Paranucleospora theridion (Microsporidia) infection dynamics in farmed Atlantic salmon Salmo salar put to sea in spring and autumn

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DOI: 10.3354/dao02464 · Source: PubMed
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The microsporidian Paranucleospora theridion (syn. Desmozoon lepeophtheirii) is a parasite of Atlantic salmon Salmo salar and also a hyperparasite of the salmon louse Lepeophtheirus salmonis. The parasite develops 2 types of spores in salmon, cytoplasmic spores in phagocytes and intranuclear spores in epidermal cells. The former type of development is assumed to be propagative (autoinfection), while the epidermal spores transfer the parasite to lice. Development in lice is extensive, with the formation of xenoma-like hypertrophic cells filled with microsporidian spores. We show that salmon are infected in the absence of lice, likely through waterborne spores that initiate infections in the gills. During summer and autumn the parasite propagates in the kidney, as evidenced by peaking normalised expression of P. theridion rRNA. Lice become infected during autumn, and develop extensive infections during winter. Lice mortality in winter and spring is likely responsible for a reservoir of spores in the water. Salmon transferred to sea in November (low temperature) did not show involvement of the kidney in parasite propagation and lice on such fish did not become infected. Apparently, low temperatures inhibit normal P. theridion development in salmon.
Dis Aquat Org
Vol. 101: 43– 49, 2012
doi: 10.3354/dao02464 Published October 10
The microsporidian parasite Paranucleospora the -
ridion (syn. Desmozoon lepeophtheirii; see S. Nylund
et al. 2011) is a parasite of Atlantic salmon Salmo
salar and also is a hyperparasite of the salmon louse
Lepeophtheirus salmonis (A. Nylund et al. 2009a,b,
S. Nylund et al. 2010). P. theridion undergoes 2 types
of development in salmon. Development occurs in
the cytoplasm of epidermal cells and phagocytes,
resulting in minute thin-walled spores assumed to be
autoinfective. These cytoplasmic stages and spores
are found in many tissues and organs and hence
appear to represent a systemic infection in suitable
host cells. A second type of sporogony is seen in epi-
dermal cells in skin and gills only, where 1 to 2 thick-
walled spores are produced inside the nucleus of the
cells. These more robust spores (‘environmental
spores’) are likely responsible for transferring the
microsporidian infection to the salmon louse, as it
feeds on salmon epidermis. Extensive propagation
may occur in greatly enlarged infected cells in the
lice, and infected cells with parasitic stages often
occupy a large part of the louse volume. How the
thick-walled spores produced in the lice infect
salmon is unknown; few salmon individuals receiv-
ing infected lice in experiments contracted the infec-
tion (A. Nylund & E. Karlsbakk, unpubl. obs.). Also,
the infection dynamics and pathology in salmon is
poorly understood. Heavy P. theridion densities in
© Inter-Research 2012 ·*Corresponding author. Email:
Paranucleospora theridion (Microsporidia)
infection dynamics in farmed Atlantic salmon
Salmo salar put to sea in spring and autumn
S. Sveen1, H. Øverland1, E. Karlsbakk1,2, A. Nylund1,*
1Department of Biology, University of Bergen, Thormohlensgt 55, 5020 Bergen, Norway
2Institute of Marine Research, PO Box 1870, Nordnes, 5817 Bergen, Norway
ABSTRACT: The microsporidian Paranucleospora theridion (syn. Desmozoon lepeophtheirii) is a
parasite of Atlantic salmon Salmo salar and also a hyperparasite of the salmon louse Lepeoph-
theirus salmonis. The parasite develops 2 types of spores in salmon, cytoplasmic spores in phago-
cytes and intranuclear spores in epidermal cells. The former type of development is assumed to be
propagative (autoinfection), while the epidermal spores transfer the parasite to lice. Development
in lice is extensive, with the formation of xenoma-like hypertrophic cells filled with microsporidian
spores. We show that salmon are infected in the absence of lice, likely through waterborne spores
that initiate infections in the gills. During summer and autumn the parasite propagates in the kid-
ney, as evidenced by peaking normalised expression of P. theridion rRNA. Lice become infected
during autumn, and develop extensive infections during winter. Lice mortality in winter and
spring is likely responsible for a reservoir of spores in the water. Salmon transferred to sea in
November (low temperature) did not show involvement of the kidney in parasite propagation and
lice on such fish did not become infected. Apparently, low temperatures inhibit normal P. theridion
development in salmon.
KEY WORDS: Microsporidian · Parasite · Hyperparasite · Salmon louse · Lepeophtheirus salmonis ·
Spore development · Temperature
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Dis Aquat Org 101: 43–49, 2012
salmon are often associated with other diseases and
diagnoses, and the parasite has been suspected of
predisposing the host to other infectious agents and
of being responsible for some of the pathology attrib-
uted to some viral diseases (S. Nylund et al. 2011). An
association has been shown between P. theridion
infection levels and the unspecific diagnosis ‘prolifer-
ative gill inflammation’ (PGI) in salmon (Steinum et
al. 2010, S. Nylund et al. 2011).
In the present study, the infection dynamics of
Paranucleospora theridion, as measured by quan -
titative real-time reverse-transcription PCR (RT-
PCR) analyses, was followed in 2 salmon farms fol-
lowing smolt sea-transfer in May and November,
hence at times of increasing and decreasing water
temperatures, respectively.
Atlantic salmon and salmon lice were collected at
intervals over a period of 1 yr from 2 farms located in
Sogn og Fjordane county, western Norway. The
smolts at Farm A were put to sea in May, while the
smolts in Farm B were sea-launched in November.
Mortality, temperature and oxygen levels were mea-
sured at both sites throughout the sampling period.
