Short report Batrachochytrium dendrobatidis in amphibians in the Netherlands and Flanders (Belgium)

REPTIELEN AMFIBIEËN VISSEN ONDERZOEK NEDERLAND

SHORT REPORT Batrachochytrium dendrobatidis in amphibians in the Netherlands and Flanders (Belgium)

A report by RAVON for Invasive Alien Species Team (TIE); Ministry of Agriculture, Nature and Food Quality

Annemarieke Spitzen – van der Sluijs, Ronald Zollinger, Wilbert Bosman, Pascale van Rooij & Frances Clare , An Martel, Frank Pasmans January 2010

STICHTING RAVON POSTBUS 1413 6501 BK NIJMEGEN www.ravon.nl B. dendrobatidis in the Netherlands

Colofon

© 2010 Stichting RAVON, Nijmegen Report nr: 2009-29 Cover: A. van Diepenbeek Text: Annemarieke Spitzen – van der Sluijs, Ronald Zollinger, Wilbert Bosman, Pascale van Rooij & Frances Clare, An Martel, Frank Pasmans Citation: A. M. Spitzen – van der Sluijs, R. Zollinger, W. Bosman, P. van Rooij, F. Clare, A. Martel, F. Pasmans. 2010. SHORT REPORT Batrachochytrium dendrobatidis in amphibians in the Netherlands and Flanders (Belgium). Stichting RAVON, Nijmegen, the Netherlands.

This study was financed by Invasive Alien Species Team (TIE), Ministry of Agriculture, Nature and Food Quality and was reviewed by Dr. T. W. Garner (IOZ, London, UK) and Dr. M. C. Fisher (Imperial College London, UK).

Data from this report may only be reproduced with the written permission of the authors. B. dendrobatidis in the Netherlands

CONTENTS

SAMENVATTING...... 1

SUMMARY ...... 3

1 INTRODUCTION ...... 5

2 METHODS...... 7

2.1 Swab collection...... 7

2.2 Study area ...... 7

2.3 Time period...... 8

2.4 Analysis for B. dendrobatidis infection ...... 8

3 RESULTS...... 9

4 DISCUSSION...... 15

4.1 Recommendations...... 17

ACKNOWLEDGEMENTS ...... 21

REFERENCES...... 23

APPENDIX 1 B. dendrobatidis in the Netherlands

Stichting RAVON

SAMENVATTING

Doel Bepalen van de aanwezigheid van de schimmel Batrachochytrium dendrobatidis, de veroorzaker van de huidziekte chytridiomycose bij amfibieën, bij vrijlevende amfibieën in Nederland en in België.

Methode Zowel vrijwilligers als professionele krachten hebben gegevens verzameld van vrijlevende dieren door volgens een vast protocol met een steriel wattenstaafje 3 – 5 keer over de onderbuik en de poten van amfibieën te strijken. Op deze manier kan DNA van de schimmel worden verkregen. In het veld werden maatregelen genomen om te voorkomen dat nog onbesmette locaties besmet zouden raken. Door middel van een TaqMan PCR analyse werden de verzamelde monsters getest op aanwezigheid van de schimmel.

In totaal werden 2.771 monsters verzameld in de periode van augustus – oktober 2008 (Amerikaanse brulkikker in België) en in de periode van maart – september 2009 (alle soorten in Nederland en in België). In alle provincies van Nederland werden monsters genomen en alle inheemse soorten, behalve de meerkikker werden bemonsterd.

Resultaten In alle provincies, behalve in Groningen, Zuid-Holland en Zeeland zijn besmette dieren gevonden. Vier procent van alle amfibieën in het onderzoeksgebied zijn geïnfecteerd met B. dendrobatidis (3,7% in Nederland; 5,4% in België). Geen van de besmette dieren vertoonde klinische verschijnselen. Voor het eerst is aangetoond dat ook in Nederland en in België de schimmel B. dendrobatidis voorkomt.

Aanbevelingen De daadwerkelijke impact van de wijdverspreide besmettingen met de schimmel op inheemse amfibie populaties blijft vooralsnog onduidelijk. Toekomstig onderzoek zal dit moeten uitwijzen.

Verder onderzoek omvat onder andere de fylogenetische analyse van de aangetroffen B. dendrobatidis stam die in deze regio is aangetroffen volgens de recent beschreven methode in Goka et al (2009). Dit onderzoek zal inzicht geven in de diversiteit en mate van gerelateerdheid van de verschillende stammen en zal het proces van binnenkomst in Nederland en België kunnen verduidelijken. Ook onderzoek aan museum exemplaren zal hier inzicht in kunnen geven. Ook de invloed van de schimmel op de inheemse soorten moet worden onderzocht door 1) het bepalen van de gevoeligheid van onze soorten voor klinische chytridiomycose door middel van ex situ experimenten, door 2) het bestuderen van de invloed van omgevingsstress op de gastheer voor infectie met B. dendrobatidis, en door 3) de invloed van B. dendrobatidis infecties op inheemse soorten op populatieniveau in situ bestuderen en tenslotte door 4) het intensieve monitoren van een selectie van soorten die een hoge mate van risico lopen daadwerkelijk aan chytridiomycose te bezwijken zoals de vroedmeesterpad

