Scrutinizing the triad of Vibrio tapetis, the skin barrier and pigmentation as determining factors in the development of skin ulcerations in wild common dab (Limanda limanda)

Ve cau e ene al. Ve Res (2019) 50:41
h ps://doi.o g/10.1186/s13567-019-0659-6
RESEARCH ARTICLE
Sc u inizing he iad o Vib io ape is,
heskin ba ie andpigmen a ion
asde e mining ac o s in hede elopmen
o skin ulce a ions inwild common dab
(Limanda limanda)
Maaike Ve cau e en1* , E elien De Swae 2, Annelies M. Decle cq1,3, Hans Pole 4, Johan Ae s5, Ba Ampe6,
Jesus L. Romalde7, F eddy Haeseb ouck1, Lisa De iese3, Annemie Decos e e1† and Koen Chie s1†
Abs ac
Recen ly, Vib io ape is was isola ed o he i s ime om skin ulce a ions in wild-caugh common dab (Limanda
limanda). To u he examine i s ole in he de elopmen o hese skin lesions, an in i o expe imen was pe o med.
The signi icance o he skin ba ie and in addi ion he di e ence be ween pigmen ed and non-pigmen ed side we e
in es iga ed. Hence, he skin o common dab was ea ed in h ee di e en ways on bo h he pigmen ed and non-
pigmen ed side. On a i s “ ea men zone”, he scales and o e lying epide mal issue we e emo ed whe eas in a sec-
ond zone only he mucus was disca ded. The hi d zone se ed as a non- ea ed zone. The ea e , ish we e challenged
wi h V. ape is. The con ol g oup was sham ea ed. Mo ali y, clinical signs, se e i y and size o he de eloping lesions
we e eco ded. All animals we e sac i iced and sampled 21 days pos -inocula ion. Signi ican ly mo e ish o he g oup
challenged wi h V. ape is died compa ed o he con ol g oup wi h he highes incidence occu ing 4 days pos -inoc-
ula ion. Fish challenged wi h V. ape is de eloped mo e se e e skin ulce a ions. In zones whe e scales and epide mal
issue we e emo ed, he ulce a ions we e mo e se e e compa ed o zones whe e only mucus was elimina ed. Ulce a-
ions occu ed mo e equen ly, we e mo e se e e and la ge on he pigmen ed side. Ou da a ep esen s p o e o V.
ape is as causa i e agen o ulce a i e skin lesions al hough p io damage o he skin seems o be a majo con ibu -
ing ac o . Fu he mo e, he pigmen ed side seemed p edisposed o he de elopmen o skin ulce a ions.
© The Au ho (s) 2019. This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License
(h p://c ea i eco mmons .o g/licen ses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium,
p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license,
and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i eco mmons .o g/
publi cdoma in/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed.
In oduc ion
Vib io ape is is a well-known pa hogen causing b own
ing disease in a ious clam species such as he manila
clam (Rudi apes philippina um) and ca pe -shell clam
(R. decussa us) [1, 2]. Only a hand ul o s udies desc ibe
he isola ion o V. ape is om skin lesions and in e nal
o gans o mo ibund cul i a ed o cap i e held aqua ic
e eb a es, namely co kwing w asse (Symphodus
melops) [3], A lan ic halibu (Hippoglossus hippoglos-
sus) [4], wedge sole (Dicologlossa cunea a) [5], Do e
sole (Solea solea) [6] and wo na i e ish species ea ed
in Chile (Genyp e us chilensis and Pa alich hys adspe -
sus) [7]. Recen ly, V. ape is was isola ed o he i s ime
om skin ulce a ions, lesions a ec ing epide mal and
de mal issues, in wild-caugh common dab (Limanda
limanda) in he Belgian pa o he No h Sea (BNS) [8].
The wild common dab popula ions in his geog aphic
a ea showed a ecen p e alence o skin ulce a ions up
o 3.5% [9]. Va ious possible causal agen s, anging om
in ec ious agen s o biological oxins, we e sugges ed as
in ol ing ac o s in he de elopmen o skin ulce a ions
Open Access
*Co espondence: Maaike.V[email p o ec ed]
†Annemie Decos e e and Koen Chie s con ibu ed equally o his wo k
1 Depa men o Pa hology, Bac e iology and A ian Diseases,
Facul y o Ve e ina y Medicine, Ghen Uni e si y, Salisbu ylaan 133,
9820 Me elbeke, Belgium
Full lis o au ho in o ma ion is a ailable a he end o he a icle
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Ve cau e ene al. Ve Res (2019) 50:41
[10–12]. Ne e heless, he e iology o hese lesions is
la gely unknown and many ques ions emain ega d-
ing hei de elopmen and possible con ibu ing ac o s
in e ms o ish heal h and en i onmen al condi ions.
Besides being a wel a e issue, skin ulce a ions in wild
ish may also pose limi a ions o he p oduc i i y o he
ma ine ecosys em [11, 13] and diminish he economic
alue o he ish [11], u ging o an unde s anding o he
cause(s) o hese lesions.
All ma ine o ganisms, including ish, li e in close con-
ac wi h hei en i onmen , con aining po en ial pa ho-
genic mic o-o ganisms [14]. Hence, he skin ac s as an
impo an ba ie be ween he ish and i s en i onmen .
The ou e mos pa o he skin consis s o he epide mal
laye and he co e ing mucus laye ; wo complex s uc-
u es o e ing mechanical, chemical and immunological
p o ec ion agains pa hogenic agen s [15, 16]. Changes in
one o bo h laye s, caused by ab asion, s ess o pollu -
an s, may inc ease he suscep ibili y o he ish o adhe -
ence and subsequen in asion o mic oo ganisms and
hence o he de elopmen o skin ulce a ions, as dem-
ons a ed o Tenacibaculum ma i imum in ou ma ine
species [17].
