Ve e ina y Mic obiology 292 (2024) 110037
A ailable online 3 Ma ch 2024
0378-1135/© 2024 The Au ho (s). Published by Else ie B.V. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/).
Use o a local anaes he ic and an isep ic wound o mula ion o he
ea men o lambs na u ally in ec ed wi h O i us
´
Alex G´
omez
a
,
1
, Delia Lacas a
a
,
*
,
1
, Ma ía Te esa Tejedo
b
, Ma a Ruiz de A cau e
a
,
Juan Jos´
e Ramos
a
, H´
ec o Ruiz
a
, Au o a O ín
a
, Se gio Villanue a-Saz
a
, Rams´
es Reina
c
,
Pablo Quílez
a
, Te esa Na a o
a
, Mai e Ve de
a
, Ma a Bo obia
a
, Pe e And ew Windso
d
a
Animal Pa hology Depa men , Ins i u o Ag oalimen a io de A ag´
on-IA2 (Uni e sidad de Za agoza-CITA), Ve e ina y Facul y o Za agoza, C/Miguel Se e 177,
Za agoza 50013, Spain
b
Ana omy, Emb yology and Animal Gene ics Depa men , CIBER CV (Uni e sidad de Za agoza-IIS), Ve e ina y Facul y o Za agoza, C/Miguel Se e 177, Za agoza
50013, Spain
c
Ins i u o de Ag obio ecnología, CSIC-Gobie no de Na a a, Mu il a 31192, Spain
d
Uni e si y o Sydney, Sydney School o . Ve e ina y Science, Camden, NSW 2570, Aus alia
ARTICLE INFO
Keywo ds:
O
Mul isol en
T ea men
Lambs
Wel a e
ABSTRACT
Con agious ec hyma (CE) is a wo ldwide highly con agious zoono ic i al skin disease o sheep and goa s.
T ea men o O i us (ORFV) in ec ion usually in ol es opical and o al an ibio ics. An anaes he ic and
an isep ic opical gel (Mul isol en® o T i-Sol en®; MS®, Medical E hics, Aus alia) has been documen ed as an
e icacious he apy o lesions om mucosal and epi helial i al in ec ions in uminan s. The p esen s udy es ed
a new ea men p o ocol o MS® o CE he apy on- a m in 150 lambs na u ally in ec ed wi h ORFV. Lambs we e
di ided in o h ee coho s o 50 lambs each (C, D and E). Coho C was ea ed wi h MS® 3 imes wi h an in e al
o 3 days be ween ea men s, coho D was ea ed daily wi h hypochlo ous acid, whils coho E se ed as
un ea ed con ols. The lambs we e examined clinically e e y wo days, weigh measu ed weekly, wi h whole
blood and s e ile swabs om ORFV lesions collec ed o haema ological analysis and speci ic ORFV PCR. Coho
C p esen ed ewe lambs displaying ORFV-associa ed lesions han o he coho s a di e en imes o he
expe imen . Fu he , lesions ea ed wi h MS® we e milde compa ed wi h o he coho s. Howe e , ollowing
cessa ion o he apy, mos o he lambs again de eloped ORFV-associa ed lesions. No di e ences be ween coho s
we e obse ed in weigh , haema ological and PCR esul s. These indings sugges ha opical ea men wi h
MS® is e ec i e o CE in ield condi ions, especially in he i s s ages o he clinical cou se, al hough ea men
wi h MS® may need o be ex ended a minimum o 4 weeks.
1. In oduc ion
Con agious ec hyma (CE), also known as O , is a highly con agious
global zoono ic i al skin disease a ec ing mainly sheep and goa s (Bala
e al., 2018). CE causes signi ican economic losses in he sheep and goa
a ming sec o , om mo ali ies in young animals and educed eed
consump ion and weigh gain. Fu he , CE is conside ed o p omo e
seconda y bac e ial in ec ions in he skin and o al mucosa ha inc ease
he use o an ibio ics (AMU), isking he gene a ion o an imic obial
esis ance (AMR). CE is a signi ican zoono ic disease in e e ina ians
and a me s (Lo a e al., 2012; Windso e al., 2017), commonly
causing lesions in con ac si es, p ima ily he hands. Lesions a e cha -
ac e ised by e y hema, papules, esicles, and some imes g anuloma ous
de ma i is ha usually can ake weeks o mon hs o heal (Nandi e al.,
2011; Spy ou and Valiakos, 2015).
CE is caused by he O i us (ORFV) om he Pox i idae amily,
Cho dopox i inae sub amily and Pa apox i us genus (Be gq is e al.,
2017). ORFV is an epi helio opic i us ha eplica es mainly in o he
cy oplasm o he s a um basale ke a inocy es (Fleming e al., 2015).
Al hough gene ally causing a sel -limi ing disease, ORFV encodes
se e al immunomodula o y p o eins pe mi ing e asion o he immune
sys em and inducing he ein ec ions o sheep and goa s (Lloyd e al.,
* Co espondence o: 177 Miguel Se e S ee , Za agoza 50013, Spain.
E-mail add esses: [email p o ec ed] (´
A. G´
omez), [email p o ec ed] (D. Lacas a), [email p o ec ed] (M. Te esa Tejedo ), [email p o ec ed] (R. Reina).