1 B. dendrobatidis in the Netherlands

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2 Stichting RAVON

SUMMARY

Goal Determine presence of the zoosporic fungus Batrachochytrium dendrobatidis, the causative agent of the amphibian skin disease chytridiomycosis, in free ranging amphibians in the Netherlands and Flanders (Belgium)

Methods Volunteers and professionals collected data from free ranging amphibians by stroking 3 – 5 times with a sterile cotton-tipped dryswab over the underside of the legs, feet and drink patch. All amphibians were individually sampled. Hygienic measures were taken to prevent cross-infection. Quantitative real-time TaqMan PCR assays were performed for the determination of B. dendrobatidis infections.

A total of 2,771 swabs were collected between August – October 2008 (American bullfrogs in Flanders) and between March – September 2009 (all species in the Netherlands and in Flanders). Samples were collected in all provinces and all species apart from the marsh frog (Rana ridibunda) were sampled.

Results B. dendrobatidis infected amphibians have been found all over the Netherlands, in all provinces apart from Groningen, Zuid-Holland and Zeeland. A total of 4% of all amphibians in the Netherlands (3.7%) and Belgium (5.4%) are infected with B. dendrobatidis. Since the majority of native amphibians have been found infected, also critically endangered, endangered and vulnerable amphibian species are infected.

None of the infected individuals showed any clinical signs associated with chytridiomycosis. We thus for the first time demonstrate the presence of B. dendrobatidis in almost all amphibian species in the Netherlands.

Discussion & recommendations No data on prevalence can be calculated due to ad hoc sampling and also the impact of B. dendrobatidis infections on native species is yet unknown.

Future research includes phylogenetic analysis of B. dendrobatidis haplotypes, using the recently method of Goka et al. (2009), which will give insight in the diversity and relatedness of the strains and the process of invasion into the Netherlands and Flanders. Secondly, the impact of B. dendrobatidis on our native amphibian fauna should be examined by 1) determining the sensitivity of our amphibian species to clinical chytridiomycosis using ex situ infection experiments 2) studying the impact of host stress on the virulence of B. dendrobatidis 3) studying the impact of B. dendrobatidis infections in native amphibians in situ and 4) intensive monitoring of populations of a selection of species at risk, a prime candidate being the midwife toad.

3 B. dendrobatidis in the Netherlands

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1 INTRODUCTION

Chytridiomycosis is an emerging infectious disease (Daszak et al., 2000) of amphibians that is causing mass mortality and population declines worldwide (Berger et al., 1998; Bosch et al., 2001; Rachowicz et al., 2006). The causative agent, the chytrid fungus, Batrachochytrium dendrobatidis, presents low host specificity (Daszak et al., 2003). The pattern of amphibian declines (Daszak et al., 1999), and genetic studies (Daszak et al., 2003; Morehouse et al., 2003) support the hypothesis that the chytrid fungus has been recently introduced into native populations, at least in Central America and Australia. Goka et al. (2009) suggest that a combination of the ‘novel pathogen hypothesis’ and the ‘endemic pathogen hypothesis’ explain the current pandemic. Chytridiomycosis is the disease state that results from a sustained cutaneous infection by B. dendrobatidis. The chytrid reproduces via waterborne zoospores, and parasitizes the mouthparts of anuran larvae, then subsequently the keratinized epidermis of post-metamorphic amphibians, but may also occur on larval caudates (Brodman & Briggler, 2008; Kriger & Hero, 2007; Berger et al., 1998; Longcore et al., 1999; Marantelli et al., 2004). B. dendrobatidis infections are associated with pathophysiological changes that lead to the disease state (chytridiomycosis) and potentially death. When chytridiomycosis occurs, electrolyte transport across the amphibian epidermis is inhibited by as much as 50%, sodium and potassium concentrations in cell plasma are reduced, and asystolic cardiac arrest can result in death (Voyles et al., 2009). Because intact skin is critical in maintaining amphibian homeostasis, disrupting the cutaneous function may be the mechanism by through which B. dendrobatidis produces morbidity and mortality across a wide range of phylogenetically distant amphibian taxa (Voyles et al., 2009).