The origin of the eggs for the salmon at Farm A was
the brood fish company Salmobreed. The smolt was
produced at a land-based site in Sogn og Fjordane
county before being put to sea in a farm in the same
county on 10 May 2009. This farm, located at a site
with the dominating current going in one direction,
consists of 4 ring nets with a diameter of 157 m and a
depth of 70 m under the rings. The previous produc-
tion at this site ended 31 October 2008. The farm was
kept empty until 10 May 2009, when new smolt was
stocked at the site. Delousing was carried out at the
site in November 2009 and April 2010. Temperature
was measured daily at 5 m depth.
The smolts that were sea-launched at Farm B were
produced at a land-based site in Hordaland county
from eggs supplied by the brood fish company Aqua-
Gen. Farm B consists of 10 steel cages (25 × 25 m),
and the depth under the farm ranged from 40 to 70 m.
A unidirectional sea current is dominating at the site.
The previous production at this site ended 17 June
2009. New production at this site started 1 October
2009, but the fish included in the present study were
sea-launched on 1 November 2009. The first samples
(n = 30) were taken at the arrival of these fish. At this
time the smolt had been exposed to full seawater, in
a well boat, for <12 h during transport from the smolt
site to Farm B. Shortly after sea-launching, mortali-
ties increased and the fish were diagnosed with
infectious pancreatic necrosis (IPN) by the responsi-
ble veterinary service. The salmon were deloused on
12 December 2009. Temperature was measured at
4 m depth.
A total of 136 salmon in 9 samples from Farm A and
130 salmon in 5 samples from Farm B were examined
(Table 1). Salmon lice Lepeophtheirus salmonis were
also collected, if present. Since the prevalence of
Paranucleospora theridion in Farm A was 100% in
both September and November, the number of fish
taken for analysis was reduced from 30 to 10 fish on
the remaining sampling dates (6 fish in June) until
the termination of the study period.
Gill and kidney tissues were sampled from all fish
and stored at −80 °C for later analysis using real-time
RT-PCR for detection of pathogens.
RNA and DNA extraction
RNA was extracted from both gills and kidney of
salmon as described by Devold et al. (2000), except
that Isol RNA Lysis Reagent (5-PRIME) was used
instead of TRIZOL (Gibco BRL). The concentration of
the resulting RNA was measured using a NanoDrop®
Sampling date Salmon Salmon length Lice
(dd/mm/yy) (n) (cm) (min.−max.) (n)
Farm A (10/05/09)
12/07/09 20 25 (21−30) 6
24/09/09 30 40 (30−49) 10
05/11/09 30 43 (29−51) 10
09/12/09 10 44 (39−50) 10
14/01/10 10 47 (40−53) 10
18/02/10 10 50 (39−57) 10
09/04/10 10 53 (48−69) 10
24/06/10 6 61 (55−70) 0a
17/08/10 10 58 (52−69) 0a
Farm B (01/11/09)
01/11/09 30 19 (16−22) 30b
04/12/09 30 21 (17−25) 10
04/01/10 30 23 (18−30) 0c/10b
01/03/10 30 21 (18−28) 10
02/09/10 10 54 (50−58) 0a
aLice present on the fish, not analysed; bLice from fish
transferred to sea 01/10/09; cDelousing of study pens,
lice from neighbouring pens
Table 1. Salmo salar and Lepeophtheirus salmonis. Samples
collected at 2 salmon farms (date of sea transfer indicated in
parentheses). n: number of specimens sampled
Author copy
Sveen et al.: Paranucleospora theridion infection dynamics in salmon
ND-1000 spectrophotometer. DNA was extracted
from whole sea lice following the addition of 2 µl sus-
pension of the archaean Halobacterium salinarum
acting as an exogenous control. The DNeasy DNA
Tissue kit (Qiagen) was used according to the manu-
facturer’s recommendations.
Extracted DNA from tissues of salmon and salmon
lice were used to obtain the partial rRNA small sub-
unit (rRNA SSU) sequence of Paranucleospora
theridion using the primers Nuc-F (5’-CGG ACA
GGG AGC ATG GTA TAG-3’) and Nuc-R5 (5’-TCC
CAT CAA TTT CCA ACG GC-3’), yielding a frag-
ment of ca. 370 to 400 bp when excluding the ends.
The PCR reaction mixture (50 µl) contained 1×
ThermoPol reaction buffer (New England BioLabs),
10 mM of each dNTP (Promega), 0.4 µM of each
primer (Invitrogen), 1 U Taq DNA polymerase (New
England BioLabs) and approximately 200 to 300 ng of
DNA template. Amplification was performed in a
GeneAmp PCR System 9700 machine (Applied Bio -
systems) with initial denaturation at 95°C for 5 min;
followed by 35 cycles of 95°C for 30 s, 53°C for 45 s,
and 72°C for 1 min; final extension at 72°C for 7 min,
and a short storage at 4°C. All PCR products were
purified with EZNA PCR cycle pure (Omega Biotek),
and sequenced using the Big Dye terminator se -
quencing kit (Applied Biosystems). Sequencing was
performed at the sequencing facility at the University
of Bergen (http://seqlab. Sequence data
were analysed and assembled using VectorNTI soft-
ware (Informax). Se quences obtained in the present
study were submitted to GenBank.
Real-time assays (TaqMan probes) for Paranucle-
ospora theridion (Nuc assay) and elongation factor
1α(EF1AAassay) (see S. Nylund et al. 2010) was
used on samples from salmon, with EF1AAas inter-
nal control (reference gene). The VersoTM 1-step
QRT-PCR kit from Thermo Scientific was used. All
assays were run in a total volume of 12.5 µl with 2 µl
template, at 45 cycles in an ABI 7500 Sequence
Detection System real-time thermocycler as de -
scribed by S. Nylund et al. (2010, 2011). The runs
were considered positive when the fluorescence
signal increased above a set threshold of 0.015.
Analyses included non-template controls (NTC),
RNA extraction and positive controls. In analyses
with the Nuc assay of sea-lice samples, the SAL
assay was used with an external control, as
described by S. Nylund et al. (2010). The repro-
ducible detection limit for the Nuc assay is at a cycle
threshold (CT) value of 37 (see S. Nylund et al.