1
Alex G´
omez and Delia Lacas a a e equal con ibu o s o his wo k.
Con en s lis s a ailable a ScienceDi ec
Ve e ina y Mic obiology
jou nal homepage: www.else ie .com/loca e/ e mic
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Ve e ina y Mic obiology 292 (2024) 110037
2
2000; Rohde e al., 2012; Buka e al., 2021). Clinical p esen a ion in
lambs o kids is cha ac e ised by papules, esicles and pus ules ha
de elop in o scabby p oli e a i e lesions, mainly a ec ing skin o he
muzzle and lip mucosae al hough may ex end o he o al mucosa and
beyond, causing mul i ocal e osions and ulce s o he nos ils, ea s,
eyelids, ee , sc o um, ul a and udde (De La Concha-Be mejillo e al.,
2003; McEl oy and Basse , 2007; Nandi e al., 2011; Spy ou and
Valiakos, 2015; Windso e al., 2017). ORFV lesions usually esol e in
app oxima ely 3–8 weeks (Nandi e al., 2011).
ORFV is mainly ansmi ed cu aneously (Spy ou and Valiakos,
2015), al hough o he in ec ion ou es a e possible (Allwo h e al.,
1987; Sa gison e al., 2007). Mo bidi y can each 100% and al hough
mo ali y is usually less han 5%, he e a e epo ed ou b eaks wi h a
90% o mo ali y a e in e y young animals (Gumb ell and McG ego ,
1997; Hosamani e al., 2009). CE ou b eaks may cause bo h signi ican
inancial losses and animal wel a e conce ns in li es ock p oduc ion,
pa icula ly in associa ion wi h in ensi e sheep and goa husband y and
comp omising he in e na ional ade in small uminan s (Windso
e al., 2017).
Con ol o CE should be based on accina ion (Zhu e al., 2022), a
ou ine p ocedu e widely conduc ed in Aus alian sheep locks using a
li e i us accine (Windso e al., 2017). The u he de elopmen o
e ec i e accines o ORFV in ec ion is an impo an p io i y, pa icu-
la ly as he e a e no uni e sally app o ed sheep o goa accines
(Lacas a e al., 2015; Windso e al., 2017). Regis e ed accines a e
pu i ied scab-based accines (Musse e al., 2008; Buka e al., 2021) and
cell cul u e-based li e-a enua ed accines (Buddle and Pul o d, 1984;
Pye, 1990; Buka e al., 2021; Zhu e al., 2022). Pu i ied scab-based
accines can e e o i ulence (Jo ge and Dellagos in, 2017) and cell
cul u e-based li e-a enua ed accines do no elici comple e p o ec i e
immuni y agains ORFV (Tan e al., 2009) and also isk e e sion o
i ulence (F iebe e al., 2004; Musse e al., 2008). Fo his eason,
p o o ypes o DNA and subuni accines based on p o eins ORFV B2L
(ORFV011 gene) and ORFV F1L (ORFV059 gene) ha e been s udied as
accines agains ORFV in ec ions (Zhao e al., 2011; Yogisha adhya
e al., 2017, 2018; Wassie e al., 2019; Zhu e al., 2022). Recen ly, a
double gene-dele ed ecombinan accine, wi h dele ions in CBP and
GIF genes and a iple gene-dele ed mu an o ORFV) wi h dele ions in
CBP, GIF and gene 121 ha e been s udied in kids (Zhu e al., 2022; Shen
e al., 2023). Howe e , hese p omising p o o ype accines a e ye o be
app o ed and egis e ed.
Despi e he widesp ead dis ibu ion o ORFV amongs sheep and goa
popula ions, he e is no e ec i e ea men agains CE (Lacas a e al.,
2023). Se e al opical an isep ics such as 10% Po assium pe mangana e
solu ion (Van De Ke k, 1954), S ibophen ( i alen an imony com-
pound) (Walde e al., 1979), 7% iodine, c eoso e dip and 3% phenol in
aseline (Beck and Taylo , 1974), Lo agen (me ac esolsul onic acid and
o maldehyde 36% (Rapun ean e al., 1975), 6% aqueous suspension o
li hium an imony hiomala e (Sande son, 1976), sodium pe mangana e
and salicylic acid (Ton is e al., 1981) and oin men (pe ola um and
mine al oil) (Lansade, 1959; La sson and Zahoo y, 1983), ha e been
sugges ed o ORFV ea men . In cases wi h seconda y bac e ial in-
ec ions, pa en e al an ibio ics, such as penicillin (Mo elmans and
Ve c uysse, 1953) and chlo amphenicol-oin men (Lansade, 1959),
ha e shown lesions imp o emen . Howe e , an imic obial he apy is
ine ec i e agains ORFV in ec ion (G eig e al., 1984) and may lead o
inc eased esis ance o an ibio ics (AMR). Addi ionally, su gical ea -
men consis ing o deb idemen and liquid ni ogen sp ay c yo he apy o
he de mis has been p o ed in lambs, esul ing in a apid esolu ion and
no ecu ence, bu an anaes hesia p o ocol was needed, and he ime pe
lamb used was mo e han i e minu es (Meynink e al., 1987, 1990). In
humans, opical imiquimod (Lede man e al., 2007) o cido o i ha e
been success ully used o ORFV ea men (McCabe e al., 2003).
