Characterization of a Brucella sp.

Strain as
a Marine-Mammal Type despite Isolation
from a Patient with Spinal Osteomyelitis in
New Zealand
W. L. McDonald, R. Jamaludin, G. Mackereth, M. Hansen, S.
Humphrey, P. Short, T. Taylor, J. Swingler, C. E. Dawson, A.
M. Whatmore, E. Stubberfield, L. L. Perrett and G. Simmons
J. Clin. Microbiol. 2006, 44(12):4363. DOI:
10.1128/JCM.00680-06.
Published Ahead of Print 11 October 2006.

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JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2006, p. 4363–4370 Vol. 44, No. 12
0095-1137/06/$08.00⫹0 doi:10.1128/JCM.00680-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Characterization of a Brucella sp. Strain as a Marine-Mammal Type

despite Isolation from a Patient with Spinal Osteomyelitis
in New Zealand䌤
W. L. McDonald,1* R. Jamaludin,1† G. Mackereth,1 M. Hansen,1 S. Humphrey,1 P. Short,2 T. Taylor,3
J. Swingler,3 C. E. Dawson,4 A. M. Whatmore,4 E. Stubberfield,4 L. L. Perrett,4 and G. Simmons5
Investigation and Diagnostic Centre, Biosecurity New Zealand, Ministry of Agriculture and Forestry, Ward St., Wallaceville,
New Zealand1; Institute of Environmental Science and Research, Ltd., Kenepuru Science Centre, 34 Kenepuru Dr., Porirua,
New Zealand2; Australian Animal Health Laboratory, Geelong, Victoria, Australia3; Veterinary Laboratories Agency,
New Haw, Addlestone, Surrey KT15 3NB, United Kingdom4; and Auckland Regional Public Health Service,
Auckland, New Zealand5

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Received 30 March 2006/Returned for modification 11 June 2006/Accepted 17 September 2006

Naturally acquired infection of humans with a marine mammal-associated Brucella sp. has only been
reported once previously in a study describing infections of two patients from Peru. We report the isolation and
characterization of a strain of Brucella from a New Zealand patient that appears most closely related to strains
previously identified from marine mammals. The isolate was preliminarily identified as Brucella suis using
conventional bacteriological tests in our laboratory. However, the results profile was not an exact match, and
the isolate was forwarded to four international reference laboratories for further identification. The reference
laboratories identified the isolate as either B. suis or B. melitensis by traditional bacteriological methods in
three laboratories and by a molecular test in the fourth laboratory. Molecular characterization by PCR,
PCR-restriction fragment length polymorphism, and DNA sequencing of the bp26 gene; IS711; the omp genes
omp25, omp31, omp2a, and omp2b; IRS-PCR fragments I, III, and IV; and five housekeeping gene fragments was
conducted to resolve the discrepant identification of the isolate. The isolate was identified to be closely related
to a Brucella sp. originating from a United States bottlenose dolphin (Tursiops truncatus) and common seals
(Phoca vitulina).

