Chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis, contributes to amphibian declines worldwide. Recently, the fungus has shown to be widely distributed in Belgium and the Netherlands, although no clinical cases of the disease have been diagnosed yet. This case report describes the first case of mortality due to chytridiomycosis in Belgium in a wild population of midwife toads (Alytes obstetricans). The presence of clinical chytridiomycosis, combined with the relatively high prevalence of the fungus in Belgium, emphasizes the urgent need for a thorough study on the impact of infection on the native amphibian populations in Belgium

Brutyn, Melanie

Ducatelle, Richard

Haesebrouck, Freddy

Maes, Sofie

Martel, An

Muijsers, Mariska

Pasmans, Frank

Van Rooij, Pascale

English

Ghent University Academic Bibliography

460 Case report Vlaams Diergeneeskundig Tijdschrift, 2010, 79INTRODUCTIONAmphibian declines have been reported worldwidesince the late eighties of the previous century. Severalcauses have been identified, such as habitat destruction,the use of pesticides and climate change (Houlahan etal., 2000). The infectious disease chytridiomycosis,caused by the fungus Batrachochytrium dendrobatidis,has caused a massive loss of amphibian biodiversity, no-tably in Central America and Australia, but also in Eu-rope (Berger et al., 1998; Bosch et al., 2001; Bielby etal., 2009; Walker et al., 2010). The fungus is restrictedto the keratinized body parts: the mouthparts in larvaeand the skin in postmetamorphs. Clinical chytridiomy-cosis is characterized by lethargy, dysecdysis and death.Epidermal hyperplasia and hyperkeratosis are the onlyconsistent histological lesions. Mortality is probablyinduced by the disturbance of the skin function, com-promising for example electrolyte homeo stasis (Voyleset al., 2009). Recently, the presence of the fungus hasbeen detected in Belgium and the Netherlands (Spitzenet al., 2010) and clinical chytridiomycosis is not un-common in captive amphibians in Belgium and the Ne-therlands (Pasmans et al., 2004; Martel et al., 2010).However, no adverse effects of B. dendrobatidis infec-tions on indigenous amphibians have been reported yet.The present paper describes the first case of mortalitydue to chytridiomycosis in native amphibians in Bel-gium.CASEA recently metamorphosed midwife toad (Alytes ob-stetricans) was found moribund under a stone (Figure1) near Marche-en-Famenne (GPS coordinates:50°08’16”N; 5°27’23”E). Necropsy did not reveal anymacroscopic lesions, except for loose skin on the ven-trum and dorsum. Samples of skin from the dorsum,pelvic patch, the middle digit of the left hindleg, liverand kidney were fixed in formalin 10%, wax embed-ded and 4 µm sections were stained using hematoxy-lin and eosin, periodic acid-Shiff (PAS) or Ziehl-Neel-sen staining. To determine the presence of iridoviruses,the PCR described by Mao et al. (1997) was carried outon a liver sample. The presence and number of B. den-drobatidis organisms in a swab from the skin were de-termined using the real time PCR described by Boyleet al. (2004).Skin lesions were similar at the ventral abdominalskin (pelvic patch), caudodorsal skin and middle digitof the left hindleg. There were diffuse epidermal hy-perplasia and hyperkeratosis, with the presence of nu-Chytridiomycosis related mortality in a midwife toad (Alytes obstetricans)in BelgiumSterfte door chytridiomycose bij een vroedmeesterpad (Alytes obstetricans)in BelgiëF. Pasmans, M. Muijsers, S. Maes, P. Van Rooij, M. Brutyn, R. Ducatelle, F. Haesebrouck, A.MartelDepartment of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, GhentUniversity, Salisburylaan 133, B9820 Merelbekefrank.pasmans@ugent.beABSTRACTChytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis, contributes to amphibian declinesworldwide. Recently, the fungus has shown to be widely distributed in Belgium and the Netherlands, althoughno clinical cases of the disease have been diagnosed yet. This case report describes the first case of mortalitydue to chytridiomycosis in Belgium in a wild population of midwife toads (Alytes obstetricans). The presenceof clinical chytridiomycosis, combined with the relatively high prevalence of the fungus in Belgium, emphasizesthe urgent need for a thorough study on the impact of infection on the native amphibian populations inBelgium.SAMENVATTINGChytridiomycose wordt veroorzaakt door de schimmel Batrachochytrium dendrobatidis