There is substantial interspecific variation in the effects of chytridiomycosis (Lips et al., 2003). In the worst cases, it may kill 90–100% of recently metamorphosed individuals of some species, causing severe reductions in their abundance or even local or global extinction (Daszak et al., 1999). At the other end of the spectrum, populations of some species seem to remain largely unaffected (Lips et al., 2003). These differences appear to be related to variation among host species in traits such as immune defences, fecundity, breeding habitat etc. (Williams & Hero, 1998; Daszak et al., 2003; Lips et al., 2003; Woodhams et al., 2006). It is also clear that, on a community level, some regions tolerate infection without obvious ill-effects (for instance South Africa; Weldon et al., 2004), while other regions undergo a rapid and catastrophic collapse in amphibian biodiversity (Australia, Central America, Spain; Skerratt et al., 2007; Lips et al., 2006; Bosch et al., 2001). While it is apparent that the emergence of chytridiomycosis has the potential to severely alter the structure of affected amphibian assemblages (Lips et al., 2006), it is not clear at this time what drives these differing population-level responses. The breadth of environments in which B. dendrobatidis may persist is wide (Walker et al., 2010). B. dendrobatidis is a generalist pathogen that is able to persist wherever susceptible hosts occur, however outbreaks of fatal chytridiomycosis seem highly related with environmental conditions (Walker et al., 2010).

The Dutch Ministry of Agriculture, Nature and Food Quality has established ‘Team Invasive Species’ (TIE). This Team acknowledges the potential risk of chytridiomycosis to amphibian biodiversity and has asked RAVON (Reptile, Amphibian and Fish Research the Netherlands, Nijmegen) to assess the risk of chytridiomycosis to native amphibians. This study is part of a broad scale study on the effects of both the American bullfrog (Rana catesbeiana) and the effects of chytridiomycosis on amphibian diversity. This study encompasses a Risk Assessment and literature studies on both topics. As agreed, a short report – this report- describes the results of the fieldwork conducted in 2008 and 2009 and laboratory analyses examining the presence of B.

5 B. dendrobatidis in the Netherlands

dendrobatidis in free ranging amphibians in the Netherlands and in Flanders (Belgium). The final report will appear early in 2010. The results will also be presented in relevant scientific journals.

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2 METHODS

Since B. dendrobatidis is a fungus that attaches to the keratin on the epidermis of metamorphosed amphibians, it can be non invasively sampled by ‘swabbing’ the skin with a cotton swab following Hyatt et al. (2007). Samples were collected in all Dutch provinces and in Flanders, by professional RAVON employees, Ghent University staff and volunteers. B. dendrobatidis has been recognized by the Office International des Epizooties (OIE) ad hoc Group on Amphibian Diseases as one of the two pathogens of particular importance (the other one being viruses belonging to the family Iridoviridae, genus Ranavirus) in international trade. To meet the OIE standards, a contra expertise on the test results was conducted by the IOZ (London) for the validation of the diagnostic assays.

In the current study, the presence of B. dendrobatidis on amphibian skin was studied and not the effects of the disease chytridiomycosis. In this report when ‘Belgium’ is mentioned, we refer to Flanders, since no samples were collected from free ranging amphibians in the Walloon provinces of Belgium.

2.1 Swab collection

All amphibian species encountered in the field were swabbed with MW102 sterile cotton-tipped dryswabTM (MWE medical wire, UK). In tadpoles, the only keratinized areas are the mouthparts. This area was swabbed very gently and only larvae that were large enough to handle without damaging were sampled. This was only done by experienced RAVON employees. When swabbing metamorphosed amphibians (juveniles, subadults and adults), the underside of the legs, feet and drink patch were comprehensively swabbed (3 to 5 times). In order to minimize the possibility of false positives, each specimen was individually sampled. The swabs were stored with one silicagel granule (VWR International, NL) in a freezer (-20 °C) until further analysis.

Animals were placed in individual bags prior to swabbing and all animals were handled using fresh disposable –non powdered vinyl- gloves. Hygienic measurements (the disinfection of dip nets, boots and other field equipment using salt or bleach) were taken to reduce the risk of transporting B. dendrobatidis among sites.

Table 1 lists the number of samples that were collected for each species, including the non- natives.

2.2 Study area

The aim of the study was to cover the whole of the Netherlands and Flanders, so calls were made to volunteers of RAVON and Hyla (a Belgium NGO) all over the Netherlands and Belgium to participate. RAVON employees took swab kits into the field during inventorying projects and additionally, sites that contain chytridiomycosis – sensitive species, such as the common midwife toad (Alytes obstetricans) were visited (Bosch et al., 2001).

7 B. dendrobatidis in the Netherlands

In 2008 and in 2009 samples of American bullfrogs (Rana catesbeiana) were taken in Flanders. Bullfrogs are known vectors of B. dendrobatidis (Daszak et al., 2004) and the species is listed on the list of ‘100 of the world’s worst invasive alien species’ (Lowe et al., 2000). Daszak et al., (2004) have demonstrated that the bullfrog is an efficient carrier of B. dendrobatidis, which adds to its capacity as threat to amphibian populations (Kats and Ferrer, 2003). In Great Britain, it is presumed that B dendrobatidis was co-introduced with either bullfrogs or Xenopus species (Cunningham et al., 2005). Bullfrogs live very close to the southern border of the Netherlands in Belgium.

2.3 Time period

Swabs were collected in 2008 from August – October (bullfrogs in Flanders) and in 2009 from March – September (all species in the Netherlands and in Flanders).

