Occurrence of Brucella ceti in striped dolphins from Italian Seas

Abstract

Brucella ceti infections have been increasingly reported in cetaceans, although a very limited characterization of Mediterranean Brucella spp. isolates has been previously reported and relatively few data exist about brucellosis among cetaceans in Italy. To address this gap, we studied 8 cases of B. ceti infection in striped dolphins (Stenella coeruleoalba) stranded along the Italian coastline from 2012 to 2018, investigated thanks to the Italian surveillance activity on stranded cetaceans. We focused on cases of stranding in eastern and western Italian seas, occurred along the Apulia (N = 6), Liguria (N = 1) and Calabria (N = 1) coastlines, through the analysis of gross and microscopic findings, the results of microbiological, biomolecular and serological investigations, as well as the detection of other relevant pathogens. The comparative genomic analysis used whole genome sequences of B. ceti from Italy paired with the publicly available complete genomes. Pathological changes consistent with B. ceti infection were detected in the central nervous system of 7 animals, showing non-suppurative meningoencephalitis. In 4 cases severe coinfections were detected, mostly involving Dolphin Morbillivirus (DMV). The severity of B. ceti-associated lesions supports the role of this microbial agent as a primary neurotropic pathogen for striped dolphins. We classified the 8 isolates into the common sequence type 26 (ST-26). Whole genome SNP analysis showed that the strains from Italy clustered into two genetically distinct clades. The first clade comprised exclusively the isolates from Ionian and Adriatic Seas, while the second one included the strain from the Ligurian Sea and those from the Catalonian coast. Plotting these clades onto the geographic map suggests a link between their phylogeny and topographical distribution. These results represent the first extensive characterization of B. ceti isolated from Italian waters reported to date and show the usefulness of WGS for understanding of the evolution of this emerging pathogen.

Fig 1. Stranding sites of striped dolphins infected by B. ceti under study, Italy, 2012–2018.

Geographical mapping was obtained by ArcGIS^® software using the geographical coordinates found from the strandings. Location data (dots): Case 1 dark green: Gallipoli Lido Pizzo, Apulian coastline, Ionian Sea (39.58 N, 18.00 E) March 11th 2012. Case 2 purple: Porto Cesareo Bacino Grande, Apulian coastline, Ionian Sea (coordinates not available) October 2nd 2012. Case 3 yellow: Alimini, Otranto, Apulian coastline, Adriatic Sea (coordinates not available) November 13th 2013. Case 4 light blue: Porto Cesareo Bacino Grande, Apulian coastline, Ionian Sea (coordinates not available) December 9th 2014. Case 5 green: Savona, on the Ligurian coastline, Ligurian sea (44.94 N, 8.18 E) February 15th 2017. Case 6 red: Ardore, on the Calabrian coastline, Ionian Sea (38.11 N, 16.12 E) May 12th 2017. Case 7 pink: Maruggio, on the Apulian coastline, Ionian Sea (40.17 N, 17.35 E) December 6th 2017. Case 8 blu: Manduria, on the Apulian coastline, Ionian Sea (40.18 N, 17.40 E) November 5th 2018. The Italy map was used under a CC BY-SA copyright from OpenStreetMap contributors (https://www.openstreetmap.org/copyright/en) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig 2. B. ceti-associated lesions in central nervous system of striped dolphins (S. coeruleoalba).

(A) Severe non-suppurative meningitis. Cerebellar meninges are infiltrated by mononuclear cells (case 2). 10x. H&E. Left upper inset: detail of the lympho-monocytic inflammatory infiltrate. 40x. H&E. (B) Severe non-suppurative meningitis (case 4). Meninges at the level of medulla oblongata are infiltrated by lympho-monocytic cells. 10x. H&E. (C) Mild non-suppurative meningoencephalitis (case 5). Chorioid plexuses are infiltrated by lympho-monocytic cells. 40x. H&E. (D) Mild non-suppurative meningoencephalitis (case 6). cerebellar meninges are infiltrated by mononuclear cells. 10x. H&E. Left upper inset: perivascular cuff characterized by the presence of lympho-monocytic cells. 20x. H&E. (E) Non-suppurative meningitis (case 7). Cerebral cortex meninges are infiltrated by mononuclear cells. 10x. H&E. (F) Non-suppurative meningitis (case 8). Meninges at the level of parietal cortex are infiltrated by mononuclear cells. 20x. H&E.

Fig 3. Maximum likelihood tree of B. ceti and B. pinnipedialis.

The tree was constructed using concatenated SNP sequences of 59 isolates and mid-point rooted. The branch colours correspond to BAPS populations and the major branches are labelled with bootstrap values.