en draagt in belangrijkemate bij tot het uitsterven van amfibieën wereldwijd. Recent werd aangetoond dat deze schimmel wijd verspreidvoorkomt in België en Nederland maar klinische gevallen van chytridiomycose werden tot nu toe niet vastgesteld.In deze casuïstiek wordt het eerste geval beschreven van chytridiomycose met dodelijke afloop bij een wildevroedmeesterpad (Alytes obstetricans) in België. De ruime verspreiding van de schimmel in België en het potentieelletale verloop van een infectie bij inheemse amfibieën benadrukken de noodzaak van een grondige studie van deimpact van de infectie op inheemse amfibieënpopulaties in België.Vlaams Diergeneeskundig Tijdschrift, 2010, 79 461merous PAS positive zoosporangia at various stages ofmaturation, confined to the keratinized epidermallayers (Figure 2). Numerous immature and mature 5-10 µm sized, thin-walled zoosporangia were invadingthe deeper portions of the corneal layers. Mature zoo -sporangia contained 1 µm sized basophilic, somewhatirregularly shaped spores. Superficial corneal layerswere loaded with empty post-discharge zoosporangiawhich tended to collapse and were colonized by rod-shaped and coccoid bacteria. Discharge tubes wereoccasionally seen. There were severe epidermal alte-rations, especially in the ventral abdominal skin. Basalkeratinocytes showed multifocal to coalescing vacuo-lar to hydropic degeneration and necrosis, with spon-giosis and frequent intra-epidermal vesicle formation.This was sometimes accompanied by partial skinsloughing and erosion. Sloughed skin fragments wereheavily colonized by bacteria. Dermal blood vesselswere congested. There was no clearly detectable dif-ference in fungal load between the various locations. In the liver, there was a diffuse microvacuolar de-generation of hepatocytes, and scattered single cellapoptosis. No histopathological lesions were noticed inthe kidney. The Ziehl-Neelsen stains were negativefor acid fast bacteria. The PCR for the detection of iridoviruses was ne-gative. In the skin swab, 1570 genomic equivalents ofB. dendrobatidis were detected. CONCLUSIONThe presence of high numbers of B. dendrobatidisorganisms, combined with the typical histological le-sions and the absence of other obvious causes of deathare conclusive evidence for clinical chytridiomycosis.Thus, this is the first published case of chytridiomy-cosis linked to mortality in indigenous amphibians inBelgium. Midwife toads (genus Alytes) are consideredhighly susceptible to chytridiomycosis (Bosch et al.,2001; Martel et al., 2010; Tobler and Schmidt, 2010).In 2009, 16% of all midwife toads examined in the Ne-therlands, were found positive for the presence of B.dendrobatidis (Spitzen-van der Sluijs et al., 2010).The relatively high prevalence of the fungus combinedwith this clinical case sheds a new light on the poten-tial risk of the presence of this emerging infectious di-sease for the native amphibians in Belgium. However,the short- and longterm effects of the presence of B.dendrobatidis at population level for the native am-phibian communities are completely unclear at present.A thorough risk assessment, taking into account amongothers strain virulence, host susceptibility and factorsthat predispose to clinical disease, is urgently needed.REFERENCESBerger L., Speare R., Daszak P., Green D.E., CunninghamA.A., Goggin C.L., Slocombe R., Ragan M.A., HyattA.D. Mc Donald K.R., Hines H.B., Lips K.R., MarantelliG., Parkess H. (1998). Chytridiomycosis causes amphibianmortality associated with population declines in the rainforests of Australia and Central America. In: Procee dingsof the National Academy of Sciences of the USA 95, 9031-9036.Bielby J., Bovero S., Sorgiu G., Tessa G., Favelli M., An-gelini C., Doglio S., Clare F.C., Gazzaniga E., Lopietra F.,Garner T.W.J. (2009). Fatal chytridiomycosis in the Thyrr-henian Painted Frog. Ecohealth 6, 27-32.Boyle D.G., Boyle D.B., Olsen V., Morgan J.A.T., HyattA.D. (2004). Rapid quantitative detection of chytridio-mycosis (Batrachochytrium dendrobatidis) in amphibiansamples using real-time Taqman PCR assay. Diseases ofAquatic Organisms 60, 133-139.Bosch J., Martinez-Solano I., Garcia-París M. (2001). Evi-dence of a chytrid fungus infection involved in the declineof the common midwife toad (Alytes obstetricans) in pro-tected areas of central Spain. Biological Conservation 97,331-337.Figure 2. Hematoxylin and eosin stained formalin fixedsection of ventral skin (drinking patch) of a midwife toad(Alytes obstetricans). Note epidermal hyperplasia and hy-perkeratosis, with the presence of numerous zoosporan-gia in the keratinized epidermal layers. There aremultifocal intra-epidermal vesicle formation (arrows),and scattered apoptosis and hydropic degeneration ofkeratinocytes  Inset: detail of zoosporangia at variousstages of maturation. Figure 1. Midwife toadlet (Alytes obstetricans) withchytridiomycosis. Note the loose, shed skin layers on theanimal’s body. The black tail remnant is typical of re-cent metamorphosis. 