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Previous Article | Next Article 
Journal of Clinical Microbiology, March 2000, p. 1258-1262, Vol. 38, No. 3
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Molecular Characterization of Brucella
Strains Isolated from Marine Mammals
Betsy J.
Bricker,1,*
Darla R.
Ewalt,2
Alastair P.
MacMillan,3
Geoff
Foster,3 and
Simon
Brew3
National Animal Disease Center, Agricultural
Research Service,1 and National
Veterinary Services Laboratories, Animal and Plant Health Inspection
Service,2 USDA, Ames, Iowa, and Central
Veterinary Laboratory, OIE Brucellosis Reference Laboratory, FAO/WHO
Centre for Brucellosis Reference and Research, Weybridge, United
Kingdom3
Received 25 May 1999/Returned for modification 22 September
1999/Accepted 30 November 1999
 |
ABSTRACT |
Recently, gram-negative bacteria isolated from a variety of marine
mammals have been identified as Brucella species by
conventional phenotypic analysis. This study found the 16S rRNA gene
from one representative isolate was identical to the homologous
sequences of Brucella abortus, B. melitensis,
B. canis, and B. suis. IS711-based DNA fingerprinting of 23 isolates from marine mammals showed all the
isolates differed from the classical Brucella species. In general, fingerprint patterns grouped by host species. The data suggest
that the marine mammal isolates are distinct types of Brucella and not one of the classical species or biovars
invading new host species. In keeping with historical precedent, the
designation of several new Brucella species may be appropriate.
| |
TEXT |
Brucellosis is a worldwide zoonotic
disease caused by gram-negative bacteria of the genus
Brucella. Currently, there are six recognized species of
Brucella based on host specificity: B. abortus (cattle), B. canis (dogs), B. melitensis (goats),
B. neotomae (desert wood rats), B. ovis (sheep),
and B. suis (pigs, reindeer, and hares). The six species
have been subdivided into 18 biovars (12, 13) based on a
panel of culture and biochemical characteristics (1, 13).
In the last five years, there have been several reports of
Brucella species isolated from marine mammals, predominantly
seals and cetaceans. Identification was based on serology, morphology, staining, metabolic phenotype, culture characteristics, and phage typing (3, 6, 7, 8, 11, 14, 15). Further characterization of
these isolates was crucial because of concerns within the agricultural community about whether marine hosts were reservoir hosts for classical
Brucella species that could be transmitted to livestock. The
purpose of this study was to genetically characterize
Brucella isolated from marine mammals and to compare them to
the classical Brucella species and biovars.
Bacteriology.
The Brucella strains used in this
report are listed in Table 1. Each
isolate was obtained from a separate animal. The history of strains
M1068/91, M644/93, M2357/93, M2466/93, 2533/93, 292/94, M336/94,
M339/94, M972/94, and M490/95 was described by Foster et al.
(7). Phenotypic characterization of these same strains was
published by Jahans et al. (11). Strain 1312, isolated from an aborted dolphin fetus, was the same strain characterized by Ewalt et
al. (6). Strains 1508 and 98-230 were isolated from different individuals within the same group of captive dolphins as
strain 1312. Strain 1508 was isolated from placental tissue following
an abortion, while strain 98-230 was isolated from the lung tissue of a
dolphin that had died of unrelated causes. Most isolates were obtained
from beach-stranded animals posthumously or from captive animals in
rehabilitation centers. Except in the two cases involving abortion,
none of the isolates were associated with disease symptoms. The
classical Brucella strains selected were the designated type
strains for species and biovars (12, 13). The strains are
available from the National Animal Disease Center (NADC) Brucella
Culture Collection and may be requested through the corresponding
author (some restrictions may apply as required by law).
Brucella strains were grown either on potato infusion agar
or tryptose serum agar plates at 37°C, with or without 5%
CO2 depending on strain requirements (1). When
cultures achieved light confluence (approximately 3 days growth), cells
were harvested by rinsing the agar surface repeatedly with saline
(0.85% NaCl in water). Alternatively, the Brucella strains
were grown in 30 ml of Trypticase soy broth with 5% bovine serum in a
37°C shaking water bath for 48 to 72 h (1). The cell
suspension was centrifuged, and the bacterial pellet was resuspended in
saline. Brucella strains grown by either method were killed
by the addition of two parts methanol to the saline suspension and were
stored in this mixture at 4°C until needed.
