Journal of Fish Diseases 2007, 30, 123–126
Short communication
Differentiation between a pathogenic and a non-pathogenic
form of Gyrodactylus salaris using PCR-RFLP
P W Kania, T R Jørgensen and K Buchmann
Section of Fish Diseases, Department of Veterinary Pathobiology, Royal Veterinary and Agricultural University,
Frederiksberg C, Denmark
Keywords: diagnosis, Gyrodactylus salaris, internal
transcribed spacer, polymerase chain reaction,
restriction fragment length polymorphism, salmo-
nids.
Forms of the monogenean ectoparasite Gyrodactylus
salaris Malmberg (found in Norway and Sweden)
are highly pathogenic to East-Atlantic salmon,
Salmo salar L., colonizing rivers draining into the
Atlantic. Several salmon stocks in Europe are as
susceptible and vulnerable as Norwegian salmon,
which is the background for implementation of
countermeasures against further spreading of the
parasite in Europe (Peeler 2004). Scottish, Irish and
Danish salmon will allow marked parasite popula-
tion increase following exposure to some of the
Norwegian forms of the parasite whereas Russian,
Finnish and Swedish salmon show limited suscep-
tibility (Lindenstrøm, Sigh, Dalgaard & Buchmann
2006). Current methods for diagnosis of the species
are based on light microscopic examination of
parasite mounts, DNA sequencing of various genes
or polymerase chain reaction-restriction fragment
length polymorphism (PCR-RFLP) (rDNA). The
PCR-RFLP method is based on differences in the
DNA banding pattern obtained following PCR
amplification of the internal transcribed spacer
(ITS) region (1300 bp) in the parasite and subse-
quent digestion by specific restriction enzymes
Correspondence K Buchmann, Section of Fish Diseases,
Department of Veterinary Pathobiology, Royal Veterinary and
Agricultural University, Stigbøjlen 7, DK-1870 Frederiksberg C,
Denmark
 2007 The Authors. (e-mail: kub@kvl.dk)
Journal compilation
 2007
Blackwell Publishing Ltd 123
(Cunningham 1997). This will allow differentiation
between different species within the genus Gyrod-
actylus but it can not differentiate between forms
within the parasite species G. salaris. The recent
discovery in Denmark of a non-pathogenic form of
G. salaris has implications for management of
disease-free zones in Europe (Jørgensen, Larsen,
Kania, Jørgensen, Bresciani & Buchmann 2006)
and the availability of a reliable diagnostic system
for detection of this form is needed. The present
work exploits the detection of a mutation in the ITS
region of the non-pathogenic form of the parasite
and the subsequent development of a PCR-RFLP
method for differentiation between the pathogenic
and non-pathogenic forms.
Rainbow trout, Oncorhynchus mykiss (Walbaum),
infected with G. salaris were obtained from a
freshwater trout farm in Jutland (western Denmark)
and used as source for production of micro-
populations on pathogen-free rainbow trout (Jør-
gensen et al. 2006). For comparison, G. salaris
obtained from Norway (River Lærdalselva form)
was kept under similar conditions (in the same
laboratory) but on susceptible Atlantic salmon from
the Danish River Skjernaa (Dalgaard, Nielsen &
Buchmann 2003). In order to compare with
another species within the genus Gyrodactylus
specimens of G. derjavini were collected from the
same laboratory (Buchmann & Uldal 1997) and
treated similarly. Water and water quality para-
meters used for all fish holding activities were
described previously (Jørgensen et al. 2006).
Specimens of G. salaris of the form isolated in
a Danish rainbow trout farm and from the
 Journal of Fish Diseases 2007, 30, 123–126
Norwegian River Laerdalselva strain obtained from
a laboratory population kept on susceptible salmon
were previously collected and described based on
the ammonium picrate glycerine technique (Malm-
berg 1970) or SEM (scanning electron microscopy)
(Jørgensen et al. 2006).
Parasites were lysed by protease treatment and
PCR was subsequently performed. Thus, parasites
conserved in 96% alcohol were dried and placed in
200 lL PCR tubes containing 7.5 lL lysis buffer
[Tween 20 (0.45%), Proteinase K (60 lL mL)1),
10 mm Tris and 1 mm EDTA] at 65 C until
complete digestion of soft parts (confirmed by
microscopy at ·50). Subsequent inactivation of the
protease was performed at 95 C for 10 min. PCR
was performed with the use of Taq polymerase
(Bioline no. BIO21040: Bioline Ltd., London, UK)
at 1.5 mm MgCl2, with a 1 min elongation time at
55 C and 32 cycles in a Biometra T3 Thermocy-
cler. The primers used were: forward primer
GyroITS1 TTTCCGTAGGTGAACCT and re-
verse primer GyroITS2 TCCTCCGCTTAGTG-
ATA (Cunningham 1997). The PCR products were
digested at 37 C for 2 h either by the use of
restriction enzymes BsuRI (Fermentas ER015:
Fermentas Inc., Hamover, MD, USA) (an isoschiz-
omer of HaeIII) or FspB (Fermentas ER1761) (an
isoschizomer of MaeI) and stopped at 80 C and
65 C (20 min), respectively. The latter enzyme
recognizes and cleaves the restriction site CTAG.
