See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/272414653

Isolation of Brucella from a White's tree frog (Litoria caerulea)

Article · January 2015

DOI: 10.1099/jmmcr.0.000017

CITATIONS READS

15 178

10 authors, including:

Emma Dale Emma Stubberfield

Department for Environment, Food and Rural Affairs Animal and Plant Health Agency
6 PUBLICATIONS   51 CITATIONS    36 PUBLICATIONS   410 CITATIONS   

SEE PROFILE SEE PROFILE

Mark Koylass Claire Elizabeth Dawson

AstraZeneca, Cambridge, UK Department for Environment, Food and Rural Affairs
41 PUBLICATIONS   767 CITATIONS    22 PUBLICATIONS   406 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Spotty liver disease View project

Brucellosis View project

All content following this page was uploaded by Emma Dale on 28 October 2015.

The user has requested enhancement of the downloaded file.

JMM Case Reports (2015) DOI 10.1099/jmmcr.0.000017

Case Report Isolation of Brucella from a White’s tree frog

(Litoria caerulea)
Adrian M. Whatmore,1 Emma-Jane Dale,1 Emma Stubberfield,1
Jakub Muchowski,1 Mark Koylass,1 Claire Dawson,1 Krishna K. Gopaul,1
Lorraine L. Perrett,1 Matthew Jones2 and Alistair Lawrie3
Correspondence 1
FAO/WHO Collaborating Centre for Brucellosis, OIE Brucellosis Reference Centre, Animal and
Adrian Whatmore Plant Health Agency (APHA), Department of Bacteriology, Woodham Lane, Addlestone,
adrian.whatmore@apha.gsi.gov.uk Surrey KT15 3NB, UK
2
IDEXX Laboratories, Grange House, Sandbeck Way, Wetherby LS22 7DN, UK
3
Lawrie Veterinary Group, 25 Griffiths Street, Falkirk FK1 5QY, UK

Received 3 November 2014
Accepted 10 January 2015
Introduction: Brucellosis is a zoonotic disease that has a significant economic, social and public
health impact in many parts of the world. The causative agents are members of the genus
Brucella currently comprising 11 species and with an expanding known host range in recent
years.
Case presentation: One of a pair of White’s tree frogs (Litoria caerulea) developed skin lesions
from which a pure growth of a haemolytic organism was obtained. The isolate was identified as
Brucella melitensis by matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry, although the colony morphology was inconsistent with this identification. Applying
the classical biotyping approach used to subdivide members of the genus Brucella, the isolate
did not correspond to any known Brucella sp. However, PCR targeting of genes specific for
members of the genus Brucella was strongly positive and 16S rRNA gene sequencing revealed a
close relationship with extant Brucella spp. In order to place the isolate more accurately, a
multilocus sequencing approach was applied, which confirmed that the isolate represented a
novel member of the emerging ‘atypical’ Brucella group, which includes isolates from human
disease, from rodents and, more recently, reported isolations from frogs in Germany.
Conclusion: This case represents the first report of isolation of a Brucella sp. from frogs outside
Germany and suggests that these isolates may be widespread. Whilst there is no evidence to date
that these isolates represent a zoonotic threat, the association of other ‘atypical’ Brucella sp. with
human disease suggests that appropriate measures should be taken to avoid unnecessary contact
with potentially infected amphibians until the zoonotic potential of this group is better understood.
Keywords: Brucella, brucellosis, frog, MALDI-ToF.

