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4, Bull. Eur. Ass. Fish Pathol., 40(1) 2020
EAFP 19TH INTERNATIONAL CONFERENCE ON DISEASES OF FISH
AND SHELLFISH SPECIAL EDITION WORKSHOP REPORT
Co-infections and multiple stressors in sh
B. Gorgoglione 1*#, C. Bailey 2#, M.D. Fast 3^, D. Bass ⁴,
M. Saraiva 5, M. Adamek 6, P. Noguera 7, S. Ciulli 8,
M. Palíková 9, I. Aguirre-Gil 10, L. Bigarré 11# and O. Haenen 12#
1 Michigan State University, East Lansing, Michigan, USA; 2 Animal Health Research Centre
(CISA-INIA), Madrid, Spain; 3 Atlantic Veterinary College, UPEI, Charloetown, Canada; 4 Centre
for Environment, Fisheries and Aquaculture Science, Weymouth, UK; 5 University of Aberdeen,
Aberdeen, UK; 6 University of Veterinary Medicine, Hannover, Germany; 7 Marine Scotland
Science, Aberdeen, UK; 8 University of Bologna, Bologna, Italy; 9 University of Veterinary and
Pharmaceutical Sciences, Brno, Czech Republic; 10 Universidad Austral de Chile, Valdivia, Chile;
11ANSES, Plouzané, France; 12 Wageningen Bioveterinary Research, Lelystad, the Netherlands
* Corresponding author and workshop leading organiser's e-mail: BartGorg@msu.edu ;
# Workshop co-organizer; ^ keynote speaker
Introduction
Fish are typically exposed to multiple physical,
chemical and biological stressors. The cumula-
tive impact of co-infections between parasites,
bacteria, viruses and (a)biotic environmental
pressures may trigger complex interactions, elic-
iting dierent pathological and immunological
outcomes than those classically assessed. New
cross-disciplinary studies aempt to measure the
impact of environmental stressors in modulating
the host response to pathogens. Scientic ad-
vances are needed to reduce pressure on natural
populations, improve sh stock management,
and to design more ecient diagnostic tools
or vaccination strategies. An EAFP-promoted
workshop, held on 10th September 2019 in Porto,
Portugal, was dedicated to sharing research
experiences on the interaction between heterog-
enous pathogens and multiple stressors in sh.
The workshop involved around 200 aendants,
opened by a keynote talk (Fast), and followed
by a further twelve oral presentations, including
three in the format of ash poster presentations.
Contributions illustrated cross-disciplinary ap-
proaches to study complex host-pathogen and
stressors interactions.
Pathological synergies in co-in-
fecting pathogens are impacted
by exposure order, and host re-
sponse to initial infection
K.S. Parrish, S.L. Purcell, S.K. Whyte, A.J.
Manning, L. Carvalho, S. Dalvin, R.G. Taylor,
M.L. Rise and M.D. Fast
In wild and cultured sh populations, expo-
sure and response to potential pathogens is
Bull. Eur. Ass. Fish Pathol., 40(1) 2020, 5
a dynamic process. Until recently our under-
standing of host-pathogen interactions was
limited to single exposures under individual
pulsed conditions. In wild and cultured salmo-
nids, the ectoparasitic copepod Lepeophtheirus
salmonis (L.s.; aka “sea lice”), is highly preva-
lent and commonly causes acute and chronic
immuno-physiological strain on its hosts in
marine environments. Studies have shown that
sea lice infection, can have signicant impacts
on immune responses of their host, such as de-
creased anti-viral responses (Barker et al., 2019),
and anti-bacterial immunity (Figueroa et al.,
2017). We have recently begun to examine the
immunomodulatory and immuno-physiological
eects sea lice infection has on the development
of disease with other parasitic pathogens, viral
and bacterial pathogens. Co-exposure of hosts
to Caligus elongatus and L.s. copepodids, showed
no impact on infection level or host impacts
compared to single infection. However, in the
case of bacterial and viral infections, prior infec-
tion with L.s. enhanced pathology and mortality
associated with subsequent infecting organisms.
This was not always linked to the intensity of sea
lice infection. In the case of co-infection with the
cold water ulcer-associated bacteria, Moritella
viscosa (M.v.), our data suggest that the order
of establishment impacted subsequent survival
(Figure 1.A), and lice abundance (Figure 1.B).
Fish exposed to co-infection with lice and M.v.
showed enhanced inammation and acute phase
response signals in skin over time. However,
sh with active lesions positive for bacterial
growth showed the opposite, inhibited inam-
matory signals in the skin, potentially linked to
a higher mortality rate. Host immunomodula-
tion through transcriptomic suppression is a
hallmark of lice infections, likely enhancing
colonisation of viral and bacterial pathogens.
