CERTAD2015Peixes Suiça Bom

RESEARCH ARTICLE
Identification of Cryptosporidium Species in

Fish from Lake Geneva (Lac Léman) in France
Gabriela Certad1*, Jean Dupouy-Camet2, Nausicaa Gantois1, Ourida Hammouma-
Ghelboun3, Muriel Pottier4, Karine Guyot1, Sadia Benamrouz1,5, Marwan Osman1,6,
Baptiste Delaire7, Colette Creusy7, Eric Viscogliosi1, Eduardo Dei-Cas1,8†, Cecile
Marie Aliouat-Denis1,4, Jérôme Follet3,9
1 Biologie et Diversité des Pathogènes Eucaryotes Emergents (BDEEP), Centre d'Infection et d'Immunité de
Lille (CIIL), Institut Pasteur de Lille, INSERM U1019, CNRS UMR 8402, Université de Lille, Lille, France,
2 Université Paris Descartes, Assistance Publique Hôpitaux de Paris, Parasitologie-Mycologie, Hôpital
Cochin, Paris, France, 3 Laboratoire de Biotechnologie et Gestion des Agents Pathogènes en Agriculture,
Institut Supérieur d’Agriculture de Lille, Lille, France, 4 Faculté de Pharmacie, Université de Lille, Lille,
France, 5 Ecologie et Biodiversité, Faculté Libre des Sciences et Technologies de Lille, Université
Catholique de Lille, Lille, France, 6 Laboratoire Microbiologie, Santé et Environnement, Centre AZM pour la
Recherche en Biotechnologie et ses Applications, Université Libanaise, Tripoli, Lebanon, 7 Service
d’Anatomie et de Cytologie Pathologiques, Groupe Hospitalier de l’Université Catholique de Lille, Lille,
France, 8 Centre Hospitalier Régional et Universitaire de Lille, Université Lille Nord de France, Lille, France,
9 Laboratoire BioMEMS, Univ.Lille, CNRS, ISEN, Univ.Valenciennes, UMR 8520, IEMN, Institut
d'Electronique de Microélectronique et de Nanotechnologie, F 59 000, Lille, France
† Deceased.
OPEN ACCESS * [email protected]
Citation: Certad G, Dupouy-Camet J, Gantois N,

Hammouma-Ghelboun O, Pottier M, Guyot K, et al.
(2015) Identification of Cryptosporidium Species in Abstract
Fish from Lake Geneva (Lac Léman) in France. PLoS
ONE 10(7): e0133047. doi:10.1371/journal. Cryptosporidium, a protozoan parasite that can cause severe diarrhea in a wide range of
pone.0133047
vertebrates including humans, is increasingly recognized as a parasite of a diverse range of
Editor: Gordon Langsley, Institut national de la santé wildlife species. However, little data are available regarding the identification of Cryptospo-
et de la recherche médicale - Institut Cochin,
ridium species and genotypes in wild aquatic environments, and more particularly in edible
FRANCE
freshwater fish. To evaluate the prevalence of Cryptosporidiumspp. in fish from Lake
Received: April 14, 2015
Geneva (Lac Léman) in France, 41 entire fish and 100 fillets (cuts of fish flesh) were col-
Accepted: June 22, 2015 lected from fishery suppliers around the lake. Nested PCR using degenerate primers fol-
Published: July 27, 2015 lowed by sequence analysis was used. Five fish species were identified as potential hosts
Copyright: © 2015 Certad et al. This is an open
of Cryptosporidium: Salvelinus alpinus, Esox lucius, Coregonus lavaretus, Perca fluviatilis,
access article distributed under the terms of the and Rutilus rutilus. The presence of Cryptosporidium spp. was found in 15 out of 41 fish
Creative Commons Attribution License, which permits (37%), distributed as follows: 13 (87%) C. parvum, 1 (7%) C. molnari, and 1 (7%) mixed
unrestricted use, distribution, and reproduction in any
infection (C. parvum and C. molnari). C. molnari was identified in the stomach, while C. par-
medium, provided the original author and source are
credited. vum was found in the stomach and intestine. C. molnari was also detected in 1 out of 100
analyzed fillets. In order to identify Cryptosporidium subtypes, sequencing of the highly
Data Availability Statement: All relevant data are
within the paper. polymorphic 60-kDa glycoprotein (gp60) was performed. Among the C. parvum positive
samples, three gp60 subtypes were identified: IIaA15G2R1, IIaA16G2R1, and IIaA17G2R1.
Funding: This work was supported by the French
National Research Agency (grant no. ANR-10-ALIA- Histological examination confirmed the presence of potential developmental stages of
004) and the regional competitiveness center, C. parvum within digestive epithelial cells. These observations suggest that C. parvum is
AQUIMER (Boulogne s/mer, France). infecting fish, rather than being passively carried. Since C. parvum is a zoonotic species,
Competing Interests: The authors have declared fish potentially contaminated by the same subtypes found in terrestrial mammals would be
that no competing interests exist.
PLOS ONE | DOI:10.1371/journal.pone.0133047 July 27, 2015 1 / 13