Introduction Brucella sp. from skin lesions on a frog housed in a tropical

Brucellosis is a zoonotic disease with a significant global animal collection in the UK.
impact. Of the 11 recognized species, Brucella melitensis,
Brucella abortus and Brucella suis, causative agents of small
ruminant, bovine and swine brucellosis, respectively, are Case report
considered the most significant animal and human path- One of a pair of White’s tree frogs (Litoria caerulea) that
ogens. In recent years, the host range of the genus has had been used as handling exhibits in a tropical animal
increased, notably with new species identified from rodents, collection presented with lesions, appearing first as swellings
marine mammals and baboons (Whatmore et al., 2014). on the frog’s lower back but progressing rather quickly from
Here, we add to this picture describing a case of isolation of small swellings to quite obvious raised, fluid-filled lesions
(Fig. 1). The lesions were incised, curetted and drained, and
Abbreviations: APHA, Animal and Plant Health Agency; MALDI-TOF a swab sent to a commercial laboratory for identification.
MS, matrix-assisted laser desorption/ionization time-of-flight mass The lesions appeared to subside after treatment with
spectrometry. enrofloxacin, but the main lesion reappeared shortly after
Downloaded from www.microbiologyresearch.org by
G 2015 The Authors. Published by SGM
IP: 93.91.26.97
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/). 1
On: Wed, 28 Oct 2015 14:56:35
A. M. Whatmore and others
Fig. 1. Appearance of lesions on L. caerulea prior to drainage and
isolation of UK8/14.
treatment and required a second drainage before resolution.
The second animal remained symptomless throughout.
Bacteriological analysis
The swab was initially received at IDEXX Laboratories for
characterization and inoculated onto blood agar with 5 %
sheep blood (Oxoid). After overnight aerobic incubation
at 36 uC, a pure growth of a haemolytic organism was
observed. Following on-target extraction using formic acid,
the isolate was examined using a Bruker Microflex LT
system (Bruker Daltonics) using the standard and security-
related (SR) databases. Using the standard database, no
reliable identification was returned (score ,1.7), but using
the SR database the MALDI Biotyper real-time classifica-
tion (RTC) software returned a secure identification of B.
melitensis with a score of 2.316, with scores >2.3 being
considered secure to the species level. The identification
was repeated three times to verify the original findings,
and each time the score was .2.3. The isolate was not
morphologically consistent with B. melitensis and was
therefore referred to the national and international Brucella
reference laboratory at the Animal and Plant Health
Agency (APHA) for further characterization.
On receipt at APHA, the isolate was labelled UK8/14 and
subcultured onto serum dextrose agar and Farrell’s medium.
The isolate grew well at 20, 30 and 37 uC with only mar-
ginally faster growth at the two higher temperatures,
forming opaque colonies not initially conforming to the
characteristic appearance of Brucella spp. On subculture, the
colonies were concave, smooth with entire edges and did
show the characteristic iridescence seen with classical Brucella
spp. on Henry illumination. The isolate was examined by a
slide agglutination test using unabsorbed Brucella antiserum
and showed strong agglutination. However, this test is con-
sidered to lack specificity, and the gold standard for
identification is a biotyping approach conventionally used
to identify Brucella isolates to the species and biovar levels
(Whatmore, 2009). By these approaches, the organism was
Downloaded from www.microbiologyresearch.org by
2 JMM Case Reports
IP: 93.91.26.97
On: Wed, 28 Oct 2015 14:56:35
excluded as a member of any of the classically described
Brucella spp., failing to agglutinate with A, M or R antisera
and failing to lyse with any of the standard Brucella typing
phage (Tb, Wb, Bk2, Fi, R/C or Iz) at a routine test dilution.
In other classical biotyping reactions, the isolate grew in the
presence of thionin at 1/50 000 (w/v) but with an atypical
uptake of dye, grew in the presence of fuchsin at 1/50 000
(w/v), produced H2S, hydrolysed urea and did not require
CO2 for growth.
Molecular analysis
Given the matrix-assisted laser desorption/ionization time-
of-flight mass spectrometry (MALDI-TOF MS) result and
two recent reports of the isolation of Brucella from frogs in
a quarantine centre and from a pet shop in Germany
(Eisenberg et al., 2012; Fischer et al., 2012), more extensive
molecular analysis was undertaken to fully characterize the
strain.
To determine whether the isolate belonged within the
genus Brucella, a real-time PCR assay based on a genus-
specific target present in the bcsp31 gene (Probert et al.,
2004) and the multiple-copy Brucella-specific insertion
sequence IS711 (Matero et al., 2011) were performed. Four
replicates were tested alongside duplicate Brucella positive
and no-template controls. The cycle threshold (Ct) values
of the replicates were all around 23.5 cycles for bcsp31 and
18.5 for IS711, in agreement with the identification of this
isolate as a member of the genus Brucella. No-template
controls were negative up to 40 cycles. The isolate was further
tested using Bruce-ladder, a multiplex PCR approach that can
differentiate all known species of Brucella (López-Goñi et al.,
2011). The isolate gave a profile of five bands distinct from
those reported from extant Brucella spp. (Fig. 2a).
Analysis of the 16S rRNA gene sequence of UK8/14 showed
over 99 % nucleotide identity to sequences of all type
strains of Brucella with the best match to Brucella inopinata.
Whilst most Brucella spp. have been reported to share
identical 16S rRNA gene sequences (Gee et al., 2004), B.
inopinata is the most variant to date, showing five nucle-
otide changes from other species type strains (Scholz et al.,
2010). UK8/14 shared these changes but additionally had
two 7 bp insertions not shared with other described Brucella
spp.
In order to place the isolate within the existing genus
of Brucella, a multilocus sequencing approach examin-
ing sequences at nine unlinked genetic loci was applied
(Whatmore et al., 2007). All nine loci were amplified
successfully by PCR, indicating a close relationship of the
isolate to Brucella. Phylogenetic placement based on con-
catenated sequence data, excluding a single gene that does
not amplify from B. inopinata, was performed, comparing
the isolate with isolates representing the known genetic
diversity of members of the genus Brucella (Fig. 2b). These
included both ‘core’ Brucella spp., comprising all the
classically described major pathogenic species, as well as
 Isolation of Brucella from Litoria caerulea

