Fib opapilloma osis and he Chelonid Alphahe pes i us 5
in G een Tu les om Wes A ica
Jessica Mon ei o,
1
Ma ga ida Dua e,
1,2
Kide
´Amadou,
3
Cas o Ba bosa,
4
Nahi El Ba ,
3
Fe nando M. Madei a,
5
Aissa Regalla,
4
Ana Dua e,
1
Luı
´s Ta a es,
1
and Ana Ri a Pa ı
´cio
6,7
1
Cen o de In es igac¸a
˜o In e disciplina em Sanidade Animal (CIISA), Faculdade de Medicina Ve e ina
´ ia, Uni e sidade de Lisboa, A enida da
Uni e sidade Te
´cnica, 1300-477 Lisboa, Po ugal
2
Ins i u o Nacional de In es igac¸a
˜o Ag a
´ ia e Ve e ina
´ ia (INIAV), 1500-310 Lisboa, Po ugal
3
Obse a oi e, Pa c Na ional du Banc d’A guin, Chami, Mau i ania
4
Ins i u o da Biodi e sidade e das A
´ eas P o egidas, A . Dom Se imio A u o Fe azze a, CP 70 Bissau, Guinea-Bissau
5
cE3c Cen e o Ecology, E olu ion and En i onmen al Changes, Facul y o Sciences, Uni e si y o Lisbon, Campo G ande 016, I749-016 Lisboa, Po ugal
6
MARE – Ma ine and En i onmen al Sciences Cen e, ISPA – Ins i u o Uni e si a
´ io, Rua Ja dim do Tabaco 34, 1149-041 Lisbon, Po ugal
7
Cen e o Ecology and Conse a ion, Uni e si y o Exe e , Pen yn TR10 9EZ, UK
Abs ac : Fib opapilloma osis (FP) is a umo igenic panzoo ic disease o sea u les, mos common in g een
u les (Chelonia mydas). FP is linked o he chelonid alphahe pes i us 5 (ChAHV5) and o deg aded habi a s
and, hough benign, la ge umou s can hinde i al unc ions, causing dea h. We analyse 108 g een u les,
cap u ed in 2018 and 2019, a key o aging g ounds in Guinea-Bissau and Mau i ania, Wes A ica, o he
p esence o FP, and use eal- ime PCR o de ec ChAHV5 DNA, in 76 indi iduals. The p e alence o FP was
mode a e; 33% in Guinea-Bissau (n= 36) and 28% in Mau i ania (n= 72), and mos u les we e mildly
a ec ed, possibly due o low human impac a s udy loca ions. Ju eniles had highe FP p e alence (35%,
n= 82) compa ed o subadul s (5%, n= 21), p obably because indi iduals acqui e esis ance o e ime.
ChAHV5 DNA was de ec ed in 83% (n= 24) o he umou biopsies, consis en wi h i s ole as ae iological
agen o FP and in 26% (n= 27) o he ‘no mal’ skin (no showing lesions) om FP u les. No ably, 45% o
he asymp oma ic u les we e posi i e o ChAHV5, suppo ing mul i ac o ial disease exp ession. We epo
he i s baselines o FP and ChAHV5 p e alence o Wes A ica g een u les, essen ial o assess e olu ion o
disease and u u e impac s o an h opogenic ac i i ies.
Keywo ds: Eme ging diseases, Fib opapilloma osis, Chelonia mydas, Chelonid alphahe pes i us 5, ChAHV5,
Guinea-Bissau, Mau i ania
INTRODUCTION
In he pas wo decades, he human popula ion has g own
exponen ially su passing se en billion indi iduals in he
yea 2019 (Uni ed Na ions 2019), leading o inc eased
in e ac ions be ween humans and wildli e, and o highe
Co espondence o: Ana Ri a Pa ı
´cio, e-mail: [email p o ec ed]
EcoHeal h
h ps://doi.o g/10.1007/s10393-021-01526-y
O iginal Con ibu ion
Ó2021 The Au ho (s)
an h opogenic p essu e on na u al ecosys ems, h ea ening
he long- e m su i al o se e al species (Deem 2015).
Clima e change, habi a deg ada ion and in ec ious diseases
a e majo h ea s o biodi e si y and ecosys ems, which can
ha e syne gis ic e ec s on one ano he (Hoegh-Guldbe g
e al. 2010). Some in ec ious diseases wi h known majo
impac s on wildli e popula ions a e chy idiomycosis in
amphibians (Scheele e al. 2017), canine dis empe in ca -
ni o es (Kennedy e al. 2000) and ib opapilloma osis in
sea u les (Wo k e al. 2004).
Sea u les, being long-li ed o ganisms ha display i-
deli y o hei eeding and b eeding habi a s, can be used as
sen inel indica o s o he heal h o ma ine ecosys ems
(Agui e and Lu z 2004; Domiciano e al. 2017). Fib opa-
pilloma osis (FP) is a umo igenic disease ha has been
epo ed in all se en species o sea u les; howe e , i is
mo e equen among g een u les (Chelonia mydas). I
was i s epo ed in 1938 in a cap i e g een u le (Smi h
and Coa es 1938), and e e since, i has been documen ed
in sea u le agg ega ions wo ldwide (Jones e al. 2016).