Brucellosis is an important zoonotic disease of humans, marine mammal strains of Brucella by isolation, PCR, and
causing a variety of vague symptoms including undulant fever, DNA sequencing (41). The two Peruvian patients were not
fatigue, malaise, joint pain, myalgia, depression, and anorexia laboratory workers, and the infection was naturally acquired.
(22). Chronic sequelae and recrudescence decades after initial Serological evidence and isolation of brucellae have been
infection also occur. Brucella may be transmitted from animals reported from a variety of marine mammals on numerous
to humans by direct contact with infected animals, ingestion of occasions from locations in the northern hemisphere. The se-
infected food products, and inhalation of aerosols. Four spe- rological prevalence ranges from 0 to 38% for cetaceans, pin-
cies of Brucella are the primary causes of infection in humans. nipeds, and mustelids (6, 24, 26, 29, 32, 34, 43). The largest
Brucella melitensis is highly infectious and is transmitted from studies of 1,855 pinnipeds from North America and 1,386 pin-
sheep and goats, B. abortus is transmitted from cattle, B. suis is nipeds and cetaceans from the North Atlantic Ocean revealed
transmitted from pigs and, infrequently, B. canis is transmitted 3.1 and 8.2%, respectively, to have serological evidence of
from dogs. Other species of Brucella have been rarely or not brucella (33, 43). Brucella have also been isolated from 31%
reported to infect humans. (54/175) of marine mammals sampled under a variety of cir-
There are only two reports in the literature of humans in- cumstances (8, 14, 17, 19, 23, 29, 31, 40, 43, 44). There are few
fected with marine mammal strains of Brucella. One report was reports of serological testing of pinnipeds and cetaceans from
of a laboratory worker who displayed symptoms consistent with the Southern Hemisphere. In the Antarctic, positive reactions
brucellosis (4). The infection was confirmed by a positive se-
were detected in 3.5% (6/17) of pinnipeds (36). A high prev-
rological response, isolation, and PCR-restriction fragment
alence of serological reactions (55.2% [32/58]) was reported in
length polymorphism (RFLP) identification of a marine Bru-
cetaceans off the coast of central Peru (45). In pinnipeds of
cella strain. Two patients originating from Peru and diagnosed
Australia, serological reactions were reported in three species,
with neurobrucellosis were also confirmed to be infected with
including 75% (9/12) of sea lions (Neophoca cinerea) (13). In
New Zealand, no serological reactions were detected in 101
* Corresponding author. Mailing address: Investigation and Diag- New Zealand sea lions (Arctocephalus hookeri) (28).
nostic Centre, Biosecurity New Zealand, Ministry of Agriculture and Brucella species isolated in New Zealand include B. ovis
Forestry, Ward St., Wallaceville, New Zealand. Phone: 61(0)4 526 from sheep and deer, B. abortus (eradicated from livestock in
5600. Fax: 61(0)4 526 5601. E-mail: wendy.mcdonald@maf.govt.nz.
1989), and B. melitensis and B. suis (both from human pa-
† Present address: Tropical and Aquatic Animal Health Laboratory,
180-202 River Blvd., Townsville, Queensland, Australia. tients). Infection of humans with the latter two species was
䌤
Published ahead of print on 11 October 2006. confirmed as having been acquired overseas (7, 39). Hitherto,

4363
4364 MCDONALD ET AL. J. CLIN. MICROBIOL.

TABLE 1. Results of brucella serology tests performed on samples from the patient at two New Zealand medical laboratories (W and M)
Result (days from onset)
a
Test Screen SAT titer Coombs titer

W M Wb M W M

First ⫹ (29) ⫹ (36) 1:1,280 (29)* ⬍1:20 (36) 1:2,560 (29) 1:640 (36)
Second NT ⫺ (153) 1:320 (40)* 1:40 (153) 1:1,280 (40) 1:160 (153)
a
NT, not tested. ⫹, Positive; ⫺, negative.
b
*, SAT titers using 2-mercaptoethanol were 1:160 and 1:80 in the first and second tests, respectively.

marine mammal Brucella strains had not been isolated in New uncooked fish bait, including pilchards (Sardina and Sardinops
Zealand. However, serum and tissue samples of stranded Hec- spp.), bonito (Sarda spp.), squid (Nototodarus sp.), and mullet
tor’s dolphins (Cephalorhynchus hectori) tested at our labora- (Mugilidae) and regularly caught snapper (Chrysophrys aura-
tory, the Investigation and Diagnostic Centre (IDC), have re- tus), kahawai (Arripis trutta), trevally (Pseudocaranx dentex),