2.4 Analysis for B. dendrobatidis infection Quantitative real-time TaqMan PCR assays were performed as described by Boyle et al. (2005) and Hyatt et al. (2007) with Bio-Rad equipment at the Faculty of Veterinary Medicine Ghent University, Department of Pathology, Bacteriology and Avian Diseases.

2.4.1 Contra expertise Contra expertise was conducted by the IOZ in London for a total of 240 samples, also using real- time TaqMan PCR, using the products and device from ‘Applied Biosystems’. One hundred and seven duplicate swabs and 133 DNA samples were analysed.

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3 RESULTS

Samples were collected across the Netherlands (blue dots in figure 1) from coastal dunes to the old woods and small brooks of Limburg and the open landscape in the province Groningen. Different water types were sampled, including garden ponds, ditches and ponds in nature reserves. Collectively, the results represent an inclusive cross section of available amphibian habitats in the Netherlands and in Flanders.

A total of 85 professionals and volunteers collected 2,771 swabs (figure 1, blue dots and table 1). The bulk (96%) of all collected swabs are from native species, 109 swabs (4%) were collected from bullfrogs (Flanders), Italian crested newts (Triturus carnifex; the Netherlands), the marbled newt (T. marmoratus; Flanders) and the African clawed frog (Xenopus laevis; Flanders).

B. dendrobatidis positive amphibians have been found across the Netherlands, in all provinces apart from Groningen, Zuid-Holland and Zeeland. A total of 4% of the amphibians sampled in the Netherlands (3.7%) and Belgium (5.4%) tested positive for B. dendrobatidis (table 1). The relatively high prevalence in Belgium driven by the high prevalence in American bullfrog samples. Eighty- eight of the 429 Belgium swabs were bullfrog samples and slightly over one fifth (20.5%) of all American bullfrogs tested positive for B. dendrobatidis (table 1).

In the Netherlands prevalence was strongly influenced by the large number of midwife toads (n = 310) that were sampled. Within this sample, a total of 48 individuals tested positive (15.5%). When in an early stage of this study midwife toads turned out to be infected, an additional study was conducted to collect supplemental samples in Limburg.

The red dots in figure 2 are locations where B. dendrobatidis was detected on amphibian hosts and the numbers in this figure correspond with the far left column in table 2. All native amphibian species were sampled, apart from the marsh frog (Rana ridibunda). The smooth newt (Lissotriton vulgaris) was the most sampled species (n = 475) of all amphibians (table 1). The common spadefoot (Pelobates fuscus) was sampled the least. This species not only has a limited distribution, but is also one of our rarest amphibians.

The alpine newt, smooth newt, midwife toad, yellow-bellied toad, common toad, treefrog, common frog, water frogs (undef.), edible frog, edible/pool frog and the pool frog are the native species that have been tested positive for B. dendrobatidis. Of the non native species, the bullfrog exhibited a high prevalence.

The contra expertise analysis conducted by the IOZ overall showed equivalent results as derived by the Faculty of Veterinary Medicine Ghent University. In 7% of the cases the results did not match, and results were interpreted conservatively (ambiguous results were treated as negative tests).

9 B. dendrobatidis in the Netherlands

Figure 1. Overview of the locations where swabs were collected in the Netherlands and in Belgium in 2008 & 2009 (blue dots). The positive locations (where B. dendrobatidis was found) are marked red.

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Table 1. Overview of the number of samples per species in the Netherlands and in Flanders, and the percentage of B. dendrobatidis positives per species per country is given.

sampled B. dendrobatidis positives mean infection rate range Species Netherlands Belgium Total Netherlands Belgium Total GE ± SE GE natives Salamandra salamandra 11 5 16 0 0 0 - - Ichtyosaura alpestris 57 248 305 5.3% (n=3) 2.0% (n=5) 2.6% (n=8) 15.8 ± 10.0 0.1 - 0.6 Triturus cristatus 80 2 82 0 0 0 - - Lissotriton helveticus 11 49 60 0 0 0 - - Lissotriton vulgaris 469 6 475 0.9% (n=4) 0 0.8% (n=4) 0.8 ± 0.3 0.3 - 1.5 Alytes obstetricans* 310 0 310 18.1% (n=56) - 18.1% (n=56) 11.5 ± 5.7 0.1 - 261 Bombina variegata 255 0 255 5.9% (n=15) - 5.9% (n=15) 312.9 ± 281.2 31.7 - 594 Pelobates fuscus 9 0 9 0 - 0 - - Bufo bufo 204 9 213 3.9% (n=8) 0 3.9% (n=8) 9.06 ±5.17 0.7 - 43.9 Bufo calamita 80 0 80 0 - 0 - - Hyla arborea 89 0 89 2.2% (n=2) - 2.2% (n=2) 26.8 ± 25.7 1.2 - 52.5 Rana arvalis 63 0 63 0 - 0 - - Rana temporaria 160 15 175 0.6% (n=1) 0 0.6% (n=1) 4.7 4.7 Rana esculenta synklepton 83 0 83 2.4% (n=2) - 2.4% (n=2) 11.1 ± 7.2 3.9 - 18.2 Rana klepton esculenta 294 0 294 3.7% (n=11) - 3.7% (n=11) 14.6 ± 9.9 0.2 - 90.6 Rana lessonae/esculenta 56 0 56 7.1% (n=4) - 7.1% (n=4) 1.1 ± 0.6 0.2 - 2.6 Rana lessonae 100 0 100 4% (n=4) - 4% (n=4) 35.2 ± 30.1 2.6 - 95.4

