Batrachochytrium dendrobatidis is a pathogen infecting amphibians at the global scale and causing their decline, but knowledge of the distribution of this pathogen is far from complete. We sampled amphibians from three species (Hyla intermedia, Rana dalmatina and Pelophylax synklepton esculentus) to evaluate whether B. dendrobatidis infects amphibians in the Po River Delta Natural Park, Northern Italy. We detected the pathogen in one population of P. sk. esculentus (prevalence: 0.33). These findings expand the known distribution of B. dendrobatidis in Italy and add further concern to the conservation of amphibians in this area

Dejean, Tony

Ficetola, Gentile Francesco

Mazzotti, Stefano

Miaud, Claude

Noferini, Andrea

Valentini, Alice

Gentile Francesco Ficetola

Alice Valentini

Claude Miaud

Andrea Noferini

Stefano Mazzotti

Tony Dejean

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<em>Batrachochytrium dendrobatidis</em> in amphibians from the Po River Delta, Northern Italy

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ISSN 1827-9635 (print) © Firenze University Press 
ISSN 1827-9643 (online) www.fupress.com/ah
Acta Herpetologica 6(2): 297-302, 2011
Batrachochytrium dendrobatidis in amphibians from the Po 
River Delta, Northern Italy
Gentile Francesco Ficetola1, Alice Valentini2, Claude Miaud2, Andrea Noferi-
ni3, Stefano Mazzotti4, Tony Dejean2,5
1 Dipartimento di Scienze dell’Ambiente e del Territorio, Università di Milano-Bicocca. Piazza della 
Scienza 1, 20126 Milano Italy. Corresponding author. E-mail: francesco.ficetola@unimib.it
2 Laboratoire d’Ecologie Alpine, UMR CNRS 5553, Université de Savoie, 73376 Le Bourget-du-Lac 
cedex, France.
3 Parco regionale del Delta del Po Emilia-Romagna. Via Mazzini 200, 44022 Comacchio (FE), Italy.
3 Museo Civico di Storia Naturale di Ferrara, Via F. De Pisis 24, 44121 Ferrara, Italy.
5 Parc naturel régional Périgord-Limousin, La Barde, 24450 La Coquille, France.
Submitted on: 2011, 30th August; revised on: 2011, 7th November; accepted on: 2011, 11th November.
Abstract. Batrachochytrium dendrobatidis is a pathogen infecting amphibians at the 
global scale and causing their decline, but knowledge of the distribution of this patho-
gen is far from complete. We sampled amphibians from three species (Hyla interme-
dia, Rana dalmatina and Pelophylax synklepton esculentus) to evaluate whether B. 
dendrobatidis infects amphibians in the Po River Delta Natural Park, Northern Italy. 
We detected the pathogen in one population of P. sk. esculentus (prevalence: 0.33). 
These findings expand the known distribution of B. dendrobatidis in Italy and add fur-
ther concern to the conservation of amphibians in this area.
Keywords. Amphibian decline, Chytridiomycosis, Emerging infectious disease, 
Management, Prevalence, Pool frogs.
The chytrid fungus, Batrachochytrium dendrobatidis (hereafter Bd), is the agent of 
chytridiomycosis, an emerging infectious disease causing amphibian decline and extinc-
tions at the global scale (e.g., Berger et al., 1998; Fisher et al., 2009). The study of dis-
tribution, prevalence and impact of Bd is becoming a central theme of amphibian con-
servation: information on Bd distribution can be used to better understand the causes 
of amphibian decline and eventually to set up management and prevention protocols 
(Andreone et al., 2008; Fisher et al., 2009). However, in several areas of the world the 
knowledge of Bd distribution remains limited. For instance, only a few studies investi-
gated the occurrence and the impact of Bd in Italy. Bd has been detected in populations 
of the yellow-bellied toad (Bombina pachypus) in the northern Apennines (Stagni et al., 
2004), in pool frogs (Pelophylax synklepton esculentus) from Piedmont and central Italy 
298 G.F. Ficetola et alii
(Simoncelli et al., 2005; Di Rosa et al., 2007), in populations of the frogs Rana latastei and 
Lithobates catesbeianus from Piedmont (Garner et al., 2004; Garner et al., 2006) and in the 