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vealed serological and molecular evidence of brucella infection and gurnard (Chelidonichthys kumu). The patient consumed
(unpublished data). raw freshly caught snapper with lemon juice and coconut
The present study describes the phenotypic and molecular cream.
characterization of a strain of Brucella isolated from a New
Zealand patient that is most closely related to isolates previ- MATERIALS AND METHODS
ously identified from marine mammals. Identification strategy. A range of phenotypic tests in addition to DNA se-
Case report. In February 2002, a 43-year-old male patient quencing of a fragment of the omp25 gene were undertaken in our laboratory in
from South Auckland in the North Island of New Zealand order to identify the isolate (02/611). After we obtained the results of the
presented with 2 weeks of symptoms of spinal osteomyelitis— in-house testing (IDC), the isolate was forwarded for confirmation to four ref-
erence laboratories, designated A, B, C, and D. Upon receipt of the results from
fever, rigors, and lumbar spinal tenderness. Blood samples
the reference laboratories, further characterization of the isolate was undertaken
were tested at two New Zealand medical laboratories (M and using molecular techniques in our laboratory (IDC) and the Veterinary Labo-
W) by a screening agglutination test using B. abortus antigen ratories Agency (United Kingdom) (VLA).
(Immunostics, Inc.; Remel), a serum agglutination test (SAT) Brucella isolates. The Brucella species examined in the present study were B.
using B. abortus antigen (Murex) with or without the addition abortus bv. 2 (Australian Animal Health Laboratory), B. abortus bv. 3 (Central
Animal Health Laboratory, New Zealand strain Tulya), B. abortus bv. 9 (CAHL
of 2-mercaptoethanol to the diluent, and a Coombs anti-bru- strain C68), B. ovis (AgResearch; NZ00/1614), B. canis (ATCC 23365), B. suis bv.
cella test using anti-human globulin (CSL Epiclone). The pa- 3 (AAHL), and B. melitensis bv. 2 (Environmental Science and Research, Ltd.;
tient tested positive with all three serological tests within 36 NZ02/820). Eight Brucella spp. of marine origin (UK1/03 and UK39/94 isolated
days of onset of illness and tested negative in the SAT and with from common seals [Phoca vitulina]; UK61/94 isolated from a gray seal [Hali-
choerus grypus]; UK9/02 and 36/94, both isolated from harbor porpoises [Phoco-
a declining titer in the Coombs test 153 days from onset of
ena phocoena]; F5/99 isolated from a bottlenose dolphin [Tursiops truncatus
illness (Table 1). United States], UK22/00 isolated from a white-sided dolphin [Lagenorphynchus
Magnetic resonance imaging revealed multifocal inflamma- acutus] and 59/94 isolated from a striped dolphin [Stenella coeruleoalba]) were all
tion involving lumbar vertebrae 1 and 4 and also the right supplied by the VLA.
ilium. Aspiration of the right iliac lesion failed to isolate an Culture. All brucella isolates were stored at ⫺70°C on cryobeads. The isolates
were resuscitated using brucella enrichment broth (Farrells medium) and incu-
organism, but after 3 days blood cultures grew a gram-negative bated at 37°C for 3 to 7 days. The broths were subcultured onto serum dextrose
bacillus. Treatment had consisted of intravenous flucloxacillin, agar and chocolate blood agar and incubated at 37°C in O2 and CO2 (1, 28).
which was changed to intravenous ceftriaxone after 3 days Phenotypic testing. Phenotypic testing was performed in our laboratory (IDC)
based on culture sensitivities. The patient was treated with using standard conventional tests for brucella (1, 30). This included growth
requirements, biochemical tests, dye sensitivity, serotyping, and phage typing.
intravenous ceftriaxone for a total of 6 weeks and made a
PCR, RFLP, and DNA sequencing. DNA was extracted from the brucella
complete recovery. The organism (02/611) was misidentified isolates by using the QIAMP DNA minikit (QIAGEN, Hilden, Germany), ac-
using traditional bacteriological methods and reported in April cording to the manufacturer’s instructions. The genetic targets for molecular
2002 as B. suis (3). characterization were the omp31 gene (differentiation of B. abortus); the IS711
Acquisition. The patient had last traveled overseas 10 years element downstream of bp26 (differentiation of “terrestrial” and “marine”
strains); the genes omp25, -2a and -2b; IS711; housekeeping genes; and specific
prior to illness. There was no previous history of a similar IRS-PCR DNA fragments (differentiation of species).
illness. The patient had no exposure to unpasteurized milk or The omp25 gene fragment was initially amplified and sequenced using oligo-
cheese and had no occupational exposure to meat or meat nucleotides designed in-house (IDC). A 50-␮l PCR mixture contained 1⫻ PCR
products. The patient had dressed two freshly killed pigs in buffer (Roche, Basal, Switzerland), 2.5 U of Taq polymerase (Roche, Basal,
Switzerland), 0.2 mM deoxynucleoside triphosphate, 0.5 mM MgCl2, 500 ng of
December 2001, which led to the belief that the patient ac-
forward primer BC1-GTTGAAGTAGCTCCCCAGTA (nucleotide position 109
quired infection while dressing the pigs. Clinical and serolog- of GenBank submission BMU33003) and 500 ng of reverse primer BC2-ACTG
ical surveys of the associated pig population and pig farmers GGTGTAACGGTACTCA (position 431 BMU33003), and 5 ␮l of genomic
were undertaken. No other cases were confirmed. After mo- DNA. The PCR was conducted in a Mastercycler gradient (Eppendorf, Ham-
lecular characterization of the organism (02/611), the patient burg, Germany) or an MJ Research Minicycler (Bio-Rad, Waltham, MA) with a
denaturation step of 96°C for 60 s; followed by 30 cycles of 96°C for 40 s, 60°C
was reinterviewed to determine a possible source of the marine for 60 s, and 72°C for 60 s; and with a final extension step of 72°C for 5 min. The
mammal source of the Brucella sp. The patient had not been PCR product was purified and sent to the University of Waikato (New Zealand)
exposed to marine mammals. The patient had been exposed to for sequencing. Chromatograms were checked for miscalls, and the identity of
VOL. 44, 2006 CHARACTERIZATION OF BRUCELLA SP. 4365

