Detection of Brucella ceti and Brucella-Associated Disease in Stranded Cetaceans in Hawaii, USA, 2000–24 Open Access

Brucella spp. bacteria infect terrestrial and marine mammals and cause zoonotic disease (Orsini et al. 2022). Clinical brucellosis has been documented in cetaceans coinfected with morbillivirus and herpesvirus (Sierra et al. 2020; Grattarola et al. 2023). Marine Brucella spp. strains have higher numbers of the mobile element insertion sequence 711 (IS711) in their genomes and fall into two clusters, Brucella pinnipedialis and Brucella ceti, differentiated by host preference, phenotype, and genotype (Cloeckaert et al. 2000; Foster et al. 2007). Brucella ceti has been found to be an important pathogen in numerous cetacean species worldwide (Guzmán-Verri et al. 2012). The first documented case of marine mammal brucellosis in Hawaii, US, was in a sperm whale (Physeter macrocephalus) neonate that stranded in 2011 (West et al. 2015). Previous multilocus sequence typing has categorized marine Brucella spp. strains of B. ceti into sequence types (ST; Whatmore et al. 2007, 2017). Assays targeting IS711 have been a successful diagnostic tool for PCR detection of Brucella spp. in infected tissues in most cases, and it is possible to differentiate among ST 23, 26, and 27, although genetic variation may limit detection in some cases (Curtiss et al. 2022). Although bacterial culture and full genome sequencing are the gold standard for Brucella spp. identification, tissue preservation, sample availability, and cost are significant barriers, especially in marine mammals for which tissues are often degraded before collection (Martino et al. 2024). The goal of our study was to screen tissues of stranded cetaceans in Hawaii for B. ceti to identify positive cases and describe pathologic lesions associated with infection.

We screened 1–16 tissues from 66 animals representing 17 species that stranded over a 24-yr period (January 2000 to July 2024; Table 1). We selected specific animals and tissues using a semitargeted approach that included consideration both of pathologic findings suggestive of Brucella sp. infection and of cetacean species for which Brucella sp. infection has been documented elsewhere (Guzmán-Verri et al. 2012; González-Barrientos and Hernández-Mora 2023). Selected cases also included animals involved in mass strandings—short-finned pilot whales (Globicephala macrorhynchus) in 2017 and pygmy killer whales (Feresa attenuata) in 2019—or from multiple solitary strandings within 1 mo—striped dolphins (Stenella coeruleoalba) in 2021. All screened cases stranded in the main Hawaiian Islands, except one false killer whale (Pseudorca crassidens) that was bycaught outside the Hawaiian Economic Exclusion Zone. The 66 individuals represented 16 odontocete species and one mysticete species. Cases included both males and females and fetal, calf, subadult, and adult age classes. Classification of carcass condition followed the 1–5 scale of Geraci and Lounsbury (2005) of fresh dead to mummified; in this study, condition generally ranged from fresh dead to moderately decomposed (1–3) except for two false killer whales and one dwarf sperm whale (Kogia sima) that were in advanced decomposition (4). We reviewed records of stranded animals for significant gross necropsy and histopathologic findings. We derived specific causes of death from descriptions of significant pathologic lesions (West et al. 2024). We selected tissue samples on the basis of tissue type and histopathologic findings for specific animals. Screened tissue types included brain, kidney, liver, lung, adrenal, spleen, pancreas, heart, skin, blubber, muscle, spinal cord, ovary or testes, placenta, lymphoid, and body fluid (amniotic–allantoic fluid, cerebrospinal fluid [CSF], feces, whole blood, or serum). Serology for Brucella-specific antibodies was not performed.

We extracted DNA from approximately 20 mg of tissue using the DNeasy blood and tissue kit (Qiagen, Germantown, Maryland, USA) according to the manufacturer’s protocol. We quantified the DNA concentration via the Qubit 1X dsDNA HS assay kit and Qubit 4 fluorometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Screening for the presence of Brucella was performed using either traditional or quantitative PCR via the QuantStudio 3 real-time PCR system (Applied Biosystems, Thermo Fisher Scientific). Primers and thermal cycler settings were adapted from Wu et al. (2017). Primers targeting a 150-base-pair (bp) piece of the IS711 gene were IS711-F 5′-TACCGCTGCGAATAAAGCCAAC-3′ and IS71-R 5′-TGAGATTGCTGGCAATGAAGGC-3′. Cycling conditions were initial denaturation of 95 C for 5 min, followed by 35 cycles of denaturation at 95 C for 15 s, annealing at 59 C for 15 s, and extension at 72 C for 30 s, with a final extension at 72 C for 7 min. For a positive control throughout the screening process we used DNA extracted from the lung tissue of a sperm whale neonate that produced a 150-bp amplicon (West et al. 2015). The PCR products that showed a 150-bp band on a 1% agarose gel were prepared for genetic confirmation using the QIAquick PCR and gel cleanup kit (Qiagen), followed by Sanger sequencing at the University of Hawaii Advanced Studies in Genomics, Proteomics and Bioinformatics laboratory (Honolulu, Hawaii, USA). Genetic sequences were aligned to the closest Brucella spp. sequence using National Center for Biotechnology Information’s nucleotide BLAST (Clark et al. 2016). All Brucella-positive cases were screened for circovirus, morbillivirus, and herpesvirus. Screening for beaked whale circovirus followed methods described by Clifton et al. (2023). Herpesvirus primers from VanDevanter et al. (1996) and thermal cycler settings from Smolarek Benson et al. (2006) were used. Cetacean morbillivirus screening used the protocol described by West et al. (2024).