Recen ly, o he inno a i e ea men s, such as (S)-HPMPA alkoxy alkyl
es e s (Dal Pozzo e al., 2007), sys emic IFN-
α
injec ion and opical
imiquimod (E ekin e al., 2017) and genis ein (iso la one) (L e al.,
2024) ha e been shown o inhibi he ORFV eplica ion and in ec ion.
Recen ly, Mul iSol en® (Dech a, UK), a local anaes he ic and an i-
sep ic wound o mula ion, also ma ke ed in some coun ies as T iSol en
(Medical E hics, Aus alia; MS®) has been ound o be an e icacious
wound he apy o mula ion app op ia e o he ea men o e osions
and ulce s in o al mucosa caused by oo and mou h disease (FMD)
(Windso e al., 2020; Lendzele e al., 2021; Roughan and Windso ,
2022). In addi ion, MS® he apy was p e iously examined in 50 lambs
in ec ed expe imen ally wi h ORFV (Lacas a e al., 2023). Al hough
MS® he apy had no e ec on weigh gain and clinical p og ession, his
was a ibu ed o ea ly opical adminis a ion o animals in ec ed by
in a-de mal inocula ion and lack o ea men o lesions in mid-la e
s ages o he disease; ORFV lesions ha ing con inued o mo e han 3
weeks (Lacas a e al., 2023). I was concluded ha u he s udies in
na u al in ec ions on- a m wi h a di e en ea men p o ocol we e
equi ed o e alua e whe he MS® could imp o e he clinical cou se o
CE.
2. Ma e ials and me hods
In he p esen s udy, 150 Lacaune neona al lambs 25–30 days old
om a comme cial sheep a m a ec ed by a CE ou b eak we e selec ed
o e alua ion o MS® ea men . All he p ocedu es we e supe ised
and app o ed by he E hics Ad iso y Commission o Animal Expe i-
men a ion (nº PI33/21), he Biosa e y Commi ee and he Occupa ional
Risk P e en ion Uni o he Uni e si y o Za agoza, in acco dance wi h
cu en egula ions ega ding hese p ocedu es. aspec s (R.D. 53/2013,
Law 31/1995, R.D. 664/1997, R.D. 1299/2006).
2.1. S udied lambs and weighing
The lambs we e selec ed ollowing p esen a ion wi h a ange o skin
and o al lesions conside ed consis en wi h a diagnosis o CE. Con i -
ma ion o ORFV in ec ion used a polyme ase chain eac ion (PCR) a -
ge ing ORFV 045 gene on swabs sampled om ORFV-associa ed skin and
o al lesions.
Subsequen ly, lambs we e andomly di ided in o 3 coho s (C, D and
E) o 50 lambs each. Lambs we e egis e ed wi h indi idual ea ags o
iden i ica ion and weigh s o all lambs we e measu ed 4 (W1), 10 (W2),
18 (W3) and 22 (W4) days pos -ini ial ea men (dp ) (Table 1).
2.2. T ea men applica ion
Animals o coho C we e ea ed wi h MS® on 3 occasions, wi h an
in e al o 3 days be ween ea men s (Table 1). Lambs we e ea ed by
sp aying 1.5 mL o MS® (Dech a, UK) using a comme cial dosing gun.
The MS® was sp ead on he ORFV-associa ed lesions and in o he
mou h. Animals o coho D we e ea ed daily wi h hypochlo ous acid
(HA) (B inasan, LEONVET, Spain), using he same echnique as in g oup
C. Animals o g oup E (con ol) we e no ea ed. All lambs we e
examined and sampled o e a 22-day pe iod (Table 1).
2.3. Clinical examina ion
The lambs we e examined a 2-day in e als (Table 1) wi h digi al
images o indi idual animal collec ed o de ailed s udy o he ype and
se e i y o he lesions h oughou he s udy. Fo each lamb, bo h on al
and la e al p o iles we e pho og aphed, including he an e io mou h
and he icini y o he gums and pala e, wi h pho os g ouped by lamb by
he inclusion o ea ag numbe s. Fo s a is ical s udy, he images we e
analysed indi idually, and lesions we e classi ied acco ding o he
pa hological na u e o he lesion, di ec ly co ela ed wi h he s age o
CE, including e y hema and/o papules in he i s s ages o CE; esicles
and/o pus ules in o mid-la e s ages o he disease; and p oli e a i e
scabby lesions in he la es s ages o CE. The se e i y o each lesion was
g aded om 0 o 4: 0 =absence; 1–3 =mild o mode a e; and 4 =
´
A. G´
omez e al.
Ve e ina y Mic obiology 292 (2024) 110037
3
se e e.
2.4. Haema ological analysis
Whole blood samples we e collec ed om he jugula ein in o EDTA
an icoagulan ubes o haema ological analysis o all s udy animals.