endemic painted frog (Discoglossus sardus) and Sardinian newt (Euproctus platicephalus) 
in Sardinia (Bovero et al., 2008; Bielby et al., 2009). Furthermore, Bd is probably a cause 
of the decline of B. pachypus, E. platicephalus and D. sardus (Stagni et al., 2004; Bovero et 
al., 2008; Bielby et al., 2009). Nevertheless, information on the presence (or absence) of Bd 
from large portions of Italy is extremely limited, and further data are required for a more 
complete assessment of the distribution of Bd; these data may also have important conse-
quences for the conservation and management of amphibian populations.
The aim of this study was to evaluate whether Bd infects amphibians in the Po River 
Delta. This area is particularly interesting for the study of Bd because it includes some 
of the most important wetland systems of Italy and hosts a rich herpetofauna (Mazzotti, 
2007; Mazzotti et al., 2007). On the other hand, the Po River Delta is one of the first areas 
of Italy that has been invaded the American Bullfrog L. catesbeianus (Albertini and Lan-
za, 1987; Ficetola et al., 2010), and this alien species is probably implicated in the global 
spread of Bd (Garner et al., 2006).
In May 2010, we sampled amphibians in three areas of the Po River Delta Regional 
Park (Table 1). One area (Bosco Mesola integral Natural State Reserve) is located in the 
north of the park, while the other two are nearby the town of Ravenna, in the central part 
of the park. Amphibians where captured by hand or using small nets. The complete body 
was comprehensively swabbed using fine-tip swabs (Medical Wire & Equipment Co. MW 
100–100). The underside of the legs, feet and drink patch was swabbed 3 to 5 five time 
following the protocol of Hyatt et al. (2007). All individuals were released in the environ-
ment immediately after swabbing.
Swab samples were stored frozen until DNA extraction. DNA extraction and real-time 
PCR were performed using the protocol by Boyle et al. (2004)slightly modified. Swab was 
cut and a 2 mm portion was put in 60 μl of PrepMan Ultra (Applied Biosystem) with 30 
to 40 mg of 0.5 mm diameter glass beads (Scientific Industries, Inc.). The samples were 
homogenised for 45 s at 30 m/s in a QIAGEN TissueLyser II (Retsch Technology GmbH) 
and centrifuged for 30 s at 14000 g. The homogenisation and centrifugation was repeated. 
Samples were then incubated at 100°C for 10 min, cooled for 2 min, then centrifuged at 
14000 g for 3 min. All the liquid was collected and stored at -20°C. The DNA extracted 
was diluted 10 times for subsequent real-time quantitative PCR assay. Real-time quan-
titative PCR reactions were performed in a iQ5 system (BioRad) following the proto-
col described by Boyle et al. (2004), but using iQ Supermix (BioRad) instead of Taqman 
Master Mix. Both samples and the negative controls were run in duplicates. PCR stand-
Table 1. Sampling locations, amphibian species and prevalence of B. dendrobatidis.
Locality Lat. Long. Species n Prevalence 95%CI
Bosco Mesola (FE) 44.83°-44.85°N 12.25°-12.26°E Rana dalmatina 2 0 0-0.667
Bosco Mesola (FE) 44.83°N 12.26°E Pelophylax sk. esculentus 3 0.33 0.039-0.823
Ravenna 44.58°N 12.27°E Pelophylax sk. esculentus 27 0 0-0.088
Bardello (RA) 44.53°N 12.24°E Hyla intermedia 28 0 0-0.085
299Batrachochytrium dendrobatidis in amphibians in Northern Italy
ards were obtained using the protocol described by Boyle et al. (2004). The isolates were 
acquired from infected Alytes obstetricans collected in Spain and cultured at the Imperial 
College of London (Fisher et al., 2009). Infection data were submitted to the Bd Global 
Mapping Project (www.bd-maps.net)
We then used the Bayesian equal-tailed Jeffreys prior intervals to estimate 95% con-
fidence intervals (CI) of Bd prevalence in collected samples (Brown et al., 2001). Jeffreys 
intervals have been shown to perform well in estimating binomial CI under a variety of 
conditions (Brown et al., 2001).