(a)

(b) B. abortus ST1

B. abortus ST5
B. abortus ST4
B. abortus ST3
B. abortus ST2
B. abortus ST6
0.02
B. melitensis ST7
B. melitensis ST10
B. melitensis ST12
B. melitensis ST11
B. melitensis ST8
B. melitensis ST9
B. neotomae ST22
B. canis ST21
B. canis ST20
B. suis ST18
B .suis ST17
B. suis ST14
B. suis ST16
B. suis ST15
B. suis ST19
B. ovis ST13

B. ceti ST23
B. pinnipedialis ST25
B. pinnipedialis ST27
B. ceti ST26
B. pinnipedialis ST24
Brucella sp. 10RB9215
Brucella sp. 83/13
Brucella sp. BO2
B. inopinata BO1
Brucella sp. 09RB8471
UK8/14
O. anthropi LMG3331T
O. intermedium LMG3301T

Fig. 2. Molecular characterization of UK8/14. (a) Comparison of the Bruce-ladder profile with members of existing Brucella
spp. and vaccine strains. Lanes: 1, 1 kb ladder; 2, B. ceti 1/94; 3, B. melitensis 63/9; 4, B. melitensis Rev1; 5, B. ovis 63/290;
6, B. abortus 86/8/59; 7, B. abortus RB51; 8, B. abortus S19; 9, B. suis 1330; 10, Brucella canis RM6/66; 11, Brucella

Downloaded from www.microbiologyresearch.org by

http://jmmcr.sgmjournals.org 3
IP: 93.91.26.97
On: Wed, 28 Oct 2015 14:56:35
A. M. Whatmore and others

neotomae 5K33; 12, Brucella microti CCM4915; 13, B. inopinata BO1; 14, PCR negative control; 15, White’s tree frog isolate
UK8/14; 16, 1 kb ladder. (b) Phylogenetic placement relative to other Brucella spp. as determined by multilocus sequence
analysis (Whatmore et al., 2007) showing division into the ‘core’ Brucella group, including all classical pathogenic species, and
the ‘atypical’ Brucella group. The relationship with the nearest phylogenetic neighbour of Brucella, Ochrobactrum, is also
shown. ST, sequence type. These sequence types include reference strains for all classical known biovars of all classical
species. The tree was constructed in MEGA5 using the Jukes–Cantor distance and the neighbour joining approach. Bar,
nucleotide substitutions per site.