Common dab and la ish in gene al, ha e a ie cely
discussed asymme ic mo phology, wi h he pigmen ed
(P) side acing he wa e column and he non-pigmen ed
(NP) side being in close in e ac ion wi h he sedimen
[18]. Based on hese di e ences, a di e gen mo phologi-
cal composi ion and possibly linked a ying suscep ibil-
i y o he de elopmen o skin ulce a ions is plausible. A
hand ul o s udies on his issue a e a ailable bu incon-
sis en anging om a clea o a sex- ela ed di e ence
ega ding he de elopmen o skin ulce a ions on bo h
sides [19–22].
In iew o his, he aim o he p esen s udy was o (i)
pinpoin he abili y o V. ape is o induce skin ulce a-
ions in common dab; (ii) assess he impac o p io skin
damage on he de elopmen o skin ulce a ions and (iii)
de e mine di e ences be ween he pigmen ed and non-
pigmen ed side on he de elopmen o skin ulce a ions.
Ma e ials andme hods
Animals andhousing acili ies
Six y common dab we e caugh du ing sho ish-
ing hauls (± 10min) using a 6m beam awl on boa d
o he esea ch essel (RV) “Simon S e in” a wo sam-
pling loca ions [L1 (N51°10.344; E2°38.699) and L2
(N51°9.886; E2°34.797)] si ua ed in he wes e n segmen
o he BNS, wi hin h ee miles o sho e. On boa d o
he RV, only ish in good condi ion and wi h a minimal
leng h o 17.5cm we e collec ed and immedia ely placed
in a su i al ank (1 × 1.2 × 1m; 640L) wi h con inu-
ous wa e enewal. Wi hin 4h a e cap u e, all ish we e
ans e ed o ci cula anks (diame e 2.6m, 4000L; 30
ish pe ank) wi h sand-co e ed bo oms (6 cm laye
hickness, 0–2mm g ain size) and illed wi h eci cu-
la ing, il e ed na u al seawa e a he Ma ine S a ion
Os end (Flande s Ma ine Ins i u e, VLIZ, Os end, Bel-
gium). The wa e quali y was moni o ed daily and kep in
p e-se anges (16.3 ± 0.4°C; pH 8.1 ± 0.0; 33.8 ± 0.2 PSU;
85.3 ± 1.1% oxygen sa u a ion). Ammonia and ni i e le -
els ne e exceeded 0.1 and 1mgL−1, espec i ely. Fish
we e ed h ee imes a week wi h chopped whi ing (Me -
langius me langus) (10g pe ish). A e an acclima iza-
ion pe iod o 18days, he ish we e anspo ed o he
expe imen al uni s using small anspo a ion boxes
(39.4 × 59.8 × 18.6cm; 30L) supplied wi h an oxygen ab-
le (JBL GmbH & Co.KG, Ge many).
Upon a i al a he expe imen al uni s, ish we e an-
domly di ided o e i e anks (1 × 1 × 0.5 m; 450 L)
wi h each ank con aining 12 ish. The anks we e illed
wi h eci cula ing and ae a ed seawa e , hal na u al
and hal a i icial (Ins an ocean, Aqua ium sys em,
F ance) and a sand laye (6cm laye hickness, 0–2mm
g ain size). Wa e pa ame e s we e moni o ed daily.
Th oughou he expe imen , he ollowing a e ages we e
eco ded: 15.3 ± 1.0°C, pH 8.4 ± 0.1; 32.5 ± 1.5 PSU and
79.5 ± 6.6% oxygen sa u a ion. Ammonia and ni i e le -
els ne e exceeded 0.2 and 1mgL−1, espec i ely. Upon
wa e enewal, a i icial seawa e was added. Fish we e
ed e e y 2days wi h chopped Pangas ca ish (Pangasius
pangasius) (10g pe ish). Feeding ime las ed o 1h
a e which he emaining eed was emo ed. One week
a e a i al, all ish we e dep i ed o ood 24h p io o
he expe imen al ial. The expe imen was app o ed by
he E hical Commi ee o he Facul y o Ve e ina y Medi-
cine and Bio-enginee ing Sciences, Ghen Uni e si y (EC
2015_89).
Bac e ial isola e
The V. ape is isola e was eco e ed in pu e cul u e om
an ulce a i e skin lesion o a common dab caugh in he
BNS [8].
Fo he expe imen al challenge, he V. ape is isola e
was cul u ed on yp ic soy aga (TSA, Sigma Ald ich
N.V., Belgium) supplemen ed wi h 1.5% NaCl and
incuba ed a 16 ± 1 °C o 3days. Colonies we e sub-
sequen ly ans e ed o 4mL yp ic soy b o h (TSB,
Sigma Ald ich) supplemen ed wi h 1.5% NaCl. A e a
24h incuba ion pe iod a 16 ± 1°C, he b o h was used
o inocula e 36 mL TSB medium supplemen ed wi h
1.5% NaCl. These cul u es we e again incuba ed o 16h
(16 ± 1°C), a e which he cul i a ed b o h was cen i-
uged (2465×g, 2 × 10min, 16°C), and he pelle e-sus-
pended in 40mL au ocla ed a i icial seawa e . Bac e ial
i e s we e e i ied by making an eigh old dilu ion se ies
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Ve cau e ene al. Ve Res (2019) 50:41
in iplica e on TSA pla es supplemen ed wi h 1.5% NaCl,
p io o adminis a ion.
