Isolation of the highly pathogenic and zoonotic agent burkholderia pseudomallei from a pet green iguana in prague, czech republic

Elschner et al. BMC Veterinary Research 2014, 10:283 Isolation of the highly pathogenic and zoonoticagent Burkholderia pseudomallei from a pet greenIguana in Prague, Czech Republic Mandy C Elschner1*, Jan Hnizdo2, Ivonne Stamm3, Hosny El-Adawy1, Katja Mertens1 and Falk Melzer1 Background: Melioidosis caused by Burkholderia (B.) pseudomallei is an endemic zoonotic disease mainly reportedfrom northern Australia and Southeast Asia. In Europe, cases of human melioidosis have been reported only frompatients travelling to endemic regions. Besides humans, B. pseudomallei has a very broad host range in domesticand wild animals. There are some reports about importation of B. pseudomallei-infected animals from endemicareas into Europe. The present report describes the first case of B. pseudomallei infection of a pet iguana in Europe.
Case presentation: In a 5-year-old pet Iguana iguana living in a private household in Prague, Czech Republic,B. pseudomallei was isolated from pus of an abscess. The isolate VB976100 was identified by Vitek®2, MALDI-TOFmass spectrometry and polymerase chain reaction as B. pseudomallei. The molecular typing resulted in multi-locussequence type 436 hitherto, which has been found only once worldwide in a B. pseudomallei strain isolated in theUSA and originating from Guatemala. The identification as internal transcribed spacer type G indicates a closerelatedness to strains mainly isolated in the Western Hemisphere. These findings support the hypothesis that theiguana became infected in this region or in a breeding facility through contact to other infected animals.
Conclusions: The present case highlights the risk of importation of the highly pathogenic and zoonoticB. pseudomallei into non-endemic regions through animal trade. Therefore, veterinarians treating animals from theseareas and physicians examining patients owning such animals should include melioidosis in differential diagnosiswhenever specific symptoms appear. Furthermore, veterinary authorities responsible for supervision of traders andpet shops should be aware of this risk of zoonotic transmission.
Keywords: Melioidosis, Burkholderia pseudomallei, Zoonoses, Iguana melioidosis have only reported from patients who had Burkholderia (B.) pseudomallei is the causative agent of been travelling to endemic regions The gram- melioidosis, an endemic disease mainly reported from negative bacterium belongs to biohazard risk group 3 northern Australia and Southeast Asia. In this area the agents, is listed as biowarfare agent, and has to be proc- disease has emerged as an important cause of morbidity essed under biosafety level 3 conditions. The disease is and mortality during the last 25 years Endemic and mainly acquired environmentally by percutaneous infec- sporadic cases of melioidosis are also reported from tion, ingestion, or inhalation. The incubation period of countries of South America, North America, Oceania, as melioidosis can vary between one day and 62 years, de- well as Aruba, Guadeloupe, Guam, Haiti, Martinique, pending on the route of infection and infection dose [.
and Puerto Rico. Increasing numbers of cases are reported Clinical studies have shown that pneumonia is the pre- from Africa []. However, in Europe cases of human dominant clinical manifestation, followed by skin and softtissue infections, as well as acute suppurative parotitis, es- * Correspondence: pecially in pediatric cases, and prostatitis in males . One 1Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, predominant risk factor is diabetes mellitus, as shown by Institute of Bacterial Infections and Zoonoses, Naumburger Strasse 96a, studies in Australia and Thailand, where up to 60% of the 07743 Jena, GermanyFull list of author information is available at the end of the article melioidosis patients are diabetic, mainly type 2 .
2014 Elschner et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly credited. The Creative Commons Public DomainDedication waiver applies to the data made available in this article,unless otherwise stated.

Elschner et al. BMC Veterinary Research 2014, 10:283 Besides humans, B. pseudomallei has a very broad host Zophobas morio larvae purchased from different breeders.
range. In domestic animals it is most commonly re- In 2011, the animal developed an abscess underneath the ported in cattle, goats and swine However, sporadic left eye, which was transected and treated antibiotically cases or small outbreaks have been reported in monkeys, without microbiological investigation. In 2013, the animal gibbons, orangutans, kangaroos, wallabies, deer, buffaloes, was admitted to the Animal Clinic, Bila Hora, Prague, cows, camels, llamas, zebras, koalas, dogs, cats, horses, Czech Republic, where again an abscess was diagnosed, mules, parrots, rats, hamsters, rabbits, guinea pigs, ground this time at the root of the tongue. After surgical interven- squirrels, seals, dolphins, crocodiles ]. Very recently, B.
tion and antibiotic treatment by marbofloxacine the pseudomallei infections in two pet iguanas in California animal recovered. One year later, in 2014 the owners pre- were reported ]. In animals, acute and chronical form of sented the animal again to the clinic because of a week- melioidosis is seen. Common symptoms in animals in- long anorexia. The clinical investigation resulted again in clude anorexia, pyrexia, coughing, skin dehydration and an encapsulated abscess located at the root of the tongue.
