Brucella ceti infection in dolphins from the Western Mediterranean sea

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figure 1

Striped dolphin, case N-301/12. A. Dolphin with clinical signs of neurobrucellosis, showing lateralization when swimming and inability to maintain equilibrium and flotation. The dolphin needed continuous holding and support. B. Histopathologic section of the brain from the same case. Thick perivascular infiltrates mainly composed of lymphocytes, plasma cells and macrophages are seen affecting leptomeninges, the neuropil surrounding the fourth ventricle and the choroid plexus (H&E).

Figure 2

Bottlenose dolphin, case N-275/12. A. Mass in the caudal peduncle, dorso-ventral view of the lesion at necropsy. B. Radiograph from the lesion in dorso-ventral view, showing periostal proliferation with ankylosis of coccygeal vertebrae. C. Longitudinal section of the affected vertebra, showing bony proliferation at the ventrolateral side of the vertebral body, leading to ankylosis. There is formation of small abscesses. Brucella ceti was cultured from one of the abscesses.

Figure 3

Immunohistochemical staining of Brucella , striped dolphin (case 372/09), spleen section. A. Section incubated with specific polyclonal antiserum to Brucella melitensis. Positivity was found in cytoplasm of cells within lymphoid follicles. B. Parallel section of the same tissue incubated with control negative antiserum. Haematoxylin counterstain.

Figure 4

Dendrograms based on MALDI-TOF analysis of protein molecular weight (A) and gas liquid chromatography analysis of the fatty acid methyl esters (B) of different Brucella extracts. Notice that in “A” the three Mediterranean strains cluster together in a single group; while in “B” they are intertwined with a B. ceti isolate from a striped dolphin from the Atlantic Ocean.

Figure 5

Molecular characterization of Brucella isolates by PCR-RFLP of omp2b locus and Multiplex PCR. Panel A: PCR-RFLP of omp2b locus, showing a common haplotype for the three B. ceti Mediterranean isolates (lanes 6–8), shared with that identified in all B. ceti Atlantic dolphin strains, irrespective of their geographic origin (lanes 4 and 5), but different from B. pinnipedialis and B. ceti porpoise reference (lanes 2 and 3, respectively) haplotypes. Panel B. Multiplex PCR using two primer pairs, identifying Mediterranean isolates as similar to those of Atlantic dolphin origin. Lane 1, 100 bp DNA ladder (Invitrogen Ltd.); lane 2, B. pinnipedialis B2/94 reference seal strain; lane 3, B. ceti B1/94 porpoise reference strain; lane 4, B. ceti B14/94 reference strain isolated from Atlantic dolphin; lane 5, B. ceti C1 strain isolated from a dolphin in the Cantabric sea (North Spain); lanes 6, 7 and 8, B. ceti Mediterranean isolates (bmarMR24, bmarMR25 and bmarMR26, respectively).

Figure 6

Cladogram based on MLVA16 analysis of Brucella isolates from marine mammals ( http://mlva.upsud.fr/brucella/ ). The dispersion of the various Brucella strains is depicted as cones proportional to the number of strains analyzed. Notice than the five B. ceti Mediterranean strains (three from this work and two reported by Garofolo et al. [15]) cluster together in a single group in a clade close to the Atlantic isolates (A1 and A2), and far from the B. ceti porpoise type (B) and B. pinnipedialis (C1, C2, C3). B. ceti bmar265 (human isolate from New Zealand) does not correspond to the B. ceti group and it is ST27. B. melitensis was used as an out-group for the analysis.

Figure 7

Brucella phylogenetic tree based on MLVA16 analysis of all the various Brucella species and strains ( http://mlva.upsud.fr/brucella/ ). Notice that the three Western (Spanish) and the Eastern (Italian) B. ceti Mediterranean strains cluster together in a single group (shadowed in gray) in a clade close to the Atlantic isolates (A1 and A2), all belonging to ST 26.