Cryptosporidium Species in Fish from Lake Geneva, France
an additional source of infection for humans and animals, and may also contribute to the
contamination of the environment with this parasite. Moreover, the risk of human transmis-
sion is strengthened by the observation of edible fillet contamination.
Introduction
Cryptosporidium, a protozoan parasite that can cause severe diarrhea in a wide range of verte-
brates including humans, is increasingly recognized as a parasite of a diverse range of wildlife
species, including mammals, birds, reptiles, amphibians, and fish [1]. Although the epidemiol-
ogy of cryptosporidiosis has been widely reported worldwide for different groups of animals,
little biological, epidemiological and molecular data are available on Cryptosporidium infection
in fish, even though the parasite has been already described and genetically characterized in
more than 20 species of both freshwater and marine fish. Cryptosporidium molnari, the only
currently recognized species infecting fish, was first identified in sea bream (Sparus aurata)
and European sea bass (Dicentrarchus labrax) [2]. Cryptosporidium scophthalmi was detected
in turbot (Psetta maxima, syn. Scophthalmus maximus) [3], but this species is still considered a
nomen nudum due to a lack of genetic data [4].
Cryptosporidium species found in other groups of vertebrates have also been identified in
fish, including C. parvum, C. hominis, C. scrofarum and C. xiaoi. Additionally, eight Cryptospo-
ridium fish genotypes, and one Cryptosporidium rat III-like genotype, have been described in
fish [4]. Recently, the species name Cryptosporidium huwi has been proposed for the piscine
genotype 1 from the guppy (Poecilia reticulata) to reflect its genetic and biological differences
from gastric and intestinal Cryptosporidium species [5].
In fish hosts, Cryptosporidium fish species and genotypes are located either in the stomach
or intestine, as attested by histological analyses. Moreover, it has been reported that the parasite
can cause clinical manifestations, such as emaciation, decrease in growth rate, anorexia, whitish
feces, abdominal swelling, and ascites [2,3]. An increase in the mortality rate associated with
Cryptosporidium infection has also been reported, particularly in larval and juvenile infected
fish [6]. A significant correlation was found between the presence of the parasite and both fish
weight and seasonality, the rate of infection being higher in fish weighing less than 100 grams
and in the spring [7]. In addition, a relationship was observed between the presence of the par-
asite and the production stage in farmed fish [7].
It is notable that many results relating to fish Cryptosporidium infection were reported in
farmed or aquarium fish [2,7,8]. However, little data are currently available regarding the
molecular identification of Cryptosporidium species and genotypes in wild fish populations
and, in particular, in edible fish. Indeed, only two studies have been conducted in Australia and
Papua New Guinea on wild marine and freshwater fish [9,10].
Therefore, the aim of our study was to evaluate the prevalence of Cryptosporidium species/
genotypes in freshwater edible fish hosts from Lake Geneva in France. Lake Geneva is located
between Switzerland and France, and is the largest freshwater reservoir in Western Europe,
with a surface area of 580 km2, a volume of 89 km3, and a maximum depth of 309 m (Fig 1).
More than 1.5 million people (in France and Switzerland) live around this lake [11]. In addi-
tion, the local fish are quite often consumed as raw preparations by the local population at
home or in restaurants located around the shores of the lake. Fish are also a source of income,
as around 150 professional fishermen are registered as active on the lake.

Fig 1. Map of the study area and sampling site (Thônon-les-Bains). Effluent from wastewater treatment plants discharged into the Lake Geneva
catchment area (CIPEL: Commission Internationale pour la Protection des Eaux du Léman).
doi:10.1371/journal.pone.0133047.g001
Materials and Methods