2010), representing <1 spore (S. Nylund et al. 2011).
The equations for the standard curves, based on
triplicates of 10-fold serial dilutions of template,
were CT= −3.51 × (logRNA dilution) + 18.38 for the
EF1AAassay (R2= 0.996), and CT= −3.53 × (logRNA
dilution) + 19.70 for the Nuc assay (R2= 0.997). Effi-
cacies (E = 101/(−slope)) were 1.921 and 1.987, respec-
tively. Analyses resulting in CTvalues in the range
37.0 to 37.9 were rerun in triplicate, and considered
positive when all were positive. The exogenous and
endogenous controls were used for calculation of
the normalised expression (NE) of SSU rRNA from
P. theridion (S. Nylund et al. 2010). This measure of
parasite density was used as a proxy of abundance,
with NE in negative samples set at 0.
Temporal change in prevalence was examined
using Fisher’s exact test (FET) in Quantitative Para-
sitology 3.0, with post hoc pairwise comparison of
samples also using FET if overall changes were sig-
nificant. The nonparametric Kruskal-Wallis test (KW;
in Statistica 10.0) was used to test for temporal
changes in Paranucleospora theridion density. Post
hoc comparisons were done with the bootstrap test
for changes in abundance in Quantitative Parasitol-
ogy 3.0 (p-values given in the text).
Farm A
Farm A was studied from 12 July 2009 to 17 August
2010. Average monthly temperature was high (ca.
16°C) in July–August 2009 followed by a gradual
decrease to a minimum of 4.5°C in March 2010. Tem-
perature increased thereafter, reaching 18.2°C in
August 2010. The behaviour and appearance of the
fish was normal throughout, except in September
2009, when abnormal behaviour was apparent, par-
ticularly highly increased jumping frequencies and
signs of skin irritation (excess mucus production).
The cause was not revealed; no disease agents were
detected by the responsible veterinary service. In
addition to gill and kidney samples, skin samples
from 7 fish were analysed for Paranucleospora
theridion (Nuc assay) this month, and high densities
of the parasite were detected.
Author copy
Dis Aquat Org 101: 43–49, 2012
Paranucleospora theridion in salmon
Paranucleospora theridion prevalence increased
significantly from July to September 2009 (to 100%),
when based on both gill (p < 0.001) and kidney (p <
0.001) sample analyses (Fig. 1A). Prevalence stayed
at 100% in the gill samples, while a few uninfected
fish occurred in the kidney samples from February to
June 2010. Parasite density (NE) also increased sig-
nificantly in the period July to September 2009 in
both tissues (p < 0.01). In the period September to
November 2009, there was a significant decrease in
density in the gills (p < 0.01) followed by further
gradual decrease until the June sample, where the
step from December to January was significant (p <
0.05). A slight increase from AprilJune to the August
sample in 2010 was not significant (p = 0.06). In the
kidney samples, parasite density did not change from
September to December 2009. A gradual decrease
occurred in the period November to April (p < 0.01).
The increase in average NE from July to Septem-
ber was 3.4-fold in the gill and 31-fold in the kidney
samples. The decrease in average NE from Septem-
ber 2009 to June 2010 was ca. 3020-fold in the gill
and ca. 540-fold in the kidney samples.
Paranucleospora theridion in lice
Prevalence of Paranucleospora theridion in lice
Lepeophtheirus salmonis varied significantly during
the period July 2009 to April 2010 (FET, p < 0.001)
(Fig. 1B). A low prevalence from July to September
2009 was followed by an increase (p < 0.001) to 100%
in the samples from November 2009 to January 2010.
A significant decrease (p = 0.001) occurred during
spring, with 2 out of 10 lice being infected in April.
P. theridion density (NE) in the lice was similarly very
low from July to September 2009, followed by a
marked increase in the November sample (p < 0.05).
Mean NE decreased from the winter samples at similar
levels (November to January) to April 2010 (p < 0.01).
Farm B
Farm B was studied from 1 November 2009 (sea
transfer) to 1 March 2010. An additional sample, col-
lected on 2 September 2010 from the same pen-pop-
ulation, was also analysed for comparison. Average
monthly temperature was highest in November
(9.7°C, maximum: 10.1°C) and gradually decreased
until March (December: 9.3°C, Janu-
ary: 7.4°C, February: 4.5°C, March:
3.9°C; minimum: 3.8°C on 22 Febru-
ary). Increasing mortality was ob -
served following sea transfer in
November and persisted through
February. This mortality was ascribed
to IPN by the responsible veterinary
Paranucleospora theridion in salmon
Prevalence of Paranucleospora the -
ridion in gill samples varied signifi-
cantly (p < 0.001) (Fig. 2). A marked
increase occurred from 13% immedi-
ately after sea transfer in November to
100% in December (p < 0.001). Preva-
lence at 100% was also observed in
January, followed by a decrease to
83% in March (p < 0.05). Mean NE
levels were very low compared to
those observed in Farm A. A signifi-
cant increase in observed mean NE
was found in the gill samples from
November to December 2009 (p <
0.02). A further increase from Decem-
Fig. 1. Paranucleospora theridion infecting Salmo salar and Lepeophtheirus
salmonis. P. theridion normalised expression (NE) and sample prevalence in
(A) farmed Atlantic salmon gill and kidney samples and (B) salmon lice in
Farm A from July 2009 to August 2010. Error bars for mean NE represent boot-
strapped 95% confidence intervals; kidney values in (A) were displaced
(+2 d) to avoid overlapping error bars
Author copy
Sveen et al.: Paranucleospora theridion infection dynamics in salmon
ber 2009 to January 2010 was not significant, but a
decrease occurred from January to March (p < 0.01).
