Abstract

Brucella ceti is an emerging zoonotic pathogen that has been recovered from several species of cetaceans in the world’s oceans over the past 20 yr. We report the recovery of B. ceti from a Sowerby’s beaked whale (Mesoploden bidens) and a long-finned pilot whale (Globicehala melas). Recovery from the testis of a long-finned pilot whale provides further evidence of potential for B. ceti infection to impact the reproductive success of cetaceans, many of which are threatened species. The addition of another two cetacean species to the growing number from which B. ceti has been recovered also further emphasizes the concern for human infections with this organism.

The isolation of Brucella from marine mammals was first reported 2 decades ago from tissues of free-ranging seals and cetaceans (Ross et al. 1994). Brucellosis has since become recognized in marine mammals inhabiting many of the world’s oceans (Nymo et al. 2011), and two species, Brucella ceti and Brucella pinnipedialis, have been described for isolates with cetaceans and seals, respectively, as preferred hosts (Foster et al. 2007).

We report the recovery of B. ceti from a Sowerby’s beaked whale (SBW) (Mesoploden bidens) and a long-finned pilot whale (LFPW) (Globicephela melas). Both animals were reported as part of the Scottish Marine Animal Strandings Scheme and necropsies were performed according to a standard protocol (Kuiken and Hartmann 1991).

Case 1 was a male SBW (M35.08) stranded on Benbecula, an island off western Scotland, in February 2008. Culture was performed on lung, liver, spleen, kidney, colorectal lymph node, brain, and small intestine.

Case 2 was a mass stranding event (MSE) of 70 LFPWs that occurred on the northern coast of the Scottish mainland at Kyle of Durness in July 2011. Nineteen animals died, and necropsies were performed with subsequent histopathologic analysis on 16 animals and bacteriologic culture on 10 animals. A mature adult male (M168.5.11) had culture performed on lung, liver, kidney, brain, testis, and pus from the left scapulohumeral joint, the latter presenting with a severe fibrino-suppurative and erosive osteoarthritis.

Tissues were cultured as previously described (Foster et al. 2002) on Columbia sheep blood agar (Oxoid, Basingstoke, UK) and Farrell’s medium, incubated at 37 C in air with 5% CO2. Colonies typical of Brucella were visible after 4 d of growth from kidney, spleen, and colorectal lymph node of the SBW and the testis and scapulohumeral joint pus of the LFPW. Cells were tiny Gram-negative cocco-bacilli that were acid-fast with the modified Ziehl-Neelsen stain. Colonies agglutinated with B. abortus antiserum (Remel, Basingstoke, UK) and grew in air without added CO2. They were urease positive, H2S negative, A dominant, grew with basic fuchsin and thionine, and lysed by phages Wb, BK2, and Iz, but not by Tb or R/C. Plaques were obtained with Fi phage.

Multilocus sequence analysis performed according to established protocols (Whatmore et al. 2007) demonstrated that both isolates shared an identical genotype consistent with B. ceti sequence type ST 26 that has been associated almost exclusively with various dolphin species (Groussaud et al. 2007).

Serum samples were tested by a competitive and two indirect enzyme-linked immunosorbent assays (cELISA and iELISA) as described previously (Dawson et al. 2008). Positive/negative cut-off values for all three ELISAs were based on those used for terrestrial mammals as validation data are difficult to obtain for cetaceans. Serum from the SBW gave positive reactions for all ELISA tests. A sample was not available for M168.11.5; however, serum from three of six other LFPWs in the MSE were positive by all three ELISAs.

Tissue samples for histopathology (SBW: brain, lung, heart, liver, spleen, kidney, adrenals, urinary bladder, and coli-rectal lymph node; LFPW: lung, heart, liver, spleen, small intestine, mesenteric lymph node, kidney, urinary bladder, testis, and dorsal and ventral skeletal muscles) were processed as described by Dagleish et al. (2007).