Expe imen al design
Following anes hesia using icaine me hanosul ona e
(MS-222; 100 mg L−1; Sigma Ald ich), a ag (T-ba
ancho ag, Floy Tag Inc., USA) was inse ed in he cau-
dal epaxial muscula u e o all ish. The weigh o he ish
(WB) was egis e ed.
On bo h he P and NP side, h ee dis inc squa e “ ea -
men zones” (± 2.3cm2) we e de ined, wi h an in e space
o 0.5cm (Addi ional ile1). In one zone, he scales and
o e lying epide mis we e emo ed by sc aping wi h a
scalpel (mechanical ea men , MT). In a second zone,
he mucus laye on op o he epide mis was emo ed
using 80% e hanol (chemical ea men , CT) [23, 24]. The
hi d zone was he non- ea men zone (NT) whe e he
skin was le in ac and he e o e se ed as he con ol
zone. The sequence o all ea men s was al e ed on each
ish as ollows: (subg oup 1) MT–CT–NT; (2) MT–NT–
CT; (3) NT–MT–CT; (4) NT–CT–MT; (5) CT–NT–MT;
(6) CT–MT–NT, esul ing in six subg oups (1–6) o 10
ish each (Addi ional ile1).
Following eco e y, all ish we e di ided in o wo
g oups; ish which we e o be challenged wi h V. ape is
and a sham- ea ed con ol g oup, including h ee and
wo ank eplica es (hence o h e e ed o as eplica es),
espec i ely. Each eplica e con ained wo ish o each
subg oup (1–6: see abo e) hence a o al o 12 ish which
we e placed in a sepa a e ank con aining 35L a i icial
seawa e (55 × 38 × 28cm) (Addi ional ile1). The bac-
e ial suspension o V. ape is was added o he wa e o
he h ee ank eplica es esul ing in a inal concen a-
ion o 3.3 × 105 colony o ming uni s (CFU)mL−1 (chal-
lenge g oup, n = 36). The ish o he wo ank eplica es
cons i u ing he con ol g oup (n = 24) we e subjec ed o
he same p ocedu es as he challenge g oup, bu wi hou
addi ion o he bac e ial suspension. A e 60min, he
ish o each eplica e we e ans e ed o he 450L ank
a e which clinical signs and mo ali y we e eco ded on
a daily basis du ing 21days pos -inocula ion (dpi). E e y
second day, s a ing 5dpi, all ish we e ne ed, clinically
inspec ed and he P and NP side o each ish pho o-
g aphed o de e mine he “g oss ulce a ion sco e” (GUS),
“ o al a ec ed a ea” (TA) and “ulce a i e a ea” (UA)
(see u he ). As soon as he humane endpoin s (ex en-
si e lesions and/o subcu aneous hemo hages, weak o
no eac ion upon handling) we e obse ed, he ish was
eu hanized using an o e dose o MS-222 (500mgL−1)
and nec opsied as desc ibed below. A 21dpi, all su i -
ing ish we e sac i iced by an o e dose o MS-222 and
nec opsied as ou lined u he .
Pos ‑mo em examina ion
All ish we e measu ed (s anda d leng h, L) and
weighed (WE) o calcula e he Ful on condi ion ac-
o [25] (KE = 100 * (WE/L3)) enabling a compa ison
wi h he body condi ion o he ish be o e he ea -
men (KB = 100 * (WB/L3)). The gende o he ish was
eco ded and bo h sagi al o oli hs we e collec ed
o de e mine he age using he me hod desc ibed by
Imsland e al. [26]. A pa asi ological examina ion was
pe o med using we moun p epa a ions o gill biop-
sies and skin mucus samples. Fish we e pho og aphed
on bo h sides o e alua e he p esence and/o ype o
lesions and o calcula e he GUS, TA and UA (see u -
he ). Finally, a ull nec opsy was pe o med whe eby
samples o he skin and in e nal o gans we e collec ed
o bac e iological and his ological examina ion.
Skin lesion assessmen
G oss ulce a ion sco e (GUS)
To quan i y he se e i y o an ulce a i e skin lesion
ha de eloped in he ea men zones (MT–CT–NT),
a sco ing sys em was applied. The “g oss ulce a ion
sco e” (GUS) was de ined as he sum o he sco es o
se en pa ame e s ela ing o he dep h, healing, ele a-
ion o he edge o he lesion, pigmen a ion in o a ound
he lesion, colo o he lesion, hemo hages a ound he
lesion and he shape o he lesion (Addi ional ile2).
Inhe en ly, ulce a ions wi h a highe GUS e lec a
mo e se e e lesion. Sco ing was conduc ed blindly
using pho og aphs o all ish collec ed e e y second
day, s a ing a 5dpi and on he day he ish died.
To al a ec ed a ea (TA) andulce a i e a ea (UA)
To quan i y he ex en o he ulce a ion, he a ea o he
ish’s skin a ec ed by he lesion was de e mined. The
“To al a ec ed a ea” (TA) o each ea men zone (MT–
CT–NT) was calcula ed using scien i ic image analysis
so wa e (ImageJ 1.4) and ep esen s he o al skin a ea
a ec ed by he ulce a ion, including he healing edge
and/o hemo hages a ound he lesion. The su ace o
he open, ac i e lesion is named he “Ulce a i e a ea”
(UA).
Bac e iological examina ion
Skin samples o all ea men zones (MT–CT–NT), as
well as samples o li e , kidney and spleen, we e inocu-
la ed on hiosul a e ci a e bile sal suc ose aga (TCBS;
Sigma Ald ich) and incuba ed o 3days a 16 ± 1 °C.
