5 minutes. The bacterial pellet was resuspended in 250 μL of water and directly sonicated in the Eppendorf tube with the aid of a titanium micro tip, at room temperature for 2 minutes. Nine hundred μL of absolute ethanol were added and the extract dried to completeness in a speed-vac centrifuge at 45°C for ~2.5 hours. Fifty μL of 70% formic acid were added to suspend the dried pellet, mixed thoroughly by pipetting and then 50 μL of acetonitrile were added and mixed. The extract was centrifuged at 14,000 rpm for 5 minutes and the supernatant transferred into a clean tube. In order to select optimal conditions for mass spectrometer analysis, several dilutions of the extract were tested. A volume of 0.5 μL of each dilution was spotted onto a steel Opti-TOF 384 plate target (ABSciex) and air-dried at room temperature. The spot sample was overlaid with 0.5 μL of matrix solution (saturated solution of alpha-cyano-4-hydroxy-cinnamic acid) in organic solvent (50% acetonitrile and 2.5% trifluoroacetic acid) and air-dried. The samples were analyzed in a MALDI-TOF on an Applied Biosystems 4800 Plus mass spectrometer. Spectra were acquired in linear positive mode, using a laser intensity of 3,800 and 500 shots/spectrum, in the m/z range 2,000 to 11,000, after external MS calibration with CalMix-5 standards (ABSciex) spotted on the same plate. Spectra were visualized using Data Explorer v.4.9 (Applied Biosystems).
Gas chromatographic analysis of fatty acid methyl esters
The three B. ceti isolates and all control strains were grown as described above. For each bacterial strain, 75 clearly separated colonies were chosen from three plates and placed in a sealed glass tube. Saponification of the samples and processing for total fatty acid methyl ester determination were carried out according to the MIDI instruction manual of Technical Note #101 (MIS, MIDI Inc., Newark, DE). Analysis was performed by gas chromatography (Agilent Technologies 6850) using a 25 m x 0,2 mm cross linked phenyl-methyl silicone fused silica capillary column HP 19091B-102 (Agilent Technologies Inc., Santa Clara, CA). A binary matrix was generated using the fatty acid profile of the tested strains.
Phylogenetic analysis and cladograms
Dendrograms based on the retention time of the fatty acid methyl esters and on the protein masses detected were constructed using an Agglomerative hierarchical clustering (AHC) algorithm, using Microsoft® Excel 2000/XLSTAT©-Pro (Version 4.07, 2013, Addinsoft, Inc., Brooklyn, NY, USA). Proximities were calculated using Squared Euclidean Distance, and aggregation was calculated using the unweighted pair-group average method. MLVA 16 phylogenetic trees based on differences in MLVA-16 was built according to the procedures described in the Brucella MLVA database .
Clinical and pathological findings
The main biopathological features of the three Mediterranean dolphins from which B. ceti strains were isolated are summarized in Table 1.
Small, multifocal, randomly distributed, non-suppurative inflammatory infiltrates were found in the liver and kidney. In addition, a small focus of granulomatous and necrotizing lymphadenitis was seen in the mesenteric lymph node. These lesions were probably associated to parasitic migrations. No other relevant microscopical findings were found in examined tissues. Brucella antigens were not detected by IHC in CNS. RT-PCR for CeMV resulted negative in all the investigated tissues.
Bacterial isolates identified as Brucella strains were obtained in high numbers from the spleen (N-372/09), vertebral abscess (N-275/12) and brain (N-301/12), respectively. The strains were isolated in the presence and absence of CO2 and selective culture media with the exception of strain from case N-301/12, whose growth was inhibited on Farrell’s medium. The isolated strains were not capnophilic, were positive for the oxidase and urease tests, and displayed a smooth type, agglutinating with both anti-A and anti-M mono-specific sera. The strains were as well lysed by the Iz but not the Tb, Wb and R/C phages, and grew on standard concentrations of both thionin and basic fuchsin. The three Mediterranean isolates were identified as B. ceti using the multiplex PCR. They were named as bmarMR24 (isolated from case N-301/12), bmarMR25 (from case N-275/12) and bmarMR26 (from case 372/09).