Iron regulation and transport are also com-
monly implicated in L.s. infection (Sutherland
et al., 2014), and acutely (eg hepcidin) observed
in lice and bacterial exposure in salmon (Martin
et al., 2006). The order of infection with L.s. and
M.v. impacted dierential expression of iron
homeostasis marker hepcidin in infected sh
skin sites away from L.s. aachment. Functional
feed properties did not have an overall inu-
ence on single and M.v. co-infection outcomes.
However, multiple functional feeds signicantly
impacted survival following viral (eg infectious
salmon anaemia) infection (eg CpG motifs) and
lice counts (eg CpGs and %EPA:DHA inclusion).
Figure 1. Differential survival (Kaplan-Meier
plot) (A), and lice counts (B) following exposure to
Lepeophtheirus salmonis (L) and/or Moritella viscosa
(M), using a commercial (C) or experimental
(E) diet. Leers on the bars denote signicant
differences in infection level (GLM; post-hoc
Bonferroni, p<0.05).
6, Bull. Eur. Ass. Fish Pathol., 40(1) 2020
In each case, feed gains in survival or reductions
in lice number were all eliminated following ad-
dition of a co-infecting pathogen. These results
demonstrate the need for a more complex ap-
proach to studying disease pathogenesis, to
improve immune potentiating approaches in
sh in the future.
The pathobiome in animal and
plant health
D. Bass, C. Tyler, H-C. Wang, B. Koskella
and G. Stentiford
A ‘pathobiome’ is the set of host-associated
organisms (encompassing prokaryotes, eukary-
otes, and viruses), linked to a reduced health
status resulting from interactions between the
members of that set of organisms and the host
(Bass et al., 2019). These interactions are moder-
ated by the environment within the host and im-
mediately surrounding it. A pathobiome com-
prises a host with anything from two non-host
lineages up to a complex symbiont community.
Microbial diversity is now known to be much
more diverse than previously thought in both
environmental and host-associated habitats.
There is a large and increasing literature report-
ing high levels of protistan, bacterial, and viral
diversity revealed by eDNA/RNA-style analyses
of host-associated (eg tissue, gut contents, skin
epibionts) and environmental samples (e.g.
water, sediment, soils) (Bass et al., 2015) (Figure
2). This diversity includes many lineages that
are (or may become) host-associated and can
form part of pathobiomes. These include cryptic,
emerging, previously unrecognised, and op-
portunistic pathogens, including commensals
switching to pathogenic modes (Overstreet and
Lo, 2016). Our appreciation of what constitutes
a ‘pathogen’ has diversied and will continue
to do so as we beer understand the complexity
of the pathogenic process. Pathobiotic mecha-
nisms include antagonism or synergism among
infecting agents, for example by one agent in-
uencing the resistance of the host to another,
or via signal sharing, metabolic interactions, etc.
Genomic contributors to pathobiotic systems
include co-existence of bacterial strains with
complementary gene content, horizontal gene
transfer via plasmids, transposons, genomic
islands, and bacteriophages. However, patho-
biotic mechanisms, particularly at the broader
symbiont community level, are poorly under-
stood. Diseases with indistinguishable clinical
signs are often thought to be caused by the
same primary pathogen, but may have dierent
aetiologies, an example being white pox disease
of the coral Acropora (Kemp et al., 2018). Even in
cases of diseases for which a primary pathogen
is identied, it is important to remember that
that pathogen is operating within a complex
microbial community, likely to modify its ac-
tivities and their results in diverse ways. This
aspect of pathogenesis and disease aetiology
Figure 2. Microbial complexity in a sh-exemplar
host-symbiont-env ironment system (Adapted from
Bass et al., 2019). Green dots represent the sh gut
microbiome (proka ryotes, eukaryotes, a nd viruses)
and its exchange with other compartments of the
habitat. Similarly for sh tissue/blood microbiome
(yellow), skin (red), and the water and sediment
microbiomes of the system (blue, brown).
Bull. Eur. Ass. Fish Pathol., 40(1) 2020, 7
is under-recognised but important for under-
standing dierent presentations of the same
disease in dierent cases; a situation frequently
complicating disease investigation. As symbi-
ont community structures are often highly vari-
able, functional traits such as gene expression
paerns at dierent levels (eg core and non-core
functions), and metabolic interactions provide
complementary perspectives with dierent pat-
terns of variability, from which to understand
pathobiotic mechanisms. Understanding host
symbiomes will be key to mitigating disease in
global food production systems (Stentiford et
al., 2017). Bodies such as the World Organisation
for Animal Health dene a list of emerging and
notiable diseases associated with infection by
specic agents, against which diagnostic tests
and management strategies can be designed.