DNA sequence of the 16S genes.
One of the genetic targets
frequently used for strain identification and strain phylogeny is the
rRNA operon, particularly the 16S rRNA gene. These genes are highly
conserved and diverge very slowly. The DNA sequences from separate
species within a genus will differ by only a few percent. Sequence
identity among 16S rRNA sequences is typically interpreted as
indicating a single species.
The 16S rRNA genes from isolate M2357/93 (common seal) and from
B. abortus biovar 1 544 were amplified by PCR with primers 16S-RNA1 and 16S-RNA2 (Table 2). These
were consensus primers (17) based on conserved regions of
sequence from other bacteria. DNA was amplified from 105 to
106 killed bacteria (see above) in a 50-µl volume
containing 60 mM Tris-HCl (pH 9.0), 15 mM
(NH4)2SO4, 1.5 mM
MgCl2, 250 µM (each) deoxynucleoside triphosphates, 200 nM each primer, and 1.0 unit Taq polymerase. The
Taq polymerase was temporarily deactivated until the first
denaturation step by the addition of TaqStart Antibody (Cat # 5400-1;
ClonTech Labs Inc., Palo Alto, Calif.). The following parameters were
used for 35 cycles of amplification: 95°C for 1.0 min, 45°C for 2.0 min, and 72°C for 2.0 min. The resulting PCR products were cloned
into the plasmid pCR 2.1 (designed for PCR products) (Cat #K2000;
Invitrogen Corp., Carlsbad, Calif.). The plasmids were replicated in
Escherichia coli DH10B (Cat #18290-015; Life Technologies,
Gaithersburg, Md.). To correct for Taq polymerase misincorporation during amplification, three positive colonies (in the
same orientation with respect to the vector) were pooled prior to
sequencing. A 0.5-µg portion of double-stranded plasmid was sequenced
by dideoxy chain termination (ABI PRISM dRhodamine Terminator Kit, Cat
# 403044; Perkin Elmer/Applied Biosystems, Foster City, Calif.) on an
ABI PRISM Model 377 Sequence Instrument (Perkin Elmer/Applied
Biosystems). Both strands were analyzed to determine the correct
sequence.
The DNA sequence of the 16S rRNA gene from isolate M2357/93 (GenBank
accession no. AF091353) (Fig. 1) was
identical to the sequence determined for B. abortus 544 (GenBank accession no. AF091354), confirming that the isolate belonged
to the genus Brucella. It should be noted that our sequence
for B. abortus 544 differed slightly from the published
sequence for B. abortus 11/19 (GenBank accession no. X13695)
(5). It is unlikely that this divergence reflected strain
differences, since our B. abortus sequence was identical to
the homogeneous 16S rRNA sequences of B. canis, B. melitensis, and B. suis, recorded in GenBank (accession no. L37584, L26166, and L26169, respectively) (K. H. Wilson and
H. G. Hills, unpublished data). The rRNA sequence of B. ovis (accession no. L26168) (K. H. Wilson, unpublished data) was nearly identical, with a single nucleotide difference.
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FIG. 1.
DNA sequence of the 16S rRNA gene from
Brucella isolate M2357/93 (common seal). A 1,412-bp sequence
of the 16S rRNA locus is presented. The sequence is identical to that
determined for B. abortus biovar 1 (strain 544) in this
study.
|
|
IS711 fingerprinting.
The presence of the mobile
genetic element IS711 (GenBank accession no. M94960, also
known as IS6501) (9) has been a useful target for
molecular characterization of classical Brucella species and
biovars (2) based on the number and distribution of
IS711 copies within the bacterial genomes. We have
previously shown that among classical Brucella species,
IS711-based fingerprints are stable, species specific
(except B. canis), and to some extent biovar specific
(2).