Samples we e collec ed p io o ea men (He0) and 22 dp (He1)
(Table 1). Haema ology was pe o med wi h an IDEXX P ocy eDx
au oma ic haema ology coun e (IDEXX labo a o ies, Wes b ook, ME,
USA). Measu ed pa ame e s included leukocy es (K/mL), e y h ocy es
(M/µL), haemoglobin (g/dL), haema oc i (%), pla ele s (K/µL), VCM
(Mean Co puscula Volume; L), HCM (Co puscula Hemoglobin Mean;
pg), MCHC (Mean Co puscula Hemoglobin Concen a ion; g/dL) and
e iculocy es (K/µL). Whi e se ies blood cells we e also e alua ed by
coun ing neu ophils (K/µL), lymphocy es (K/µL), monocy es (K/µL),
basophils (K/µL), and eosinophils (K/µL).
2.5. PCR and i us cul u e
Fo he de ec ion o ORFV i al DNA in in ec ed skin and mucous
memb anes, samples we e collec ed om ORFV-associa ed lesions using
s e ile swabs and we e p ese ed in Dulbecco’s Modi ied Eagle Medium
(DMEM) (Del alab). Samples we e ob ained om all animals p io o
ea men (P0) and on 10 (P1) and 22 (P2) dp (Table 1). Nucleic acid
ex ac ion was pe o med manually (E.Z.N.A.® Blood DNA Ki , Omega
Bio- ek). The ex ac ed DNA samples we e s o ed a −80 ◦C. Fo PCR,
p ime s o ORFV 045 gene ( o wa d p ime : 5´-CCTACTTCTCG-
GAGTTCAGC-3´; e e se p ime : 5´- GCAGCACTTCTCCTCGTAG-3´) we e
used in ampli ica ion on a FAST 7500 cycle (Applied Biosys ems).
S e ile swabs collec ed a P0, P1 and P2 we e submi ed o incuba ion
wi h p ima y issue cul u es om o ine skin ib oblas s (OSF). B ie ly,
swabs we e imme sed in 2 mL o DMEM supplemen ed wi h 2% oe al
bo ine se um, 1% glu amine and 1% an ibio ics (Sigma Ald ich, S .
Louis, Missou i, USA) and hen added o OSF. Cells we e incuba ed a
37◦C, 5% CO2 a mosphe e o 7 days. DNA ex ac ion was pe o med in
cells using E.Z.N.A® Blood DNA Mini Ki (Omega, bio- ek). PCR was
pe o med as desc ibed abo e.
2.6. S a is ical analysis
All he da a collec ed we e in eg a ed in o a s a is ical ma ix o he
SPSS STATISTICS 26.0 p og am (IBM Co p., A monk, NY, USA). The
absence/ p esence o lesions was codi ied as 0/1 and compa isons
among ea men g oups we e ca ied ou by Pea son Chi-squa e es .
G ade o lesions (0−4) we e conside ed as a quan i a i e a iable,
desc ibed by means and s anda d de ia ion (SD). Since dis ibu ions
we e no no mal (as assessed by he Shapi o-Wilks es ), compa isons
among ea men g oups we e pe o med by he K uskal-Wallis es
(nonpa ame ic es ). Fo compa isons o ini ial weigh among ea -
men g oups, one-way Ano a was applied. When conside ing weigh a
ollowing assessmen s, Ano a was conduc ed, wi h weigh s om
p e ious assessmen s used as co a ia es. P- alues <0.050 we e consid-
e ed s a is ically signi ican . The Bon e oni co ec ion was applied in
mul iple pai wise compa isons.
3. Resul s
3.1. Weigh ing
A 4 dp , he mean o al weigh was 6.616 ±1.6397 (SD) kg. The
compa ison be ween he means o he coho s e ealed no signi ican
di e ences a he commencemen o he s udy: 6.904 ±1.9083 (SD) kg
in coho C ( ea ed wi h MS®); 6.436 ±1.3717 (SD) kg in coho D
( ea ed wi h hypochlo ous acid); and 6.491 ±1.5751 (SD) kg in coho
E (con ol g oup). The p og ession o he a e age weigh o he lambs
pe coho du ing he s udy is displayed (Fig. 1). Signi ican di e ences
we e no ound (p >0.05) be ween coho s. Howe e , he a e age
weigh o coho C ea ed wi h MS® was highe h oughou he en i e
s udy, eaching i s maximum di e ence wi h he o he wo g oups on
days 18 and 22 dp , a e he hi d ea men wi h MS®.
3.2. Clinical examina ion
A commencemen o he s udy, all he animals displayed clinical
signs consis en wi h CE, wi h a ia ions in loca ion, numbe and
pa hological na u e o he lesions. Following ea men , he de elop-
men o lesions in each coho di e ed. Coho C ea ed wi h MS®
con ained ewe lambs wi h ORFV-associa ed lesions han o he coho s
a di e en pe iods o he s udy (Table 2). A 2 dp , coho C (MS®
g oup) displayed a lowe pe cen age o animals wi h e y hema/papules
and p oli e a i e scabby lesions han coho E (con ol g oup). A 6 dp ,
coho C displayed a lowe pe cen age o animals wi h e y hema/
Table 1
T ea men , clinical examina ion, weighing and sampling schedule.