non-natives Xenopus laevis 0 10 10 - 0 0 - - Triturus marmoratus 0 1 1 - 0 0 - - Triturus carnifex 11 0 11 0 - 0 - - Lithobatus catesbeianus 0 88 88 0 20.5% (n=18) 20.5% (n=18) 8.9 ± 3.8 0.8 - 18.6

Total 2342 433 2775 4.7% (n=110) 5.3% (n=23) 4.8% (n=133) 18.5 ± 7.3 0.1 - 594

* including the introduced individuals in Artis Zoo and in Haarlem.

11 B. dendrobatidis in the Netherlands

4

5

6

3

1 2 7

10 9

8 11

13 12

14 15 16

23 24 17

22 19 21 18 25 20

Figure 2. All locations where B. dendrobatidis infected amphibians were found. The numbers correspond with those in table 2.Site 19 consists of 2 red dots, each dot is a square kilometer.

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No. Fig. Coun

2. -try Salamandra salamandra Mesotriton alpestris Triturus cristatus Lissotriton helveticus Lissotriton vulgaris Alytes obstetricans Bombina variegata Pelobates fuscus Bufo bufo Bufo calamita Hyla arborea Rana arvalis Rana temporaria Rana esculenta synklepton Rana lessonae Rana klepton esculenta Rana lessonae/esculenta catesbeiana** Rana Triturus marmoratus** ● ○ 1 NL ○ (26) ○ (18) ● (39;1) ○ (1) ○ (14) (21;6) (11) ○ 2 NL ○ (16) ● (4;3)* Table 2. Overview of all (31) positive locations and 3 NL ● (30;1) species. Numbers in the 4 NL ● (5;1) ● (25;2) left most column 5 NL ○ ○ ○ ● (1) (2) (3) (4;1) correspond with the ○ ● 6 NL ○ (69) ○ (8) (1) (34;4) numbers in figure 2. 7 NL ● (4;1) ○ (7) ○ 8 NL ○ (7) ○ (1) ● (30;1) ○ open circle (2) Sampled species, not 9 NL ○ (8) ○ (1) ● (17;2) infected. In brackets the 10 NL ● (14;3) ○ (9) number of sampled ○ ○ ○ 11 NL ○ (7) ○ (16) ○ (1) ○ (2) ○ (5) ● (17;2) ● (20;3) (2) (5) (12) individuals 12 NL ○ (3) ○ (13) ○ (4) ● (5;1) ● ● closed circle 13 NL (14;1) Sampled species, infected. ● 14 NL ○ (2) ○ (3) In brackets the number of (26;2) 15 B ● (4;2) sampled individuals and the number of positives 16 NL ○ (10) ○ (1) ● (1;1) ○ (1) ● (26;1) ○ (8) ○ 17 NL (25 ● (21;1) ○ (1) * introduced from France ) ** exotic species ● 18 NL ○ (2) (42;12) ● ○ 19 NL ○ (21) ○ (17) ● (68;8) ● (8;2) (32;2) (10) ○ ● 20 NL (4) (59;31) ○ ● 21 NL ○ (7) ● (30;2) (11) (2;1) ● ○ 22 B ○ (16) ○ (2) (33;5) (1) 23 B ● (70;14) 24 B ● (10;2) ● 25 NL ○ (2) ○ (1) ○ (1) (11;1)

13 B. dendrobatidis in the Netherlands

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4 DISCUSSION

The prevalence of chytridiomycosis is known to show seasonal variation (e.g. Kriger & Hero, 2007a) and can also be influenced by climatic variables (e.g. Walker et al., 2010) and by the species specific characteristics (e.g. Kriger & Hero, 2007b). Within the Iberian peninsula (southern France and Spain), climatic variables weakly influence the prevalence of infection, however strongly determine the presence/absence of fatal chytridiomycosis (Walker et al. 2010). In the current study, sampling was performed ad hoc and opportune and aimed at sampling all native species nationally for a first ‘snap-shot’ of B. dendrobatidis absence/presence. This sampling strategy was successful in determining whether there is B. dendrobatidis in the Netherlands and Flanders, and what species are infected. However, this sampling strategy is inadequate for answering more detailed questions on the prevalence, dynamics and effects of infection at specific infected sites.

B. dendrobatidis was detected on amphibians in nine provinces and on all native amphibian species apart from the common spadefoot and marsh frogs, the latter of which were not sampled. However, B. dendrobatidis has been detected on marsh frogs previously and on a congener of P. fuscus (T.W.J. Garner pers. comm.) In this study the fungus was detected on amphibians inhabiting a variety of water bodies and in different geographical regions. Overall, we can cautiously state that B. dendrobatidis can be potentially infect all amphibian species across the Netherlands.