We screened for the presence of Brucella in 468 tissues from the 66 individuals; results indicated that 21/66 (32%) of these individuals were positive for a strain of Brucella in one to five tissues each (Table 1). Brucella was detected by PCR in five Delphinidae species, including pelagic and nearshore species, and most commonly in striped dolphins. All 10 striped dolphins (100%) and 5/6 pygmy killer whales were positive (83%). The other six positive individuals represented six cetacean species in which we had screened one individual of each species: dwarf sperm whale, Fraser’s dolphin (Lagenodelphis hosei), humpback whale (Megaptera novaeangliae), Longman’s beaked whale (Indopacetus pacificus), rough-toothed dolphin (Steno bredanensis), and spinner dolphin (Stenella longirostris). The species that we found to be Brucella positive have previously been identified as susceptible to brucellosis in studies conducted in other parts of the world (Guzman-Verri et al. 2012). Brucella was not detected in the tissues of false killer whales (n=7), melon-headed whales (Peponocephala electra, n=12), short-finned pilot whales (n=5), pygmy sperm whales (n=4), pantropical spotted dolphins (Stenella attenuata, n=2), Risso’s dolphin (Grampus griseus, n=1), bottlenose dolphin (Tursiops truncatus, n=1), or Blainville’s beaked whale (Mesoplodon densirostris, n=1). Considering that the recovery rates of cetacean species in Hawaii are extremely low (near 1% overall, combining nearshore and pelagic populations; K.L.W. pers. comm.), our findings cannot exclude brucellosis in additional species that are often seen in mixed pods of cetaceans. A limited number of individuals representing certain cetacean species was screened in this study, for example, a single humpback whale was tested. Negative Brucella findings in a low number of individuals of any given species may not reflect infection rates among the greater population.

Of the samples screened, 45/468 (9.6%) tested positive for Brucella. Positives were detected in brain and spinal cord (19/66), lung (7/91), CSF (4/4), marginal lymph node (3/36), spleen (3/46), liver (3/53), feces (2/10), meninges (1/6), adrenal (1/4), aortic lymph node (1/3), and mesenteric lymph node (1/18). Brucella was not detected in blubber (n=4), heart (n=13), kidney (n=35), skeletal muscle (n=4), ovary (n=1), pancreas (n=3), penis (n=1), placenta (n=1), skin (n=3), testis (n=5), thymus (n=2), tongue (n=1), umbilicus (n=3), and lymph nodes not mentioned above (n=34). Brucella was detected in brain tissue in 77.8% of the positive individuals for which brain tissue was tested. Lung tissue was positive in 33% of the positive individuals. Inflammatory lesions associated with Brucella spp. infection were most observed in brain and lung tissues (Table 1). In 17/21 positive cases examined, infection was associated with severe respiratory and neurologic disease (Fig. 1A, B) and contributed to death. Previously, B. ceti infection has been reported in multiple Delphinidae species and has been associated with both clinical neurologic symptoms observed in live stranded animals and occasional hydrocephalus with enlargement of lateral ventricles in dead animals (Granados-Zapata et al. 2022). The range of tissues in which we detected Brucella is not unexpected. Brucella has been isolated from CSF, cerebrum, and lymph nodes, as well as other organs (West et al. 2015; Granados-Zapata et al. 2022). Brucella sp. was detected in tissues of a humpback whale fetus (MN8248) that presented in breech position, a condition that probably resulted in the death of the mother and calf. Gross and histopathologic examinations of tissues were not completed for this case in our study because of the decomposition of tissues. Brucella sp. also has been described as the cause of necrotizing placentitis in a late-term abortion and maternal death of a dwarf sperm whale (Hernández-Mora et al. 2021).

Over the 24.5-yr examination period in our study, the highest number of positive cases occurred in 2019 and 2021, representing 5/21 (24%) and 3/21 (14%), respectively, of all positive cases detected over the study duration. Brucella was detected in strandings that occurred on four Hawaiian Islands, with most cases documented in Maui (n=12), followed by Oahu (n=7; Fig. 2). The high incidences in 2019 and 2021 were probably due to the prolonged mass stranding event on Maui involving pygmy killer whales in 2019 and the unusually high frequency of the three striped dolphin strandings on Maui (n=2) and Oahu (n=1) in a 29-d period in 2021. Other positive cases over the study duration were temporally sporadic and included a humpback whale fetus in breech position in a case of dystocia from Molokai and a rough-toothed dolphin that stranded on Hawaii Island in 2000.