MLVA16, protein and fatty acid polymorphisms and phylogeny
During the last decade B. ceti strains have been isolated from stranded dolphins of both the Atlantic and Pacific Oceans . Information about B.ceti strains from the Mediterranean Sea is scarce. Recently, a B. ceti strain showing phenotypic characteristics of Atlantic B. ceti strains was isolated from a striped dolphin in the Tyrrhenian littoral of Italy . Also, two other B. ceti strains have been characterized from the Italian Southern Apulian Coast . MLVA analysis of the two isolates assigned them to a novel genotype within cluster A. Here we confirm and extend these observations, endorsing the presence of B. ceti infecting and causing pathology in at least two dolphin species (S. coeruleoalba and T. truncatus) inhabiting the Mediterranean Sea. The multilocus sequence analysis of these three B. ceti Mediterranean strains indicates that they also belong to the dolphin type of the ST26 cluster.Up to now, all B. ceti Mediterranean strains stem in a separate branch from the main MLVA16 A1 and A2 clusters of B. ceti isolates from dolphins inhabiting the Atlantic Ocean. Although the number of analysed Mediterranean B. ceti strains by MLVA16 is still low to draw a definitive branching order, the taxonomical position of these newly defined strains  is supported by proteome analysis and, to a less extent, by fatty acids analysis, two robust techniques used in bacterial taxonomy  and useful for characterization of Brucella-,,,. Therefore, it seems that the B. ceti isolates described here belong to a particular genotype prevalent in the Mediterranean Sea. The close relationship between Mediterranean and Atlantic B. ceti strains keeps important parallelism with the phylogenetic and taxonomical studies on the striped and bottlenose dolphin populations in both seas. Indeed, there is strong evidence of dispersal of bottlenose dolphin populations between both seas, keeping a population structure and genetic diversity in concordance with the boundaries that coincide with transitions between different habitat regions . In the case of striped dolphins, there is also evidence of some genetic flow between the Mediterranean and Atlantic populations; albeit, this is significantly more restricted than in bottlenose dolphins. This is mainly due to ecological and behavioral factors that limit the exchange between these two S. coeruleoalba populations across Gibraltar Strait . Considering this, the phenotypic and genetic structure from both North Atlantic and Mediterranean B. ceti isolates is not unexpected, as populations of S. coeruleoalba and T. truncatus share similar habitats and feed resources, and may then share various microorganisms, including B. ceti.
Infection by B. ceti is common in cetaceans but only a small proportion of infected cetaceans display clinicopathological signs associated to brucellosis, suggesting that many infected cetaceans overcome infection, perhaps remaining as carriers and potential Brucella shedders . This is in sharp contrast with the absence of obvious disease in seals or walrus infected with B. pinnipedialis. Several clinico-pathological entities have been associated to B. ceti infection in a relevant fraction of the stranded dolphins and porpoises , with neurobrucellosis being one of the most significant signs present ,,. One of our Mediterranean striped dolphins (N-301/12) presented unequivocally B. ceti induced non-suppurative meningoencephalitis with B. ceti being isolated from the CNS, and regarded as the primary cause of death. This is in agreement with the suggested higher susceptibility of this dolphin species for developing neurobrucellosis in comparison to other cetaceans .
B. ceti has been found to invade joints and cause chronic inflammatory lesions, and has been frequently isolated from these lesions in cetaceans ,,. In line with these observations, a causal relationship was hypothesized between the discospondylitis of the peduncle observed in the bottlenose dolphin case (N-275/12) and the B.ceti strain isolated from that lesion. This discospondylitis could be causing a disabling condition, but this lesion was probably not live-threatening. Primary cause of death in this dolphin was attributed to a mycotic encephalitis caused by Cunninghamella bertholletiae.