This time the abscess was sampled for microbiologicalanalysis. As soon as the first suspicion of a B. pseudomallei Case presentation infection was announced, the veterinary authority was in- The female Iguana iguana was purchased by private formed, and it mandated the immediate euthanization and owners as a young animal from a pet shop in Prague in incineration of the animal. One veterinary assistant was 2009. It was kept alone in a terrarium, but was also allowed bitten by the iguana when he tried to fix the maxilla for to move freely outside the cage in the flat (Figure . The sampling of the abscess by the veterinarian. The skin was owners had a very close and tender contact to the animal, injured although he wore gloves. Finally, the wound was such as hand feeding and kissing. Usually the iguana was cured and the assistant was prophylactically treated with fed with special pellet formulation for iguanas, fresh amoxicillin-clavulanate for 10 days in a clinic specialized green salad, vegetables and fruits, and once a month by for infectious diseases. Also the owners, not showing any Figure 1 The infected animal: 5-year-old female green iguana (Iguana iguana).

Elschner et al. BMC Veterinary Research 2014, 10:283 clinical signs, were referred to a physician for medical Germany) with very high score values of >2.600 corre- examination and received the same treatment. Serological sponding to highly probable species identification.
investigations were not induced in the contact persons, For confirmation of B. pseudomallei and further char- because the physicians could not find a laboratory provid- acterization, the culture was sent to the Federal Research ing this analysis. Unfortunately, no information about Institute for Animal Health, Jena, Germany. The isolate follow up examinations of the contact persons is available.
VB976100 was cultured under biosafety level 3 condi-tions and the typical shape of colonies was seen on Isolation, identification, and molecular characterisation of Ashdown-Agar after 72 h (Figure Molecular identification was performed by real-time For microbiological diagnosis, the pus from the abscess PCR detecting B. mallei/pseudomallei-specific sequences was submitted on a swab with Amies transport medium of the fliC gene and B. pseudomallei-specific se- (D&D Laborservice, Henningsdorf, Germany) to the Vet quences of the orf2 gene, which belongs to the type III Med Labor GmbH, Ludwigsburg, Germany. The sample secretion system .
was cultured on MacConkey agar (BioMerieux, Nürtin- Multi-locus sequence typing (MLST), the predominant gen, Germany), tryptic soy agar with 5% sheep blood, method for molecular subtyping of B. pseudomallei was chocolate agar (both BD, Heidelberg, Germany) and in- performed by comparison of 7 housekeeping genes cubated at 36°C under atmosphere with 5% CO2 for The isolate was typed as sequence type (ST) 436 by the 24 hours, and afterwards for 24 hours in ambient atmos- B. pseudomallei database of MLST.Net and data phere. Additionally, brain heart infusion enrichment have been deposited there. This ST has been only broth was inoculated and sub-cultured for 24 hours on isolated in California from a patient originating from the same agar media as above. Heavy growth of metallic Guatemala in 2012 ]. However, for an isolate of un- gleaming navel forming colonies was visible in pure known origin, MLST alone is not adequate for determin- culture after 48-h incubation on all culture media. The ation of the geographic origin Therefore, we isolate VB976100 could also be grown at 42°C, but not performed the 16S-32S internal transcribed spacer (ITS) at 4°C. It showed a positive Oxidase reaction (VWR, typing For identification of the ITS type, the Darmstadt, Germany) and was tested negative for cata- obtained sequence data were aligned to known ITS lase (Sigma-Aldrich Chemie GmbH, Munich, Germany).
types A, B, C, D, E, and G, available data at GenBank Short gram-negative rods with bipolar "safety pin" ap- (FJ981703-F981726) using the Software Geneious 6.1.8.
pearance were visible after Gram staining (Figure (Biomatters Development Team). Our B. pseudomallei Identification with Vitek®2 (BioMerieux) resulted in B.
isolate VB976100 was typed as ITS type G (613 base pseudomallei with 95% probability, corresponding to a pairs), and the ITS sequence was annotated and submitted very good validity of identification. The same identification to GenBank under the accession number KP069478.
result was obtained with MALDI-TOF mass spectrometry(Biotyper Microflex LT, Bruker Daltonics GmbH, Bremen, ConclusionsImport of B. pseudomallei infected animals from en-demic areas has been previously reported in sheep, goatsand pigs from Aruba In the 1970s a B. pseudomal-lei outbreak was reported in the zoo of Paris, and the in-fection was spread to other zoos and equestrian clubs.