Fish sampling
A total of 41 adult fish were purchased directly on the shores of the lake from local fishermen
of Thônon-les-Bains (geographic coordinates: 46° 22' 0" North, 6° 29' 0" East), or Sechex, a
small village located eight kilometers West of Thônon-les-Bains, in November 2011 (fall) and
April 2013 (spring) (Fig 1). The weight, size, sex, origin and sexual maturity of each individual
were determined (Table 1). For each fish, scratchings of the gastric and intestinal epithelia were
performed after dissection, and the cells were preserved in the fixative RCL2 and stored at
-20°C. A section of the stomach and bowel were also fixed in 10% buffered formalin. One hun-
dred additional fillets (only cuts of fish flesh without viscera) of European perch (Perca fluviati-
lis) were purchased from the fishermen of Thônon-les-Bains, or St Gingolph (27 km Eeast of
Thônon-les-Bains near the Swiss border, geographical coordinates: 46° 23' 0" North, 6° 40' 0"
East) to evaluate potential contamination with Cryptosporidium spp. at this location. Slices of
2–3 mm were sampled and stored at—20°C in RCL2. No approval from Institutional Animal
Care and Use Committee or ethics committee was necessary as no experiments that involved
alive fish were performed. All fish examined were bought dead from professional fishermen,
fishmongers and supermarkets selling fresh fish for consumption. Therefore, no sacrificial

method was required. No fish sampled in this work was captured in a protected area and conse-
quently, our sampling protocol did not need any specific permission for the location. Finally,
no specimen included in the present work involved endangered or protected species.
Table 1. Freshwater fish specimens collected in Lake Geneva.
Fish code Fish species Common name Size (cm) Weight (g) Sexual maturity Sex Presence of other parasites
5301 Salvelinus alpinus Arctic char 40 610 ND Male Cestoda
5307 Esox lucius Northern pike 37 328 ND Female Cestoda
5308 Esox lucius Northern pike 35 328 No Female Cestoda
5309 Lota lota Burbot 29 134 ND Male Cestoda
5310 Lota lota Burbot 24 96 No Female Nematoda
5311 Coregonus lavaretus European whitefish 31 276 No Female No
5312 Coregonus lavaretus European whitefish 33 264 ND Male No
5313 Coregonus lavaretus European whitefish 29 232 ND Male Cestoda
5316 Coregonus lavaretus European whitefish 22 84 ND ND Cestoda
5317 Perca fluviatilis European perch 11 16 ND Male Cestoda
5320 Perca fluviatilis European perch 11 16 ND ND Cestoda
5321 Perca fluviatilis European perch 42 1500 Yes Female Acantocephala
5322 Perca fluviatilis European perch 29 318 No Female Trematoda digenea
5323 Perca fluviatilis European perch 26 220 Yes Female Cestoda
5324 Perca fluviatilis European perch 21 124 No Female Trematoda digenea
5325 Rutilus rutilus Roach 18 42 No Female Cestoda
5326 Lota lota Burbot 31 222 No Female No
5327 Esox lucius Northern pike 53 1800 Yes Female Cestoda
5328 Lota lota Burbot 25 97 ND Male Microsporidia
5329 Coregonus lavaretus European whitefish 40 54 Yes Female Cestoda
5332 Coregonus lavaretus European whitefish 40 54 Yes Female No
5333 Coregonus lavaretus European whitefish 39 54 Yes Female No
5334 Esox lucius Northern pike 69 2600 ND Male Monogenea
5335 Squalius cephalus European chub 52 2300 Yes Female Monogenea
5336 Esox lucius Northern pike 60 2000 Yes Female Monogenea
5337 Perca fluviatilis European perch 27 30 Yes Female Acantocephala
5338 Abramis brama Common bream 50 2000 ND Male Trematoda digenea
5339 Perca fluviatilis European perch 15 45 Yes Female Trematoda digenea
5341 Perca fluviatilis European perch 16 56 Yes Female No
Fish from 5301 to 5326 were purchased from the fishermen of Thônon-les-Bains; Fish from 5327–5341 were purchased from the fishermen of Sechex
ND: Not determined.
doi:10.1371/journal.pone.0133047.t001

DNA extraction
Genomic DNA extraction was performed on 96-well plates, using the NucleoSpin Kit
(Macherey-Nagel, GmbH & Co KG, Germany) according to the manufacturer’s protocol. The
final DNA elution was 100 μl.
Primer design and nested PCR