Prevalence and mean NE based on analyses of the
kidney samples was low, and did not vary signifi-
cantly in the period November to March. However, a
sample from the same fish group on 2 September
2010, i.e. 6 mo later, showed 100% prevalence of
P. theridion in the kidney samples, but mean NE
was low (not significantly different from the March
Paranucleospora theridion in lice
Analyses of lice from Farm B during the study
period (November to March) revealed only a single
positive specimen, in the November sample (NE of
Paranucleospora theridion was low).
Partial rRNA SSU sequences of Paranucleospora
theridion were obtained in order to verify the identity
of the templates detected by the Nuc assay. The
following sequences were obtained (GenBank ac -
cession numbers): Farm A: salmon: September (HM
231163), November (HM231161 and HM231162),
January (HM231159 and HM231160); salmon lice:
November, December, January, February (HM23
1155 to HM231158, respectively); Farm B: salmon:
January (HM231164). It was not possible to sequence
P. theridion from the single weakly positive salmon
louse detected in Farm B. The 10
sequences did not show any variation.
Paranucleospora theridion shows
2 developmental cycles in Atlantic
salmon. One type of development
(type I) has been observed in the cyto-
plasm of phagocytes and in epidermal
cells, and results in the production of
numerous small thin-walled spores
with a very short polar tube. Infected
cells have been observed to degener-
ate, hence releasing these small
spores into the host organism (S.
Nylund et al. 2010). These spores are
considered autoinfective, likely being
phagocytised by phagocytes where
the short polar tube may be responsible for the deliv-
ery of the sporoplasm to the phagocyte cytoplasm
and hence initiating a new cycle of development.
Real-time PCR studies on P. theridion tissue tropism
supports such an interpretation; the parasite pro-
duces a systemic infection and only type I develop-
ment has been observed in tissues other than the epi-
dermis. Type II development occurs in the nuclei of
epidermal cells, where 1 to 2 thick-walled spores
with a long polar tube are produced. These spores
(‘environmental spores’) have been considered
responsible for transferring the infection to salmon
lice, since these copepods must devour large num-
bers of them when feeding on the salmon skin.
Development of the microsporidian in the lice repre-
sents a third type of sporogony, and may result in the
copepod being packed with P. theridion spores (Free-
man et al. 2003, Freeman & Sommerville 2009, A.
Nylund et al. 2009a,b,c, S. Nylund et al. 2010). How-
ever, the events in the P. theridion life cycle leading
to transmission from lice to salmon are unknown. No
likely mode of direct spore release from infected lice
to the salmon has been described; there is no evi-
dence for spore transmission via the lice mouth or
release via the gut (Freeman & Sommerville 2009, S.
Nylund et al. 2010). Hence it appears likely that
infected lice release their spore contents to the water
The present study showed that salmon put to sea in
November (Farm B) may become infected immedi-
ately by the parasite, as evidenced by real-time PCR
positive gill samples. Since no direct contact with lice
can be responsible for the infections appearing in
Fig. 2. Paranucleospora theridion infecting Salmo salar. P. theridion nor-
malised expression (NE) and sample prevalence in farmed Atlantic salmon gill
and kidney samples in Farm B from November 2009 (sea transfer) to March
2010, with an additional observation in September 2010. Error bars for NE
represent bootstrapped 95% confidence intervals
Author copy
Dis Aquat Org 101: 43–49, 2012
Farm B, the likely source is waterborne spores. Lice
collected at the farm (other pens) at this time were
also generally uninfected, and the single louse found
positive for the parasite showed a very weak signal
(November sample). It appears likely that this posi-
tive signal represents salmon tissues in the louse gut.
In Farm A, infections in the lice showed a marked
increase in prevalence and NE during the period
September to November, which likely represents
infections established through ingestion of epidermal
intranuclear spores from the salmon. The propaga-
tion of the parasite in the lice results in xenoma-like
hypertrophic cells visible through the exoskeleton of
live lice (e.g. Freeman et al. 2003), and such lice were
commonly observed during winter in the present
study (Farm A).
There was a clear difference in the observed infec-
tion dynamics in salmon between Farm A (stocked 10
May) and Farm B (stocked 1 November). In Farm A
an increase in Paranucleospora theridion density in
gill samples in summer was accompanied by in -
creased amounts of the parasite in the kidney. During
the period autumn-winter-spring, the parasite den-
sity decreased in the gill samples, while remaining
high from September to December in the kidney
before declining in the spring. Hence kidney infec-
tions occurred with a propagation of the parasite that
is likely critical for the initiation of a secondary
intranuclear developmental sequence in the epider-
mis. Intranuclear epidermal spores are considered
infective to the salmon lice (see previous paragraph).
In Farm B, increasing densities of the parasite were
observed in gill samples after sea transfer and they
peaked in January, but were not accompanied by
elevated levels of the parasite in the kidney and sea
lice from the fish that remained uninfected. It
appears likely that this difference is due to a failure
in the parasite development at low temperatures dur-
ing winter; autoinfection is inhibited and spores that
may infect the lice are not produced. Since seawater
temperature was 10°C after sea transfer, such tem-
peratures seem to arrest the initial development of P.
theridion infections in salmon. There was no evi-
dence for propagation of the parasite in the kidney,
as observed in Farm A and by S. Nylund et al. (2010).
In Farm A, temperatures below 10°C were not seen
until late in November, and were 14.5 to 17.1°C in the
period July to September when parasite propagation
was evidenced in the salmon, and 10.5 to 15.2°C in
the period September to November when parasite
propagation occurred in salmon lice.
Microsporidian development may be highly tem-
perature-dependent (see Sanchez et al. 2000). Kaba -
tana takedai infections in salmonids do not develop
at 9°C, and are reduced at 11°C. Normal develop-
ment occurs at 13 to 17°C (Zenke et al. 2005). Antonio
& Hedrick (1995) found that Nucleospora salmonis
infections in Chinook salmon Oncorhynchus tsha -
wytscha are not completely arrested at 9°C but infec-
tions are less severe and mortality low. In these cases
the stages in the development of the parasites
affected by temperature is not known. Beaman et al.