Days pos - ea men
-1 0 2 4 6 8 10 12 15 18 22
MS® ea men 1 TS 2 TS 3 TS
Clinical examina ion CE 0 CE 1 CE 2 CE 3 CE 4 CE 5 CE 6 CE 7 CE 8 CE 9 CE 10
Weighing W1 W2 W3 W4
Haema ological analysis He 0 He 1
PCR P0 P1 P 2
Abb e ia ions: Days pos - ea men : days pos - i s ea men o MS® and hypochlo ous acid; MS®: Mul isol en; CE: clinical examina ion; W: Weighing; He: hae-
ma ological analysis om whole blood samples; P: PCR a ge ing ORFV 045 gene om swabs o skin lesions.
No e: Animals om g oup D we e ea ed daily wi h hypochlo ous acid and animals om g oup E we e no ea ed. In hese g oups, he clinical examina ion and
sampling we e pe o med as desc ibed in he able.
Fig. 1. P og ession o he a e age weigh s o he h ee MS® (blue), HA (yel-
low) and con ol (black) coho s h oughou he s udy whe e all lambs we e
in ec ed na u ally by ORFV. E o ba s: 95% CI o he mean.
´
A. G´
omez e al.
Ve e ina y Mic obiology 292 (2024) 110037
4
papules and esicles/pus ules han coho E. A 12 dp coho C dis-
played a lowe pe cen age o animals wi h e y hema/papules han he
con ol g oup. A 18 dp , coho E displayed he lowes pe cen age o
indi iduals wi h e y hema/papules, wi h coho D (HA g oup) he
highes , and coho C (MS ® g oup) in e media e be ween he o he wo
coho s. All di e ences we e s a is ically signi ican (p <0.005).
Coho C displayed a lowe mean se e i y (0–4 scale) in all ca ego ies
o ORFV-associa ed lesions han he o he wo coho s a he di e en
pe iods o he s udy (Table 3 ). A 2dp , coho C displayed a lowe mean
se e i y o e y hema/papules han he o he coho s; whe eas o p o-
li e a i e scabby lesions, coho C displayed a lowe mean se e i y han
coho E (con ol). A 6 dp , coho C displayed a lowe mean se e i y o
e y hema/papules han o he coho s, and o esicles/pus ules, coho
C displayed a lowe mean se e i y han coho E. A 12 dp , coho C
displayed a lowe mean se e i y o e y hema/papules han coho E.
Howe e , a 18 dp , he lowes mean se e i y o e y hema/papules was
coho E, he highes was in coho D (HA g oup); wi h coho C be ween
he o he coho s. All di e ences we e s a is ically signi ican (p <
0.005). A 15 dp , coho C p esen ed a lowe mean se e i y o p oli -
e a i e scabby lesions han he o he g oup, al hough mul iple com-
pa isons ailed o ind signi ican di e ences, wi h he global p- alue
unde 0.05 (p=0.047).
3.3. Haema ological analysis
Analysis o hemog am and leukog am pa ame e s, including con-
cen a ions and pe cen ages o o al leukocy es, we e ound wi hin he
no mal anges in all lambs sampled. In addi ion, no signi ican di e -
ences (p >0.05) we e de ec ed be ween he h ee coho s a any o he
sampling pe iods.
3.4. PCR and i us cul u e
Posi i e and nega i e PCR esul s o sampled swabs ob ained a P0,
P1 and P2 ound no signi ican di e ences be ween coho s (P >0.05)
(Table 4). Swabs submi ed o incuba ion wi h p ima y issue cul u es
om o ine skin ib oblas s (OSF) showed posi i e esul s in he h ee
g oups h oughou P0, P1 and P2, wi h no signi ican s a is ical di e -
ences obse ed be ween g oups (p >0.05) (Table 4).
4. Discussion
Con agious ec hyma is a highly con agious zoono ic i al skin dis-
ease causing signi ican economic losses in global sheep and goa pop-
ula ions (Lo a e al., 2012; Bala e al., 2018). Despi e wo ldwide
dis ibu ion and signi ican economic impac , he e a e ew egis e ed
accines (Lacas a e al., 2015) o con ol his disease in some coun ies
(Buddle and Pul o d, 1984; Pye, 1990; Buka e al., 2021; Zhu e al.,
2022), wi h conce ns ha can e e o i ulence o elici incomple e
immune p o ec ion (F iebe e al., 2004; Musse e al., 2008; Tan e al.,
2009; Jo ge and Dellagos in, 2017). P o o ypes o DNA and subuni
accines (Zhao e al., 2011; Yogisha adhya e al., 2017, 2018; Wassie
e al., 2019; Zhu e al., 2022; Shen e al., 2023) ha e been expe imen-
ally s udied wi h p omising esul s, ye equi e app o al. Fu he , some
opical an isep ic o mula ions ha e been sugges ed o ORFV ea men
(Van De Ke k P., 1954; Lansade, 1959; Beck and Taylo , 1974; Rapun-
ean e al., 1975; Sande son, 1976; Walde e al., 1979; La sson and
Zahoo y, 1983), al hough, gene ally, he esul s we e no p omising.