However, such legislation does not currently
take in to account broader symbiont proling
of hosts, and the role that the symbiome may
play in disease outbreaks.
Expecting the unexpected: an
analysis of multiple stressors
and their physiological conse-
quences for rainbow trout
C. Bailey, E. Wernicke von Siebenthal, K. Re-
hberger, A. Ros, E.L. Herzog and H. Segner
The purpose of this study was to disentangle
the molecular and organism level reactions of
rainbow trout, Oncorhynchus mykiss, to the com-
bined impact of two environmental stressors oc-
curring in the natural habitat of salmonids. Fish
were either exposed to: 1) a myxozoan parasite,
Tetracapsuloides bryosalmonae (T.b.) causing pro-
liferative kidney disease (PKD); 2) an estrogenic
endocrine-disrupting compound ethinylestradi-
ol (EE2); or 3) a combination of both (T.b.xEE2).
To evaluate the chronic impact of these stressors
exposures the sh response was investigated
for 130 days post-exposure (dpe). In T.b. only
exposed sh a greater parasite burden was
observed at every time point, in contrast to sh
exposed to T.b.xEE2. Moreover, in these sh at
90 dpe there was an increase in mRNA levels of
immune genes in the anterior kidney that had
shown to be reactive during PKD pathogenesis
in our earlier studies (Bailey et al., 2017) (blimp1,
igm-sec, il-10 and nkef) and increased patho-
logical alterations in primary and secondary
lymphoid organs (anterior kidney, posterior
kidney and spleen) relative to the EE2 exposed
sh and T.b.xEE2 sh (Wernicke von Siebenthal
et al., 2018). While in T.b.xEE2 sh there was
lower parasite burden and lower expression of
immune genes, but clear evidence of EE2 in the
liver in terms of pathology and vitellogenesis
production in contrast to the other treatments
(Wernicke von Siebenthal et al., 2018). At 130
dpe, RNA-seq was applied to the posterior
kidney of all experimental groups, this was a
time when parasite intensity in the sh kidney
started to decrease. A greater number of dier-
entially expressed genes (DEGs) across major
physiological and immunological processes
was seen in the T.b.xEE2 group, not observed
in or that could be deduced from either of the
single stressor groups (Bailey et al., 2019). All
groups exhibited a low intensity immune re-
sponse, in contrast to what was reported for
advanced PKD. This might suggest a trade-o
where the host increases investment in recovery/
resolution processes over immune responses at
a later stage of disease. In the T.b.xEE2 group
several T cell related genes (tbet, cd8α, foxp3-1,
foxp3-2, cd3e and mhc-II) were downregulated
(Figure 3.A) (Bailey et al., 2019). This could in-
8, Bull. Eur. Ass. Fish Pathol., 40(1) 2020
dicate that T.b.xEE2 might be having a greater
immunomodulating role than in T.b. infected
sh, leading to a reduction in PKD associated
immune response and aenuating the disease
impact (Bailey et al., 2019). Alternatively, as a
greater number of immune genes were upregu-
lated (mcsf, c, notch1, bcl6b, sigirr, kit and nramp-
α) in these sh this may have translated into a
lower parasite burden and reduced pathological
alterations (Figure 3.B). In conclusion, the EE2
eects were paradoxical, that they exerted both
immunosuppressive and immunoenhancing
actions as reported in mammals. This is likely
inuenced by the pathogen (chronic/acute), the
EE2 concentration, and physiological status of
the host.
Saprolegniosis:
who is out there?
M. Saraiva and P. van West
Saprolegnia is a eukaryotic pathogen of sh,
endemic to all freshwater habitats around the
world. Due to their lamentous hyphal struc-
ture oomycetes are often described as fungal-
like microorganisms. Saprolegnia parasitica (S.p.)
causes devastating infections to freshwater sh,
leading to Saprolegniosis (Figure 4.A) (van
West, 2006). S.p. tends to favour sh, whereas
S. diclina prefers to infect sh eggs, although
both species are found on all freshwater stages
of both salmon and trout. S.p. was previously
thought to be a secondary pathogen, but this
is not the case since Saprolegnia is capable of
actively suppressing the host immunity and
expressing several eector proteins that can
translocate into host cells, of which one is able
to degrade sh RNA (Belmonte et al., 2014).