(1999) found that normal development of Loma
salmonae is interrupted by temperatures below 9°C
as well as above 20°C, when xenoma formation in the
gills is inhibited. At low temperature, the parasite
arrives in the heart where an initial merogony nor-
mally occurs, but xenoma formation in the gills does
not occur and the gills eventually become PCR-nega-
tive by 4 wk post-challenge (Sanchez et al. 2000,
2001). Apparently, normal proliferation in the heart is
arrested by low temperatures. This observation
appears similar to the present one, in the sense that a
propagative phase is inhibited so the subsequent
production of environmental spores does not occur.
This interpretation assumes that the lack of infections
in the sea lice from Farm B was due to an absence of
infective epidermal spores in the salmon. Following
infection (detected in the gills), the density of the
parasite also increased in kidney samples (Farm A).
It seems likely that epidermal cells become infected
through contact with waterborne spores, injecting
sporoplasms into the cytoplasm of the cells. This first
cycle of cytoplasmic development in epidermal cells
likely releases autoinfective spores that are taken up
by phagocytes (see S. Nylund et al. 2010). Antigen-
trapping phagocytes moving to the kidney are likely
responsible for initiating infections there and the
observed parasite propagation. It is interesting that
gill inflammation with phagocytosis of the autoinfec-
tive spores may be important for the spread of the
parasite from the gills to the kidney and other tissues.
Paranucleospora theridion infections have been
found to be closely associated with PGI (Steinum et
al. 2010, S. Nylund et al. 2011).
Some of the salmon in Farm A, put to sea on 10
May, had already become infected in our first sample
of 12 July. However, a clear increase in prevalence
shows that transmission of the parasite to salmon also
occurred at this site during autumn. Signs of irritation
were apparent in the salmon in September, and
exposure to Paranucleospora theridion spores may
possibly be responsible. Further experimental stud-
ies are needed on the effects of P. theridion infections
on salmon, including potential effects on the immune
system caused by parasite exploitation of phago-
Author copy
Sveen et al.: Paranucleospora theridion infection dynamics in salmon
cytes, possibly affecting susceptibility to other dis-
ease agents and prognosis following other infections
(see S. Nylund et al. 2011).
Antonio DB, Hedrick RP (1995) Effect of water temperature
on infections with the microsporidian Enterocytozoon
salmonis in chinook salmon. Dis Aquat Org 22: 233−236
Beaman HJ, Speare DJ, Brimacombe M (1999) Regulatory
effects of water temperature on Loma salmonae
(Microspora) development in rainbow trout. J Aquat
Anim Health 11: 237−245
Devold M, Krossøy B, Aspehaug V, Nylund A (2000) Use of
RT-PCR for diagnosis of infectious salmon anaemia virus
(ISAV) in carrier sea trout Salmo trutta after experimen-
tal infection. Dis Aquat Org 40: 9−18
Freeman MA, Sommerville C (2009) Desmozoon lepeoph-
therii n. gen., n. sp., (Microsporidia: Enterocytozoonidae)
infecting the salmon louse Lepeophtheirus salmonis
(Copepoda: Claigidae). Parasites Vectors 2: 58
Freeman MA, Bell AS, Sommerville C (2003) A hyperpara-
sitic microsporidian infecting the salmon louse, Lepeoph-
theirus salmonis: an rDNA-based molecular phyloge-
netic study. J Fish Dis 26: 667−676
Nylund A, Watanabe K, Nylund S, Arnesen CE, Karlsbakk E
(2009a) New pathogen − old disease. Norsk Fiskeopp-
drett 34(2): 44−49 (in Norwegian with English Abstract)
Nylund A, Watanabe K, Nylund S, Arnesen CE, Karlsbakk E
(2009b) Salmon lice act as vectors for a new species of
microsporidian. Norsk Fiskeoppdrett 34(6a): 20−23 (in
Norwegian with English Abstract)
Nylund A, Watanabe K, Nylund S, Sævareid I, Arnesen
CE, Karlsbakk E (2009c) Salmon lice biological vec-
tor for salmon parasite. Naturen 133: 217−222 (in Nor-
wegian with English Abstract)
Nylund S, Nylund A, Watanabe K, Arnesen CE, Karlsbakk E
(2010) Paranucleospora theridion n. gen., n. sp.
(Microsporidia, Enterocytozoonidae) with a life cycle in
the salmon louse (Lepeophtheirus salmonis, Copepoda)
and Atlantic salmon (Salmo salar). J Eukaryot Microbiol
57: 95−114
Nylund S, Andersen L, Sævareid I, Plarre H and others
(2011) Diseases of farmed Atlantic salmon Salmo salar
associated with infections by the microsporidian Para -
nucleospora theridion. Dis Aquat Org 94: 41−57
Sanchez JG, Speare DJ, Markham RJF (2000) Normal and
aberrant tissue distribution of Loma salmonae (Micro -
spora) within rainbow trout, Oncorhynchus mykiss (Wal-
baum), following experimental infection at water tem-
peratures within and outside of the xenoma-expression
temperature boundaries. J Fish Dis 23: 235−242
Sanchez JG, Speare DJ, Markham RJF, Wright GM,
Kibenge FSB (2001) Localization of the initial develop-
mental stages of Loma salmonae in rainbow trout
(Oncorhynchus mykiss). Vet Pathol 38: 540−546
Steinum T, Kvellestad A, Colquhoun DJ, Heum M, Moham-
mad S, Nygaard Grøntvedt R, Falk K (2010) Microbial
and pathological findings in farmed Atlantic salmon
Salmo salar with proliferative gill inflammation. Dis
Aquat Org 91: 201−211
Zenke K, Urawa S, Fujiyama I, Yokoyama H, Ogawa K
(2005) Effects of water temperature on infection of the
microsporidian Kabatana takedai in salmonids. Fish
Pathol 40: 119−123
Editorial responsibility: Dieter Steinhagen,
Hannover, Germany
Submitted: December 15, 2011; Accepted: March 12, 2012
Proofs received from author(s): August 9, 2012
Author copy
  • ... In salmon, the parasite infects different cell types such as gill and skin epithelial cells, blood vessel endothelial cells, polymorphonuclear leucocytes and macrophage-like cells (Nylund et al. 2010;Weli et al. 2017). The transmission route of the parasite has not been fully elucidated, but it has been suggested that the microsporidian spores possibly infect the salmon gills first and then spreads to other tissues and organs (Nylund et al. 2010;Sveen et al. 2012). It is likely that the sea lice would ingest the parasite spores whilst feeding on the epithelial cells of the skin of infected salmon (Sveen et al. 2012). ...