462 Vlaams Diergeneeskundig Tijdschrift, 2010, 79Houlahan J.E., Findlay C.S., Schmidt B.R., Meyer A.H.,Kuzmin S.L. (2000). Quantitative evidence of global am-phibian population declines. Nature 404, 752-755.Mao J.H., Hedrick R.P., Chinchar V.G. (1997). Molecularcharacterization, sequence analysis, and taxonomic posi-tion of newly isolated fish iridoviruses. Virology 229,212-220.Martel A., Van Rooij P., Vercauteren G., Baert K., VanWaeyenberghe L., Debacker P., Garner T.W., Woeltjes T.,Ducatelle R., Haesebrouck F. (2010). Developing a safeantifungal treatment protocol to eliminate Batracho-chytrium dendrobatidis from amphibians. Medical Myco-logy, in press.Pasmans F., Zwart P., Hyatt A.D. (2004). Chytridiomycosisin the Central American bolitoglossine salamander (Boli-toglossa dofleini). Veterinary Record 154, 153.Spitzen-van der Sluijs A., Zollinger R., Bosman W., vanRooij P., Clare F., Martel A., Pasmans F. (2010). Short re-port: Batrachochytrium dendrobatidis in amphibians in theNetherlands and Flanders (Belgium). A report by RA-VON for invasive Alien Species Team (TIE): Ministry ofAgriculture, Nature and Food Quality.Tobler U., Schmidt B.R. (2010). Within- and among-popu-lation variation in chytridiomycosis-induced mortality inthe toad Alytes obstetricans. PLoS One 5: e10927.Voyles J., Young S., Berger L., Campbell C., Voyles W.F.,Dinudom A., Cook D., Webb R., Alford R.A., SkerrattL.F., Speare R. (2009). Pathogenesis of chytridiomycosis,a cause of catastrophic amphibian declines. Science 326,582-585.Walker S.F., Bosch J., Gomez V., Garner T.W.J., Cunning-ham A.A., Schmeller D.S., Ninyerola M., Henk D.A., Gi-nestet C., Arthur C-P. and Fischer M.C. (2010). Factorsdriving pathogenicity vs. prevalence of amphibian pan-zootic chytridiomycosis in Iberia. Ecology letters 13, 372-382.Uit het verledenMUSEUM VAN HET BELGISCH TREKPAARD TE VOLLEZELEVollezele, nu deelgemeente van Galmaarden, in het Pajottenland niet zo ver van Henegouwen, herbergteen klein museum dat zonder twijfel een van de sympathiekste in het land is. In het vroegere gemeente-huis(je) wordt de geschiedenis geïllustreerd van de bloeitijd van het Belgisch Trekpaard. Het kleine dorpherbergde in de glorietijd niet minder dan drie wereldvermaardehengstenhouderijen. ‘Wereldvermaard’ is hier letterlijk tenemen. In 1910 werden 34.599 Belgische paarden uitgevoerd,waaronder 1600 naar Amerika. De uitvoer strekte van Argenti-nië tot Rusland.Een belangrijk aantal van de beste en duurste hengsten wasuit Vollezele afkomstig. Pionier Remi Van Der Schueren, ge-vestigd in dat dorp, had niet enkel fokkerstalent, hij begreepook dat men het product goed moest promoten en dat gebeurdevooral op internationale tentoonstellingen en wedstrijden.Samen met een tweetal andere hengstenhouders stichtte hij in1885 de vereniging van het Belgisch Trekpaard. In minder dangeen tijd vormde deze organisatie drie lokale strekkingen, de‘Kolossen uit de Mahaignestreek’, de ‘Grijzen uit het Nijvelse’en de ‘Dikke uit de Denderstreek’ om tot een succesvol uni-form ras te komen. Van Der Schueren’s Orange I en Brillantwaren daarin voortrekkers. Het monsterachtig grote succesduurde echter niet veel langer dan een halve eeuw…Een bezoekje is zonder meer aan te bevelen. Wel moet datvooraf aangevraagd worden bij de vrijwilligers die het mu-seumpje openhouden (dat kan via de site, gemakkelijk te vinden als je Vollezele en trekpaard intikt opGoogle).De illustratie hierbij toont het voorplat van een museumboekje opgesteld door Philippe van Dixhoorn,telg uit een Zeeuws-Vlaams hengstenhoudersgeslacht gevestigd te Vollezele en verdienstelijke stichter vanhet museum.Luc Devriese

Chytridiomycosis related mortality in a midwife toad (Alytes obstetricans) in Belgium

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Chytridiomycosis related mortality in a midwife toad (Alytes obstetricans) in Belgium

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