In the Netherlands, the yellow-bellied toad is a critically endangered species, the tree frog and fire salamander are both endangered species and the midwife toad, palmate newt, crested newt are three vulnerable species (van Delft et al., 2007), all of which have tested positive for infection with B. dendrobatidis in this study. We did not detect B. dendrobatidis on the common spadefoot (endangered), but only a few individuals (n=9) were sampled. It is known that chytridiomycosis can be lethal to many species of amphibians, including the common toad (Garner et al., 2009) and extensive studies from Spain show that the midwife toad and fire salamander are at direct risk from rapid population decrease owing to the effects of chytridiomycosis (Bosch et al., 2001; Bosch & Martínez-Solano, 2006). On the other hand, shifts in species composition have been reported in Spain, where the common toad population is increasing due to a competitive advantage after the extirpation of midwife toads (Bosch & Rincón, 2008).

One needs to sample 60 individuals per species per location to be 95% certain of detecting 1 positive animal when the apparent prevalence is ≥5% and when using a qPCR test with perfect specificity (Skerratt et al., 2008). Sampling effort at all locations fell short of this level of effort. Sampling at this level is often impossible, since in many locations 60 individuals per species cannot be captured. This implies that some of the now ‘negative’ locations could be falsely categorized as negative and therefore it is likely that what we have underestimated the true distribution of B. dendrobatidis over the Netherlands and Flanders

Sampling was also biased among potential hosts. For example the data set includes an overrepresentation of midwife toad samples. When in an early stage of the study it was found out that this B. dendrobatidis sensitive species was infected, more samples were collected at and near positive locations, likewise for the bullfrogs in Flanders.

This year, RAVON received many reports from amphibian mass mortalities over the country. In most cases, animals had died due to frozen ponds. Reports of from common toads dying in

15 B. dendrobatidis in the Netherlands

amplexus, even up to 130 individuals in one pond did sound more alarming, but this instantaneous mortality is not a characteristic of the seasonal envelope for lethal chytridiomycosis. In Europe, chytridiomycosis has the tendency to be expressed in newly metamorphosed animals (Walker et al., 2010). Until now, no reports of mass mortalities in recently metamorphosed amphibians have been reported within our study region. When amphibians metamorphose, the amount of keratin in the epidermis greatly increases and helps to explain the observation that tadpoles are largely unaffected by infection, while high mortality occurs following the metamorphosis of infected individuals (Berger et al. 1998). However, mortality can also occur in larval stages (Garner et al., 2009). Fatal infections are also known in adult amphibians, but their symptoms are unclear. Clinical signs of chytridiomycosis include abnormal posture, loss of righting reflex, lethargy, and rapid progression to death (Cunningham et al., 2005; Simoncelli et al., 2005). Gross lesions are usually not apparent, but increased epidermal sloughing, epidermal ulceration and reddening (hyperaemia) of the digital and ventral skin have been reported (Daszak et al., 1999; Berger et al., 1998). Simoncelli et al. (2005) did not observe abnormal posture, reflex loss and lethargy in the field-collected water frogs and found gross skin lesions, such as excessive sloughing and roughening of the superficial epidermis, in only 5% of the individuals examined. Therefore it is important to realise that symptoms observable for the human eye are not always present and seemingly healthy animals may still be infected. In the present study, none of the Bd- positive amphibians showed any signs of sickness or reduced condition. However, this does not mean that Bd-infected animals are not suffering from chytridiomycosis, as the effects of infection may be manifested over a long period and may lead to (as yet) undocumented reductions in lifespan and breeding success. Some clinical remarks were made on the collected datasheets such as ‘very skinny, white rash visible’, ‘blood on mouth’, ‘external characteristics of a fungus infection’ or ‘film over body’ were noted. However, all these animals were shown to not be infected by B. dendrobatidis. Thus we can make no reasonable conclusions linking our detections of B. dendrobatidis to either disease or host population dynamics.

Garner et al. (2009) showed in their study with Bufo bufo (common toad) that a B. dendrobatidis infection of larvae imposes a significant cost, as the probability of surviving through metamorphosis significantly reduces as well as a reduction in mass at metamorphosis. When post- metamorphic toads were exposed to B. dendrobatidis, this resulted in infection however body size was a more important determinant for survival than was dose. Interestingly, some of these individuals do not exhibit infection when they die, suggesting that they have cleared their infections however have incurred substantial costs that lead to mortality nevertheless. Mortality not only varies with the condition of the individual and dose, but also with the pathogenicity of the strain (Berger et al., 2005) and some European strains of B. dendrobatidis have been found to be highly virulent (Fisher et al. 2009b). These results emphasize the complex reaction of amphibians on a B. dendrobatidis infection and illustrate the challenge for clarifying the true impact of B. dendrobatidis infections on amphibians and amphibian populations.