the recently described ‘atypical’ Brucella spp. that include
the recently described B. inopinata (Wattam et al., 2012).
These isolates are genetically divergent from the ‘core’ or
classical Brucella spp. but have been described as Brucella
spp. based on a much closer relationship to the ‘core’
Brucella group than to the next closest phylogenetic
neighbours (Ochrobactrum spp.).
Isolate UK8/14 clearly fell within the ‘atypical’ Brucella
clade, which includes the B. inopinata type strain (De et al.,
2008; Scholz et al., 2010) as well as a number of isolates yet
to be formally described taxonomically. These include strain
83/13, a representative of a group of isolates from Australian
rodents (Tiller et al., 2010a), strain BO2, described as a B.
inopinata-like isolate from a human infection (Tiller et al.,
2010b), and two additional isolates, 10RB9251 and 09RB8471.
Interestingly, the latter isolates represent the first Brucella-like
isolates from amphibians described recently in wild-caught
African bullfrogs from Tanzania (Eisenberg et al., 2012).
Isolate UK8/14 is thus clearly a member of the ‘atypical’
Brucella group most closely related to a strain isolated
previously from frogs (09RB8471).
Conclusions
Whilst this isolate would be excluded as Brucella by con-
ventional phenotyping approaches, molecular analysis con-
firmed that the isolate belongs to the rapidly expanding
group of ‘atypical’ Brucella spp. Although the species-level
MALDI-TOF MS identification turned out to be inaccurate,
probably reflecting both the known homogeneity of the
group and the poor representation of extant Brucella spp. in
the commercial databases, MALDI-TOF MS was useful in
identifying as a potential Brucella an isolate that would
immediately be excluded by conventional phenotyping. In
order to avoid ‘missing’ such isolates, diagnostic or reference
laboratories should consider the use of 16S rRNA gene
sequencing and/or the presence of IS711, considered specific
for Brucella spp. (Whatmore & Gopaul, 2012), in their
routine identification procedures. In addition, the reaction
with unabsorbed Brucella antiserum may be a useful screen,
despite the acknowledged lack of specificity, provided that
confirmatory testing is then pursued. With regard to
MALDI-TOF MS, it should be noted that, according to
our findings and as reported elsewhere (Cunningham &
Patel, 2013), only users with the SR database installed and
enabled would be able to identify this isolate as a Brucella sp.
Whilst MALDI-TOF MS might provide a useful initial
screen, particularly as the strain would be excluded as a
Downloaded from www.microbiologyresearch.org by
4 JMM Case Reports
IP: 93.91.26.97
On: Wed, 28 Oct 2015 14:56:35
Brucella sp. by conventional biotyping, the identification as
the highly pathogenic and zoonotic B. melitensis would have
significant implications. Clearly, it is therefore valuable to
use molecular approaches, such as multilocus sequence
analysis, to help accurately place these isolates in context
with extant Brucella strains.
One of the classical characteristics of Brucella is the ability
to survive and replicate intracellularly. Whilst testing of
this characteristic was outside the scope of this case report,
it would be of interest to examine this property. Whilst
some of the emerging atypical Brucella spp. appear to
possess this capability (Jiménez de Bagüés et al., 2014),
others, such as BO2, which, like UK8/14, fails to react with
either monospecific A or M antisera, and has a novel O-
polysaccharide biosynthetic pathway (Wattam et al., 2012),
appear unable to replicate intracellularly (Wattam et al.,
2014). LPS is considered the major virulence factor of
classic Brucella spp. (Lapaque et al., 2005), and thus any
modifications in its structure may impact host–pathogen
interactions. Whole-genome sequencing currently in pro-
gress should provide a route to understanding the
structure of UK8/14 LPS.
This is the first report outside Germany of the isolation
of such strains from amphibians and both confirms the
rapidly expanding host range of the genus and suggests
that such isolates may be widely distributed. The lesions
observed in this case appeared superficially similar to those
reported in a previous case in another frog species (Leptopelis
vermiculatus). Whilst there is no evidence to date to suggest
that Brucella isolates associated with amphibians are
pathogenic for humans, many members of the genus
represent significant zoonotic pathogens (Godfroid et al.,
2011). It is worth noting that the fact that strain UK8/14 was
untypeable using the monospecific sera (A or M dominant)
commonly used to classify Brucella spp. is suggestive of
modifications in the LPS that may compromise the ability to
detect any infection serologically, as tests are based largely on
detection of antibodies against the O-polysaccharide of the
LPS (Zygmunt et al., 2012). Thus, it is possible that any
human infections with such organisms would not be
detected by routine serodiagnostic approaches. The associa-
tion of other ‘atypical’ Brucella isolates (B. inopinata and B.
inopinata-like organisms) with serious human infections (De
et al., 2008; Tiller et al., 2010b) suggests that appropriate
measures should be taken to avoid unnecessary contact with
potentially infected amphibians until the zoonotic potential
of this emerging group is better understood.