    Full-text available
    Gill disease of farmed Atlantic salmon (Salmo salar ) in the marine environment has emerged as a significant problem for the salmon aquaculture industry. Different types of marine salmon gill disease reported include amoebic gill disease (AGD), parasitic gill disease, viral gill disease, bacterial gill disease, zooplankton (cnidarian nematocyst)‐associated gill disease, harmful algal gill disease and chemical/toxin‐associated gill disease. The term ‘multifactorial gill disease’ is used when multiple distinguishable types of disease (as opposed to an obvious single primary type) are present. When gill disease is non‐specific, it is referred to as ‘complex gill disease’ (CGD) or ‘complex gill disorder’. These two terms are often used interchangeably and are overlapping. The significance of many infectious and non‐infectious agents that may be associated with CGD is often unclear. In this review, we summarise aspects of the different types of gill disease that are relevant to the epidemiology of gill disease and of CGD in particular. We also tabulate simultaneously occurring putative pathogens to explore the multifactorial nature of gill disease.
  • ... Exceptions to this included T. bryosalmonae and S. destruens, which were absent in river-collected Atlantic salmon in this study. The consistent prevalence of P. theridion (aka Desmozoon lepeophtherii, a candidate causative agent of proliferative gill inflammation) across sampling locations in this study aligns with the widespread occurrence of its alternate sea louse host (Lepeophtheirus salmonis) in eastern Canada (Carr and Whoriskey 2004;Sveen et al. 2012). High prevalence among wild Atlantic salmon in this study suggests that P. theridion is not highly Teffer et al. ...
    Full-text available
    Infectious agents are key components of animal ecology and drivers of host population dynamics. Knowledge of their diversity and transmission in the wild is necessary for the management and conservation of host species like Atlantic salmon ( Salmo salar). Although pathogen exchange can occur throughout the salmon life cycle, evidence is lacking to support transmission during population mixing at sea or between farmed and wild salmon due to aquaculture exposure. We tested these hypotheses using a molecular approach that identified infectious agents and transmission potential among sub-adult Atlantic salmon at marine feeding areas and adults in three eastern Canadian rivers with varying aquaculture influence. We used high-throughput qPCR to quantify infection profiles and next generation sequencing to measure genomic variation among viral isolates. We identified 14 agents, including five not yet described as occurring in Eastern Canada. Phylogenetic analysis of piscine orthoreovirus showed homology between isolates from European and North American origin fish at sea, supporting the hypothesis of intercontinental transmission. We found no evidence to support aquaculture influence on wild adult infections, which varied relative to environmental conditions, life stage, and host origin. Our findings identify research opportunities regarding pathogen transmission and biological significance for wild Atlantic salmon populations.
  • ... Paranucleospora theridion), has recently been described [31,40]. D. lepeophtherii is believed to have a complex life cycle involving both Lepeophtheirus salmonis and Atlantic salmon [31], although salmon have been found to be infected with the microsporidian in the absence of lice [41]. The true significance of this parasite as a gill pathogen is still unclear as it is frequently the most prevalent agent detected in gill samples, even in gills with no reported pathologies [7,9]. ...
    Full-text available
    Gill diseases are a growing health challenge in salmon farming worldwide, but many gaps remain in our knowledge. Gill diseases are generally complex and multifactorial disorders, often with presumable spatial and temporal distribution patterns, but are highly difficult to effectively prevent and control. The term complex gill disease (CGD) includes a wide range of clinical disease presentations on the gills of farmed salmon; usually, CGD presents from the end of summer to early winter. The pathogens involved include Neoparamoeba perurans, Tenacibaculum maritimum, Candidatus Piscichlamydia salmonis, Candidatus Branchiomonas cysticola, Desmozoon lepeophtherii (syn. Paranucleospora theridion) and viruses, such as the Atlantic salmon paramyxovirus (ASPV) and salmon gill poxvirus (SGPV). Amoebic gill disease (AGD) is perhaps the most significant disease in terms of gill health and economic impact. AGD results in high mortality, reduced production performance and impaired fish welfare. This review summarizes and analyses CGD research, outbreaks and treatment, with a focus on AGD, as well as on knowledge gaps and avenues for future research.
  • ... B. cysticola (83%-100%). The pattern of infection in relation to D. lepeophtherii corresponds with the infection pattern observed by Sveen, Øverland, Karlsbakk, and Nylund (2012), wherein, the gill load of D. lepeophtherii was highest in autumn followed by a decline over the winter months. In this study, the decline in gill load continued through the following summer until the end of the sampling. ...
  • ... B. cysticola (83%-100%). The pattern of infection in relation to D. lepeophtherii corresponds with the infection pattern observed by Sveen, Øverland, Karlsbakk, and Nylund (2012), wherein, the gill load of D. lepeophtherii was highest in autumn followed by a decline over the winter months. In this study, the decline in gill load continued through the following summer until the end of the sampling. ...
