Brucella ceti has been isolated from dolphins, whales and porpoises, and has important zoonotic potential.1,2,3 Pathologic lesions associated with Brucella infection in cetaceans have been well documented and include non-suppurative meningitis or meningoencephalitis, arthritis, reproductive lesions, in utero pneumonia and placentitis.2,4,5,6 Lesions are often reported as chronic, yet without confirmatory polymerase chain reaction (PCR), pathologists are often met with difficulty in differentiating between primary Brucella infections or other infectious causes. This is due, at least in part, to confounding factors such as poor carcass condition, underlying immune suppression, and co-infections, but may also be attributable to variability in tissue tropism and pathogenicity of different Brucella types. The objective of this study was to determine if pathologic lesions vary with B. ceti sequence types (ST) and cetacean age groups. We hypothesize that infection with the ST 23 and ST 26 genotypes is associated with chronic, multi-organ infections in non-perinatal cetaceans while infection with the ST 27 genotype is associated with abortion and perinatal death. An additional hypothesis is that the Brucella-specific mobile genetic element, insertion sequence 711 (IS711) gene, is an accurate marker for the 9 locus multilocus sequence type (MLST) for B. ceti sequence types 23, 26 and 27 in porpoises, dolphins and whales.7,8,9 Cases included in this study were stranded cetaceans with Brucella sp. infections previously confirmed via PCR using 16S primers and with archived histopathology report or hematoxylin and eosin-stained slides available for review. Species included were Tursiops truncatus, Delphinus delphis, Stenella coeruleoalba, and Balaenoptera acutorostrata. Characterization of sequence type was performed using a nine loci MLST, as previously described, and by PCR using three separate IS711 primers designed to be specific to ST23, ST26, and ST27.8 Sensitivity and specificity of the IS711 primer sets to assign ST type using both conventional PCR and SYBR qPCR detection methods was compared using results from the 9-MLST as the gold standard. Comparisons between lesion and ST type were performed using Fisher’s exact tests, followed by odds ratios (OR) where appropriate.
Preliminary statistical analysis indicates that meningoencephalitis is more likely to occur in cetaceans infected with ST 23 or 26, and arthritis more likely with ST 26. ST 27 was significantly associated with in utero pneumonia and identified primarily in perinates. Interestingly, endometritis and epididymitis cases were associated with ST 26 infections; however, orchitis was noted in animals infected with all three sequence types. Minke whales were infected with ST 26. In addition, results demonstrate that the IS711 ST-specific PCR assays may provide a more rapid method for distinguishing among B. ceti STs of pathogenic importance.
In conclusion, these results indicate that there are strain differences in the pathogenesis of Brucella sp. infections in cetaceans. Few cases with confirmed reproductive tract infections were available for analysis. Routine sampling of reproductive tissues from stranded cetaceans for Brucella PCR and histopathology is encouraged to better evaluate the relationship between Brucella sequence type in reproductive tract infections and the pathogenesis of abortion and fetal loss.
The authors wish to thank the many stranding network institutions that contributed diagnostic samples for this project and Nadia Ahmed for laboratory assistance.
* Presenting author
+ Student presenter
1. Foster G, et al. 2007. Brucella ceti sp. nov. and Brucella pinnipedialis sp. nov. for Brucella strains with cetaceans and seals as their preferred hosts. International Journal of Systematic and Evolutionary Microbiology. 57:2688–2693.
2. Guzmán-Verri C, et al. 2012. Brucella ceti and brucellosis in cetaceans. Frontiers in Cellular and Infection Microbiology. 2:3.
3. Nymo IH, Tryland M, Godfroid J. 2011. A review of Brucella infection in marine mammals, with special emphasis on Brucella pinnipedialis in the hooded seal (Cystophora cristata). Veterinary Research. 42(1):93.
4. González-Barrientos R, et al. 2010. Pathology of striped dolphins (Stenella coeruleoalba) infected with Brucella ceti. Journal of Comparative Pathology. 142(4):347–352.
5. Colegrove KM, et al. 2016. Fetal distress and in utero pneumonia in perinatal dolphins during the Northern Gulf of Mexico unusual mortality event. Diseases of Aquatic Organisms. 119(1):1–16.
6. Colegrove KM, et al. 2016. Brucella infections in North American cetaceans: pathology, genetics, and unanswered epidemiologic questions. IAAAM 47th Annual Conference Proceedings, Virginia Beach, VA.
7. Wu Q, et al. 2017. Identification of three Brucella ceti genotypes in bottlenose dolphins (Tursiops truncatus) using a multiplex SYBR Green real-time PCR. Aquatic Mammals. 43.3.
8. Zygmunt MS, et al. 2010. Novel IS711-specific chromosomal locations useful for identification and classification of marine mammal Brucella strains. Journal of Clinical Microbiology. 48(10):3765–3769.
9. Whatmore AM, Perrett LL, MacMillan AP. 2007. Characterisation of the genetic diversity of Brucella by multilocus sequencing. BMC Microbiology. 7(1):34.