Lo agen was e ec i e in o al mucosa lesions bu less e ec i e in hai ed
skin (Rapun ean e al., 1975). Only S ibophen educed lesion se e i y in
8–10 days a e applica ion (Walde e al., 1979), al hough a de ailed
lesion ypi ica ion was no pe o med o de e mine in which ype o
lesion his d ug had he g ea es e ec . Addi ionally, an imony
Table 2
P esence o lesions (%) by ype, days pos - i s ea men and coho . Only e-
sul s wi h s a is ically signi ican di e ences a e shown.
Type o lesion Dp G oup p- alue
Con ol HA MS®
E y hema/papules
2 68.1%
a
81.8%
a
38.3%
b
<0.001
6 72.7%
a
84.4%
a
47.8%
b
0.001
12 88.1%
a
76.2%
a,b
61.9%
b
0.020
18 25.6%
b
53.7%
a
31.6%
a,b
0.024
Vesicles/pus ules 6 56.8%
a
37.8%
a,b
28.3%
b
0.020
P oli e a i e scabby lesions 2 25.5%
b
4.5%
a
4.3%
a
0.001
Abb e ia ions: Dp : days pos - i s ea men o MS® and hypochlo ous acid.
Con ol: in ec ed and no ea ed; HA: ea ed daily wi h hypochlo ous acid;
MS®: ea ed wi h Mul isol en 3 imes wi h an in e al o 3 days. a,b: Di e en
le e s in a ow mean signi ican di e ences (p<0.05).
Table 3
Se e i y o lesions (g aded 0–4) o lesion- ype, by days pos -ini ial ea men and coho .
Type o lesion Dp
G oup
p- alue Con ol HA MS®
Mean SD Mean SD Mean SD
E y hema/papules
2 0.74
a
0.57 1.02
a
0.66 0.47
b
0.83 <0.001
6 1.43
a
0.70 1.38
a
0.72 1.04
b
0.87 0.014
12 1.07
b
0.56 0.88
a,b
0.59 0.74
a
0.67 0.040
18 0.26
b
0.44 0.66
a
0.69 0.37
a,b
0.63 0.012
Vesicles/pus ules 6 0.64
b
0.61 0.38
a,b
0.49 0.30
a
0.51 0.015
P oli e a i e scabby lesion 2 0.32
b
0.63 0.05
a
0.21 0.04
a
0.20 0.001
15 0.57 0.73 0.97 1.04 0.44 0.55 0.047
Abb e ia ions: Dp : days pos -ini ial ea men o MS® o hypochlo ous acid; Con ol: in ec ed and no ea ed; HA: ea ed daily wi h hypochlo ous acid; MS®: ea ed
wi h Mul isol en 3 imes wi h an in e al o 3 days; SD: s anda d de ia ion. a,b: Di e en le e s in a ow show signi ican di e ences (p<0.05).
Table 4
Resul s in pe cen age o PCR a ge ing ORFV 045 gene. Samples we e collec ed
using s e ile swabs p ese ed in DMEM (Del alab) om ORFV-associa ed lesions.
Sample Resul
-1 dp
p- alue G oup
Con ol HA MS®
Swabs Posi i e 65.00% 64.70% 91.70% 0.06
Vi us cul u e Posi i e 81.80% 73.30% 75,.00% 0.874
Sample Resul
10 dp
p- alue G oup
Con ol HA MS®
Swabs Posi i e 80,00% 87.50% 100.00% 0.345
Vi us cul u e Posi i e 27.30% 33.30% 45.80% 0.404
Sample Resul
22 dp
P- alue G oup
Con ol HA MS®
Swabs Posi i e 46.20% 46.70% 53.30% 0.911
Vi us cul u e Posi i e 66.70% 62.50% 53.80% 0.780
Abb e ia ions: Dp : days pos - i s ea men o MS® and hypochlo ous acid;
Con ol: g oup in ec ed and no ea ed; HA: g oup ea ed daily wi h hypo-
chlo ous acid; MS®: g oup ea ed wi h Mul isol en 3 imes wi h an in e al o 3
days.
´
A. G´
omez e al.
Ve e ina y Mic obiology 292 (2024) 110037
5
compounds used as he apy can cause ca dio oxici y and panc ea i is
(Sunda and Chak a a y, 2010). The e o e, no e ec i e ea men s o
i al in ec ions a e a ailable o use in a m condi ions, wi h local an-
isep ics and an ibio ics o en used, assuming hese may assis con ol o
seconda y bac e ial in ec ions. Recen s udies ha e con i med he e i-
cacy o he wound he apy o mula ion Mul isol en® (MS®) o
educing pain and has ening he healing o skin and mucosal lesions in
sheep and ca le (Windso e al., 2020; Lendzele e al., 2021; Roughan
and Windso , 2022). Thus, p io o his s udy, MS® was examined as a
he apeu ic ea men in 50 lambs expe imen ally in ec ed by
in a-de mal inocula ion wi h ORFV. Al hough MS® did no imp o e he
clinical p og ession o CE in lambs, i was conside ed ha his could
ha e been due o he b ie possibly inadequa e ea men p ocedu e
(Lacas a e al., 2023).