An alignment of the 18S rRNA gene sequences obtained from Cryptosporidium isolates charac-
terized in fish (GenBank accession numbers: FJ769050, HM243547, HM243548, HM243549,
HM243550, JF285332, JF285333, AY524773, HM989832, HM989833, HM989834, HM991857,
GQ925452) [7] was performed using the BioEdit v7.0.1 package (http://www.mbio.ncsu.edu:
BioEdit/bioedit.html). After identification of a target DNA fragment for nested 18S PCR com-
mon to all sequences, two sets of generate primers were selected within the hypervariable
region. These degenerate primers were modified from those proposed by Ryan et al [12]. The
external primer pair JerExtF (5’-GACATATCWTTYAAGTTTCTGACC-3’) (base pair posi-
tion 292) and JerExtR (5’-CTGAAGGAGTAAGGAACAACC-3’) (base pair position 1007)
amplified a DNA fragment of 784 bp. The internal primer pair JerIntF (5’-CCTATCAGCTTT
MGACGGTAGG-3’) (base pair position 289) and JerIntR (5’-TCTAAGAATTTCACCTCTG
ACTG-3’) (base pair position 851) resulted in the amplification of a DNA fragment of 588 bp.
For the first round of amplification, the PCR mixture contained 10 μl of DNA, 1x HotStarTaq
Plus buffer, 2 mM MgCl2, 0.4 μM for each primer, 200μM dNTP each and 1.5U HotStarTaq
Plus DNA polymerase (Qiagen Inc., Valencia, California) in a final volume of 50 μl. The PCR
conditions were as follows: a denaturation step at 94°C for 10 min, followed by 40 cycles of
94°C for 45 sec, annealing at 67°C for 45 sec, and extension at 72°C for 1 min. The post-exten-
sion was completed at 72°C for 5 min. The second PCR amplification was performed in a 50 μl
reaction volume containing 2 μl of the primary PCR product, 1xHotStarTaq Plus buffer, 3 mM
MgCl2, 0.4 μM for each primer, 200 μM dNTP each and 1.5 U HotStarTaq Plus DNA polymer-
ase. The PCR conditions were identical to those in the first round. Nested 18S PCR reactions
were conducted using a PTC 200 thermocycler (MJ Research, Waltham, USA). The PCR prod-
ucts were analyzed on a 2% agarose gel and visualized by ethidium bromide staining.
DNA sequencing and analysis

To identify Cryptosporidium species at the molecular level, positive nested 18S PCR products
were purified and sequenced directly on both strands, using the forward and reverse primers
from the second round, by the company Genoscreen (Institut Pasteur de Lille, France). The
sequences obtained were aligned using the BioEdit v7.0.1 package, and then compared with the
sequences of Cryptosporidium published on the NCBI server (http://www.ncbi.nlm.nih.gov/
BLAST/) using the basic local alignment search tool (BLAST) program. Isolates genotyped as
C. parvum were further subtyped using a second nested PCR that amplifies a fragment of the
60 kDa glycoprotein (gp60) gene, as described [13]. The amplified DNA fragments were puri-
fied, sequenced, and analyzed as described above.
Histological analysis
The stomach and intestine of the fish were removed, fixed in 10% buffered formalin, and paraf-
fin-embedded specimens were sectioned to a thickness of 5 μm to be processed using standard
staining techniques (Hematoxylin & Eosin). Inflammation in digestive sections was scored as
follows: 0, no inflammation; +1, moderate inflammation, focally distributed; +2, moderate
inflammation, widely distributed; +3, severe inflammation, widely distributed throughout the

section. The sections were examined by a pathologist using a Leica DMRB microscope
equipped with a Leica digital camera connected to an Imaging Research MCID analysis system
(MCID Software, Cambridge, UK).
Nucleotide sequence accession numbers