However, it is likely that other microbes, such
as true fungi, play a major role in the initia-
tion of infection and possibly in increasing the
severity of saprolegniosis. Fungal–bacterial
interactions vary depending on species, strain
and environments, and they can be endosym-
biotic, synergistic or antagonistic. In general,
polymicrobial infections are harder to treat
due to increased resistance to antimicrobial
therapy, as such, polymicrobial diseases can
have increased mortality compared with their
monomicrobial counterparts. These mixed
communities possess an intercellular form of
communication, often referred to as quorum
Figure 3. Impact of multiple stressor exposure
(T.b.+EE2) on T cell-related genes at 130 dpe in the
posterior kidney. T cell-associated genes shown to
be sig nicantly downregu lated (A); im mu ne genes
shown to be signicantly upregulated (B).
Bull. Eur. Ass. Fish Pathol., 40(1) 2020, 9
sensing (QS). QS is used to monitor the local
environment to bring about a concerted change
in behaviour benecial to the community. QS
molecules were characterised in several true
fungal species (Barriuso et al., 2018) and are
involved in growth regulation, stress resist-
ance, morphogenesis, antibiotic production,
motility, sporulation and biolm formation,
some produce quorum-sensing inhibitors that
prevent communication, reducing their com-
petitors’ virulence (Rasmussen et al. 2005).
More than 300 pure isolates were obtained
from sh farms and hatcheries around Scot-
land. The second most abundant/represented
genus was Mortierella (Figure 4.B), a soil fungus
with species known to eectively transform a
series of sh-toxic diterpenes and their chlo-
rinated analogues into nontoxic metabolites
(Kutney et al., 1985). It is speculative at this
stage, but we believe this mechanism might
aid Saprolegnia to infect sh. Metagenomic
analysis is being undertaken to understand
what species of bacteria and eukaryotes are
present in dierent seings (before, during and
after infection). Characterising the complex
interactions that occur in these mixed-species
communities will undoubtedly increase our
understanding of host-pathogen relationships,
the mechanisms that underly infection and
help design/discovery of novel sustainable
control strategies.
Figur e 4. Atlantic salmon i nfected with Saprolegnia parasitica (A); fungal community found on sh in Scotla nd
(B).
10, Bull. Eur. Ass. Fish Pathol., 40(1) 2020
Multi-causal eel diseases in the
Netherlands
O. Haenen, M. Voorbergen-Laarman, E. van
Gelderen, A. Davidse, I. Roozenburg, R.
Vloet, S. van Beurden and M. Engelsma
Eel disease is seen as one of the factors in the
decline of wild eel stocks (Haenen, 2019). Wild
eels are farmed for consumption, and some are
restocked into the wild against the eel popula-
tion decline. This implies the transmission of
pathogens at least from wild to farmed eel,
and possibly back. In the past, global trade of
stocking eels caused the introduction of eel para-
sites and pathogens from South East Asia into
West Europe. Examples are the swim bladder
nematode Anguillicoloides crassus (Van Banning
and Haenen, 1990), gill trematodes Pseudodac-
tylogyrus anguillae and P. bini (Buchmann et
al., 1987), and Herpesvirus anguillae (AngHV-1)
(Davidse et al., 1999). In our diagnostics, farmed
eel suered regularly from multi-causal infec-
tions, like Pseudodactylogyrus spp. with stress
through poor water quality or handling as basis,
with single or double viral infections by the
AngHV-1, Eel Virus European (EVE), or Eel
Virus European X (EVEX) (Haenen et al, 2002;
Van Beurden et al., 2012), and single or double
bacterial infections with e.g. Pseudomonas anguil
-
liseptica, Vibrio vulnicus (Figure 5), Edwardsiella
tarda, or Aeromonas salmonicida. The water tem-
perature thereby is an important factor, with an
optimum per virus, and a positive correlation
for most bacteria and parasites in replication.
It is recommended, to recruit healthy wild eels
regionally, avoid global trade of live eels, avoid
stress in eel farming, and check farmed eels for
the absence of diseases before they are restocked
regionally into the wild.
Flavobacterium branchiophi-
lum co-infection can increase
pathological changes during koi
sleepy disease caused by carp
edema virus infection in carp
M. Adamek, F. Teitge, V. Jung-Schroers, M.
Heling, D. Gela, V. Piackova, M. Kocour and
D. Steinhagen
Koi sleepy disease (KSD) aecting common
carp is an often-fatal gill condition. Carp edema
virus (CEV) is treated as the causative agent
of KSD, however the disease often seems to
have multifactorial causes (Way et al., 2017).
Therefore, we hypothesised that CEV infection
Figure 5. Farmed European eel from a Dutch eel farm with skin lesions, and extreme septicaemia with
haemorrhages due to a severe triple infection with two viruses (EVEX, AngHV-1) and (Vibrio vulnicus/
navarrensis).