In he p esen s udy, a new MS® ea men p o ocol was applied,
using 150 Lacaune lambs om a comme cial sheep a m a ec ed by a
na u al CE ou b eak. The p o ocol in ol ed applica ion o he MS® on 3
occasions wi h an in e al o 3 days be ween ea men s. The esul s
indica ed ha he coho ea ed wi h MS® p esen ed wi h ewe lambs
displaying ORFV-associa ed lesions han o he coho s a di e en imes
o he s udy (Table 2). The ype o lesion ha p esen ed he mos sig-
ni ican di e ences be ween g oups was e y hema/papules, a lesion
obse ed in he ini ial phase o he clinical cou se o CE (Nandi e al.,
2011; Spy ou and Valiakos, 2015). The coho ea ed wi h MS® showed
a lowe numbe o animals wi h e y hema/papules on 2, 6, 12 and 18
dp han he o he wo coho s, sugges ing ha MS® can educe he
e y hema/papules i i is applied in an ea ly s age o CE. Howe e ,
esicles/pus ules and p oli e a i e scabby lesions we e obse ed in a
signi ican ly lowe numbe o animals only in 6 and 2 dp , espec i ely.
These esul s may sugges ha , al hough MS® appea s o educe he
esicles/pus ules and p oli e a i e scabby lesions a e ea men , hey
p oli e a e again when ea men is discon inued. These indings e lec
he p olonged clinical cou se o CE and sugges ha mul iple ea men s
wi h MS® could p oduce be e esul s in con olling he p esence o
hese ypes o lesions. T ea men wi h MS® could be ex ended o a
minimum o 4 weeks, he a e age pe iod necessa y o esolu ion o
ORFV lesions (Nandi e al., 2011).
Fu he , he se e i y o each lesion- ype was e alua ed, wi h g ading
om 0 o 4. The coho ea ed wi h MS® displayed lowe mean se e i y
sco es in all ypes o ORFV-associa ed lesions han he o he coho s wi h
he same lesion- ype and days as desc ibed abo e, wi h he excep ion
ha on 15 dp , when he coho ea ed wi h MS® displayed milde
lesions ca ego ised as p oli e a i e scabby lesions, han in o he coho s
(Table 3). The indica ions we e ha MS® likely educed he se e i y o
ORFV-associa ed lesions, especially o e y hema/papules and p oli e -
a i e scabby lesions. The indings concluded ha in his, MS® he apy
educed bo h he numbe and se e i y o o lesions, especially imme-
dia ely a e ea men . Howe e , i appea ed ha a e emo al o he
he apeu ic gel solu ion, mos o he lambs again de eloped ORFV-
associa ed lesions in o he loca ions. I is well-known ha in a na u al
CE ou b eak, ORFV lesions usually esol e wi hin 3–8 weeks (Haig e al.,
2002; Nandi e al., 2011) and du ing clinical p og ession o CE, ORFV
eplica es in he ke a inocy es o s a um basale (Fleming e al., 2015;
Windso e al., 2017). Whils opical applica ion o MS® appea s capable
o imp o ing he healing o e up ed lesions, he i us con inues o
mul iply in he s a um basale and new lesions will likely appea du ing
he p olonged 4–6 weeks o CE disease (Be gq is e al., 2017). Fo his
eason, i is ecommended ha applica ion o he p oduc con inues on
epea ed occasions du ing he p e-healing phase as i may assis con-
olling he de elopmen o he sub-acu e and ch onic-ac i e ORFV
lesions.
The apy wi h a single dose o MS® has been showed o be e icacious
o ea ing e osions and ulce s in o al mucosa and on he ee o animals
a ec ed by oo and mou h disease (FMD) (Windso e al., 2020; Lend-
zele e al., 2021; Roughan and Windso , 2022). Foo and mou h disease
i us (FMDV) eplica es p incipally in he s a um spinosum, comp ising
he mos supe icial laye s o he epi helium and mucosa, wi h lesions
commencing as esicles hen p og essing o e osions and ulce s (Alex-
ande sen e al., 2003). These cha ac e is ics enable opical applica ion
o MS® o eadily con ac he i us and esol e he clinical p og ession
o FMD mo e quickly han o he he apies, wi h p e ious obse a ions
sugges ing a i icidal e ec o MS® agains FMDV due o he low pH o
he p oduc (2.7–2.9) o lidocaine concen a ion anging om
0.5 mg/mL (0.05%) o 100 mg/mL (10%) (Windso e al., 2020; Lend-
zele e al., 2021; Roughan and Windso , 2022). In con as , ORFV a ec s
he s a um basale, he deepes laye o he epide mis and o al mucosa,
and he p incipal lesions a e papules, esicles, pus ules and p oli e a i e
lesions, wi h he la e enc us a ions po en ially comp omising he
pene a ion o MS® in o he basal epi helium and deli e ing he i icidal
e ec . In p e ious s udies, MS® has no been e ec i e in educe ORFV
i al load in- i o (Lacas a e al., 2021, 2023). In he p esen s udy, 48
and 53.8% o swabs o he g oup ea ed wi h MS® om 10 and 22 dp ,
espec i ely, we e PCR posi i e on i us cul u e, sugges ing ha ORFV
lesions may p e en MS® om inac i a ing he i us in i o (Table 4).
