Integrated approach to Brucella ceti infection in stranded Mediterranean dolphins

Introduction:

Brucella ceti is an emerging zoonotic pathogen causing neurological, reproductive, and systemic disease in cetaceans, representing a public health concern due to increasing humans-marine mammals interactions. Despite growing reports worldwide, critical gaps persist in cetacean-specific diagnostics and identification of genomic markers for epidemiological tracing. This study addresses these limitations to advance disease understanding in cetaceans.

Methods:

Thirty cetaceans with suspected Brucella infection stranded along the western Mediterranean coast (2011- 2021) were investigated combining bacteriological culture, qPCR, histopathology, and serological testing via a commercially available blocking ELISA, to assess diagnostic strategies for epidemiological surveillance and phylogenetic understanding. Whole-genome sequencing (WGS) assessed genetic diversity and phylogenetic relationships among circulating strains.

Results:

B. ceti infection was confirmed in nearly half of the investigated dolphins by culture and qPCR, with minor discrepancies underscoring their complementary utility. Culture remained the gold standard, while qPCR provides a valuable screening tool. Infection was detected exclusively in striped dolphins (Stenella coeruleoalba), consistent with their elevated susceptibility in the Mediterranean Sea.

A commercial blocking ELISA, originally developed for terrestrial livestock, was validated for cetaceans, using panels from culture-positive and brucellosis-free dolphins, achieving 100% diagnostic specificity and filling a key gap in marine brucellosis serology.

Two circulating sequence types, ST26 and ST49, were identified, clustering with Mediterranean strains from Spain and Italy. SNP analysis revealed minimal variation between isolates from the same animal, whereas comparisons between different animals showed both closely related and divergent strains, contributing to a genomic reference dataset for future epidemiological SNP thresholds.

Histopathological examination revealed lesions exclusively in the CNS, with no reproductive or osteoarticular findings reported.

Conclusions:

This study demonstrates the superiority of an integrated multi-method approach for marine brucellosis surveillance. Validation of blocking ELISA for cetaceans and genomic characterization of circulating strains represents significant advances for wildlife health monitoring and zoonotic risk management.