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

Case Report Isolation of Brucella from a White’s tree (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 1FAO/WHO Collaborating Centre for Brucellosis, OIE Brucellosis Reference Centre, and Adrian Whatmore Plant Health Agency (APHA), Department of Bacteriology, Woodham Lane, Addlestone, [email protected] Surrey KT15 3NB, UK 2IDEXX Laboratories, Grange House, Sandbeck Way, Wetherby LS22 7DN, UK 3Lawrie Veterinary Group, 25 Griffiths Street, Falkirk FK1 5QY, UK

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 and with an expanding known host range in recent years. Case presentation: One of a pair of White’s tree (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 until the zoonotic potential of this group is better understood. Received 3 November 2014 Accepted 10 January 2015 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

G 2015 The Authors. Published by SGM This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/). 1 A. M. Whatmore and others

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- Fig. 1. Appearance of lesions on L. caerulea prior to drainage and of-flight mass spectrometry (MALDI-TOF MS) result and isolation of UK8/14. 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 treatment and required a second drainage before resolution. molecular analysis was undertaken to fully characterize the The second animal remained symptomless throughout. strain. To determine whether the isolate belonged within the genus Brucella, a real-time PCR assay based on a genus- Bacteriological analysis specific target present in the bcsp31 gene (Probert et al., The swab was initially received at IDEXX Laboratories for 2004) and the multiple-copy Brucella-specific insertion characterization and inoculated onto blood agar with 5 % sequence IS711 (Matero et al., 2011) were performed. Four sheep blood (Oxoid). After overnight aerobic incubation replicates were tested alongside duplicate Brucella positive at 36 uC, a pure growth of a haemolytic organism was and no-template controls. The cycle threshold (Ct) values observed. Following on-target extraction using formic acid, of the replicates were all around 23.5 cycles for bcsp31 and the isolate was examined using a Bruker Microflex LT 18.5 for IS711, in agreement with the identification of this system (Bruker Daltonics) using the standard and security- isolate as a member of the genus Brucella.No-template related (SR) databases. Using the standard database, no controls were negative up to 40 cycles. The isolate was further reliable identification was returned (score ,1.7), but using tested using Bruce-ladder, a multiplex PCR approach that can the SR database the MALDI Biotyper real-time classifica- differentiate all known species of Brucella (Lo´pez-Gon˜i et al., tion (RTC) software returned a secure identification of B. 2011). The isolate gave a profile of five bands distinct from melitensis with a score of 2.316, with scores >2.3 being those reported from extant Brucella spp. (Fig. 2a). considered secure to the species level. The identification Analysis of the 16S rRNA gene sequence of UK8/14 showed was repeated three times to verify the original findings, over 99 % nucleotide identity to sequences of all type and each time the score was .2.3. The isolate was not strains of Brucella with the best match to Brucella inopinata. morphologically consistent with B. melitensis and was Whilst most Brucella spp. have been reported to share therefore referred to the national and international Brucella identical 16S rRNA gene sequences (Gee et al., 2004), B. reference laboratory at the Animal and Plant Health inopinata is the most variant to date, showing five nucle- Agency (APHA) for further characterization. otide changes from other species type strains (Scholz et al., On receipt at APHA, the isolate was labelled UK8/14 and 2010). UK8/14 shared these changes but additionally had subcultured onto serum dextrose agar and Farrell’s medium. two 7 bp insertions not shared with other described Brucella The isolate grew well at 20, 30 and 37 uC with only mar- spp. ginally faster growth at the two higher temperatures, In order to place the isolate within the existing genus forming opaque colonies not initially conforming to the of Brucella, a multilocus sequencing approach examin- characteristic appearance of Brucella spp. On subculture, the ing sequences at nine unlinked genetic loci was applied colonies were concave, smooth with entire edges and did (Whatmore et al., 2007). All nine loci were amplified show the characteristic iridescence seen with classical Brucella successfully by PCR, indicating a close relationship of the spp. on Henry illumination. The isolate was examined by a isolate to Brucella. Phylogenetic placement based on con- slide agglutination test using unabsorbed Brucella antiserum catenated sequence data, excluding a single gene that does and showed strong agglutination. However, this test is con- not amplify from B. inopinata, was performed, comparing sidered to lack specificity, and the gold standard for the isolate with isolates representing the known genetic identification is a biotyping approach conventionally used diversity of members of the genus Brucella (Fig. 2b). These to identify Brucella isolates to the species and biovar levels included both ‘core’ Brucella spp., comprising all the (Whatmore, 2009). By these approaches, the organism was classically described major pathogenic species, as well as