Acknowledgements
The Brucella research and surveillance activities at APHA are
supported by the UK Department of Environment, Food and Rural
Affairs (Defra).
References
Cunningham, S. A. & Patel, R. (2013). Importance of using Bruker’s
security-relevant library for biotyper identification of Burkholderia
pseudomallei, Brucella species and Francisella tularensis. J Clin
Microbiol 51, 1639–1640.
De, B. K., Stauffer, L., Koylass, M. S., Sharp, S. E., Gee, J. E.,
Helsel, L. O., Steigerwalt, A. G., Vega, R., Clark, T. A. & other
authors. (2008). Novel Brucella strain (BO1) associated with a
prosthetic breast implant infection. J Clin Microbiol 46, 43–49.
Eisenberg, T., Hamann, H. P., Kaim, U., Schlez, K., Seeger, H.,
Schauerte, N., Melzer, F., Tomaso, H., Scholz, H. C. & other authors.
(2012). Isolation of potentially novel Brucella spp. from frogs. Appl
Environ Microbiol 78, 3753–3755.
Fischer, D., Lorenz, N., Heuser, W., Kämpfer, P., Scholz, H. C. &
Lierz, M. (2012). Abscesses associated with a Brucella inopinata-like
bacterium in a big-eyed tree frog (Leptopelis vermiculatus). J Zoo
Wildl Med 43, 625–628.
Gee, J. E., De, B. K., Levett, P. N., Whitney, A. M., Novak, R. T. &
Popovic, T. (2004). Use of 16S rRNA gene sequencing for rapid
confirmatory identification of Brucella isolates. J Clin Microbiol 42,
3649–3654.
Godfroid, J., Scholz, H. C., Barbier, T., Nicolas, C., Wattiau, P.,
Fretin, D., Whatmore, A. M., Cloeckaert, A., Blasco, J. M. & other
authors. (2011). Brucellosis at the animal/ecosystem/human interface
at the beginning of the 21st century. Prev Vet Med 102, 118–131.
Jiménez de Bagüés, M. P., Iturralde, M., Arias, M. A., Pardo, J.,
Clooeckaert, A. & Zygmunt, M. S. (2014). The new strains Brucella
inopinata BO1 and Brucella species 83-210 behave biologically like
classic infectious Brucella species and cause death in murine models
of infection. J Infect Dis 210, 467–472.
Lapaque, N., Moriyon, I., Moreno, E. & Gorvel, J. P. (2005). Brucella
lipopolysaccharide acts as a virulence factor. Curr Opin Microbiol 8,
60–66.
López-Goñi, I., Garcı́a-Yoldi, D., Marı́n, C. M., de Miguel, M. J.,
Barquero-Calvo, E., Guzmán-Verri, C., Albert, D. & Garin-Bastuji, B.
(2011). New Bruce-ladder multiplex PCR assay for the biovar typing
of Brucella suis and the discrimination of Brucella suis and Brucella
canis. Vet Microbiol 154, 152–155.
Matero, P., Hemmilä, H., Tomaso, H., Piiparinen, H., Rantakokko-
Jalava, K., Nuotio, L. & Nikkari, S. (2011). Rapid field detection assays
Downloaded from www.microbiologyresearch.org by
http://jmmcr.sgmjournals.org
IP: 93.91.26.97
On: Wed, 28 Oct 2015 14:56:35
View publication stats
Isolation of Brucella from Litoria caerulea