Of note, in the prolonged stranding event in 2019, 10 live pygmy killer whales stranded on Maui. Four were euthanized. A dead calf was found nearby. Two live pygmy killer whales stranded 26 d later at the same location and were euthanized. All animals that stranded on the first date had bronchopneumonia, and two adults and the calf tested positive for Brucella in the brain and pulmonary marginal lymph nodes. Haplotypes suggested that the animals that died during the initial stranding event were nonresidents (Baird et al. 2024). Both animals that stranded later had meningoencephalitis and pneumonia, and the brain of FA-7995 and lung of FA-2937 tested positive for Brucella. The adult animals that initially stranded were in good body condition, in contrast to emaciation in the animals that stranded later, suggestive of disease progression and impairment of neurologic function. Using drone technology, Currie et al. (2021) estimated a 27% total body mass loss when feeding was disrupted in the two pygmy killer whales that stranded later during the stranding event. In Italy, poor body condition was observed in >50% of 20 striped dolphins that tested positive for Brucella. Inability to feed due to neurologic disease was suggested as the cause of the poor body condition (González-Barrientos and Hernández-Mora et al. 2023; Grattarola et al. 2023).

Striped dolphins appear to have been especially susceptible to clinical brucellosis in Costa Rica, where Brucella infection has been a common cause of cetacean strandings, as well as in Italy (González-Barrientos and Hernández-Mora 2023; Grattarola et al. 2023). In our study in Hawaii, striped dolphins also appeared to be especially susceptible to clinical brucellosis (Table 1). We observed neurobrucellosis in seven subadults of 10 striped dolphins that died of the condition; this agrees with findings in an Italian study in which 6/8 juvenile striped dolphins had been diagnosed with neurobrucellosis (Grattarola et al. 2023). In our study, only one other subadult animal, a rough-toothed dolphin, tested Brucella positive. The other positive cases detected in this study were six adults, one calf, and one fetus (Table 1). These findings suggest species-specific and age-specific pathogenicity and pathology of Brucella spp. infection in cetaceans. Our approach to determining cases and tissues for screening was limited to cases with suggestive pathologic changes, especially inflammation in the nervous, respiratory, and reproductive organ systems, and we might have missed atypical chronic cases of brucellosis.

Clinical brucellosis has been documented in cetaceans coinfected with morbillivirus and herpesvirus (Sierra et al. 2020; Grattarola et al. 2023). In our study, coinfections in a Longman’s beaked whale, a Fraser’s dolphin, and a pygmy killer whale contributed to respiratory and neurologic inflammatory disease, complicating determination of the pathogen that was primarily responsible for death. Additional pathogens detected in animals positive for Brucella were circovirus (n=6), followed by morbillivirus (n=3) and herpesvirus (n=1). A targeted surveillance study of 10 cetacean species that stranded across the western and central Pacific Ocean previously detected beaked whale circovirus in 50% of cases, but the health impact on cetaceans was unknown (Clifton et al. 2023). Morbillivirus has been determined as the cause of death on the basis of lesions and virus presence in multiple organs of a Fraser’s dolphin that was also positive for Brucella and circovirus (West et al. 2021; Clifton et al. 2023). Grattarola et al. (2023) found morbillivirus coinfection in cases of systemic brucellosis and documented lymphoid depletion suggestive of immunosuppression in striped dolphins. Tissues from a Longman’s beaked whale with signs of chronic inflammatory disease tested positive for morbillivirus, herpesvirus, circovirus, and Brucella (West et al. 2013; Landrau-Giovannetti et al. 2020). The sperm whale neonate, which was the original case of brucellosis that served as the positive control in this study, was positive for Brucella, morbillivirus, herpesvirus, and circovirus and had meningitis and pneumonia (West et al. 2015; Clifton et al. 2023; K.L.W. pers. comm.). A study of 88 cases that included the neonate sperm whale had identified ST 27 Brucella primarily in perinates and associated with reproductive tract inflammation and meningoencephalitis (Curtiss et al. 2022). Except for the sperm whale neonate used as the positive control, the STs of Brucella identified in this study are not known. Our findings indicate brucellosis as a disease threat to cetaceans in Hawaii, with high prevalence in striped dolphins. Additional research focused on understanding genetic diversity of cetacean Brucella strains, infectious cycles, and strain- and host-specific pathogenicity and virulence is warranted.

We thank the Pacific Islands Marine Mammal Stranding Response Network and the Health and Stranding Lab team for necropsy support. All work was conducted under National Oceanic and Atmospheric Administration (NOAA) National Marine Fisheries Service permit 18786 and 24359. Funding for stranding response and necropsy operational support was received from the NOAA John H. Prescott Marine Mammal Rescue Assistance Program over many years between 2006 and 2024. We are also grateful to HDR Inc. and the US Commander Pacific Fleet Environmental Readiness Division and to the US Fish and Wildlife Service Zoonotic Disease Initiative for funding this project.