In the course of this outbreak, termed "l'affaire du jardindes plantes", two individuals died. Imported horses fromIran or an imported panda from China were assumed assources of infection B. pseudomallei infections in green iguanas were already detected in California, USA, in 2007 and 2012.
The bacteria were isolated from the abscess, as well asfrom the animal housing In both cases the isolateswere typed as ST 518 and ITS type G. The authorssuggested that the animals had been infected in CentralAmerica, where most imported iguanas in the USA origin-ate from. Furthermore, these cases already highlighted the Figure 2 Gram-stain of B pseudomallei isolate VB976100, phase possible risk of human infections because the infection in contrast microscopy with a 100 x oil immersion objective the iguanas was not curable in spite of refractory antibiotic (Microscope Leica DM4000B).
treatment. In one of these cases after the incubation

Elschner et al. BMC Veterinary Research 2014, 10:283 Figure 3 Colonies of B. pseudomallei isolate VB976100 on Ashdown agar showing the typical pink crinkled colonies after 72 h incubation.
period of around 1.5 years an abscess was diagnosed. In physician could misdiagnose or overlook the suspicion the clinical course of the present reported B. pseudomallei of melioidosis. The utility of a post exposure prophylaxis infection, the animal developed abscesses at the age of 2, 4 in contact persons should be discussed with the physi- and 5 years. However, there was no known history of any cians. Recommendations for prophylaxis and treatment kind of trauma, which potentially could have triggered the are available from Bossi et al. development of abscesses []. Unfortunately, only in Personal safety can be improved by wearing eye pro- 2014 bacteriological examination was initiated. It is specu- tection and suitable gloves during sampling and treat- lative but possible that after an incubation period of 2 years ment of possibly affected animals. For the serological the first abscess was caused by a B. pseudomallei infection.
investigations in non-endemic areas IgG ELISA could be These finding strongly supports the presumption, that in useful to identify chronically infected persons Since these animals a long incubation period is seen, bearing a no standardized commercial tests are available only spe- risk of long not recognized phase of shedding and here- cialized laboratories can perform such investigations.
with infection risk for contact persons.
Although we are unable to prove the source of infec- The iguana was purchased as a young animal in 2009, tion in the present case, veterinarians involved in treat- and unfortunately, it was not possible to determine the ment of animals and competent veterinary authorities origin of this animal. However, the ST 436 was hitherto responsible for supervision of traders should be aware of described for one B. pseudomallei strain, isolated in this potential risk.
USA, originating from Guatemala On the otherhand, the ITS type G of B. pseudomallei is a less com- AbbreviationsB: Burkholderia; MLST: Multi-locus sequence typing; ITS: Internal transcribed mon type in endemic regions, such as Australia and spacer typing; ST: Sequence type; PCR: Polymerase chain reaction.
Southeast Asia. This type seems to be associated withisolates found only in sporadic melioidosis regions like Competing interests Africa and South America These findings sup- The authors declare that they have no competing interests.
port the hypothesis that the iguana became infected in Authors' contributions this region or in a breeding facility through contact to MCE designed the study, coordinated the investigations, substantially other infected animals originating from this area.
contributed to analysis and evaluation of ITS sequence data, and wrote themanuscript. JH performed the clinical examination of the iguana, collected Our report emphasizes the risk of importation of B.
the samples and substantially contributed to the manuscript by drafting the pseudomallei into non-endemic regions through animal clinical and epidemiological section. IS performed isolation and bacteriological trade. It is possible that for patients presenting charac- identification of the B.pseudomallei isolate and substantially contributed to themanuscript by drafting the bacteriological methodological section. HEA, KM teristic symptoms of melioidosis but with absolutely no performed the MLST and ITS, and contributed substantially to evaluation and history of travelling to endemic regions, the attending analysis of the sequence data. FM, KM, HEA have been substantially involved in Elschner et al. BMC Veterinary Research 2014, 10:283 the design of the study and discussion of results. All authors revised the Sutmoller P, Kraneveld FC, Van Der Schaaf A: Melioidosis (Pseudomalleus) manuscript critically for technical and scientific content and approved the final in sheep, goats, and pigs on Aruba (Netherland Antilles). J Am Vet Med Assoc Dance DA: Melioidosis: the tip of the iceberg? Clin Microbiol Rev 1991, We are grateful to K. Nitzsche, P. Marten and K. Fischer for excellent technical White NJ: Melioidosis. Lancet 2003, 361(9370):1715–1722.
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