The 18S rRNA nucleotide sequences obtained in this study were deposited in the GenBank
database under the accession numbers KP939333-KP939354.
Results
The molecular analysis of digestive tissues identified the presence of Cryptosporidium spp. in
15 out of 41 fish, representing a frequency of 37%. The fish species Arctic char (Salvelinus alpi-
nus) (4/6), Northern pike (Esox lucius) (2/5), European whitefish (Coregonus lavaretus) (4/11),
European perch (Perca fluviatilis) (4/12), and roach (Rutilus rutilus) (1/1) were identified as
potential new hosts for Cryptosporidium spp. (Table 2).
The sequence analysis of the 18S rDNA locus identified two species of Cryptosporidium, dis-
tributed as follows: 13 C. parvum (87%), 1 C. molnari (7%), and 1 mixed infection (C. molnari
and C. parvum) (7%). In 9 of the 15 infected fish, the presence of Cryptosporidium spp. was
found either in the stomach or intestine, while in the 6 remaining infected fish, Cryptosporid-
ium spp. were present in both organs. The selective extraction of DNA from these organs, fol-
lowed by nested 18S PCR and sequencing, confirmed the presence of C. molnari only in the
stomach of fish, while C. parvum was found in both stomach and intestine. Among the stom-
ach samples, two were positive for C. molnari, and 10 were positive for C. parvum. Among the
intestinal samples, eight were positive for C. parvum only. The 18S rRNA gene sequences of 5
out of 19 isolates of C. parvum found either in the stomach or intestine were 100% identical to
that of a previously described species of C. parvum (GenBank: KJ939305 [7]), while 14 isolates
exhibited single nucleotide polymorphisms (SNPs). It is common to identify sequence differ-
ences and variations such as single nucleotide polymorphisms (SNPs) that can be associated
to genetic diversity according to the degree of homology. SNPs were distributed as follows:
only 1 SNP for 8 isolates, 2 SNPs for 4 isolates, 3 SNPs for 1 isolate, and 4 SNPs for one isolate
(Table 2). All SNPs identified in the C. parvum isolates corresponded to transition mutations.
The two isolates identified as C. molnari were identical but showed 9 SNPs in comparison to
the C. molnari reference sequence (GenBank: HM243550[7] (Table 2). In particular, 5 SNPs
were associated with transition mutations, and 4 SNPs (in positions 322, 330, 378, 384) were
associated with transversion mutations between adenine and thymine (A/T). The SNPs could
not be associated with a specific sampling site (gastric vs. intestinal site) or with a specific fish
species.
In order to identify Cryptosporidium subtypes, sequencing of the highly polymorphic
60-kDa glycoprotein (gp60) was performed. Partial sequences of the gp60 gene were subse-
quently obtained for 13 isolates identified as C. parvum. Three different subtypes were identi-
fied as follows: IIaA15G2R1 (6/13), IIaA17G2R1 (5/13), and IIaA16G2R1 (2/13) (Table 2).
Following histological examination of sections either from the stomach or intestine, the
presence of Cryptosporidium-like bodies within the cells of the digestive epithelium was con-
firmed in samples from 10 C. parvum-positive fish (Fig 2A, 2B and 2C; Table 3). An inflamma-
tory reaction with leukocyte infiltration was observed occasionally. The presence of other
intestinal parasites, identified as nematodes, was confirmed in histological sections of two fish
(Fig 2D, Table 2). The histological analysis of the remaining fish was not possible due to autoly-
sis of tissues.

Table 2. Cryptosporidium species and subtypes in wild freshwater fish from Lake Geneva identified at the 18S rDNA and GP60 loci.
Code Fish species Fish common Organ Cryptosporidium Percentage of identity with SNP** SNP** GP60
name species 18S reference sequence * position
5302 Salvelinus Arctic char Intestine C. parvum 99.8% 347 T/C NA
alpinus
5303 Salvelinus Arctic char Intestine C. parvum 99.6% 347 T/C IIaA17G2R1
alpinus
435 C/T
5304 Salvelinus Arctic char Stomach C. parvum 99.8% 390 G/A IIaA15G2R1
alpinus
Intestine C. parvum 99.8% 145 A/G IIaA15G2R1
5305 Salvelinus Arctic char Intestine C. parvum 99.6% 300 T/C IIaA15G2R1
alpinus
507 A/G
5307 Esox lucius Northern pike Stomach C. molnari 98.3% 314 A/T NA
322 T/A
324 T/C
329 C/T
341 A/G
370 A/T
376 A/T
377 C/T
506 G/A
Intestine C. parvum 99.4% 244 G/A IIaA17G2R1
347 T/C
496 T/C
5308 Esox lucius Northern pike Stomach C. molnari 98.3% 314 A/T NA
322 T/A
324 T/C
329 C/T
341 A/G
370 A/T
376 A/T
377 C/T
506 G/A
5311 Coregonus European Stomach C. parvum 100% - - IIaA15G2R1
lavaretus whitefish
Intestine C. parvum 99.8% 437 T/C IIaA17G2R1
5312 Coregonus European Stomach C. parvum 99.6% 324 T/C IIaA17G2R1
475 T/C
Coregonus European Intestine C. parvum 99.2% 87 A/G -
151 A/G
390 G/A
491 T/C
5314 Coregonus European Stomach C. parvum 99.8% 235 A/G -
5316 Coregonus European Stomach C. parvum 99.8% 390 G/A -
(Continued)