The SBW had a single moderately well-demarcated focus of necrosis infiltrated by lymphocytes and Gitter cells within the gray matter of the head of the caudate nucleus. A distinct lymphocytic perivascular cuff was adjacent to this lesion. Small amounts of perivascular serum leakage were present in the white matter of the thalamus and a mild-to-moderate lymphocytic infiltrate expanded the meninges over the occipital lobe of the cerebrum and cerebellum, respectively. A nematode parasite (presumed larval) was present in the subcapsular area of the renal cortex, surrounded by moderate numbers of lymphocytes, eosinophils, and occasional multinucleated giant cells. Lungs displayed severe generalized congestion with abundant protein-rich fluid in alveoli and large bronchi and excess mucus in some airways. Marked hepatic periportal congestion and marked corticomedullary congestion of adrenal glands were also noted. A small number of swollen, hypereosinophilic myofibers with loss of cross-striations were noted in both dorsal and ventral skeletal muscle.

Histologic interpretation of the LFPW tissues was hindered by moderate-to-marked autolysis. However, protein-rich fluid was present in many airways and alveoli in the lungs, and there was a small number of swollen, eosinophilic myofibers with loss of cross-striations in dorsal and ventral skeletal muscle samples.

Although there was no correlation between presence of B. ceti and histopathologic lesions in individual tissues in the SBW, the nonsuppurative, necrotizing encephalitis is reminiscent of that described in other species infected with Brucella, some of which have only small numbers of colonies recovered from brain tissue (González et al. 2002). It is possible that brain culture was not sufficiently sensitive to detect Brucella.

The Brucella-infected scapulohumeral joint in the LFPW is the first record of this pathology in a cetacean stranded in Scotland, although a similar Brucella-infected lesion was reported in a striped dolphin (Stenella coeruleoalba) from Costa Rica (González-Barrientos et al. 2010). Although there are few synovial joints in cetaceans, these findings, coupled with Brucella-infected atlanto-occipital joints in dolphins (Dagleish et al. 2007), indicate such joints should be a focus of attention for B. ceti infection. Isolation from testis strengthens the argument for sexual transmission as a possible route of infection in cetaceans and raises further concerns about potential impact upon reproductive success (Foster et al. 2002).

It has been suggested that cetacean MSEs often have a lead animal that other pod members follow ashore (Geraci and Lounsbury 2005). One of us (G.F. unpubl.) postulated that if the lead animal strands because of Brucella meningoencephalitis, this may lead to the stranding and death of other animals, many of whom may not be infected with B. ceti. Although microbiologic or histopathologic evidence of neurobrucellosis was not detected from any animals sampled during the 2011 MSE, not all carcasses were examined. The recovery of Brucella from one animal, however, provides conclusive evidence that LFPW can be infected by B. ceti. Furthermore, three of six animals involved were positive for Brucella antibodies in all three ELISA tests, indicating the pod possibly contained other infected animals. During MSE investigations, samples should be examined from the lead animals and as many other carcasses as possible and include brain and cerebrospinal fluid, because such fluid can be useful sample materials for Brucella isolation from cetaceans with neurobrucellosis (Hernández-Mora et al. 2008).

Brucella ceti is an emerging zoonosis for which there have been four documented human cases: a UK laboratory researcher who developed bacteremia while working with a B. ceti strain (Brew et al. 1999) subsequently identified as ST 26 (Groussaud et al. 2007), two patients with neurobrucellosis in the United States (Sohn et al. 2003), and a case of spinal osteomyelitis in New Zealand (McDonald et al. 2006). The latter cases belonged to ST 27 (Whatmore et al. 2008). Our findings further emphasize the zoonotic risk from contact with a wide range of cetacean species.