    Gill diseases are a complex and multifactorial challenge for marine farmed Atlantic salmon. Co‐infections with putative pathogens are common on farms; however, there is a lack of knowledge in relation to the potential effect co‐infections may have on pathology. The objective of this study was to determine the prevalence and potential effects of Neoparamoeba perurans, Desmozoon lepeophtherii, Candidatus Branchiomonas cysticola, Tenacibaculum maritimum and salmon gill poxvirus (SGPV) during a longitudinal study on a marine Atlantic salmon farm. Real‐time PCR was used to determine the presence and sequential infection patterns of these pathogens on gill samples collected from stocking until harvest. A number of multilevel models were used to determine the effect of these putative pathogens on gill health (measured as gill histopathology score), while adjusting for the effect of water temperature and time since the last freshwater treatment. Results indicate that between 12 and 16 weeks post‐seawater transfer (wpst), colonization of the gills by all pathogens had commenced and by week 16 of marine production each of the pathogens had been detected. D. lepeophtherii and Candidatus B. cysticola were by far the most prevalent of the potential pathogens detected during this study. Detections of T. maritimum were found to be significantly correlated with temperature showing distinct seasonality. Salmon gill poxvirus was found to be highly sporadic and detected in the first sampling point, suggesting a carryover from the freshwater stage of production. Finally, the model results indicated no clear effect between any of the pathogens. Additionally, the models showed that the only variable which had a consistent effect on the histology score was N. perurans.
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    Compared to the higher fungi (Dikarya), taxonomic and evolutionary studies on the basal clades of fungi are fewer in number. Thus, the generic boundaries and higher ranks in the basal clades of fungi are poorly known. Recent DNA based taxonomic studies have provided reliable and accurate information. It is therefore necessary to compile all available information since basal clades genera lack updated checklists or outlines. Recently, Tedersoo et al. (MycoKeys 13:1–20, 2016) accepted Aphelidiomycota and Rozellomycota in Fungal clade. Thus, we regard both these phyla as members in Kingdom Fungi. We accept 16 phyla in basal clades viz. Aphelidiomycota, Basidiobolomycota, Blastocladiomycota, Calcarisporiellomycota, Caulochytriomycota, Chytridiomycota, Entomophthoromycota, Glomeromycota, Kickxellomycota, Monoblepharomycota, Mortierellomycota, Mucoromycota, Neocallimastigomycota, Olpidiomycota, Rozellomycota and Zoopagomycota. Thus, 611 genera in 153 families, 43 orders and 18 classes are provided with details of classification, synonyms, life modes, distribution, recent literature and genomic data. Moreover, Catenariaceae Couch is proposed to be conserved, Cladochytriales Mozl.-Standr. is emended and the family Nephridiophagaceae is introduced.
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    Microsporidia are diverse opportunistic parasites abundant in aquatic organisms with some species hyperparasitic in digenean parasites. In the current study, we describe a unique microsporidian parasite, Ovipleistophora diplostomuri n. sp. that has a tropism for both the bluegill sunfish Lepomis macrochirus , and its digenean parasite Posthodiplostomum minimum . Though the microsporidium first infects a fish, the subsequent infection causes hypertrophy of the metacercarial wall and degeneration of the P. minimum metacercariae within the fish tissue. Genetic analysis placed this species within Ovipleistophora and ultrastructural characteristics were consistent with the genus, including the presence of dimorphic spores within sporophorous vesicles. Meronts did not have a surface coat of dense material, which has been previously reported for the genus. This is the first Ovipleistophora species described that does not have a tropism for ovary. Genetics demonstrated that O. diplostomuri n. sp. groups closely within fish microsporidia and not other species known to be hyperparasitic in digeneans, suggesting that it evolved from fish-infecting microsporidians and developed a secondary tropism for a common and widespread digenean parasite. The high genetic identity to Ovipleistophora species demonstrates the close relationship of this unique microsporidian with other microsporidia that infect ovary.
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    Gill disease (GD) in farmed Atlantic salmon may be due to the impact of several agents that may interact. We followed the infections of four gill disease-associated agents during the first year of production in six farms; examining the correlation between their densities in the gills. These farms were located in an area with high risk of developing gill disease, and three of the farms were diagnosed with GD in the autumn. In the present study we show temporal changes in pathogens in salmon from all 6 farms, revealing recurring infection patterns by Desmozoon lepeophtherii and Candidatus Branchiomonas cysticola (C. B. cysticola), and large variation without an apparent pattern in densities of Ichthyobodo salmonis. Infections with the microsporidian D. lepeophtherii show a clear seasonal pattern, and high densities are associated with GD. Ca. B. cysticola appears not to be directly connected to the disease, albeit densities of this bacterium may coincide with high D. lepeophtherii densities. Amoebae (Paramoeba spp.) were only sporadically detected with low densities. Salmon with low condition had elevated densities of D. lepeophtherii and Ichthyobodo spp., while no such pattern was seen for the other pathogens. Challenge studies are needed to substantiate the role of D. lepeophtherii in GD, but realistic challenge model (i.e. bath) has so far not been developed. Based on the present molecular screening, we suggest that D. lepeophtherii infections both may be responsible for GD, and cause runting in farmed salmon populations. Statement of relevance This is an epizootiological study of Atlantic salmon and gill diseases. There is much known about pathogen in connection with GD outbreak, less is known about the occurrence of potential pathogen in healthy farmed salmon or how such infections develop over time. High densities of the microsporidian parasite Paranucleospora theridion in the gills coincide with gill disease, making P. theridion a possible primary source to this condition. Densities of Paranucleospora theridion, Candidatus Branchiomonas cysticola and Ichthyobodo salmonis may be correlated in farmed salmon populations with gill disease. P.theridion and I. salmonis densities are elevated in Atlantic salmon with low condition (‘runts’).