Table 2. (Continued)
Code Fish species Fish common Organ Cryptosporidium Percentage of identity with SNP** SNP** GP60
name species 18S reference sequence * position
5318 Perca fluviatilis European Stomach C. parvum 99.6% 300 T/C IIaA15G2R1
perch
507 A/G
5320 Perca fluviatilis European Stomach C. parvum 99.8% 211 C/T -
perch
5322 Perca fluviatilis European Stomach C. parvum 99.8% 27 G/A IIaA16G2R1
perch
Intestine C. parvum 100% - - IIaA16G2R1
5323 Perca fluviatilis European Stomach C. parvum 100% - - IIaA15G2R1
perch
5325 Rutilus rutilus Roach Stomach C. parvum 100% - - IIaA17G2R1
Intestine C. parvum 100% - - -
* The reference sequences for C. parvum and C. molnari are: KJ939305 and HM243550, respectively.
**SNP: Single nucleotide polymorphism
NA: Not available.
The potential contamination of fish flesh with Cryptosporidium spp. was evaluated. One
hundred fish fillets of European perch (Perca fluviatilis) were analyzed by nested PCR and
sequencing. The presence of C. molnari was detected in only one fillet. The 18S rRNA gene
sequence of this C. molnari isolate was identical to that of the two isolates found in the stomach
of two Northern pike (Table 2).
Discussion
This study reports the first epidemiological and molecular data on the presence of Cryptospo-
ridium in fish in France. The overall frequency of Cryptosporidium spp. in fish sampled from
Fig 2. Stained sections of the digestive tract of fish. A. Presence of round bodies suggestive of the
developmental stages of C. parvum was observed in the apical position (arrows) within the epithelial cells of
gastric glands. Bar = 25 μm. B. Presence of round bodies suggestive of the developmental stages of C.
parvum observed in the apical position (arrows) within the intestinal epithelial cells. Bar = 75 μm. C. Presence
of round bodies suggestive of the developmental stages of C. molnari observed in the apical position (arrows)
within the epithelial cells of gastric glands. Bar = 25 μm. D. Section of a nematode in the intestinal mucosa,
surrounded by a severe inflammatory reaction. Bar = 200 μm. Hematoxylin & Eosin staining.
doi:10.1371/journal.pone.0133047.g002

Table 3. Histological examination of digestive organs of different fish species from Lake Geneva infected by Cryptosporidium spp.
Fish Fish species Fish common Organ Histological examination* Cryptosporidium species (18S
code name rDNA)
5302 Salvelinus alpinus Arctic char Intestine Inflammation: +1 C. parvum
Intracellular Cryptosporidium-like bodies
5303 Salvelinus alpinus Arctic char Intestine ND C. parvum
5304 Salvelinus alpinus Arctic char Stomach Inflammation: 0 Intracellular C. parvum
Cryptosporidium-like bodies
Intestine ND C. parvum
5305 Salvelinus alpinus Arctic char Intestine Inflammation: +1 C. parvum
Intracellular Cryptosporidium-like bodies
5307 Esox lucius Northern pike Stomach ND C. molnari
Intestine ND C. parvum
5308 Esox lucius Northern pike Stomach Zones of autolysis C. molnari
Inflammation: 0 Intracellular Cryptosporidium-like
bodies
5311 Coregonus European whitefish Stomach ND C. parvum
lavaretus
Intestine Zones of autolysis C. parvum
bodies
5312 Coregonus European whitefish Stomach Zones of autolysis C. parvum
lavaretus
bodies
Intestine Zones of autolysis C. parvum
bodies
5314 Coregonus European whitefish Stomach Inflammation: 0 Intracellular Cryptosporidium-like C. parvum
lavaretus bodies
5316 Coregonus European whitefish Stomach Inflammation: 0 Intracellular Cryptosporidium-like C. parvum
lavaretus bodies
5318 Perca fluviatilis European perch Stomach Inflammation: +3 C. parvum
Presence of a nematode
5320 Perca fluviatilis European perch Stomach Autolysis C. parvum
5322 Perca fluviatilis European perch Stomach ND C. parvum
Intestine Inflammation: +3 C. parvum
Presence of a nematode
5323 Perca fluviatilis European perch Stomach Inflammation: 0 Intracellular Cryptosporidium-like C. parvum
bodies
5325 Rutilus rutilus Roach Stomach Inflammation: 0 Intracellular Cryptosporidium-like C. parvum
bodies
Intestine Inflammation: 0 Intracellular Cryptosporidium-like C. parvum
bodies
ND: Not done