Bull. Eur. Ass. Fish Pathol., 40(1) 2020, 11
may promote infections of gills with secondary
pathogens, which subsequently increase the
severity of pathology. We analysed the possi-
bility of an interaction between infections with
Flavobacteria and CEV during the development
of clinical KSD. We examined gill samples of
carp and koi from Germany and Hungary and
performed infection experiments and antibiotic
treatment of KSD aected sh. The amounts
of Flavobacteria and CEV were evaluated by
qPCR. The typing of Flavobacteria was per-
formed by isolating the bacteria by culturing
method using Cytophaga Agar Base and by
molecular cloning and sequencing of the 16S
amplicon. Pathogenesis was monitored by anal-
ysis of sh behaviour and by gill histology. The
co-infection with CEV and Flavobacteria was
diagnosed in samples collected from common
carp aquaculture. There was a positive correla-
tion (r=0.7318, p<0.0001) between CEV and Fla-
vobacteria loads (Figure 6.A) in gills. Especially
individuals with higher CEV loads (>105 virus
copies/250 ng DNA) had statistically higher
Flavobacteria loads in gills when compared to
CEV-free sh. Infection experiments indicated
a rapid transfer and progress of CEV and avo-
bacterial co-infections. Culturing and molecular
methods identied Flavobacterium branchiophi-
lum (F.b.) as the possible Flavobacteria species
causing co-infections during KSD. Both CEV
and Flavobacteria infections progressed faster
in KSD-susceptible koi strain when compared
to a KSD-resistant common carp strain (Amur
wild carp), which could indicate possible co-
dependency of the pathogens. Importantly,
further experiments with an antibiotic treatment
preventing F.b. infection conrmed that CEV is
the main pathogen in KSD, as the presence of the
bacterial co-infection was not required for KSD
pathogenesis. However, CEV-F.b. co-infection
could be responsible for increased levels of epi-
thelial cells hyperplasia, epithelium lifting and
proliferation of the intra-lamellar cellular mass,
when compared to single CEV infection (Figures
6.B and 6.C). Therefore, F.b. co-infection could
worsen pathological changes recorded during
KSD outbreaks. Our results suggest that CEV
gill infection may facilitate co-infections with
other pathogens (Adamek et al., 2018).
Simultaneous and sequential
co-infection paerns modulat-
ing rainbow trout response to
BCWD and IHN
B. Gorgoglione, D.R. Jones, D.W. Leaman
and A.R. Wargo
Figure 6. Correlation analysis between CEV and Flavobacteria loads in gills of common carp farmed in
Germany and Hungary (A). Gill pathology in koi aected by CEV with avobacterial co-infection, treated
with 18 mg/l of orfenicol (B), or untreated (C); H&E.
12, Bull. Eur. Ass. Fish Pathol., 40(1) 2020
Few studies have addressed co-infections with
multiple pathogen species in salmonids. Those
conducted have shown increased suscepti-
bility and pathogenesis (Kotob et al., 2017;
Nicholson et al., 2019), or enhanced or pro-
longed immune responses (Gorgoglione et
al., 2019). Flavobacterium psychrophilum (F.p.),
causing bacterial cold-water disease (BCWD),
and Salmonid novirhabdovirus (IHNV), causing
infectious hematopoietic necrosis (IHN), are
major pathogens aecting rainbow trout in
aquaculture, although lile is known about
their interaction. Our study investigated
how F.p. and IHNV co-infections alter the
host’s ability to respond to these pathogens.
Seventy sh per treatment underwent the
following pathogen exposures: single IHNV;
sequential F.p.+IHNV; single F.p.; simultane-
ous F.p.+IHNV; negative control. Trout were
maintained in a ow-through system at 15°C
in individual 0.8 l tanks. For single infections
sh were respectively IP-injected with 50 μl
F.p. suspension, with sterile TYES medium
(F.p. control), bath-challenged with IHNV
(C-genogroup), or with sterile medium (IHNV
control). Fish allocated to the sequential co-
infection group were infected with bacteria,
and after 48 h with INHV, using the same
dosage as single infections. Trout organs were
sampled at 1,3,5,7,9 and 11 dpe. The simultane-
ous F.p.+IHNV co-infection induced the fastest
and most exacerbated pathology, with mor-
tality starting at 5 dpe and peaking between
6-7 dpe. These trout showed typical clinical
signs of IHNV, including marked exophthal-
mos (Figure 7), anaemia, ascites often with
diuse haemorrhaging to abdominal organs,
and a higher splenosomatic index at every
time-point. The sequential infection group
exhibited pathological signs, although milder,
and with mortality starting at 8-9 dpe. A much
milder gross pathology was observed in the
single infection groups, with no mortality up
to 11 dpe. Our results are in line with similar
synergistic pathology and mortality results
described upon F.p.+IHNV co-infections, from
a dierent infection methodology (Ma et al.,
2019). Sequential infections are likely to be
more biologically relevant, thus more suitable
to study pathobiological dynamics during
co-infections, and beer quantiable immune
responses, when compared to simultaneous
co-infections where the combined pathoge-
netic eect of multiple pathogens exacerbates
the pathology and drastically reduces host sur-
vival. Studies on heterogenous co-infections
Fi gure 7. Necropsy of rainbow trout sequentially co-infected with Flavobacterium psychrophilum and IHNV,
showing exacerbated exophthalmos and ascites.