TABLE 2. Comparison of biochemical, serotyping, phage typing, growth in the presence of dyes, and other growth requirements of the
brucella isolate 02/611 from four laboratories with results reported in the literature for marine isolates of brucellaa

Test or test Result for marine Result for bottlenose Result at laboratory:
laboratory location mammal brucella dolphin (F5/99) IDC (repeat) A (repeat) B C

CO2 dependency ⫹ (38/59)b

⫺ ⫺ c
⫺ ⫺ ⫺
H2S production ⫺ (29/29) ⫺ ⫺ ⫹w (⫺) ⫺ ⫺
Oxidase ⫹ (13/13) ⫹ ⫹ ⫹
Catalase ⫹ (19/19) ⫹ ⫹ ⫹
Urease production ⫹ (40/40) ⫹ ⫹d ⫹ ⫹ ⫹
Growth on:
Thionin (1:25,000) ⫹ (9/9) ⫹ ⫾ ⫺
Thionin (1:50,000) ⫹ (34/34) ⫹ ⫹ ⫹ ⫹
Thionin (1:100,000) ⫹ (4/4) ⫹ ⫹ ⫹
Basic fuchsin (1:50,000) ⫹ (34/34) ⫹ ⫹ ⫹ ⫹
Basic fuchsin (1:100,000) ⫾/⫹ (3/3) ⫹ ⫹ ⫹
Safranin O (1:5,000) ⫹ (5/5) ⫹w
Safranin O (1:10,000) ⫹ (33/33) ⫹

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Thionin blue (1 mg/ml) ⫹ (8/8) ⫹ ⫹
I-erythritol (1 mg/ml) ⫹ (2/2) (5/5, 2 ␮g/ml) ⫹ ⫹ ⫹
Penicillin (5 IU/ml) ⫹ (8/8) ⫹ ⫹ (10 ␮m disk) ⫹
DL-Ornithine decarboxylase ⫺ (22/22)e ⫺ ⫹ (⫺)
L-Arginine dihydrolase ⫺ (22/22)e ⫺ ⫹ (⫺)
L-Lysine decarboxylase ⫺ (22/22)e ⫺ ⫹ (⫺)
D-Galactose ⫹ (11/22)f ⫹ ⫹
Agglutination with:
Monospecific sera A ⫹ (22/41) ⫺ ⫺ ⫹ ⫺ ⫺
Monospecific sera M ⫹ (2/41) ⫹ ⫹ ⫺ (? rough) ⫹ ⫹
Sera A and M ⫹ (17/41)
Weybridge, United Kingdomg NL (7/28) CL PL (NL) NL NL
PL (17/28)
CL (4/28)
Tbilisi, Georgiag NL (27/30) NL NL NL NL
PL (3/30)
g
Berkeley, CA PL (5/22) CL (NL) CL
CL (17/22)
Firenze, Italyg NL (13/27) NL (NL) NL
PL (14/27)
gh
R/C NL (28/28) CL NL (plq) NL
a
⫹, Positive; ⫺, negative. NL, no lysis; PL, partial lysis; CL, complete lysis; plq, evident plaques.
b
Brucella isolates from pinnipeds require CO2.
c
Growth was enhanced by CO2.
d
Positive within 10 min.
e
Tetrazolium reduction.
f
Brucella isolates from cetaceans utilize D-galactose.
g
Phages were tested at the routine test dilution.
h
R/C, is a derivative of phage R; phage R was obtained by simultaneous incubation of a number of brucella phages with B. abortus.