* Inflammation in digestive sections was scored as follows: 0, no inflammation; +1, moderate inflammation, focally distributed; +2, moderate inflammation,
widely distributed; +3, severe inflammation, widely distributed throughout the section.
Lake Geneva was high, reaching 37%. Previous studies have reported a high prevalence of Cryp-
tosporidium spp. in fish, but mainly in juvenile marine fish. For instance, Sitjà-Bobadilla et al.
reported 100% C. scophthalmi prevalence in juvenile turbot in Europe [7]. In contrast, a recent

study in Australia found no Cryptosporidium isolates in freshwater fish [9], while a Cryptospo-
ridium prevalence of 0.2% was found in wild freshwater species in Papua New Guinea [10].
Therefore, even if the comparative data is scarce, this is to our knowledge the first time that
Cryptosporidium has been detected at a very high prevalence in freshwater fish.
Five new species of fish hosts for Cryptosporidium were identified: Arctic char (Salvelinus
alpinus), Northern pike (Esox lucius), European whitefish (Coregonus lavaretus), European
perch (Perca fluviatilis) and Roach (Rutilus rutilus). Although it is generally accepted that the
prevalence of Cryptosporidium is higher in juvenile fish, all of the fish analyzed in our study
were adults, according to size and weight, and according to sexual maturity when this parame-
ter could be determined (Table 1).
Two species of Cryptosporidium were detected in fish hosts: C. molnari and C. parvum. C.
molnari was identified in freshwater aquaculture fish [14], but this is the first time that this par-
asite species has been found in wild freshwater fish. The 18S rRNA gene sequences of the 3 C.
molnari isolates identified in our study were 98% identical to those of the C. molnari reference
sequences collected from the databases. Interestingly, these three sequences amplified from dif-
ferent individuals presented the same points of mutation, suggesting the circulation of the
same parasite isolates in the lake environment.
A matter of importance to public health was the high rate of detection of C. parvum among
fish hosts, as this species is the most common source of zoonotic infections [4]. Previous stud-
ies in Papua New Guinea and Australia also reported consistent detection of C. parvum in fish
[9,10]. We speculate that the presence of C. parvum, and in particular the IIa subtype, in fish
samples from Lake Geneva could be due to waterborne contamination with human and animal
waste. In fact, the zoonotic C. parvum IIa subtype family has predominantly been found in
calves and humans in North America, Europe, and Australia [15,16]. In addition, even if we
did not search for the presence of Cryptosporidium in the lake water, it is well known that Cryp-
tosporidium oocysts are found in groundwater, lakes, rivers, estuaries, and oceans, as a conse-
quence of the great amount of feces from humans, pets, and domesticated or wild animals that
is discharged, dumped, or carried in runoff into these waters [17]. In particular, in Lake
Geneva, an increase in fecal bacteria of human and animal origin was described in sediment
contaminated with wastewater treatment plant effluent, suggesting the presence of both
human and animal sources of fecal pollution in the lake environment [11]. In parallel, it has
been suggested that when fecal bacteria is present in water, Cryptosporidium could be present
as well, and even though water quality monitoring and water treatment can reduce the presence
of pathogens, they do not ensure absolute safety, due to the fact that Cryptosporidium oocysts
are highly resistant [18].
Partial sequences of the gp60 gene subsequently amplified from C. parvum isolates
allowed the identification of three different subtypes belonging to the IIa family, as follows:
IIaA15G2R1, IIaA16G2R1, and IIaA17G2R1. The IIaA15G2R1 subtype has also been identi-
fied consistently in Papua New Guinea in mackerel scad, Decapterus maracellus [10], a wild
marine fish. The subtypes IIaA15G2R1 and IIaA17G2R1 have been identified in cattle [19], the
first of which is the most dominant zoonotic Cryptosporidium subtype infecting dairy cattle
and humans in industrialized countries [4]. Indeed, the IIaA15G2R1 subtype represents up to
75% of the identified Cryptosporidium subpopulation in French calves [16]. On the other hand,
the IIaA16G2R1 subtype has been identified in diarrheic calves [20–22] and also in wild boars
(Sus scrofa) [23]. In rural areas, it is well known that animals can cohabit with livestock, often
by sharing grazing and water sources. Other zoonotic Cryptosporidium species already identi-
fied in marine fish such as C. hominis C. xiaoi and C. scrofarum were not found in this study
[4].