V. ape is was iden i ied by a PCR-based me hod using
REP1D and REP2D sequences [27].
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Ve cau e ene al. Ve Res (2019) 50:41
His ological examina ion
Tissue samples o all skin ea men zones (MT–CT–
NT), as well as samples o gill, li e , spleen, in es ine,
kidney and hea we e ixa ed o 24h in a phospha e-
bu e ed 4% o maldehyde solu ion. Tissues we e p o-
cessed acco ding o s anda d echniques, sec ioned
(5µm) and s ained wi h hema oxylin and eosin (H&E).
Immunohis ochemis y
Demons a ion o V. ape is in a subse o skin samples
(a leas 20% o he ish pe g oup) by means o immu-
nohis ochemis y was pe o med as desc ibed p e i-
ously [8]. In sho , 5µm issue sec ions we e incuba ed
wi h an ibodies agains V. ape is (1:500). Subsequen ly,
bound an ibodies we e isualized using he DAKO
En ision + Sys em/HRP diaminobenzidine (DAB+)
s aining ki (DakoCy oma ion, Glos üp, Denma k).
Da a p ocessing ands a is ical analysis
To in es iga e he di e ence in mo ali y be ween he
con ol and challenge g oup, a logis ical eg ession was
used. Hos -speci ic da a namely leng h, weigh , body
condi ion, gende and age ga he ed du ing clinical
examina ion was analyzed using a linea mixed model.
To s udy he ole o V. ape is and he e ec o he
h ee ea men s in he de elopmen o skin ulce a-
ions, he o al expe imen al ollow up was di ided in o
h ee dis inc pe iods: 0–5dpi, 6–15dpi and 16–21dpi.
Fo each pe iod, a dis inc ion was made be ween
ish ha died be o e and ish ha su i ed un il he
end o he expe imen al pe iod (21dpi). The ish ha
died be o e he end o he expe imen al pe iod we e
excluded om he s a is ical analysis o es ima ing
he di e ences in GUS, TA and UA. Thus, only he su -
i ing ish we e included in he analyses on 5, 15 and
21dpi. The esul ing da a o ish ha died du ing he
expe imen we e epo ed in a desc ip i e manne . Fo
es ima ing he di e ence in GUS, TA and UA, a linea
mixed model (p oc GLIMMIX) was applied, ollowed
by pai wise compa isons using a Tukey–K ame adjus -
men o mul iple es ing. GUS, TA and UA we e used
as esponse a iables. Di e en ea men s (MT–CT–
NT), g oups, eplica es, and subg oups ( a iable o de
o he ea men s) and side (P o NP) we e implemen ed
as co- a iables. Analysis on di e ences be ween P and
NP side was pe o med in he same manne wi h side
and g oup as esponse a iables. In e ac ion e ec s
we e s udied o es ima e g oup-dependency. In all
models, ank was included as a andom in e cep .
S a is ical esul s we e conside ed o be signi ican
when p- alues we e lowe han 0.05. A p- alue be ween
0.05 and 0.1 was conside ed as a end. All s a is ical
analyses we e pe o med using SAS 9.4 and g aphs
we e cons uc ed using R S udio.
Resul s
The main ish cha ac e is ics a e lis ed in Table1. The
mean leng h o he ish was 21.7 ± 2.8cm in he chal-
lenge and 22.8 ± 3.0cm in he con ol g oup. In o al, 21
males and 39 emales we e included wi h 16 males and
20 emales and 5 males and 19 emales in he challenge
and he con ol g oup, espec i ely. The mean age o ish
included in he challenge g oup (35.4 ± 12.6mon hs) was
sligh ly lowe , albei no signi ican ly, compa ed o he age
o he con ol g oup (37.4 ± 12.0 mon hs) (p = 0.6703).
The ish om he challenge (KB = 0.99 ± 0.1) and con ol
(KB = 1.0 ± 0.2) g oup displayed a simila body condi ion
a he beginning o he s udy (KB; p = 0.3958) emaining
s able h oughou he o al expe imen al pe iod (KB − KE)
wi h no di e ence be ween challenged and con ol ish
(p = 0.6144).
In one ish o he con ol g oup, a pa asi e esembling
Le nan h opus spp. was no ed on he gills. In wo ish o
he challenge g oup, a pa asi e wi h a mo phology simila
o Te ahymena spp. was ound on he skin mucus albei
in low numbe s.
Leng h (p = 0.2190), weigh (WB: p = 0.1635; WE:
p = 0.2191), body condi ion (KB − KE: p = 0.6144) and age
(p = 0.6703) o ish included in he con ol g oup showed
no signi ican di e ences wi h ish om he challenge
g oup and we e he e o e no implemen ed in u he
analyses. Fu he mo e, gende and pa asi ic load we e
nei he implemen ed in u he analyses.
A signi ican ly highe mo ali y was ound in he chal-
lenge g oup (55.6%) compa ed o he con ol g oup
(16.7%) (p = 0.0060). The main mo ali y peak was
encoun e ed on 4dpi whe eby 12 ou o 36 challenged
ish died (Addi ional ile3).
No mac oscopic abno mali ies we e seen in he gills
and in e nal o gans om he ish o he challenge o he
con ol g oup upon nec opsy. These indings we e con-
i med by his ological examina ion.
Ch onological changes inskin lesions ollowingchallenge
wi hV. ape is
Non‑ ea ed (NT‑) zone
No ulce a ions de eloped nei he in he challenge no in
he con ol g oup. V. ape is was isola ed om he NT-
zone in wo challenged and wo con ol ish a 21dpi.