Bull. Eur. Ass. Fish Pathol., 40(1) 2020, 13
are likely to lead to insights regarding disease
management in the eld, where co-infection
is common.
As a multifunctional organ, gills are the primary
site for respiration, osmoregulation, acid-base
balance and metabolism of circulating hor-
mones and xenobiotics. Gill diseases in farmed
Atlantic salmon have been on the rise in recent
years becoming a serious welfare concern and
causing substantial economic losses. From the
eects of well-characterised agents, eg Neop-
aramoeba perurans causing amoebic gill disease
(AGD), to the combined response to AGD and
other biological or non-biological factors, gill
health represents a major challenge for salmon
industry (Herrero et al., 2018). Aimed to support
early actions on management strategies, disease
control schemes implemented at farm level
include periodical monitoring for scoring gross
macroscopic changes associated with AGD and
proliferative gill disease (PGD). Salmon gills
from geographically diverse Scoish aquacul-
ture sites were examined to explore the un-
derlying molecular events associated with the
progression of the above-mentioned conditions.
After in situ macroscopic scoring of over 200
sh, with data recorded from both surfaces
of all 8 arches. The gill arch with the highest
PGD score was sampled for histopathology
and RNA-seq analysis. The histological evalu-
ation and scoring (Mitchell et al., 2012) was
performed on digitalised images of routine
H&E stained sections, while extracted RNA
from a subset of 43 sh presenting low (=1) or
medium score (=3) PGD scores were analysed
by whole transcriptome analysis using RNA-
seq. For each sh, 20 M reads were generated
and mapped to the Atlantic salmon genome.
The predominant histopathological features
included hyperplastic and proliferative lesions,
frequently associated with a degree of lamel-
lar fusion, inammatory reaction, presence of
amoeba and relevant vascular lesions. Changes
in gross morphology and histopathology were
not consistent with each other but were rather
linked to the sampling location. Results from
RNA-seq analysis on same individuals showed
a similar trend. The minimal common responses
between dierent sites suggest spatio-temporal
location represents an additional important
factor inuencing the gill response. Histopathol-
ogy scores, and dierences in gene expression,
were driven by the site rather than by PGD
scores, clustering together based on the sample
origin. This suggests that macro and micro
scores may inform on the overall progression of
gill damage, but not on underlying pathology,
providing support for a complex and multifac-
torial aetiology of the gill condition.
Multiple co-infections and envi-
ronmental stressors as causes of
chronic mortalities in juvenile
sturgeons
S. Ciulli, E. Volpe, G. Tura, E. Zavaa, A.
Renzi, T. Preo, A. Toan, O. Mordenti, F.
Errani, R. Sirri, G. Sarli, P. Serratore and L.
Mandrioli
Sturgeon farming is an increasing activity with
great interest in Huso huso species for caviar
Gill histopathology scoring vs
gross morphology and transcrip-
tome analysis in farmed Atlantic
salmon
P. Noguera, E. Król, A. Douglas, R. Bicker-
dike, V. Valdenegro, K. Gajardo and S.A.M.
Martin
14, Bull. Eur. Ass. Fish Pathol., 40(1) 2020
production and restocking. Currently, few
data on H. huso health issues and on stur-
geon pathology are available. We examined
an episode of chronic mortality in juvenile H.