RAVON has an extensive monitoring network for amphibians, but despite this intensive monitoring, subtle or even massive mortalities may occur unnoticed. This is particularly true for post-metamorphic die-offs, as recently metamorphosed amphibians are difficult to detect even when living. Furthermore, the effects of massive die-offs on recruitment may only be noticed after a few years. These findings emphasize the need for year-round intensive monitoring of infected locations, as well as year-on-year assessment of the size of infected populations. It is also clear that environmental variability has an effect on the incidence of chytridiomycosis, and there may be as-yet undetected temporal as well as spatial variation in the amount of disease within the Netherlands.

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Data were collected using swabs and these swabs were analysed with quantitative real-time TaqMan PCR. Both techniques have proven to be the most reliable techniques for sampling of B. dendrobatidis in field conditions. Boyle et al. (2004) developed the real-time PCR TaqMan assay that accurately detects and quantifies one genomic equivalent (GE) which is equivalent to one zoospore in a diagnostic sample. The different collection and assay protocols were compared by Hyatt et al. (2007). Swabbing is consistently as good as, or even better than collecting toe clips, washing the animals or filtering the water. Another advantage is that collecting swabs is a non- invasive technique, applicable under field and laboratory conditions. Comparing TaqMan PCR and histology, demonstrates a much higher sensitivity for the PCR technique. Hyatt et al. (2007) developed a set of protocols for the OIE that can be used to detect B. dendrobatidis in amphibians (table 3). The manual for the detection of B. dendrobatidis by the OIE is in preparation (http://www.oie.int/eng/en_index.htm; accessed 05 Jan 2010), and the pan-European project RACE (Risk Assessment of Chytridiomycosis to European Amphibian Biodiversity) has recently produced guidelines for detecting B. dendrobatidis, as well as Hygiene protocols (appendix 1).

Table 3. Recommended detection protocol by Hyatt et al. (2007)

4.1 Recommendations

The genetic diversity and endemic nature of B. dendrobatidis detected in the study of Goka et al. (2009) suggests a host–parasite co-speciation between Japanese amphibians and B. dendrobatidis, in which each of the fungal strains possesses a specific natural host. Such co-evolution has also been suggested to explain the relative low virulence of B. dendrobatidis and chytridiomycosis in Africa (Weldon et al., 2004). Anthropogenic disturbance of the environment and artificial transportation of amphibians into new habitats undoubtedly carry the chytrid fungus from its native habitats into non-native habitats as has been proven by Walker et al. (2008). Under these conditions, the alien fungus must switch to a new host amphibian if it is to survive, and unnatural combinations of amphibians and fungal strains that have not undergone a co-evolutionary process may explain the

17 B. dendrobatidis in the Netherlands

resulting pandemic (Goka et al., 2009). Therefore organisations and individuals that work with amphibians should practice disinfection techniques in the field. At the same time, we should study the dispersal routes of B. dendrobatidis and try to allocate the most important routes of dispersal in the framework of risk management. A useful risk assessment tool has been developed by St- Hilaire et al. (2009). This tool is developed for field biologists and other individuals who visit multiple water bodies to determine their risk of spreading B. dendrobatidis via their activities. The tool also identifies disinfection strategies to reduce the risk of anthropogenic spread of B. dendrobatidis.

When amphibians are part of a reintroduction program, extensive screening for diseases of the translocated individuals before translocation is strongly recommended, with emphasis on a screening for chytridiomycosis (Walker et al., 2008).

Remarkably, B. dendrobatidis has been found from the north of the Netherlands to the south of Flanders and from east to west. This, alongside the lack of evidence for chytridiomycosis could indicate that B. dendrobatidis is not a recently introduced pathogen in the region, or that the infection is (yet) relatively mild. Recent data from other regions of Europe (the Pyrenees) show that B. dendrobatidis is still spreading into uninfected populations. In the Pyrenees, the use of phylogenetic analysis has shown that B. dendrobatidis has been only introduced one into the region, and there is a striking homology with North American strains of B. dendrobatidis suggesting a link between the two continents (Walker et al. 2010). Strains of B. dendrobatidis should be recovered from the infected populations and studied using the same set of molecular markers that are being used by RACE in order to identify the potential sources of the isolates that are infecting Dutch and Flemish populations of amphibians. In order to determine the time and location of the emergence or introduction of B. dendrobatidis, it is also important to examine archived specimens housed in museums and research institutions. This can be reliably done by the non-destructive sampling technique (swabbing) which enables qPCR analysis of ethanol-preserved museum specimens for B. dendrobatidis (Soto-Azat et al., 2009).