the DNA sequences was analyzed by comparison to DNA sequences within The omp2a gene was amplified using the primers 2aA and 2aB (11). The
GenBank using BLASTN (2; http://www.ncbi.nlm.nih.gov/BLAST). reaction mixture and cycling parameters were as described above for the second
The omp25 gene was later amplified using the primers 25A and 25B that omp25 PCR. The amplified DNA was digested by using four restriction endo-
encompassed the in-house designed primers and provided additional single nu- nucleases: HinfI, PstI, AluI, and BanI. The digestion mixture and incubation was
cleotide polymorphisms for RFLP testing and DNA sequencing (11). A 50-␮l as described for the omp25 digestion.
PCR mixture contained 1⫻ HotStar master mix (QIAGEN), 0.1 ␮M concentra- The omp2b gene was amplified using the primers 2bA and 2bB and the
tions of each primer, and 5 ␮l of genomic DNA. The PCR was conducted with conditions described previously (11). The amplified DNA was digested by using
a denaturation step at 95°C for 15 min; followed by 35 cycles of 95°C for 1 min, six restriction endonucleases: BglII, EcoRI, HaeIII, HinfI, KpnI, and TaqI.
58°C for 2 min, and 70°C for 3 min; followed by a final extension step of 70°C for The omp2a and -2b PCR products from 02/611 and F5/99 were sent to Monash
10 min. The amplified fragment was digested with EcoRV. A 20-␮l digestion University (Australia) for sequencing. After editing and compilation of the
reaction contained 5 ␮l of DNA, 10 U of enzyme (New England Biolabs, Beverly, contigs, the omp2a and -2b DNA sequences were phylogenetically analyzed by
MA; Promega Corp., Madison, WI), 100 ␮g of bovine serum albumin, and the creating a phylogram from a neighbor-joined consensus tree of Kimura distance
corresponding 1⫻ buffer for at least 3 h at 37°C in a water bath. Digests were matrices created from 500 bootstraps of the CLUSTAL W aligned sequences
electrophoresed in a 3% agarose gel in 1⫻ TAE (40 mM Tris–acetate–2 mM (16, 42) (BioManager by ANGIS [http://www.angis.org.au]; TreeView [Win32];
EDTA). http://taxonomy.zoology.gla.ac.uk/rod/rod.html).
The bp26 gene and downstream IS711 element was amplified using the primers For the IS711 RFLP analysis, genomic DNA was digested with EcoRI (5).
26A and 26B (9). The reaction mixture was as described for the second omp25 Digested DNA was separated on 0.8% agarose gels and Southern blotted by
PCR. The PCR was conducted with a denaturation step of 95°C for 15 min; using a vacuum method. The membrane-bound DNA was probed using a digoxi-
followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 60 s, genin (DIG)-labeled IS711 probe, and the hybridized probe was detected using
and extension at 72°C for 60 s; followed by a hold at 72°C for 5 min. anti-DIG monoclonal antibodies. The membrane was immersed in CSPD chemi-
The omp31 gene was amplified using the primers 31sd and 31ter (46). The luminescence substrate to allow visualization of the probe hybridization by ex-
reaction mixture and cycling parameters were as described for the bp26 PCR. posure to X-ray film. The resulting images were analyzed by using Bionumerics
4366 MCDONALD ET AL. J. CLIN. MICROBIOL.

software (Applied Maths, Belgium), and a dendrogram analysis of the profiles

was produced using the coefficient of Jaccard to calculate similarities between
the fingerprint patterns.
Isolate 02/611 was also characterized by using a multilocus sequence typing
approach currently under development at the VLA (A. M. Whatmore, L. L.
Perrett, and A. P. Macmillan, submitted for publication). Fragments of six genes
(dnaK, gyrB, glK, trpE, cobQ, and omp25) were amplified by PCR using a proof-
reading polymerase and sequenced from either end. The sequences obtained
were compared to a database of equivalent sequences from 200 strains repre-
senting all known Brucella species and biovars.
The IRS-PCR fragments were amplified using the primer pairs I1/I2 (PCR I),
III1/III2 (PCR III), and IV1/IV2 (PCR IV) (10). The reaction mixture was the
same as described for the second omp25 PCR. The PCR was conducted with a
hold step at 95°C for 15 min; followed by 40 cycles of 95°C for 1 min, 62°C for
2 min, and 72°C for 2 min; followed by a final hold at 72°C for 10 min.