huso in Italy, showing inverted and circular
swimming, hyperactivity to stimuli alternated
to prolonged resting on the boom. Samples
from the brain, kidney, spleen, gill, skin were
collected for bacteriological and virologic anal-
yses. Cytology of coelomatic eusions was
performed and the main organs were sampled
for histology. Body shape deformities, epaxial
muscle softening, multifocal ulcerative der-
matitis and a sero-haemorrhagic coelomatic
eusion, which at cytological examination
showed to be septic due to the presence of
Gram-negative rods and pigmented-wall
hyphae were observed. Bacteriology indi-
cated septicaemia, due to Aeromonas veronii,
Shewanella spp. and Citrobacter freundii. No
viral growth was obtained in WSSK-1 cells, a
known suitable cell line for sturgeon herpes-
viruses, nor investigated viruses (viz Betano-
davirus and sturgeon nucleo-cytoplasmic large
DNA viruses) were detected by PCR (Ciulli et
al., 2016). Histology showed rarefaction and
disorganisation of hematopoietic lymphoid
tissue. Other ndings, such as glomerular
regeneration, sporadic intratubular mineral
deposits in the renal parenchyma (Figure 8.A)
and splenic vessel hyalinosis (Figure 8.B),
were detected and interpreted as a response
to negative environmental impacts, as previ-
ously reported in sturgeons (Romanov et al.,
2006). The histology conrmed septicaemia,
showing bacterial aggregates and pigment-
ed-wall hyphae in several tissues. Current
evidence suggests that pathological ndings
result from the sum of environmental stressors
and multiple co-infections. Despite some of
the bacteria found in this study being previ-
ously associated with mortality outbreaks
in several sh species (Gholamhosseini et
al., 2018; Volpe et al., 2019), other primary
causes could be present, such as Herpesvirus
or Adenovirus. In the absence of other primary
biological agents, prolonged environmental
stressors could be considered the cause of
immune system impairment, facilitating the
entry of opportunistic bacteria and mycetes.
Indeed, the nephrocalcinosis and vessel hya-
linosis suggested the presence of predisposing
Figure 8. Nephrocalcinosis in kidney of sturgeon (A); spleen with vascular hyalinosis and depletion of the
white and red pulp (B). H&E.
Bull. Eur. Ass. Fish Pathol., 40(1) 2020, 15
environmental stressors. Ongoing analyses,
including electron microscopy and metagen-
omics, are further investigating the presence
of viral pathogens that could have triggered
the impairment of the H. huso immune system.
Component causes of severe gill
damage in rainbow trout farmed
under conditions of RAS
M. Palíková, I. Dyková, I. Papežíková and J.
Mareš
Figure 9. Macroscopic and histopathological ndings in rainbow trout from RAS: marked gill swelling
(A); hyperplasia of gill epithelium (“clubbing”) (B); inammatory changes in gill laments with prevailing
lymphocytes (C); eosinophilic granular cells predominating in inammatory inltrate along gill laments
axis (D); gill lament segments with lamellar fusion and channels with bacteria aggregates (E). H&E.
16, Bull. Eur. Ass. Fish Pathol., 40(1) 2020
In the fall of 2018, a rainbow trout farm using
an indoor recirculating aquaculture system
(RAS) reported disease issues, with mortality
among stocks of marketing weight. To assess
the health condition of the entire system, we
combined detailed examinations with a series
of tests of water quality and evaluation of
water treatment eects. During ve repeated
samplings we performed necropsies, histo-
pathological examination, and aempted the
isolation of bacteria and amoebae. RAS water
temperature ranged between 15.0 and 18.7°C.
Clinical signs included frequent jumping
of sh with grossly visible lesions, includ-
ing marked gill swelling (Figure 9.A). The
presence of Ichthyophthirius multiliis (0–20
specimens in a microscopic eld at 50X mag-
nication) was only found in gills at the rst
sampling. Histopathological lesions were con-
stantly including lamellar hypertrophy (like
that described after exposure to high ammonia
levels), lamellar fusion, and enormous hyper-
plasia of the epithelium in the distal part of
laments (“clubbing”) (Figure 9.B). Chronic
branchitis was found in a few instances (Figure
9.C), characterised by a dense accumulation of
eosinophilic granular cells along the vascular
axis of gill laments (Figure 9.D). Bacterial
clusters seen at the gill histological exami-
nation were identied upon the microbio-
logical examination (Figure 9.E), revealing the
multiple presences of Pseudomonas koreensis,
Aeromonas hydrophila, A. eucrenophila and A.
bestiarum (isolated using BA and TYES media).
Amoebae isolation aempts were in contrast
negative. Hydrochemical analysis revealed
hypersaturation (oxygen up to 200%), corre-
sponding to the presence of gas bubbles on ns
at one sampling point. Therapeutic measures
taken by the farmer included water ozonation,
daily application of peracetic acid, or addition
of salt and formaldehyde in a long-term bath.
Based on retrieved results, the exact cause of
gill damage could not be identied, thus it
can be speculatively aributed to the conjunct
action of bacteria and I. multiliis, in combina-
tion with the long-term exposure to multiple
chemicals used for treatment in combination
with gas hypersaturation.