It is known that amphibians can be infected with B. dendrobatidis for a longer period of time without showing any negative symptoms and that some species can clear infections (e.g. Simoncelli, 2005; Woodhams et al., 2007; Kriger & Hero, 2006). Simultaneously, it is likely that changes in environmental variables may trigger the onset of catastrophic mortalities by reducing amphibians condition and herewith increasing susceptibility to infections (Walker et al., 2010). Garner et al. (2009) show that the survival of the common toad (Bufo bufo) after infection by B. dendrobatidis is dependent on host condition as well as life history stage. Water quality can negatively influence amphibian growth and development (e.g. Gutleb et al., 2007; Relyea, 2009). The relation between aquatic parameters and environmental variability upon the virulence of B. dendrobatidis on amphibians is an important topic of further studies.

A spatial analysis on prevalence data should be conducted to relate B. dendrobatidis distribution with environmental data and species characteristics for framing the results in the right perspective and for a view through on the detrimental effects of the fungus on specific species and/or sites.

There is not only a species-specific variation in amphibian responses to B. dendrobatidis exposure, but species-level responses may also vary (Fisher et al., 2009a). Some populations of species known to be susceptible to fatal chytridiomycosis may not experience detectable mortality or declines even when harbouring infected individuals (Briggs et al., 2005). Additionally, a seasonal effect of B. dendrobatidis infections (Retallick et al., 2004; Berger et al., 2004; Bosch et al., 2001; Drew et al., 2006) exists, with a higher prevalence detected with decreasing temperatures. The

18 Stichting RAVON presence of chytridiomycosis is more likely at sites where average summer temperature is below 30°C (Drew et al., 2006), although all these associations (temperature, altitude, precipitation) have been noted primarily in tropical regions, in Australia, in montane areas of central Spain, or in laboratory settings. B. dendrobatidis responds to decreasing temperatures by increasing infectivity. The cooler the temperatures, the greater the number of zoospores are produced per zoosporangium, and they remain infectious for a longer period of time (Woodhams et al., 2008). The lack of studies on the relation of the presence and virulence of B. dendrobatidis in our temperate (lowland) climate zone complicates the assessment of the risk of B. dendrobatidis infections currently found in the Netherlands and in Flanders. It is therefore recommended to conduct both field, experimental and modelling studies to further clarify this topic.

Since chytrid infections are new for the Netherlands and Flanders, we should assess the impact of this skin disease on amphibian survival rates. Both ex situ experiments (infection tests) and in situ monitoring, with emphasis on vulnerable species like the midwife toad, are required to obtain the necessary insight for the appropriate risk management. In these experiments the B. dendrobatidis strains that occur in the Netherlands and Flanders should be used, and these strains should be isolated first. Pathogenicity varies with strain and Fisher et al. (2009b) demonstrated variation in the virulence of different strains of B. dendrobatidis in Europe. In this study the researchers infected amphibians with three different strains of B. dendrobatidis and found differences in lethal and sub- lethal responses of the animals. Interestingly, one lineage of B. dendrobatidis recovered from Mallorca demonstrates low virulence relative to other European lineages, suggesting that the may be widespread variation in virulence. Conducting experiments with B. dendrobatidis strains not found in the Netherlands and/or Flanders, would therefore make the results difficult to interpret.

When fatalities occur, they generally occur during, or just after, metamorphosis. Intensive monitoring with the aid of volunteers is necessary to study if this phenomenon occurs in infected populations of the Netherlands and in Flanders. Amphibians can be relatively long lived animals, up to 20 years for newts and salamanders and up to 10 years for frogs and toads (species descriptions in: Creemers & van Delft, 2009), although most individuals do not reach the age of five, so increased mortality of metamorphosed amphibians will potentially, without extra monitoring, only be noticed after several years. Monitoring the effects of B. dendrobatidis presence on adults survival, reproductive success and population development should be monitored by capture-mark-recapture studies at several sites with varying prevalence (from none to substantial) over a number of years.

If the problem of B. dendrobatidis presence in this region is compared with a traffic light, the light is orange. We know now that the fungus is widely present, so far no casualties have been reported, but also in other countries fatalities have occurred only after a certain period of time (Bosch & Martínez-Solano, 2006).

In conclusion, we have shown that B. dendrobatidis widely infects amphibians across the Netherlands and Belgium. The challenge is now to define the true risks of B. dendrobatidis to our native amphibians, to minimize these risks if possible and to protect this vulnerable group from a potentially catastrophic event that could result in a significant loss of biodiversity.

19 B. dendrobatidis in the Netherlands

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ACKNOWLEDGEMENTS

The study is conducted by RAVON in close cooperation with the Faculty of Veterinary Medicine Ghent University, Laboratory of Veterinary Bacteriology and Mycology & Division of Poultry, Exotic Companion and Laboratory Animals Department of Pathology, Bacteriology and Avian Diseases, the Institute of Zoology (IOZ, dr. T. W. J. Garner), London and with Dr. Matthew Fisher (Imperial College Faculty of Medicine, Department of Infectious Disease Epidemiology (London), the Central Bureau of Fungus Cultures (dr. A. de Cock), Radboud University and the amphibian department ‘Hyla’ of Natuurpunt, a Flemish NGO. All volunteers are greatly acknowledged for their help.

21 B. dendrobatidis in the Netherlands

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