RESULTS

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Phenotypic characterization. Isolate 02/611 is a gram-nega-
tive coccobacillus that is nonmotile, grows aerobically, and is
oxidase, catalase, and urease positive, features typical of the
genus Brucella (Table 2). The rapid metabolism of urea was
suggestive of B. suis and with positive results for L-arginine
dihydrolase, L-lysine decarboxylase, DL-ornithine decarboxyl-
ase, and growth on I-erythritol, the isolate was tentatively iden-
tified as B. suis, although it was atypical in that it grew on basic
fuchsin at 1:25,000.
To confirm the identity of the brucella isolate, it was for-
warded to four reference laboratories, and the results of their
analyses are presented in Table 1. Reference laboratory A
identified the isolate as B. suis biovar 3, and laboratories B and
C identified it as B. melitensis biovar 1.
Molecular characterization. At the time of the submission of
isolate 02/611, brucella isolates were routinely tested in our
laboratory (IDC) using the in-house designed omp25 PCR, and
sequencing BlastN analysis conducted in April 2002 of the
partial DNA sequence from the forward primer (BC1) re-
vealed that the isolate was most closely related to the following
Brucella spp. (no. of matching nucleotides): B. suis (398/403),
B. melitensis (398/403), B. abortus (398/403), B. neotomae (398/
403), B. ovis (397/403), and B. canis (396/404). The E value was
0 for each Brucella spp. The next lowest value was 1e-126 for
Ochrobacterum anthropi. Reference laboratory D returned an
identification of B. suis biovar 5 using molecular tests; however,
details on the methodology were not forthcoming from this lab-
oratory.
Further molecular characterization was undertaken in our
laboratory (IDC), including an updated BLASTN analysis con-
ducted in June 2005. omp25 sequences from marine isolates of
brucella were submitted to GenBank in September 2004. The
latter BLASTN analysis of the compiled sequence obtained
with the forward and reverse primers (25A and 25B) now
revealed 02/611 to be a 100% match (642/642 nucleotides) to
B. cetaceae accession numbers AY484523 and AY484520 and
B. pinnipediae accession number AY484522.
The omp25 PCR amplified a 700-bp DNA fragment from all FIG. 1. Restriction digestion of omp2a PCR product with HinfI
Brucella spp., and upon digestion with EcoRV, only the B. (A), PstI (B), AluI (C), and BanI (D). Lanes: 1, B. abortus bv. 2; 2, B.
melitensis isolate remained uncut. Isolate 02/611 produced a P1 abortus bv. 9; 3, B. ovis; 4, B. canis; 5, B. suis bv. 3; 6, B. melitensis; 7,
02/611; 8, marine mammal brucella (seal UK1/03); 9, marine mammal
pattern (11).
brucella (porpoise UK9/02); 10, marine mammal brucella (United
The bp26 PCR amplified a 1,900-bp fragment from brucella States bottlenose dolphin F5/99); 11, marine mammal brucella (striped
isolates from marine mammals, including pinnipeds and ceta- dolphin UK59/94); 12, B. ovis; 13, VI marker (Boehringer-Mannheim).
ceans, and a smaller fragment of 1,029 bp was amplified from
VOL. 44, 2006 CHARACTERIZATION OF BRUCELLA SP. 4367

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FIG. 2. Phylogram comparing the omp2a and -2b genes from 02/611 with the omp2a and -2b genes from marine and terrestrial isolates of
brucella. Internal nodes are labeled with the percent bootstrap values.