Chemical ea men (CT‑) zone
In he con ol g oup, nine ou o he 24 ish de eloped
skin ulce a ions in he CT-zone. One o hese ish died
a 4 dpi wi h a se e e ulce a ion wi h a GUS o 9, TA o
3.1cm2 and UA o 3.2cm2. In wo ou o eigh su i ing
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Ve cau e ene al. Ve Res (2019) 50:41
Table 1 Main ish cha ac e is ics (leng h, weigh , body condi ion, gende and age) pe ank eplica e and pe g oup as well as mo ali y
WB: weigh a he beginning o he expe imen al pe iod, WE: weigh a he end o he expe imen al pe iod, KB: Ful on condi ion index a he beginning o he expe imen al pe iod, KE: Ful on condi ion index a he end o
he expe imen al pe iod, M: male ish, F: emale ish.
G oup Replica e No. Leng h (cm) Weigh (g) Body condi ion Gende Age (mon hs) Mo ali y
WBWEKBKE< 5dpi < 15dpi < 21dpi
Challenge 1 12 22.6 ± 3.4 119.8 ± 51.7 116.5 ± 50.2 0.99 ± 0.09 0.96 ± 0.1 5 M/7 F 41.0 ± 14.8 2 1 0
2 12 20.6 ± 2.2 86.6 ± 26.6 85.9 ± 30.4 0.96 ± 0.09 0.9 ± 0.09 6 M/6 F 29.7 ± 5.6 6 2 0
3 12 21.7 ± 2.8 105.7 ± 39.3 106.3 ± 39.3 1.0 ± 0.08 0.99 ± 0.1 5 M/7 F 35.0 ± 13.4 8 1 0
Mean 21.7 ± 2.9 104.0 ± 41.7 103.8 ± 42.1 0.99 ± 0.1 0.96 ± 0.09 16 M/20 F 35.4 ± 12.6
Con ol 1 12 22.8 ± 2.9 124.3 ± 56.4 120.1 ± 54.6 1.0 ± 0.2 0.97 ± 0.08 2 M/10 F 38.5 ± 7.8 0 0 1
2 12 22.9 ± 3.2 130.2 ± 58.3 128.8 ± 61.1 1.0 ± 0.1 0.99 ± 0.09 3 M/9 F 36.3 ± 15.5 2 0 1
Mean 22.8 ± 3.0 127.3 ± 56.2 124.3 ± 56.7 1.0 ± 0.2 0.98 ± 0.08 5 M/19 F 37.4 ± 12.0

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Ve cau e ene al. Ve Res (2019) 50:41
ish, he ulce a ion had healed a 21dpi, he o he six ish
showed signs o healing. In he challenge g oup, 16 ou
o he 36 ish de eloped skin ulce a ions in he CT-zone.
Nine and h ee ish died be o e 5 and 15dpi, espec i ely.
The a e age GUS, TA and UA sco es o he CT-zone
ulce a ions a e lis ed in Table2.
The di e ence be ween he GUS, TA and UA o he
ulce a ions in he CT-zone o su i ing ish we e no
s a is ically signi ican be ween he con ol and chal-
lenged ish h oughou he en i e expe imen al pe iod,
based on he analyses a 5 dpi (GUS: p = 0.0729; TA:
p = 0.6443; UA: p = 0.6933), 15 dpi (GUS: p = 0.7525; TA:
p = 0.7976; UA: p = 0.9136) and 21 dpi (GUS: p = 0.9968;
TA: p = 1.0000; UA: p = 0.9814).
Mechanical ea men (MT‑) zone
The GUS, TA and UA sco es o MT-zone ulce a ions a e
lis ed in Table3.
0–5 dpi
Two ish o he con ol g oup died a 4 dpi. One ish
showed small ulce a ions wi h a GUS o 6.5 ± 2.1 and
TA o 0.9 ± 0.7cm2. The o he ish was eu hanized as
i showed ex ensi e hemo hages and la ge and se e e
ulce a ions. These ulce a ions had a GUS o 8.5 ± 2.1 and
TA o 4.3 ± 2.3cm2. V. ape is was no isola ed om any
ish. Immunohis ochemical analysis o a skin ulce a ion
o he second ish on he P side e ealed small numbe s
o comma-shaped immunoposi i e cells a he edge o
he ulce a ion on op o he s a um spongiosum (de mis)
no in ading he muscula issue.
Me ely all ish o he con ol g oup (n = 22) ha we e
ali e a he end o he i s expe imen al pe iod showed
ulce a ions wi h a GUS o 5.4 ± 4.0. The TA o he ulce -
a ions was 1.1 ± 1.3 cm2 whe eby 0.2 ± 0.3 cm2 was
obse ed o be ac i e (UA).
In he challenge g oup, 16 ish died be o e 5dpi, mainly
on 4 dpi (Table 1). The obse ed skin ulce a ions in
hese ish had a GUS o 9.3 ± 2.3, wi h a TA and UA o
2.1 ± 1.2cm2 and 1.5 ± 1.3cm2, espec i ely. F om h ee
o i e sampled ish, V. ape is was isola ed om he
ulce a ion. One o he ish ha died a 5 dpi ha bo ed a
pu e cul u e o V. ape is in he spleen. Immunohis o-
chemical analyses o he ulce a ions o eigh sampled ish
e ealed he p esence o comma-shaped immunoposi i e
cells. The cells we e loca ed a he ulce a ed su ace and
in unde lying de mal issue. In all cases, he immunopos-
i i e cells in il a ed in o he muscula issue supe icially,
wi h occasional obse a ions o he cells in he muscula
connec i e issue (Figu e1).