2 JMM Case Reports 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 http://jmmcr.sgmjournals.org 3 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 Brucella sp. by conventional biotyping, the identification as the recently described B. inopinata (Wattam et al., 2012). the highly pathogenic and zoonotic B. melitensis would have These isolates are genetically divergent from the ‘core’ or significant implications. Clearly, it is therefore valuable to classical Brucella spp. but have been described as Brucella use molecular approaches, such as multilocus sequence spp. based on a much closer relationship to the ‘core’ analysis, to help accurately place these isolates in context Brucella group than to the next closest phylogenetic with extant Brucella strains. neighbours (Ochrobactrum spp.). One of the classical characteristics of Brucella is the ability Isolate UK8/14 clearly fell within the ‘atypical’ Brucella to survive and replicate intracellularly. Whilst testing of clade, which includes the B. inopinata type strain (De et al., this characteristic was outside the scope of this case report, 2008; Scholz et al., 2010) as well as a number of isolates yet it would be of interest to examine this property. Whilst to be formally described taxonomically. These include strain some of the emerging atypical Brucella spp. appear to 83/13, a representative of a group of isolates from Australian possess this capability (Jime´nez de Bagu¨e´s et al., 2014), rodents (Tiller et al., 2010a), strain BO2, described as a B. others, such as BO2, which, like UK8/14, fails to react with inopinata-like isolate from a human infection (Tiller et al., either monospecific A or M antisera, and has a novel O- 2010b), and two additional isolates, 10RB9251 and 09RB8471. polysaccharide biosynthetic pathway (Wattam et al., 2012), Interestingly, the latter isolates represent the first Brucella-like appear unable to replicate intracellularly (Wattam et al., isolates from amphibians described recently in wild-caught 2014). LPS is considered the major virulence factor of African bullfrogs from Tanzania (Eisenberg et al., 2012). classic Brucella spp. (Lapaque et al., 2005), and thus any Isolate UK8/14 is thus clearly a member of the ‘atypical’ modifications in its structure may impact host–pathogen Brucella group most closely related to a strain isolated interactions. Whole-genome sequencing currently in pro- previously from frogs (09RB8471). gress should provide a route to understanding the structure of UK8/14 LPS. Conclusions This is the first report outside Germany of the isolation Whilst this isolate would be excluded as Brucella by con- of such strains from amphibians and both confirms the ventional phenotyping approaches, molecular analysis con- rapidly expanding host range of the genus and suggests firmed that the isolate belongs to the rapidly expanding that such isolates may be widely distributed. The lesions group of ‘atypical’ Brucella spp. Although the species-level observed in this case appeared superficially similar to those MALDI-TOF MS identification turned out to be inaccurate, reported in a previous case in another frog species ( probably reflecting both the known homogeneity of the vermiculatus). Whilst there is no evidence to date to suggest group and the poor representation of extant Brucella spp. in that Brucella isolates associated with amphibians are the commercial databases, MALDI-TOF MS was useful in pathogenic for humans, many members of the genus identifying as a potential Brucella an isolate that would represent significant zoonotic pathogens (Godfroid et al., immediately be excluded by conventional phenotyping. In 2011). It is worth noting that the fact that strain UK8/14 was order to avoid ‘missing’ such isolates, diagnostic or reference untypeable using the monospecific sera (A or M dominant) laboratories should consider the use of 16S rRNA gene commonly used to classify Brucella spp. is suggestive of sequencing and/or the presence of IS711, considered specific modifications in the LPS that may compromise the ability to for Brucella spp. (Whatmore & Gopaul, 2012), in their detect any infection serologically, as tests are based largely on routine identification procedures. In addition, the reaction detection of antibodies against the O-polysaccharide of the with unabsorbed Brucella antiserum may be a useful screen, LPS (Zygmunt et al., 2012). Thus, it is possible that any despite the acknowledged lack of specificity, provided that human infections with such organisms would not be confirmatory testing is then pursued. With regard to detected by routine serodiagnostic approaches. The associa- MALDI-TOF MS, it should be noted that, according to tion of other ‘atypical’ Brucella isolates (B. inopinata and B. our findings and as reported elsewhere (Cunningham & inopinata-like organisms) with serious human infections (De Patel, 2013), only users with the SR database installed and et al.,2008;Tilleret al., 2010b) suggests that appropriate enabled would be able to identify this isolate as a Brucella sp. measures should be taken to avoid unnecessary contact with Whilst MALDI-TOF MS might provide a useful initial potentially infected amphibians until the zoonotic potential screen, particularly as the strain would be excluded as a of this emerging group is better understood.

4 JMM Case Reports Isolation of Brucella from Litoria caerulea

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