Pathogen interactions during
experimental co-infection with
Piscirickesia salmonis and
Piscine Orthoreovirus in Salmo
salar
I. Aguirre-Gil, A. Mancilla, M. Navarrete,
E. Paredes, R. González-Stegmaier and R.
Enríquez
Piscine orthoreovirus (PRV) infections are wide-
spread in Chilean salmon farms, estimating that
over 60% of freshwater Atlantic salmon is pre-
dominantly infected with PRV-1 (SUBPESCA,
2018). In this scenario, co-infections with other
highly prevalent viruses or bacteria are likely
to occur. Clinical signs could be misdiagnosed
or be more severe due to dierent responses
triggered during simultaneous infections with
two or more pathogens. We evaluated the eect
of the natural exposure to PRV-1 of a smolt
population of Atlantic salmon on the outcome
of a cohabitation challenge with Piscirickesia
salmonis (P.s.), the most relevant bacterial patho-
gen for the Chilean salmon industry (Rozas and
Enríquez, 2014). From a population of PRV-1
positive salmon smolts (100 g), 84 shedder sh
were intraperitoneally injected with 5.2x10
6
CFU P.s. and placed together with 156 PRV-1
positive cohabitant sh to reach 35% pressure
Bull. Eur. Ass. Fish Pathol., 40(1) 2020, 17
of infection. Samples were taken at 14, 21, and
30 dpe to evaluate viral loads and immune
gene expression in the head kidney by qPCR
and RT-PCR. For histology, heart, head kidney,
liver, spleen, and gill were sampled. Viral loads,
and Type I Interferon transcripts, decreased
signicantly from 14 to 21 dpe and up to 30 dpe
(Figure 10). This was accompanied by a rise in
the percentage of P.s. positive co-habitants and
IL-8 expression from 14 to 21 dpe, with a drop
at 30 dpe. Our results indicate that the bacteria
pressed against the viral infection, decreasing
the viral load in the host. Histopathological
examination showed only mild to moderate
lesions compatible with initial SRS or HSMI
in liver and heart, and only in samples at 21
dpe. Tissue examination at 14 and 30 dpe did
not exhibit pathological evidence aributable
to any disease. The previous natural exposure
to PRV-1 did not increase the severity of SRS
in a population of salmon smolts exposed to
P.s. cohabitation challenge, however, the viral
loads dropped as the proportion of P.s. posi-
tive sh increased. Co-infection with these two
biological agents was achieved, but only mild
to moderate clinical signs were observed, at
21 dpe.
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... We extended our investigation to fry and fingerling of red hybrid tilapia from farms with a remote anamne- (Table S2). Polymicrobial infections with bacteria, parasites and viruses are common in aquaculture and often occurring during natural disease outbreaks, being fish exposed to a rich microbiome and concomitant stressors in their aquatic environment (Gorgoglione et al., 2020). Additionally, polymicrobial infection patterns including bacteria, parasites and TiLV were recently reported from field investigations and laboratory experimental challenge (Amal et al., 2018;Basri et al., 2020;Nicholson et al., 2017Nicholson et al., , 2020Surachetpong et al., 2017). ...
... Other emerging fish viruses were also rapidly discovered in many countries worldwide after their transboundary spreading was initially documented during coinfection patterns with more virulent pathogens. For example, Carp oedema virus (Piscipoxvirus; CEV) is becoming another emerging global threat to cyprinids and other fish species (Way et al., 2017), often detected in co-infection patterns involving Cyprinid herpesvirus-3 (KHV), Rhabdovirus carpio (SVCV) or ectoparasites, which can lead to high mortality (Gorgoglione et al., 2020;Haenen et al., 2016;Lewisch et al., 2015;Sauerwald et al., 2020;Zhang et al., 2017). ...
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Proliferative kidney disease (PKD), caused by the myxozoan endoparasite Tetracapsuloides bryosalmonae, is of serious ecological and economical concern to wild and farmed salmonids. Wild salmonid populations have declined due to PKD, primarily in rivers, in Europe and North America. Deep lakes are also important habitats for salmonids, and this work aimed to investigate parasite presence in five deep Norwegian lakes. Kidney samples from three salmonid species from deep lakes were collected and tested using real‐time PCR to detect PKD parasite presence. We present the first detection of T. bryosalmonae in European whitefish in Norway for the first time, as well as the first published documentation of the parasite in kidneys of Arctic charr, brown trout and whitefish in four lakes. The observed prevalence of the parasite was higher in populations of brown trout than of Arctic charr and whitefish. The parasite was detected in farmed, but not in wild, charr in one lake. This suggests a possible link with a depth of fish habitat and fewer T. bryosalmonae‐infected and PKD‐affected fish. Towards a warmer climate, cold hypolimnion in deep lakes may act as a refuge for wild salmonids, while cold deep water may be used to control PKD in farmed salmonids.
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