A 5 dpi, he emaining indi iduals o he challenge
g oup (n = 20) all showed ulce a ions wi h a GUS o
Table 2 Mean g oss ulce a ion sco e (GUS), o al a ec ed a ea (TA, cm2) and ulce a i e a ea (UA, cm2) o he ulce a ions in
he chemically ea men zones in challenged and con ol ish
Values a e subdi ided acco ding o pe iod wi h 0–5, 6–15 and 16–21days pos -inocula ion (dpi). A subdi ision was made be ween ish ha died du ing he
expe imen al pe iod (D) and ish ha su i ed in ha pe iod (S).
NA: no a ailable.
G oup Dead
o su i ing 0–5dpi 6–15dpi 16–21dpi
GUS TA UA GUS TA UA GUS TA UA
Challenge D 4.5 ± 5.2 1.6 ± 2.9 1.6 ± 2.9 NA NA NA NA NA NA
Con ol D 7.8 ± 2.6 1.5 ± 0.9 1.1 ± 1.2 6.7 ± 0.6 1.7 ± 0.5 1.3 ± 1.1 NA NA NA
Challenge S 7.0 ± 1.0 0.8 ± 0.4 0.6 ± 0.6 7.8 ± 4.9 0.7 ± 0.6 0.4 ± 0.7 5.6 ± 1.1 0.4 ± 0.3 0.1 ± 0.2
Con ol S 8.4 ± 5.0 0.9 ± 0.6 0.7 ± 0.7 6.5 ± 2.5 0.3 ± 0.2 0.2 ± 0.1 4.3 ± 2.5 0.3 ± 0.2 0.1 ± 0.3
Table 3 Mean g oss ulce a ion sco e (GUS), o al a ec ed a ea (TA) and ulce a i e a ea (UA) o he ulce a ions in he
mechanically ea men zones in challenged and con ol ish
Values a e subdi ided acco ding o pe iod wi h 0–5, 6–15 and 16–21days pos -inocula ion (dpi). A subdi ision was made be ween ish ha died du ing he
expe imen al pe iod (D) and ish ha su i ed in ha pe iod (S).
NA: no a ailable.
G oup Dead
o su i ing 0–5dpi 6–15dpi 16–21dpi
GUS TA (cm2)UA (cm2) GUS TA (cm2)UA (cm2) GUS TA (cm2)UA (cm2)
Challenge D 9.3 ± 2.3 2.1 ± 1.2 1.5 ± 1.3 8.0 ± 3.6 1.7 ± 0.9 1.0 ± 0.8 NA NA NA
Con ol D 7.5 ± 2.1 2.6 ± 2.4 1.8 ± 2.8 NA NA NA 3.8 ± 3.3 0.6 ± 0.6 0.1 ± 0.1
Challenge S 8.5 ± 2.3 1.5 ± 0.9 1.1 ± 0.9 7.0 ± 2.6 1.2 ± 0.9 0.4 ± 0.6 6.0 ± 2.8 1.1 ± 0.7 0.3 ± 0.5
Con ol S 5.4 ± 4.0 1.1 ± 1.3 0.2 ± 0.3 4.4 ± 3.5 0.8 ± 0.8 0.1 ± 0.2 4.5 ± 2.8 0.7 ± 0.7 0.1 ± 0.2
Page 7 o 13
Ve cau e ene al. Ve Res (2019) 50:41
8.5 ± 2.3. The ulce a ions had a TA o 1.5 ± 0.9cm2 and
UA o 1.1 ± 0.9cm2.
The GUS (p = 0.0035) and UA (p < 0.0001) o he su i -
ing ish a 5dpi we e signi ican ly highe in he challenge
g oup compa ed o he con ol g oup (Figu es2A and
C). The TA did no show a signi ican di e ence be ween
challenge and con ol g oups (p = 0.6682) (Figu e2B).
6–15 dpi
None o he con ol ish died. The GUS a 15dpi was
4.4 ± 3.5. TA and UA o he ulce a ions 0.8 ± 0.8cm2 and
0.1 ± 0.2cm2, espec i ely.
In he challenge g oup, ou ou o 20 emaining ish
died be ween 6 and 15 dpi. Ulce a ions we e ound in all
ish (GUS 8.0 ± 3.5). The TA and UA we e 1.7 ± 0.9 cm2
and 1.0 ± 0.8cm2, espec i ely. In wo o he ou ish,
V. ape is was isola ed om he li e . V. ape is was no
e ie ed om he ulce a ions on he MT-zone. In wo o
he ou ish, comma-shaped immunoposi i e cells we e
isualized in he skin ulce a ion using immunohis o-
chemical s aining.
The emaining challenged ish (n = 16) all showed
ulce a ions wi h GUS, TA and UA alues o 7.0 ± 2.6,
1.2 ± 0.9cm2 and 0.4 ± 0.6cm2, espec i ely.
A 15dpi, he GUS o he ulce a ions o he challenge
g oup was signi ican ly highe compa ed o he con ol
g oup (p < 0.0001) (Figu e2A). A s a is ically signi ican
di e ence was no ed o he TA and UA be ween he
challenge and con ol g oup (p < 0.0001 and p = 0.0002,
espec i ely) (Figu es2B and C).
