Abstract
Brucellosis is one of the most important bacterial zoonoses recorded in free-ranging and managed cetaceans worldwide.1 It is caused by facultative intracellular gram-negative bacteria of the Brucella genus, which have evolved to evade the immune system, causing persistent infection and replication within the host.2 While not host-specific, Brucella ceti and Brucella pinnipedialis are two species with general phylogenetic association, respectively, among cetaceans and pinnipeds.3 Subclinical to chronic, severe disease has been documented in stranded cetaceans globally, including reproductive lesions and neurobrucellosis;1,4,5 however, the occurrence and associated pathology have not been well-characterized for the northeastern Pacific Ocean.
A total of 685 stranded cetaceans from 34 species were documented by The Marine Mammal Center (TMMC, Sausalito, California) from 1999 to 2023. We aimed to review prevalence and gross and microscopic findings associated with Brucella infection among stranded cetaceans. Tissue samples were collected for serology, histopathology, culture, and molecular analyses. Selected tissues (SNC, CSF, joint synovium/capsule/fluid, lung, heart, lymph nodes, reproductive tract) were submitted for culture and/or PCR/qPCR from (n=22; 10 Pacific white-sided dolphins (Lagenorhynchus obliquidens), three long-beaked common dolphins (Delphinus capensis), four harbor porpoises (Phocoena phocoena), four striped dolphins (Stenella coeruleoalba), and one short-beaked common dolphin (Delphinus delphis). Serum samples from selected suspect cases were analyzed using the Brucella card agglutination test/Brucella abortus/suis buffered acidified plate antigen (BAPA), n=9; three gray whales (Eschrichtius robustus), one Dall’s porpoise (Phocoenoides dalli), one harbor porpoise, one Pacific white-sided dolphin, one long-beaked common dolphin, one Risso’s dolphin (Grampus griseus), and one striped dolphin. Positives were further tested by the fluorescence polarization assay (FPA).
Three animals were BAPA positive (3/9, 33.33%). Nine animals (9/22, 40.90%) were confirmed Brucella-positives via culture (n=3), qPCR (n=2), and culture and PCR/qPCR (n=4), including four L. obliquidens, two S. coeruleoalba, two D. capensis, and one D. delphis. Whole genome sequencing was performed in four of those cases; isolates belonged to B. ceti group two. Among these; one D. capensis and one S. coeruleoalba were BAPA/FPA positive (173.9/176.7 and 163.8/171.2 delta mP, respectively) and one G. griseus was BAPA positive/FPA negative (−1.8/−3.4 delta mP). Three additional cases will be tested.
Main gross findings from Brucella-positive cases included: meningeal congestion/thickening/abscesses (6/9; 66.66%), synovial congestion/thickening/remodeling/granulomas/ulceration at the atlantooccipital joint (5/9; 55.55%), fat/skeletal muscle atrophy (5/9; 55.55%), lymphoid hyperplasia/lymphadenitis (5/9; 55.55%), increased/cloudy CSF (2/9; 22.22%), hydrocephalus (1/9; 11.11%), and fibrinous pericarditis (1/9; 11.11%). Main histopathological results included amniotic aspiration (fetal distress), bronchointerstitial granulomatous pneumonia, and neutrophilic granulomatous meningoencephalitis in a male neonate pacific white-sided dolphin; features described in peri/neonatal brucellosis.6,7 Lymphoplasmacytic meningoencephalitis/ependymitis, epicardial fibrosis, and lymphocytic epicarditis/myocarditis were noted in a subadult female pacific white-sided dolphin, as typically documented in chronic cases of cetacean brucellosis.1,4,8,9 The histopathology for the remaining positive cases is ongoing.
The confirmed Brucella cases found in this study contribute to the knowledge of the distribution and pathology of B. ceti in cetaceans in the northeastern Pacific Ocean. Ongoing investigation includes a survey of Brucella antibodies for additional stranded cetaceans (n=70) and characterization of Brucella distribution across tissues in positive cases.
Acknowledgements
The authors thank the staff, volunteers, and interns from The Marine Mammal Center for their help recovering carcasses, conducting necropsies, and samples collection. We acknowledge Dr. Kathleen Colegrove and Dr. David S. Rotstein for support with histopathological diagnosis.
*Presenting author
Literature Cited
1. Guzmán-Verri C, Gonzáles-Barrientos R, Hernández-Mora G, Morales J-A, Baquero-Calvo E, Chaves-Olarte E, Moreno E. Brucella ceti and brucellosis in cetaceans. Front Cell Infect Microbiol. 2012;2:3.
2. Ahmed W, Zheng K, Zheng-Fei L. Establishment of chronic infection: Brucella’s stealth strategy. Front Cell Infect Microbiol. 2016;6:30.
3. Whatmore AM, Dawson C, Muchowski J, Perrett LL, Stubberfield E, Koylas M, Foster G, Davison NJ, Quance C, Sidor IF, Field C, St. Leger J. Characterisation of North American Brucella isolates from marine mammals. PLoS One. 2017;12(9):e0184758.
4. Buckle K, Roe WD, Howe L, Michael S, Duignan PJ, Burrows E, Ha HJ, Humphrey S, McDonald WL. Brucellosis in endangered Hector’s dolphins (Cephalorhynchus hectori). Vet Pathol. 2017;54(5):838–845.
5. Hernandez-Mora G, Gonzáles-Barrientos R, Morales JA, Chaves-Olarte E, Guzmán-Verri C, Baquero-Calvo E, De-Miguel MJ, Marin CM, Blasco JM, Moreno E. Neurobrucellosis in stranded dolphins, Costa Rica. Emerg Infect Dis. 2008;14(9):1430–1433.
6. Colegrove KM, Venn-Watson S, Litz J, Kinsel MJ, Terio KA, Fougeres E, Ewing R, Pabst DA, McLellan WA, Raverty S, Saliki J, Fire S, Rappucci G, Bowen-Stevens S, Noble L, Costidis A, Barbieri M, Field C, Smith S, Carmichael RH, Chevis C, Hatchett W, Shannon D, Tumlin M, Lovewell G, McFee W, Rowles TK. Fetal distress and in utero pneumonia in perinatal dolphins during the Northern Gulf of Mexico unusual mortality event. Dis Aquat Organ. 2016;119(1):1–16.
7. Hong CB, Donahue JM, Giles RC, Jr, Poonacha KB, Tuttle PA, Cheville NF. Brucella abortus-associated meningitis in aborted bovine fetuses. Vet Pathol. 1991;28(6):492–496.
8. Davison NJ, Brownlow A, Doeschate MT, Dale EJ, Foster G, Muchowski J, Perrett LL, Rocchi M, Whatmore AM, Dagleish MP. Neurobrucellosis due to Brucella ceti ST26 in three Sowerby’s beaked whales (Mesoplodon bidens). J Comp Pathol. 2021;182:1–8.
9. González-Barrientos R, Morales JA, Hernández-Mora G, Barquero-Calvo E, Guzmán-Verri C, Chaves-Olarte E, Moreno E. Pathology of striped dolphins (Stenella coeruleoalba) infected with Brucella ceti. J Comp Pathol. 2010;142:347–352.