The major disease concern for livestock brucellosis is abortion, but infertility also occurs. Reproductive success of cetaceans, many of which are threatened species according to the International Union for Conservation of Nature Red List (IUCN 2015) may be crucial to their survival. It is important to be aware of the potential impact that Brucella infection could have on distinct populations, particularly if introduced to naïve groups, should they exist. Although abortion has been reported in captive bottlenose dolphins (Ewalt et al. 1994), there have been only two reports of note from free-ranging cetaceans. One report was an Atlantic white-sided dolphin (Lagenorhynchus acutus) with evidence of having aborted that had B. ceti cultured from several organs (Foster et al. 2002). A live-stranded striped dolphin with meningoencephalitis had a fetus of estimated 7-mo gestation (González-Barrientos et al. 2010); B. ceti was recovered from necrotic foci within the placenta, but significant pathologic changes were not detected in the fetus. Epididymitis and orchitis have been reported in harbor porpoises (Phocoena phocoena) (Dagleish et al. 2008). Strandings investigations are opportune in nature and restricted largely to available material that washes ashore or live strands in suitable condition for necropsy. Evidence of the impact of B. ceti on reproductive success in free-ranging cetaceans represents a major challenge and is likely to rely on additional input from researchers from other fields involved in cetacean studies.

Our report increases the number of cetacean species in Scottish waters from which Brucella has been cultured to nine, including harbor porpoise; minke whale (Balaenoptera acutorostrata); and common dolphin, Atlantic white-sided dolphin, striped dolphin, white-beaked dolphin (Lagenorhynchus albirostris) (Foster et al. 2002), and bottlenose dolphin (G.F. unpubl.). This number is likely to increase if sufficient animals from other hosts are examined.

Postmortem investigations were carried out under the aegis of the UK Cetacean Strandings Investigation Programme, which is jointly funded by the Department for Environment Food and Rural Affairs (Defra) and the Scottish Government Marine Directorate. Brucella research and surveillance at Animal and Plant Health Agency is supported by Defra. We thank Jakub Muchowski for valuable technical support. We thank Rob Penrose of Marine Environmental Monitoring and Matt Perkins of the Zoological Society of London for assistance with the necropsy performed on the pilot whale; Clare Underwood and Valerie Forbes of the Moredun Research Institute for histologic preparations; and Emma Jane Dale of Animal Health and Veterinary Laboratories Agency for assistance with phenotypic characterization of Brucella isolates.