brucella isolates from terrestrial animals. The amplified fragment produced a fingerprint profile identical to that of the F5/99
from isolate 02/611 corresponded in size to the marine mammal United States bottlenose dolphin isolate (Fig. 3). Each genome
isolates of brucella. contained 25 copies of the IS711 element sized between 20,000
The omp31 PCR amplified 900 bp from all Brucella spp. tested and 1,520 bp. The profiles were similar to those described for
(including isolate 02/611) with the exception of B. abortus. brucella isolated from marine mammals (5).
Products of 1,230 bp were produced upon amplification of Sequencing of fragments from the five housekeeping genes
the omp2a and omp2b genes of all species of Brucella. The and omp25 revealed that the most closely related sequences
RFLP patterns produced after restriction digestion of omp2a were those of marine mammal isolates and B. suis. A report
are shown in Fig. 1. The omp2a RFLP pattern produced by the fully describing this technique is in preparation, but isolate
human isolate 02/611 matched the RFLP patterns produced by 02/611 differed from each of the B. suis biovar type strains by
the isolate F5/99 originating from a bottlenose dolphin and the a minimum of four base differences. Of the five distinct marine
isolate UK1/03 originating from a common seal. The combined mammal genotypes seen to date, isolate 02/611 varied from
omp2a and -2b profile of RFLP patterns produced by 02/611 four of these by two to four nucleotides but was an exact match
was identical to that produced by F5/99. All other Brucella spp. with F5/99 (United States bottlenose dolphin) (Table 3). De-
differed by at least one RFLP pattern. spite our having examined some 50 marine mammal isolates by
DNA sequencing of the omp2 genes of isolate 02/611 re- this approach, this genotype has only been previously seen in
vealed that the isolate contained one omp2a and one omp2b this single isolate.
and that both genes were 100% identical in sequence to the PCR I specific for B. pinnipediae amplified a 300-bp frag-
omp2a and -2b genes, respectively, of F5/99. The omp2a gene ment from 02/611, UK1/03 (common seal), and F5/99 (United
from 02/611 was also 100% identical to the omp2a genes from States bottlenose dolphin). PCR III and IV specific for B.
two common seal isolates (GenBank accessions AF003819 and cetaceae did not amplify DNA fragments from 02/611, F5/99,
DQ059380) but not to the omp2b genes from these two iso- or UK1/03. A 400-bp fragment was amplified from UK9/02
lates. Phylogenetic analysis showed 02/611 and F5/99 to be (porpoise) using PCR III, and a 300-bp fragment was amplified
identical and closely related to isolates from common seals from UK59/94 (striped dolphin) using PCR IV.
(Fig. 2). Phenotypic reevaluation. Following the conflicting identifi-
IS711 RFLP molecular characterization of isolate 02/611 cation and results from follow-up molecular testing, reevalua-
4368 MCDONALD ET AL. J. CLIN. MICROBIOL.

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FIG. 3. IS711 fingerprint patterns of Brucella species tested in the present study.

tion of the phenotypic profile for isolate 02/611 with repeat 34). Similarly, there is evidence to suggest that marine mam-
testing of L-arginine dihydrolase, L-lysine decarboxylase, and mal strains of Brucella can cause a range of symptoms in
DL-ornithine decarboxylase revealed that it was consistent with humans, including headaches, malaise, severe sinusitis, sei-
the phenotypic results for marine brucella isolates reported in zures (4, 41), and spinal osteomyelitis. It is also possible that
the literature (Table 1) (8, 14, 18, 19, 21, 25, 31, 44). In addi- marine mammal strains of Brucella may cause only mild symp-
tion, isolate 02/611 produced the same phenotypic results as toms in humans, and thus the disease goes undiagnosed or
those reported for the brucella isolate from the United States misdiagnosed. In this report the patient was symptomatic and
bottlenose dolphin (13). serologically positive since testing was within the early phase of
infection. However, in the previous report only one of the
DISCUSSION males had a single positive titer to brucella; both males had a
longer history of symptoms when tested (41).
This is the second published report of humans with naturally Zoonotic disease of humans with brucella has been com-
acquired infection originating from a marine mammal strain of
monly associated with B. melitensis, B. abortus, B. suis, and B.
Brucella and the first report of a male presenting with spinal
canis. Given the reported rarity of infection of humans with
brucellosis due to a marine mammal strain. The earlier report
marine mammal strains of Brucella, and the high-risk exposure
of two males with naturally acquired infection with a marine
to pigs within the incubation period of disease, it is not sur-
Brucella spp. were described as having neurological symptoms
prising that initial interpretation of the phenotypic results led
(41). Marine mammal strains of Brucella have been reported to
to a preliminary identification of B. suis. After results were
cause a range of clinical symptoms with varying severity in
pinnipeds and cetaceans ranging from asymptomatic carriage obtained from the molecular tests, a review of the phenotypic
to orchitis, abortions, and meningeoencephalitis (16, 23, 31, results found these consistent with identification of the isolate
as a marine mammal strain of Brucella. Classification of marine
mammal strains of Brucella as B. pinnipediae and B. cetaceae
TABLE 3. Comparison of 02/611 sequences with B. suis and marine has been proposed as species originating from pinnipeds and
mammal brucella sequences showing all bases variable cetaceans, respectively (12). These two species can generally be
between the isolates differentiated on the basis of CO2 dependency (B. pinnipediae)
Gene polymorphisma and metabolism of D-galactose (B. cetaceae) (20). The human
Brucella isolate isolate most closely resembled the isolate from the United
glk dnaK gyrB trpE cobQ omp25
States bottlenose dolphin, and both would be classified as B.
Human isolate 02/611 AGG GACC GGC AA G C
B. suis bv. 1 ... .G.T A.T GG . . cetaceae based on the phenotypic results. The phenotypic test
B. suis bv. 2 G.. .G.T ..T GG . . results do not correlate with the results from the specific PCR
B. suis bv. 3 ... .G.T AAT GG . . test (I) and sequencing of the omp2a gene since both genotypic
B. suis bv. 4 ... .G.T AAT GG . .
B. suis bv. 5 ... .GA. ... GG . . test results indicate that the human and bottlenose dolphin
Marine mammal brucella 36/94 ..A AG.. ... G. . . isolates are more closely related to B. pinnipediae than B.
Marine mammal brucella 39/94 .A. .G.. ... G. . .
Marine mammal brucella 59/94 ... .G.. ... G. A T cetaceae. In addition, the variation in results obtained from
Marine mammal brucella 61/94 ... .G.. ... G. . . repeat biochemical testing indicates that identification to the
Marine mammal brucella F5/99 ... .... ... .. . .
species level using phenotypic traits may not be reliable.
a
Residues identical to 02/611 are indicated as a dot. Restriction patterns of the PCR-amplified brucella omp2a
VOL. 44, 2006 CHARACTERIZATION OF BRUCELLA SP. 4369