The esul s sugges ha he clinical imp o emen in he lambs na u ally
in ec ed wi h ORFV and ea ed wi h MS® a e likely due p olonged
pain- elie ing and wound healing e ec s ollowing blockage o local
nocicep o s du ing ea men o ORV lesions. In addi ion, ewe sec-
onda y in ec ions occu ed ollowing he applica ion o MS®, as eco -
ded in p e ious s udies (Lacas a e al., 2021, 2023), imp o ing wound
healing, an impo an a ibu e o MS® he apy (Windso e al., 2020;
Lendzele e al., 2021). Al hough i emains con o e sial whe he p o-
ision o some o ms o analgesia educes acu e in lamma ion, he e a e
nume ous s udies demons a ing imp o ed immune sys em unc ion in
di e en animal species (Ya deni e al., 2009; Cab al e al., 2015;
Amodeo e al., 2018; DeMa co and Nunamake , 2019).
The esul s ob ained in he p esen s udy indica e ha opical
ea men wi h MS® is e ec i e o CE in ield condi ions, especially in
he ea ly s ages o he clinical cou se, and ha i would likely o be
bene icial o p olong he he apy o a minimum o 4 weeks o educe he
de elopmen o new ORFV lesions. Fu he , his s udy ein o ces he
hypo hesis ha whils MS® may no pene a e o he s a um basale o
p oli e a i e lesions and inac i a e ORFV, he e is me i in he p oposal
ha his mul i-modal anaes he ic and an isep ic combina ion inhibi s
in lamma ion in i al skin diseases, imp o ing wel a e and assis ing he
con ol o seconda y in ec ions, p omo ing he healing o ORFV and i al
lesions wi hou p omo ion o AMR isks.
Au ho con ibu ions
Concei ed and designed he expe imen s (D.L., P.A.W and M.R.);
pe o med he ea men and sample collec ion (H.R, A.G., D.L., M.R., P.
Q., M.V., M.B., and J.J.R); did he labo a o y examina ion (S.V., A.O., R.
R., and T.N.); w o e he manusc ip (A.G.); did he s a is ical analysis
(M.T.T.); did he p ojec managemen (D.L. and J.J.R.); e iewed he
manusc ip (D.L., P.A.W., S.V., M.T.T., A.O., M.B., M.V., H.R. and T.N.).
All au ho s ha e ead and ag eed on he manusc ip .
Funding
This esea ch was suppo ed by unding and p oduc om he
Aus alian company Animal E hics P y L d. The wo k was also suppo ed
by he A ag´
on Go e nmen and he Eu opean Social Fund (A15_17R,
Cons uyendo A ag´
on 2016–20) and P ojec CONECTIM unded by
Gobie no de Na a a (PC052-053).
CRediT au ho ship con ibu ion s a emen
Pe e And ew Windso : W i ing – e iew & edi ing, Valida ion,
Resou ces, Me hodology, Funding acquisi ion, Concep ualiza ion. Te -
esa Na a o: In es iga ion. Pablo Quílez: In es iga ion. Ma a Bo -
obia: In es iga ion. Mai e Ve de: In es iga ion. ´
Alex G´
omez: W i ing –
´
A. G´
omez e al.

Ve e ina y Mic obiology 292 (2024) 110037
6
o iginal d a , In es iga ion, Fo mal analysis, Da a cu a ion. Au o a
O ín: W i ing – e iew & edi ing, Me hodology, In es iga ion. H´
ec o
Ruiz: W i ing – e iew & edi ing, Me hodology, In es iga ion. Rams´
es
Reina: In es iga ion. Se gio Villanue a-Saz: W i ing – e iew & edi -
ing, Me hodology, In es iga ion. Ma ía Te esa Tejedo : W i ing – e-
iew & edi ing, Valida ion, Fo mal analysis, Da a cu a ion. Delia
Lacas a: W i ing – e iew & edi ing, Supe ision, P ojec adminis a-
ion, Me hodology, In es iga ion, Funding acquisi ion, Concep ualiza-
ion. Juan Jos´
e Ramos: P ojec adminis a ion, Me hodology,
In es iga ion. Ma a Ruiz de A cau e: Me hodology, In es iga ion,
Concep ualiza ion.
Decla a ion o Compe ing In e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Da a a ailabili y
The da a ha suppo he indings o his s udy a e a ailable om he
co esponding au ho upon eques .
Acknowledgemen s
We would like o hank Albe o Bo dalde, he a me who owns he
a m whe e he s udy was ca ied ou , o his help and suppo . Likewise,
we would like o hank he labo a o y echnician Ma iangeles Los ao
and he in e n s uden s o he Ruminan Clinical Se ice (SCRUM) o he
Facul y o Ve e ina y o he Uni e si y o Za agoza, especially Helena,
Lucía and Ma ina.
Ins i u ional e iew boa d s a emen
The s udy was conduc ed in acco dance wi h he Decla a ion o
Helsinki, and app o ed by he Ins i u ional Re iew Boa d (o E hics
Commi ee) o he Uni e si y o Za agoza (P ojec Licence PI 33/21,
2021) o s udies in ol ing animals.
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