Twenty-three isolates from seven marine host species located in four
widely dispersed geographic locations were fingerprinted by Southern
blot analysis of EcoRI-digested DNAs from each marine mammal
isolate as previously described (2). Fingerprints were generated with a DIG-labeled IS711 probe. The probe was
prepared by PCR amplification of an 842-bp fragment containing the
entire IS711 element from 75 pg of the plasmid pBO31-I1
(10) per manufacturer's instructions (PCR DIG Probe
Synthesis Kit, Cat #1636-090; Boehringer Mannheim Biochemicals Corp.,
Indianapolis, Ind.). The primers used for PCR are listed in Table 2.
The labeled probe was denatured by boiling in hybridization buffer (TBS
[100 mM Tris, pH 7.5, and 150 mM sodium chloride] containing 1%
blocking reagent [Cat #1096 176; Boehringer Mannheim Biochemicals]
and 5% sodium dodecyl sulfate) then allowed to anneal with the
membrane blot overnight at 65°C. The membrane was washed, blocked,
reacted with anti-DIG-Alkaline Phosphatase-conjugated Fab
antibody, and washed as described in the manufacturer's
instructions for the DIG Luminescent Detection Kit (Cat no. 1 363 514, Boehringer Mannheim Biochemicals). Luminescence was induced with
LumiPhos 530 impregnated substrate sheets (Cat # 78043) per
manufacturer's instructions (Schleicher & Schuell, Keene,
N.H.). Luminescence was recorded on Kodak X-OMAT film.
The results, shown in Figure 2,
demonstrated that IS711 occurred in every isolate and that
each genome contained at least 25 or more copies. There was a general
similarity in the fingerprints, but among the 23 isolates, 10 different
fingerprints emerged that differed by at least one copy or locus. The
fingerprint patterns tended to group by host species, with the
exception of the single harp seal isolate (98-633), which was identical
to three ringed seal isolates (98-630, 98-631, and 98-632) and one
harbor seal isolate (98-473). There was some variability within a host
species (e.g., common seal isolates M2357/93, M490/95, and M972/94).
Minor variations could be found within a limited geographic location. For example, both 98-473 and 96-408 were isolated from harbor seals in
the Puget Sound, and isolates 1312, 98-230, and 1508 were obtained from
a small fleet of captive bottlenose dolphins maintained in California.
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FIG. 2.
Southern blot analysis of Brucella isolates
obtained from marine mammal hosts probed with IS711 DNA.
EcoRI genomic digests of 23 Brucella strains were
electrophoresed, blotted onto nylon membrane, and probed with
DIG-labeled IS711 DNA. Sizes of fragment are given in
kilobase pairs on the right side. H. porpoise, harbour porpoise; c.
dolphin, common dolphin; c. seal, common seal; h. seal, harbor seal; r.
seal, ringed seal; b. dolphin, bottlenose dolphin; and std, size
standard (DIG-labeled lambda DNA cut with HindIII).
Lanes 1 through 9 contain DNA obtained from hosts in the eastern North
Atlantic Ocean or North Sea; lanes 10 through 19 and 21 through 23 contain DNA from hosts found in the eastern Pacific Ocean, Canada, and
the western Atlantic Ocean. The arrow indicates a 1.7-kbp fragment
common to most isolates. Isolate M336/94 (common seal) is not pictured
but has a pattern identical to common seal isolates M972/94 and
M339/94.
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|
To see how the distribution of IS711 copies within the
genomes of marine mammal isolates differed from the classical
Brucella species and biovars, two representative marine
mammal isolates were directly compared to members from each of the six
Brucella species (Fig. 3). For
the five biovars of B. suis, the biovar with the most
IS711 copies was used. The data showed that the fingerprints
from the Brucella isolates derived from marine mammals were
quite distinct from the fingerprints of the classical
Brucella species. The marine mammal isolates had
significantly more copies of IS711 than all classical
species except B. ovis. The fingerprint patterns differed
extensively from the B. ovis fingerprint.
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FIG. 3.