16–21dpi
Be ween 16 and 21 dpi, wo con ol ish died. The
ulce a ions in he MT-zone had a GUS o 3.8 ± 3.3, TA
o 0.6 ± 0.6cm2 and UA o 0.1 ± 0.1 cm2. V. ape is was
no isola ed and immunohis ochemical analysis did no
e eal immunoposi i e cells in he ulce a ions.
The emaining con ol ish (n = 20) we e sac i iced a
21dpi, o which 14 and six ish had ulce a ions on bo h
sides o only on he P side, espec i ely. The ulce a ions
had a GUS o 4.5 ± 2.8. The TA was 0.7 ± 0.7cm2 whe eby
0.1 ± 0.2 cm2 was obse ed o be ac i e (UA). F om one
ish, V. ape is was isola ed om an ulce a ion in he MT-
zone. This was no con i med by immunohis ochemical
s aining.
None o he 16 emaining challenged ish died. A
21 dpi, he ulce a ions ound in he sac i iced ish
had a GUS, TA and UA o 6.0 ± 2.8, 1.1 ± 0.7cm2 and
0.3 ± 0.5cm2, espec i ely. V. ape is was isola ed om
he ulce a ions o ou ish. The immunohis ochemical
analysis did no e eal immunoposi i e cells in he ulce -
a ions o any o he sampled ish.
A 21 dpi, a end o a highe GUS o ulce a ions
o challenged compa ed o con ol ish (p = 0.0600)
was no ed (Figu e 2A). The TA (p = 0.0030) and UA
(p = 0.0006) emained signi ican ly highe in he MT-
zone ulce a ions o he challenge g oup compa ed o he
con ol g oup (Figu es2B and C).
The a e age GUS, TA and UA sco es o he ulce a ions
in he MT-zones we e signi ican ly highe compa ed o
he CT-zones a 5 dpi (GUS: p = 0.0058; TA: p = 0.0097;
UA: p = 0.0353), 15dpi (GUS: p = 0.0035; TA: p = 0.0029;
UA: p = 0.0410) and 21 dpi (GUS: p = 0.0026; TA:
p = 0.0028; UA: p = 0.0333). These esul s we e independ-
en om he g oup (con ol o challenge).
His ological examina ion con i med he g oss appea -
ance o he lesions. Ulce a ions we e cha ac e ized by
ocal loss o epide mal and/o de mal issue. The de -
mis was o en in il a ed by mild o mode a e amoun s
o in lamma o y cells. Unde lying muscle issue was
egula ly degene a ed and/o in il a ed by in lamma o y
cells. Pa ially healed ulce a ions we e mainly ypi ied by
a one o wo cell-laye ed epide mis o e lying a dis up ed
de mal issue wi hou he p esence o scales and/o scale
pocke s. In lamma o y cells we e p esen in mode a e
amoun s and we e localized mainly in de mal and mus-
cula issue. Hemo hages we e a he mode a e. These
obse a ions we e simila in he challenge and he con-
ol g oup and in lesions occu ing on he P and NP side.
Ch onological changes inskin lesions on hepigmen ed
andnon‑pigmen ed side
Since he main di e ence be ween he challenge and con-
ol g oup in he de elopmen o skin ulce a ions was
Figu e1 Immunohis ochemical esul s demons a ing he
p esence and loca ion o comma‑shaped immunoposi i e
cells (b own). Example o a skin ulce a ion o a challenged ish ha
died a 4 days pos -inocula ion. In he exposed de mal issue and
in e s i ial space o he unde lying muscula issue comma-shaped
immunoposi i e cells a e no ed. The inse shows a highe
magni ica ion o poin ou he in asion o he immunoposi i e cells
in he in e s i ial spaces o he muscle. Scale ba = 100 µm.
Page 8 o 13
Ve cau e ene al. Ve Res (2019) 50:41
0
5
10
GUS
5 dpi
***
0
5
10
15 dpi
***
0
5
10
21 dpi
*
A
0
1
2
3
4
TA
(cm²)
5 dpi
0
1
2
3
4
15 dpi
***
0
1
2
3
4
21 dpi
***
B
0
1
2
3
4
UA
(cm²)
5 dpi
***
0
1
2
3
4
15 dpi
***
0
1
2
3
4
21 dpi
***
C
Figu e2 O e iew o skin lesion assessmen o mechanically ea ed zones du ing he expe imen al pe iod. Compa ison o he main
pa ame e s be ween challenge (g ey) and con ol (whi e) g oups a 5, 15 and 21 days pos -inocula ion (dpi). Only he ish ha we e ali e a he end
o he expe imen al pe iod (21 dpi) a e depic ed. Th ee as e isks indica e a signi ican di e ence be ween challenge and con ol g oup, one as e isk
ep esen s a end. A G oss ulce a ions sco e (GUS) we e signi ican ly highe in challenge g oup a 5 and 15 dpi. A 21 dpi he same end was
isible. B To al a ec ed a ea (TA) was highe in challenge g oup compa ed o he con ol g oup a 15 and 21 dpi. C Ulce a i e a ea (UA) was highe
in challenge g oup compa ed o he con ol g oup du ing he en i e expe imen al pe iod.
Page 9 o 13
Ve cau e ene al. Ve Res (2019) 50:41
ound in he MT-zones, only hese zones a e discussed
when compa ing he ulce a ions in he pigmen ed (P)
and non-pigmen ed (NP) sides o he ish.
A 5, 15 and 21dpi, he di e ence be ween bo h sides
was no dependen on he g oup he ish belonged o
(con ol o challenge). The e o e, in he s a is ical analy-
sis, he esul s o he con ol and challenged ish we e
aken oge he .