Since C. parvum is a zoonotic species, fish potentially contaminated by the same subtypes
infecting terrestrial mammals would be an additional source of infection for humans and other
animals, and may also contribute to the contamination of the environment with this parasite.
However, it is not clear if fish are only carriers of C. parvum, or if C. parvum can develop its life
cycle and multiply in this fish host.
In order to clarify this question, histological analysis of digestive tissues from C. parvum-
positive fish was performed, and round bodies suggestive of C. parvum developmental stages
were observed in an apical position within the cells, either in the stomach or intestine. These
observations suggest that C. parvum is actually infecting fish, rather than being passively car-
ried. Fluorescent-antibody staining assay using an anti-Cryptosporidium antibody (Crypto Cel
immunofluorescence test, Cellabs, Brookvale, New South Wales, Australia) was tried to con-
firm the detection of Cryptosporidium oocysts from fish tissues but unfortunately, no signal
was detected. This failure was probably due to the vulnerability of the oocyst antigens to forma-
lin as was already described, particularly after more than one month of formalin fixation which
was the case in our study [14]. Mild to moderate inflammation was occasionally found in gas-
trointestinal tissues, but we could not determine whether it was Cryptosporidium that was caus-
ing this reaction, since co-infection with other parasites was present. In some cases, the
histological analysis of fish was not possible due to autolysis of tissues.
Furthermore, to evaluate a potential contamination of fish fillets with Cryptosporidium spp.,
100 fish fillets of European perch (Perca fluviatilis) were analyzed by nested 18S PCR and
sequencing, and the presence of C. molnari was detected in fillets from one individual. Fillet
contamination with C. molnari could occur as a consequence of evisceration of the infected
fish during the cleaning and preparation process. Although previous studies have shown no
conclusive evidence of transmission of fish-hosted Cryptosporidium to mammals [24], the
presence of the parasite also in fillets clearly highlights the risk of Cryptosporidium infection to
humans, either when handling fish or consuming raw or undercooked fish carrying zoonotic
species of Cryptosporidium. In our study, only C. molnari, apparently a non-pathogenic species
for humans, was isolated from perch fillets. However, C. parvum isolated from the fish digestive
tract could certainly be present in fish fillets. Further studies should be done to clarify this
aspect.
One study in Maryland consistently reported that urban anglers are at a risk of contracting
cryptosporidiosis from exposure received while fishing and consuming caught fish with a
mean probability of infection of almost one [25]. Another study showed that blue crabs can
transfer C. parvum oocysts to people who handle the crustaceans [26]. In addition, it has been
reported that immunosuppressed patients are at risk of contracting cryptosporidiosis, either by
contact with fish during preparation and handling, or by consumption of undercooked fish
[27].
It was not unexpected to find Cryptosporidium in fish from Lake Geneva, as this parasite
has already been found to be responsible for a human outbreak occurring in 2003 due to the
contamination of the water supply network, in the nearby city of Divonne-les-Bains, affecting
more than 700 individuals [28]. In addition, in Switzerland, a study reported the presence of C.
parvum in samples collected from the drinking water distribution system in alpine rural
regions, and it was suspected that the drinking water was contaminated by grazing cattle [29].
However, future studies should be conducted to detect the presence of the parasite in the lake
environment.
In conclusion, these findings suggest that the transmission of Cryptosporidium could poten-
tially occur in the interfaces between human, livestock, and fish populations. In fact, the wide
host range of Cryptosporidium spp., together with the high output of oocyst shedding, allows a
high level of contamination of the environment [23]. In particular, for fish hosts, the dispersion

and transmission of zoonotic parasites would be facilitated by the aquatic habitat of the host
that could potentially release fully sporulated oocysts contributing to the perpetuation of Cryp-
tosporidium circulation. Finally, fish may be a good sentinel for the detection of water contami-
nation caused by sewage or agricultural runoff.
Acknowledgments
We wish to thank Dr. Slapeta for kindly providing us with the DNA from Cryptosporidium
molnari, used as a positive control in our PCR, the CIPEL (Commission Internationale pour la
Protection des Eaux du Léman) for its assistance in the design of Fig 1 and the Fish-Parasites
Network.
Author Contributions
Conceived and designed the experiments: GC JDC KG EDC CMA JF. Performed the experi-
ments: GC JDC NG OHG MP KG SB MO BD CC EDC. Analyzed the data: GC NG KG SB EV
CMA JF. Contributed reagents/materials/analysis tools: CC. Wrote the paper: GC JDC EV
CMA JF.
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RESEARCH ARTICLE

Identification of Cryptosporidium Species in

Citation: Certad G, Dupouy-Camet J, Gantois N,

PLOS ONE | DOI:10.1371/journal.pone.0133047 July 27, 2015 1 / 13

PLOS ONE | DOI:10.1371/journal.pone.0133047 July 27, 2015 2 / 13

Materials and Methods

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Table 1. Freshwater fish specimens collected in Lake Geneva.

PLOS ONE | DOI:10.1371/journal.pone.0133047 July 27, 2015 4 / 13

Primer design and nested PCR

DNA sequencing and analysis

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Nucleotide sequence accession numbers

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ND: Not done

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PLOS ONE | DOI:10.1371/journal.pone.0133047 July 27, 2015 11 / 13

PLOS ONE | DOI:10.1371/journal.pone.0133047 July 27, 2015 12 / 13

PLOS ONE | DOI:10.1371/journal.pone.0133047 July 27, 2015 13 / 13

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