Southern blot analysis of two marine mammal isolates and
six classical Brucella species probed with IS711
DNA. EcoRI genomic digests of eight Brucella
strains were electrophoresed, blotted onto nylon membrane, and probed
with DIG-labeled IS711 DNA. Sizes of fragment are given in
kilobase pairs on the right side. The arrow indicates the location of
the marine-mammal-specific copy of IS711 used for PCR
identification.
|
|
The observation that the marine mammal isolates had more copies of
IS711 per genome and showed greater diversity in
localization of the elements suggests that IS711 elements at
some time were more active in the marine mammal isolates than in most
terrestrial isolates. Whether this activation is ongoing or related to
host adaptation needs to be examined. Despite the differences observed in IS711 fingerprints, there were greater similarities among
the isolates from marine animals.
PCR amplification of a DNA sequence specific for marine mammal
isolates.
The fingerprints shown in Fig. 2 revealed many fragments
that appeared to be common among the isolates from marine mammals but
showed no corresponding fragment among the classical isolates (Fig. 3).
We wanted to see if at least one of these fragments was from the same
locus in each genome and, if so, whether it could serve as a marker for
the marine mammal isolates as a group. To promote cloning of the
target, we selected a small, 1.7-kb EcoRI fragment (Fig. 2).
No corresponding copy was apparent in other Brucella strains
(Fig. 3). An EcoRI genomic library of strain M644/93
(dolphin) was created in pBluescript II SK(+) (Cat #212205; Stratagene,
La Jolla, Calif.) and was probed for IS711 elements as
previously described (2). The DNA flanking the
IS711 copy in the selected clone was sequenced. The sequence
data was used to develop a locus-specific PCR primer (Table 2).
To determine if this specific copy of IS711 was present in
other marine mammal isolates, PCR amplification was performed by using
one primer homologous to IS711 and the locus-specific
primer. The cycling parameters were 95°C for 2.0 min, followed by
95°C for 10 s, 50°C for 10 s, and 72°C for 1.0 min, for
a total of 30 cycles. The predicted product of 375 bp was amplified
from all marine mammal isolates (data not shown). No product was
amplified from the classical Brucella species and biovars
with the exception of B. ovis. Examination of several
B. ovis isolates by PCR routinely produced a small quantity
of product at the predicted size, suggesting partial but not total
homology to the PCR primers (data not shown). Surprisingly, DNA from
isolate 98-230 (bottlenose dolphin) did not demonstrate a 1.7-kbp
IS711 fragment by Southern blot analysis (Fig. 2) but did
amplify large quantities of the DNA sequence targeted by the
locus-specific probe.
The demonstration of one and possibly many IS711 loci common
to all marine mammal isolates but not their terrestrial counterparts could indicate that the marine mammal isolates may have arisen from a
single progenitor strain that differed from the current terrestrial
strains. The discovery that B. ovis showed partial homology
to the primer specific for marine mammal isolates was unexpected. It
may suggest that the marine mammal isolates are closest
phylogenetically to B. ovis, or it may be that a copy inserted into the same locus in B. ovis as in the marine
mammal isolates by an independent event. Further characterization of the marine mammal isolates at other loci should help illuminate phylogeny.
The results of this study complicate the issue of how to name the new
isolates. While the classical Brucella have been grouped into species, ribotyping and other genetic analyses suggest that the
Brucella genus is actually monospecific (16). For
practical reasons, the species designations have been retained
(4). Based on their 16S DNA sequences, the marine mammal
isolates appear to belong to the same Brucella monospecies
as the classical Brucella strains. If the historical
precedence is continued, the marine mammal isolates appear to comprise
several new species of Brucella corresponding to diverse
marine mammal hosts, and some species may contain more than one subtype.
| |
ACKNOWLEDGMENTS |
We thank Tonia McNunn and Chad McFadden for technical assistance in
preparing purified genomic DNAs. We also thank Allen Jensen for his
assistance in the preparation of Brucella cultures.
USDA, Agricultural Research Service, CRIS project 3625-32000-031-00D
funded this study.
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
USDA/ARS/National Animal Disease Center, 2300 N. Dayton Ave., P.O. Box
70, Ames, IA 50010. Phone: (515) 663-7310. Fax: (515) 663-7458. E-mail:
bbricker{at}nadc.ars.usda.gov.
| |
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Journal of Clinical Microbiology, March 2000, p. 1258-1262, Vol. 38, No. 3
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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