Products (digested and non-digested) were analysed
on 2% agarose gels (TBE buffer) with ethidium
bromide. A total of 33 ITS clones (see below) from
the Danish form were tested as described above.
Products for sequencing were cloned into the
pCR2.1-TOPO vector and transformed into
chemically competent TOP 10 cells (Kit no.
K4500–40; Invitrogen, Carlsbad, CA, USA). Plas-
mids were purified with a QiaprepSpin Miniprep
Kit (Cat. no. 27106; Qiagen, Venlo, The Nether-
lands). Sequencing was performed with a BigDye
1.1 (Applied Biosystems no. 4337450; Applied
Biosystems, Foster City, CA, USA) and using
specific primers. Six cloned ITS products from five
individuals (Danish form of G. salaris) were
analysed in an ABI-310 automated sequencer. A
total of 33 ITS clones from these Danish parasites
were further analysed by PCR-RFLP with MaeI.
The morphometric measurements of the hard
parts in both the Danish and Norwegian parasite
forms have been presented previously and fall
 2007 The Authors. within the same range. No morphological differ-
Journal compilation
 2007
Blackwell Publishing Ltd 124
P W Kania et al. Diagnosis of a non-pathogenic Gyrodactylus salaris
ences (except for a slightly larger ventral bridge)
could be detected with this technique (Jørgensen
et al. 2006). Sequencing of the ITS region from the
two types of G. salaris was performed previously
and they were identical except for a base substitu-
tion (G1100 to A1100) in the Danish parasite form
(GenBank accession number DQ823390) com-
pared with the Norwegian Lærdalselva form
(Jørgensen et al. 2006). However, in the present
work it was shown that two types of ITS sequences
(the mutated and the non-mutated ITS regions)
occurred within individual non-pathogenic para-
sites. PCR was performed on lysates from whole
parasites and all amplicons from both the Danish
parasite form and the Norwegian River Lærdalselva
form revealed bands of 1300 bp. The PCR-RFLP
technique performed on the two forms [using
restriction enzyme BsuRI (HaeIII)] displayed the
same banding pattern (four distinct bands) as
described by Cunningham (1997). However, the
number of restriction sites of the type CTAG is
diminished from three to two by the substitution.
Therefore, when the parasites (Danish and
Norwegian forms) were subjected to PCR-RFLP
using restriction enzyme FspB (MaeI) the Laerdals-
elva strain could be clearly differentiated from the
Danish form. Thus, five bands were found in the
former parasite and four in the Lærdalselva form
(Fig. 1). Subsequent work showed that the five
Figure 1 Polymerase chain reaction and restriction fragment
length polymorphism of the rDNA internal transcribed spacer
(ITS) region (1300 bp) of Gyrodactylus spp. Visualisation of the
DNA banding pattern of two different forms of G. salaris
(pathogenic and non-pathogenic) and G. derjavini in a 2%
agarose gel (with ethidium bromide) following digestion using
FspB (MaeI) of the ITS sequences. 1–3: Undigested, 1: Danish
G. salaris, 2: Norwegian G. salaris, 3: G. derjavini. 4–6: HaeIII
digested, 4: Danish G. salaris, 5: Norwegian G. salaris, 6:
G. derjavini. 7–9: FspB (MaeI) digested, 7: Danish G. salaris, 8:
Norwegian G. salaris, 9: G. derjavini, 10: DNA marker.
 Journal of Fish Diseases 2007, 30, 123–126
bands obtained from the non-pathogenic G. salaris
represented a combination of two different ITS-
sequences: a mutated and a non-mutated type. This
was confirmed by digestion with MaeI of isolated
clones of the 1300 bp ITS region from the Danish
non-pathogenic G. salaris form. This produced
either (i) three DNA fragments (495, 475 and
330 bp) or (ii) four DNA fragments (495, 475, 203
and 127 bp) (Fig. 1). These latter bands were
identical to the pattern of the pathogenic G. salaris.
Thus, it was shown (by performing PCR-RFLP
with MaeI on 33 clones of ITS fragments obtained
from five individual parasites) that both the
mutated and the non-mutated ITS-sequences
occurred in individual parasites of the Danish
parasite form. This indicated a chimaeric occur-
rence of both mutated and non-mutated ITS
regions in one and the same parasite. On average
24% (range 0–43%) of the clones were non-
mutated (Table 1). Sequencing of six of these
clones confirmed this combined occurrence of the
different types of ITS in the non-pathogenic
parasite. Based on ITS sequence information from
this work and published records of Norwegian
G. salaris, G. teuchis, G. truttae and G. derjavini
(GenBank accession numbers AJ249350,
AJ132260, AJ132259, AJ249350) banding patterns
using FspB (MaeI) were predicted and showed a
distinct banding pattern between these species and
forms (data not shown). However, Hae III digestion
is still needed to differentiate another Danish
G. salaris variant with GenBank accession number
AJ515912 (Lindenstrøm, Collins, Bresciani, Cun-
ningham & Buchmann 2003) from the mutated
type found in this work.