We obtained samples from 60 individuals from 3 species: Italian treefrog (Hyla inter-
media), agile frog (Rana dalmatina) and pool frogs (P. sk. esculentus) (Table 1). One adult 
pool frog from Bosco Mesola was positive to Bd; the amount of Bd DNA in the positive 
frog was 0.24 genome equivalents.
The detection of Bd infection in amphibians from the Po River Delta adds evidences 
to the presence of this pathogen in multiple areas of mainland Italy, and improves our 
knowledge of its distribution. Bd prevalence in our populations was probably not high, 
as we detected only one positive sample. On the other hand, in the Bosco Mesola area 
we captured a small number of individuals, therefore the confidence intervals associat-
ed to the prevalence in this area remain wide (Table 1). In this area, further sampling is 
required to assess the actual prevalence of Bd in pool frogs and in other amphibians. Fur-
thermore, sampling should cover multiple seasons, because the prevalence and intensity of 
infection are strongly affected by climatic conditions and may vary seasonally (Pullen et 
al., 2010; Savage et al., 2011).
It should be noted that the only positive sample was from P. sk. esculentus. This result 
was analogous to the findings of Federici et al. (2008) from Piedmont (north-western Ita-
ly): out of 10 species analyzed, they detected Bd in pool frogs only. As this species may 
coexist with Bd without suffering a direct decline, it might act as a reservoir for the patho-
gen, spreading it to other species (Simoncelli et al., 2005; Di Rosa et al., 2007).
The Po River Delta hosts important populations of amphibians (Mazzotti et al., 2007), 
but unfortunately these are threatened by multiple factors, including the salinization of 
breeding wetlands, the isolation of wetlands, and alien invasive species (Ficetola et al., 
2004; Mazzotti, 2007). As a consequences, some threatened amphibians, such as the spa-
defoot toad Pelobates fuscus and the Italian agile frog R. latastei are nearly extinct in the 
area (Mazzotti, 2007). The presence of Bd is a further threat to amphibians, and increas-
es the complexity of management. For instance, captive breeding and translocation pro-
grams are ongoing in the study area for the conservation of several amphibians (Costa 
and Gattelli, in press). The potential impact of Bd should be taken into account during 
these actions, because translocating animals may increase the spread of the pathogen. It 
would be particularly interesting testing whether the declining P. fuscus and R. latastei are 
infected by Bd. Furthermore, a more systematic application of precautionary measures 
to avoid the dissemination of these emerging diseases through human activities is highly 
desirable (Speare et al., 2004; Dejan et al., 2010; Phillott et al., 2010). To date, information 
on the spread of Bd in Italian amphibians remains limited. It is thus important to expand 
the sampling of this pathogen and to evaluate whether it is implicated in the decline of 
other Italian amphibians (Bonardi et al., 2011).
300 G.F. Ficetola et alii
AkNOWLEDGEMENTS
We thank Gregoire Noirjean (PNRPL) for help in the field. This article was written as part of 
a research partnership on amphibian disease between the Parc Naturel Régional Périgord-Limousin, 
the University of Savoie, the University of Milano-Bicocca and the Parco Regionale del Delta del Po 
Emilia-Romagna. Funding was provided by the European Union (FEDER Limousin), the Conseil 
Régional du Limousin and the Association Nationale de la Recherche et de la Technologie (ANR) 
and EU program Biodiversa (RACE). GFF was funded by a by a scholarship of the University of 
Milano Bicocca. The Parco Regionale del Delta del Po Emilia-Romagna provided the authorization 
to perform sampling.
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Batrachochytrium dendrobatidis in amphibians from the Po River Delta, Northern Italy

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