Similarly, death of the other striped dolphin (N-372/09) was linked to CeMV-related encephalitis and not to B. ceti infection (isolated retrospectively only from spleen, without evidence of lesions related to Brucella). CeMV infections of the brain have been unambiguously linked with epizootic disease and deaths of Mediterranean striped dolphins in the past ,.
As a corollary to these findings, it appears mandatory to establish adequate differential diagnoses for the aetiological agents that may affect disoriented live-stranded dolphins. This is becoming even more relevant when a number of cetaceans in the Mediterranean Sea show a rapid decline , mainly due to by-catch, the presence of pollutant contamination, and the pressure of infectious diseases that threaten the health of free-ranging cetaceans . Brucellosis, as a contagious disease, can be an additional factor hampering the conservation efforts of cetaceans at local and global scale. Our study confirms the relevance of B. ceti as a cetacean pathogen in the Mediterranean, the severity of pathological signs in stranded S. coeruleoalba dolphins, and gives insight on the phylogenetic structure of these B. ceti Mediterranean isolates. The Mediterranean B. ceti strains isolated so far form a distinct phylogenetic cluster, close to that of B. ceti strains isolated from dolphins inhabiting the Atlantic Ocean. In addition to seriously compromising the wellbeing of marine mammals, the B. ceti strains possess all the current molecular virulence determinants and therefore, are potential pathogens for other animals, including humans .
B. ceti has been isolated for the first time from the Spanish Mediterranean Sea, expanding the known range of this species. Neurobrucellosis with non-suppurative meningoencephalomyelitis in a striped dolphin and spondylitis in a bottlenose dolphin were the main clinicopathological features of these cases. In a third case, B. ceti was isolated from the spleen of a striped dolphin. The omp2b haplotype was common for all three B. ceti Mediterranean isolates and multiplex characterization showed that they were similar to the Atlantic dolphin type B. ceti B14/94 strain. Following MLVA16 analysis the three B. ceti Spanish Mediterranean strains clustered together in a distinct clade with the two reported B. ceti isolated in the Mediterranean Italian littorals, and close to the Atlantic A1 dolphin cluster. This taxonomical position was supported by protein and fatty acid analyses. Collecting appropriate samples for testing for Brucella has to be included in necropsy protocols in stranded dolphins.
The work did not include experimental procedures. Handling of live and dead cetaceans (species included in CITES 2 list) was done with official governmental permission. Medical treatments on live dolphins were applied following established procedures for these species.
FA provided veterinary treatment to live-stranded dolphins. MIA, LP, and MD performed necropsies and histopathologic examinations. LP performed immunostaining and RT-PCR for CeMV. JMB and PMM performed bacterial isolation and species identification. MB and RGB performed immunostaining for Brucella. NRV confirmed bacteriological identification of isolates received from Spain and performed MLVA analysis. CGV, CCD and ECO designed, analysed and performed lipidomics and molecular analysis of isolates and reference strains. EM designed, performed and analysed proteomics data, and integrated all results from taxonomic studies. All authors helped to draft specific parts of the manuscript. MD, EM and JMB were responsible of the design of the study and assembled the final draft of the manuscript. All authors approved the final version of the manuscript.
We thank Departament de Medi Natural from the Government of Catalonia for economical support. We thank Bruno Lomonte, Instituto Clodomiro Picado, University of Costa Rica for his assistance in the proteomic analysis. Research at the CITA is funded by INIA (RTA2011-00103-00-00), MICIN (AGL2011-30453-C04), and consolidated group A14 from Gobierno de Aragón. This work was partially funded by grants FIDA-2014 UNA, FS-CONARE UNA/UCR. We wish to acknowledge M.J. de Miguel, S. Serrano, M. Uriarte, B. Pérez and A. Neira for the excellent technical assistance.
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