LITERATURE CITED

Brew
SD
,
Perrett
LL
,
Stack
JA
,
MacMillan
AP
,
Staunton
NJ.
1999
.
Human exposure to Brucella recovered from a sea mammal
.
Vet Rec
144
:
483
.
Dagleish
MP
,
Barley
J
,
Finlayson
J
,
Reid
RJ
,
Foster
G.
2008
.
Brucella associated pathology in the testicle of a harbour porpoise (Phocoena phocoena)
.
J Comp Pathol
139
:
54
59
.
Dagleish
MP
,
Barley
J
,
Howie
FE
,
Reid
RJ
,
Herman
J
,
Foster
G.
2007
.
Isolation of Brucella species from a diseased atlanto-occipital joint of an Atlantic white-sided dolphin (Lagenorhynchus acutus)
.
Vet Rec
160
:
876
878
.
Dawson
CE
,
Perrett
LL
,
Stubberfield
EJ
,
Stack
JA
,
Farrelly
SSJ
,
Cooley
WA
,
Davison
NJ
,
Quinney
S.
2008
.
Isolation and characterization of Brucella from the lungworms of a harbor porpoise (Phocoena phocoena)
.
J Wildl Dis
44
:
237
246
.
Ewalt
DR
,
Payeur
JB
,
Martin
BM
,
Cummins
DR
,
Miller
WG.
1994
.
Characteristics of a Brucella species from a bottlenose dolphin (Tursiops truncatus)
.
J Vet Diagn Invest
6
:
448
452
.
Foster
G
,
MacMillan
AP
,
Godfroid
J
,
Howie
F
,
Ross
HM
,
Cloeckaert
A
,
Reid
RJ
,
Brew
S
,
Patterson
IAP.
2002
.
A review of Brucella sp. infection of sea mammals with particular emphasis on isolates from Scotland
.
Vet Microbiol
90
:
563
580
.
Foster
G
,
Osterman
B
,
Godfroid
JG
,
Jacques
I
,
Cloeckaert
A.
2007
.
Brucella ceti sp. nov. and Brucella pinnipedialis sp. nov. for Brucella strains with cetaceans and seals as their preferred hosts
.
Int J Syst Evol Microbiol
57
:
2688
2693
.
Geraci
JR
,
Lounsbury
VL.
2005
.
Marine mammals ashore: A field guide for strandings
.
2nd Ed
.
National Marine Aquarium in Baltimore
,
Baltimore, Maryland
. .
González
L
,
Patterson
IAP
,
Reid
RJ
,
Foster
G
,
Barberán
M
,
Blasco
JM
,
Kennedy
S
,
Howie
FE
,
Godfroid
J
,
MacMillan
AP
,
et al
.
2002
.
Chronic meningoencephalitis associated with Brucella sp. infection in live-stranded striped dolphins (Stenella coeruleoalba)
.
J Comp Pathol
126
:
147
152
.
González-Barrientos
R
,
Morales
JA
,
Hernández-Mora
G
,
Baquero-Calvo
E
,
Guzmán-Verri
C
,
Chaves-Olarte
E
,
Moreno
E.
2010
.
Pathology of striped dolphins (Stenella coeruleoalba) infected with Brucella ceti
.
J Comp Pathol
142
:
347
352
.
Groussaud
P
,
Shankster
SJ
,
Koylass
MS
,
Whatmore
AM.
2007
.
Molecular typing divides marine mammal strains of Brucella into at least three groups with distinct host preferences
.
J Med Microbiol
56
:
1512
1518
.
Hernández-Mora
G
,
González-Barrientos
R
,
Morales
JA
,
Chaves-Olarte
E
,
Guzmán-Verri
C
,
Baquero-Calvo
E
,
De-Miguel
MJ
,
Marin
CM
,
Blasco
JM
,
Moreno
E.
2008
.
Neurobrucellosis in stranded dolphins, Costa Rica
.
Emerg Infect Dis
14
:
1430
1435
.
International Union for Conservation of Nature (IUCN)
.
2015
.
The IUCN red list of threatened species. 2015.2
. http://www.iucnredlist.org.
Accessed August 2015.
.
Kuiken
T
,
Hartmann
M.
1991
.
Dissection techniques and tissue sampling
.
In:
Proceedings of the first ECS workshop on cetacean pathology
,
Leiden, the Netherlands
,
13–14 September
, pp.
1
39
.
McDonald
WL
,
Jamaludin
R
,
Mackereth
G
,
Hansen
M
,
Humphrey
S
,
Short
P
,
Taylor
T
,
Swingler
J
,
Dawson
CE
,
Whatmore
AM
,
et al
.
2006
.
Characterisation of a Brucella sp. as a marine-mammal type despite isolation from a patient with spinal osteomyelitis in New Zealand
.
J Clin Microbiol
44
:
4363
4370
.
Nymo
IH
,
Tryland
M
,
Godfroid
JG.
2011
.
A review of Brucella infection in marine mammals with special emphasis on Brucella pinnipedialis in the hooded seal (Cystophara cristata)
.
Vet Res
42
:
93
.
Ross
HM
,
Foster
G
,
Reid
RJ
,
Jahans
KL
,
MacMillan
AP.
1994
.
Brucella species infection in marine mammals
.
Vet Rec
134
:
359
.
Sohn
AH
,
Probert
WS
,
Glaser
CA
,
Gupta
N
,
Bollen
A
,
Wong
J
,
Grace
E
,
McDonald
W.
2003
.
Human neurobrucellosis with intracerebral granuloma caused by a marine mammal Brucella sp
.
Emerg Infect Dis
9
:
485
488
.
Whatmore
AM
,
Dawson
CE
,
Groussaud
P
,
Koylass
MS
,
King
A
,
Shankster
SJ
,
Sohn
AH
,
Probert
WS
,
McDonald
WL.
2008
.
Marine mammal Brucella genotype associated with human infection
.
Emerg Infect Dis
14
:
517
518
.
Whatmore
AM
,
Perrett
LL
,
MacMillan
AP.
2007
.
Characterisation of the genetic diversity of Brucella by multilocus sequencing
.
BMC Microbiol
7
:
34
.