gene of isolate (02/611) produced an overall pattern classifica- Bingham, unpublished data). The organism was isolated post
tion of I as described previously (12). Pattern I has been asso- mortem from the retropharyngeal and mandibular lymph
ciated with isolates from a hooded seal (Cystophora cristata), a nodes of three piglets. On initial investigation, pigs were im-
gray seal (Halichoerus grypus), 10 common seals, and an otter plicated as the likely source of infection based on the presump-
(Lutra lutra), whereas cetacean isolates have produced overall tive identification of the isolate as B. suis and the patient’s
patterns classified as J or K (12). Restriction patterns of the contact with pigs in the timeframe of infection. There was no
PCR-amplified brucella omp2b gene of isolate 02/611 pro- serological evidence of infected pigs in trace herds (3). How-
duced an overall pattern classification similar to O and P; ever, a low-prevalence transient antibody response probably
however, an EcoRI pattern 3 was observed which has not been would not have been detected. Further testing of pigs for
previously noted. Pattern O has been associated with two com- Brucella spp. is being conducted. Lymph nodes (retropharyn-
mon seals, and pattern P has been associated with a hooded geal, submaxillary, gastrohepatic, internal iliac, and inguinal)
seal. Overall, patterns L, M, and N have been associated with collected from 74 chopper pigs sent to an abattoir from 22 pig
cetacean isolates, including a bottlenose dolphin with a com- farms were pooled and tested in our laboratory (IDC) by the
plete omp2a, opm2b profile as M-J. It is interesting that the nested omp25 PCR for Brucella sp. All tested negative. The
overall patterns of the bottlenose dolphin (United States F5/ New Zealand patient was known to have eaten raw snapper.
99) tested in the present study also matched the I (O/P) profile. The two Peruvian patients were reported to have consumed

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The omp2a and -2b genes are 85% homologous, and both the unpasteurized cheese and raw shellfish, and it was speculated
human isolate (02/611) and the bottlenose dolphin isolate (F5/ that the source of their infection with brucella may have been
99) were determined to have one omp2a gene and one omp2b other than marine mammals (41).
gene. The majority of isolates from pinnipeds have also been This case highlights the need for follow-up testing of unusual
found to contain one omp2a and one omp2b gene compared to phenotypic results of brucella isolates and proves that the
the majority of isolates from cetaceans that have two omp2b molecular characterization techniques described in the litera-
genes (12). ture are very useful tools for differentiating Brucella species.
The close relationship of isolate 02/611 and the marine This case also reinforces earlier warnings that marine mammal
mammal isolate F5/99 was supported by two other approaches. strains of Brucella may be an emerging zoonotic disease, and
First, the two isolates shared an identical profile as determined consideration needs to be given to marine sources when bru-
by IS711 fingerprinting. Second, the isolates were identical to cella infections are being diagnosed.
each other based on housekeeping gene sequence data, and
ACKNOWLEDGMENTS
the most closely related organisms are other marine mammal
brucella. Thus, evidence from a variety of approaches supports We thank Susan Taylor and Christopher Mansell for providing in-
the observation that the closest known relative of isolate 02/611 formation on the serological testing of the patient.
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