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    Full-text available
    The microsporidian Paranucleospora theridion was discovered in Atlantic salmon Salmo salar suffering from proliferative gill disease in a marine farm in western Norway in 2008. The parasite develops in cells of the reticuloendothelial system, cells important for normal immune function. The aim of this study was to see if P. theridion could play a part in some of the diseases with unclear causes in salmon production in Norway, i.e. proliferative gill disease (PGI), pancreas disease (PD), heart and skeletal muscle inflammation (HSMI) and cardiomyopathy syndrome (CMS). P. theridion was present in all areas with salmon farming in Norway, but high prevalence and densities of the parasite in salmon and salmon lice were only seen in southern Norway. This region is also the main area for PGI and PD in Norway. Quantification of pathogens associated with PGI, PD, HSMI and CMS diagnoses showed that P. theridion levels are high in southern Norway, and may therefore play a role in susceptibility and disease development. However, among the different diagnoses, fish with PGI are particularly heavily infected with P. theridion. Therefore, P. theridion appears as a possible primary agent in cases with high mortality in connection with PGI in western Norway.
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    Full-text available
    Proliferative gill inflammation (PGI) is an important cause of loss in seawater-farmed Atlantic salmon in Norway. Several microbes have been associated with PGI, including the commonly but not exclusively observed inclusions (epitheliocysts) within the gill lamellae related to infection with 'Candidatus Piscichlamydia salmonis'. Atlantic salmon transferred in the spring of 2004 to 12 seawater farms situated in mid- and southwest Norway were sampled throughout that year. Outbreaks of PGI, as evaluated by clinical examination, histology, and mortality data, were diagnosed in 6 of 7 farms in southwest Norway but not in the 5 farms studied in mid-Norway. Generally, mortality started 3 to 5 mo after seawater transfer and outbreaks lasted at least 1 to 3 mo. 'Ca. P. salmonis' was detected by real-time PCR only in fish from PGI-affected farms and our results indicate an association between 'Ca. P. salmonis' load and PGI severity. Likewise, although widely distributed in all 12 farms studied, epitheliocyst prevalence and number per fish as observed by histology appears associated with PGI prevalence and severity. However, the occurrence of epitheliocysts showed no association with molecular detection of 'Ca. P. salmonis', suggesting that at least 1 other organism is responsible for many of the observed inclusions. A microsporidian, Desmozoon lepeophtherii, was identified at high prevalence regardless of fish and farm PGI status, but at higher loads in fish with PGI. Our results support a multifactorial etiology for PGI in which 'Ca. P. salmonis', an unidentified epitheliocyst agent, and the microsporidian are contributing causes. No evidence for the involvement of Atlantic salmon paramyxovirus in PGI development was identified in the present study. High water temperatures and ectoparasites probably exacerbated mortality.
  • Article
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  • Article
    Full-text available
    A microsporidian was previously reported to infect the crustacean parasite, Lepeophtheirus salmonis (Krøyer, 1837) (Copepoda, Caligidae), on farmed Atlantic salmon (Salmo salar L.) in Scotland. The microsporidian was shown to be a novel species with a molecular phylogenetic relationship to Nucleospora (Enterocytozoonidae), but the original report did not assign it to a genus or species. Further studies examined the development of the microsporidian in L. salmonis using electron microscopy and re-evaluated the molecular findings using new sequence data available for the group. Here we report a full description for the microsporidian and assign it to a new genus and species. The microsporidian infects subcuticular cells that lie on the innermost region of the epidermal tissue layer beneath the cuticle and along the internal haemocoelic divisions. The mature spores are sub-spherical with a single nucleus and an isofilar polar filament with 5-8 turns in a double coil. The entire development is in direct contact with the host cell cytoplasm and is polysporous. During early merogony, a diplokaryotic nuclear arrangement exists which is absent throughout the rest of the developmental cycle. Large merogonial plasmodia form which divide to form single uninucleate sporonts. Sporogonial plasmodia were not observed; instead, binucleate sporonts divide to form two sporoblasts. Prior to final division, there is a precocious development of the polar filament extrusion apparatus which is associated with large electron lucent inclusions (ELIs). Analyses of DNA sequences reveal that the microsporidian is robustly supported in a clade with other members of the Enterocytozoonidae and confirms a close phylogenetic relationship with Nucleospora. The ultrastructural findings of the precocious development of the polar filament and the presence of ELIs are consistent with those of the Enterocytozoonidae. However, the confirmed presence of an early diplokaryotic stage and a merogonial plasmodium that divides to yield uninucleate sporonts instead of transforming into a sporogonial syncitium, are features not currently associated with the family. Yet, analyses of DNA sequence data clearly place the microsporidian within the Enterocytozoonidae. Therefore, due to the novelty of the copepod host, the ultrastructural findings and the robust nature of the phylogenetic analyses, a new genus should be created within the Enterocytozoonide; Desmozoon lepeophtherii n. gen. n. sp. is proposed.
  • Article
    The emergence of infectious salmon anaemia virus (ISAV) in Canada and Scotland and frequent new outbreaks of the disease in Norway strongly suggest that there are natural reservoirs for the virus. The main host for the ISA virus is probably a fish occurring in the coastal area, most likely a salmonid fish. Since sea trout is an abundant species along the Norwegian coast, common in areas where ISA outbreaks occur, and possibly a life-long carrier of the ISA virus, a study was initiated to evaluate reverse transcriptase polymerase chain reaction (RT-PCR) for diagnosis of the virus in experimentally infected trout. Several tissues (kidney, spleen, heart and skin) were collected from the trout during a 135 d period. The following diagnostic methods for detection of the ISA virus were compared: cell culture (Atlantic Salmon Kidney, ASK cells), challenge of disease-free salmon with blood from the infected trout, and RT-PCR. The RT-PCR was the most sensitive of these methods. With the help of this technique it was possible to pick out positive individuals throughout the experimental period of 135 d. Challenge of disease-free salmon were more sensitive than cell culture (ASK cells). These 2 latter methods require use of the immunofluorescent antibody test (IFAT) or RT-PCR for verification of presence of ISA virus.