for Bacillus anthracis, Brucella spp., Francisella tularensis and Yersinia
pestis. Clin Microbiol Infect 17, 34–43.
Probert, W. S., Schrader, K. N., Khuong, N. Y., Bystrom, S. L. &
Graves, M. H. (2004). Real-time multiplex PCR assay for detection of
Brucella spp., B. abortus, and B. melitensis. J Clin Microbiol 42,
1290–1293.
Scholz, H. C., Nöckler, K., Göllner, C., Bahn, P., Vergnaud, G.,
Tomaso, H., Al Dahouk, S., Kämpfer, P., Cloeckaert, A. & other
authors. (2010). Brucella inopinata sp. nov., isolated from a breast
implant infection. Int J Syst Evol Microbiol 60, 801–808.
Tiller, R. V., Gee, J. E., Frace, M. A., Taylor, T. K., Setubal, J. C.,
Hoffmaster, A. R. & De, B. K. (2010a). Characterization of novel
Brucella strains originating from wild native rodent species in North
Queensland, Australia. Appl Environ Microbiol 76, 5837–5845.
Tiller, R. V., Gee, J. E., Lonsway, D. R., Gribble, S., Bell, S. C.,
Jennison, A. V., Bates, J., Coulter, C., Hoffmaster, A. R. & De, B. K.
(2010b). Identification of an unusual Brucella strain (BO2) from a
lung biopsy in a 52 year-old patient with chronic destructive
pneumonia. BMC Microbiol 10, 23.
Wattam, A. R., Inzana, T. J., Williams, K. P., Mane, S. P., Shukla, M.,
Almeida, N. F., Dickerman, A. W., Mason, S., Moriyón, I. & other
authors. (2012). Comparative genomics of early-diverging Brucella
strains reveals a novel lipopolysaccharide biosynthesis pathway. MBio
3, e00246–12.
Wattam, A. R., Foster, J. T., Mane, S. P., Beckstrom-Sternberg, S. M.,
Beckstrom-Sternberg, J. M., Dickerman, A. W., Keim, P., Pearson, T.,
Shukla, M. & other authors (2014). Comparative phylogenomics and
evolution of the Brucellae: a path to virulence. J Bacteriol 196,
920–930.
Whatmore, A. M. (2009). Current understanding of the genetic
diversity of Brucella, an expanding genus of zoonotic pathogens.
Infect Genet Evol 9, 1168–1184.
Whatmore, A. M. & Gopaul, K. K. (2012). Recent advances in
molecular approaches to Brucella diagnostics and epidemiology. In
Brucella: Molecular Microbiology and Genomics, pp. 57–88. Edited
by I. Lopez-Goni & D. O’Callaghan, D. (eds), Norwich, UK: Caister
Academic Press.
Whatmore, A. M., Perrett, L. L. & MacMillan, A. P. (2007).
Characterisation of the genetic diversity of Brucella by multilocus
sequencing. BMC Microbiol 7, 34.
Whatmore, A. M., Davison, N., Cloeckaert, A., Al Dahouk, S.,
Zygmunt, M. S., Brew, S. D., Perrett, L. L., Koylass, M. S.,
Vergnaud, G. & other authors. (2014). Brucella papionis sp. nov.
isolated from baboons (Papio spp.). Int J Syst Evol Microbiol 64,
4120–4128.
Zygmunt, M. S., Jacques, I., Bernardet, N. & Cloeckaert, A. (2012).
Lipopolysaccharide heterogeneity in the atypical group of novel
emerging Brucella species. Clin Vaccine Immunol 19, 1370–1373.
5