Table 1 The frequency of mutated (G1100 to A1100 substitution)
and non-mutated internal transcribed spacer (ITS) regions in 33
clones obtained by cloning ITS Polymerase chain reaction
(PCR)-products from five individuals of the Danish non-
pathogenic Gyrodactylus salaris form. Data obtained by perform-
ing PCR-restriction fragment length polymorphism and MaeI
digestion of ITS-clones with subsequent agarose gel visualisation
Mutated Non-mutated
ITS clone ITS clone
Specimen (%) (%)
1 5 (100) 0 (0)
2 4 (80) 1 (20)
3 6 (75) 2 (25)
4 6 (75) 2 (25)
5 4 (57) 3 (43)
Total 25 (76) 8 (24)
 2007 The Authors.
Journal compilation
 2007
Blackwell Publishing Ltd 125
P W Kania et al. Diagnosis of a non-pathogenic Gyrodactylus salaris
Different species within the genus Gyrodactylus
can be separated by the use of PCR-RFLP. Thus,
G. salaris, G. teuchis, G. derjavini and G. truttae
from various salmonids can be differentiated on
their banding pattern following PCR of the ITS
region and subsequent digestion with various
restriction enzymes (Cunningham 1997). Diagnosis
of different forms of parasites within one species
may be time consuming due to the necessary
sequencing of several gene products including
intergenic spacers (IGS) and mitochondrial genes
(COI) (Collins & Cunningham 2000; Meinila,
Kuusela, Zietara & Lumme 2002, 2004; Sterud,
Mo, Collins & Cunningham 2002; Cunningham,
Collins, Malmberg & Mo 2003; Hansen, Bach-
mann & Bakke 2003; Hansen, Olstad, Bakke &
Bachmann 2004; Jørgensen et al. 2006). However,
the recent finding by Jørgensen et al. (2006) that
the non-pathogenic form of G. salaris found in
Danish rainbow trout farms has a base substitution
in the ITS region, allowed us to create a PCR-RFLP
for easy differentiation between the Norwegian
pathogenic form from the River Lærdalselva and the
Danish non-pathogenic form. The technique is
based on the absence of one of three CTAG
restriction sites in the ITS-region of the non-
pathogenic form compared with the Norwegian
form. Thus, the use of the restriction enzyme FspB
(MaeI) (which recognizes and cleaves the CTAG
site) for digestion of the 1300 bp ITS sequence and
subsequent agarose gel electrophoresis allows a clear
difference in banding pattern to be visualized.
Moreover, other important gyrodactylids infecting
salmonids in Europe such as G. truttae, G. derjavini
and G. teuchis have ITS sequences, which also
produce clear differences following FspB (MaeI)
digestion. Thus, Fsp(MaeI) restriction creates three
fragments (495, 475, 330 bp) of the mutated ITS-
sequence and four fragments (495, 475, 202,
127 bp) of the wild type ITS. The non-pathogenic
parasite is a chimaeric form and contains both the
mutated and the wild type ITS in its genome.
Therefore PCR-RFLP of this entire genome will
create five bands (495, 475, 303, 202, 127 bp)
Total which represent a combination between the above
clones
tested mentioned types. It is noteworthy that individuals
of the non-pathogenic form contain both the
5
mutated and the non-mutated type of the G. salaris
5
8 ITS region. This is not readily explained but may
8 reflect a recent mutation in part of the ITS repeat
7
regions, which has not yet been adjusted to the rest
33
of the repeat regions. Alternatively, it could be
 Journal of Fish Diseases 2007, 30, 123–126
speculated that the worm represents a hybrid
between a ÔclassicalÕ G. salaris and another as yet
undescribed species or form containing the sub-
stitution. Due to the ability of Gyrodactylus to
produce a population (clone) of only one founder
individual (even hybrids) this could explain the
occurrence of various forms and variants within
G. salaris (sensu lato). However, the relatively low
rate of clones exhibiting the substitution supports
the notion that this non-pathogenic form is formed
through mutation and not by hybridization
between two species. Lindenstrøm et al. (2003)
found another chimaeric occurrence of ITS regions
in a similarly non-pathogenic form of G. salaris in
Denmark. However, that form differed from the
parasite type treated in the present work and could
readily be detected by the PCR-RFLP method
(HaeIII digestion) described by Cunningham
(1997). Thus, it cannot be excluded that several
other types and forms of G. salaris may exist and
even have other variations in the ITS region. These
may then call for other diagnostic techniques to be
developed. However, the new method presented
here will act as a supplement to current diagnostics
for detection of the non-pathogenic form contain-
ing the G1100 to A1100 substitution.
Acknowledgements
This work was supported by a grant from the
Danish Research Council to the research school
SCOFDA at the Royal Veterinary and Agricultural
University and by a grant from the Danish Ministry
of Environment.
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Received: 28 August 2006
Revision received: 25 September 2006
Accepted: 5 October 2006