0–5dpi
Me ely all su i ing ish (n = 42) de eloped ulce a-
ions on bo h P and NP sides excep o ou ish wi h
only an ulce a ion on he P side. The GUS o he ulce a-
ions de eloped on P and NP side a 5 dpi was 8.6 ± 1.8
and 6.1 ± 4.1, espec i ely. TA and UA o ulce a ions
we e 2.2 ± 0.9cm2 and 1.0 ± 1.1cm2 on he P side and
0.6 ± 0.6cm2 and 0.5 ± 0.6 cm2 on he NP side, espec-
i ely. Gene ally, he ulce a ions on he P side we e sig-
ni ican ly mo e se e e (GUS, p = 0.0321) and la ge (TA,
p = 0.0066) compa ed o he NP side (Figu es3A and B).
The pa o he ulce a ions ha was ac i e (UA) was simi-
la be ween he P and NP side (p = 0.1580) (Figu e3C).
6–15 dpi
A 15 dpi, all ish (n = 38) had de eloped an ulce a ion
on he P side while only 24 ish displayed an ulce a ion
on he NP side. The GUS o he ulce a ion on he P side
was signi ican ly highe (7.5 ± 2.3) compa ed o he GUS
o he ulce a ions on he NP side (3.5 ± 3.1) (p = 0.0058)
(Figu e3A). The TA o he ulce a ions was 1.6 ± 0.7cm2
on he P side whe eby 0.4 ± 0.6 cm2 was ound o be
ac i e (UA). On he NP side, TA o he ulce a ions was
0.3 ± 0.4cm2 wi h UA o 0.1 ± 0.2cm2. The TA was sig-
ni ican ly highe in P side compa ed o he NP side
(p = 0.0017) and he same end was p esen upon con-
side ing he UA (p = 0.0538) (Figu es3B and C).
16–21 dpi
O he su i ing ish (n = 36), 29 had de eloped an ulce -
a ion on bo h sides and 7 ish showed ulce a ions only on
he P side. The GUS o he ulce a ions on he NP side was
3.6 ± 2.6. The TA and UA o he la e we e 0.4 ± 0.3cm2
and 0.1 ± 0.1cm2, espec i ely. The ulce a ions on he P
side had a GUS o 6.7 ± 2.1, TA o 1.4 ± 0.6cm2 and UA
o 0.3 ± 0.5 cm2. Compa ing ulce a ions on he P side
and NP side, a 21dpi, ulce a ions on he P side we e
gene ally mo e se e e (p = 0.0126) and had a la ge su -
ace (TA; p = 0.0029) compa ed o he ulce a ions on
he NP side (Figu e3). The UA showed a simila end
(p = 0.0528).
Discussion
The esul s o he p esen s udy s ongly poin owa ds
he in ol emen o V. ape is in skin ulce a ions since
mo e common dab inocula ed wi h a bac e ial suspen-
sion o V. ape is (3.3 × 105CFUmL−1, an inocula ion
dose compa able o ano he s udy [28]) de eloped skin
ulce a ions compa ed o con ol ish ha we e sham-
ea ed. Fu he mo e, skin ulce a ions we e signi ican ly
mo e se e e and co e ed a la ge su ace in he chal-
lenged ish. Following bac e iological analysis, V. ape is
was isola ed om skin ulce a ions o eigh ish in o al
and using immunohis ochemical s aining, comma-
shaped immunoposi i e cells, p esumed o be V. ape-
is we e isualized in hese skin ulce a ions. No ably, V.
ape is was isola ed p edominan ly om he ulce a ions
o ish ha died a 4dpi. This obse a ion was again sup-
po ed by immunohis ochemical s aining whe e mainly
in hose ish he comma-shaped immunoposi i e cells
we e isualized.
To de e mine he ole o he skin o mucus as a p o-
ec i e ba ie agains in ec ion and coloniza ion by V.
ape is, h ee ea men s we e applied on he skin. A e
inocula ion wi h V. ape is, ulce a ions de eloped p e-
dominan ly in he mechanically ea ed zone whe e scales
and o e lying epide mis we e emo ed. Mo eo e , he
ulce a ions in his zone we e mo e se e e compa ed o
ulce a ions in chemically ea ed zone whe e he mucus
was locally emo ed using e hanol. In he non- ea ed
zone ha se ed as con ol zones, no skin ulce a ions
de eloped.
The mechanical ea men emo es he scales and o e -
lying epide mis which lea es he de mal issue exposed
he eby esembling wounds in lic ed by p eda o s o
ishing gea a sea [29]. Exposu e o de mal issue could
acili a e he adhe ence o V. ape is o, e.g. exposed
ib onec in and collagen. The i ulence ac o (s) o V.
ape is ha could possibly be in ol ed in his p ocess
a e no ye elucida ed, al hough adhesion componen s
such as pili we e desc ibed in s ains pa hogenic o mol-
lusks [1, 30]. The adhesion may be ollowed by he u -
he des uc ion and in asion o he hos issue. Based
on immunohis ochemical s aining in he p esen s udy,
i was shown ha comma-shaped immunoposi i e cells,
p esumed o be V. ape is, we e able o in ade he de mis
and adjacen muscula issue, which is o en media ed by
se e al enzymes deg ading he ex acellula ma ix. In V.
ape is isola es pa hogenic o mollusks, 87 di e en p o-
eins including phospholipases and p o eases we e dem-
ons a ed in hei sec e ome [30]. Howe e , a p esen ,
no such da a a e a ailable o V. ape is isola es collec ed
om ish.
The chemical ea men emo ed he mucus laye ,
which has a ious p o ec i e p ope ies, bo h mechanical