Fib opapillomas may be ound on he lippe s, he so
skin a ound he ca apace, he ocula and o al egions and
in ex eme cases, he in e nal o gans (Jones e al. 2016).
Tumou s can cause di icul ies in sigh , eeding and
swimming and cause in e nal p essu e, which may culmi-
na e in o gan dys unc ion and/o physiologic imbalances
(Page-Ka jian 2019). The ae iology o he disease, al hough
no ully unde s ood, is linked o an alphahe pes i us, he
chelonid alphahe pes i us 5 (ChAHV5, Domiciano 2019),
as i s DNA is ound in mos FP lesions (Al a o-Nu
´n
˜ez and
Gilbe 2014; He bs e al. 2004). ChAHV5 DNA has also
been de ec ed in g ossly FP- ee u les, sugges ing ha he
de elopmen o umou s is no due o i us in ec ion alone,
bu o he ac o s may be implica ed, such as hos –pa ho-
gen–en i onmen in e ac ions, hos immuni y, i al load
and se e i y o i us a ian (He bs e al. 2008). Phylo-
gene ic analysis es ima es ha mode n a ian s o ChAHV5
appea ed be o e he widesp ead FP phenomenon (Pa ı
´cio
e al. 2012), sugges ing ha ecen ou b eaks a e linked o
human-induced en i onmen al changes. Some s udies e-
po ed highe FP p e alence in human-al e ed en i on-
men s (Van Hou an e al. 2010; Kelle e al. 2014), u he
suppo ing his hypo hesis.
The p e alence o he disease and i s se e i y di e s
spa ially and empo ally (Pa ı
´cio e al. 2016), wi h some
epo s o p e alence peaks su passing 90%, leading o high
mo ali y (dos San os e al. 2010). The e is an age-e ec on
disease p e alence; ju eniles a e mo e a ec ed (Jones e al.
2016; Pa ı
´cio e al. 2016). Reg ession o he umou s has
been epo ed (Agui e e al. 1999, Benne e al. 1999,
Limpus e al. 2005; Hi ama and Eh ha 2007; Chaloupka
e al. 2009; Machado Guima a
˜es e al. 2013), and cycles o
epidemic ou b eaks ollowed by eg ession may be com-
mon (Pa ı
´cio e al. 2016).
Geog aphically, he e a e majo gaps on he knowledge
o FP p e alence, pa icula ly in Wes A ica. Lesions
compa ible wi h ib opapillomas ha e been obse ed in
s anded g een u les om Mau i ania, Senegal, The
Gambia, Guinea-Bissau (Gi a d 2015) and Cape Ve de
(Ma ins e al. 2020), and Ca y e al. (2009) ound no
e idence o FP among nes ing g een u les in Guinea-
Bissau. Howe e , o his da e, he e we e no assessmen s o
FP on o aging animals in he egion. E en in he wide
Eas e n A lan ic, only one s udy o da e cha ac e ized FP
and he in ec ion o ChAHV5 among diseased and
asymp oma ic o aging g een u les, om he P incipe
Island, Gul o Guinea (Dua e e al. 2012). In Wes A ica,
he wo main eeding si es o g een u les a e Guinea-
Bissau and Mau i ania. The Bijago
´s A chipelago, in Gui-
nea-Bissau, hos s he hi d la ges g een u le popula ion
in he A lan ic Ocean, six h wo ldwide, wi h an a e age o
25,436 nes s pe yea (2013–2016, Pa ı
´cio e al. 2018), as
well as impo an o aging g ounds o imma u e g een
u les. In Mau i ania, he Banc D’A guin Na ional Pa k
suppo s high le els o ma ine p oduc i i y and a key o -
aging a ea o he ep oduc i e g een u les om he Bi-
jago
´s and o imma u e g een u les (Ca dona e al. 2009;
Godley e al. 2010). Al hough hei o igin is unknown,
mos likely, a g ea p opo ion o hese imma u e u les
o igina es om he la ge ooke y in he Bijago
´s (Pa ı
´cio
e al. 2017).
Guinea-Bissau and Mau i ania a e key ‘ho spo s’ o
he g een sea u le in he A lan ic Ocean; howe e , un il
his da e, no da a we e a ailable conce ning he p e alence
o FP, o he p e alence o in ec ion by he ChAHV5, in
o aging u les om ei he o hese si es. To suppo local
conse a ion e o s and con ibu e o ill-in iden i ied
geog aphic in o ma ion gaps, we i) assessed he p esence,
se e i y and p e alence o FP, ii) in es iga ed whe he
ChAHV5 in ec ion was ubiqui ous among symp oma ic
and asymp oma ic indi iduals, and iii) explo ed he ela-
ionship be ween FP and body size (a p oxy o age), on
o aging g een u les om Wes A ica.
J. Mon ei o e al.
MATERIALS AND METHODS
S udy Si e
This s udy was conduc ed a wo si es, he sou h o he
Na ional pa k o he Banc D’A guin, in Mau i ania (PNBA,
N19.58°, W16.42°, Fig. 1), and he wes e nmos islands o
he Bijago
´s A chipelago in Guinea-Bissau, Unhocomo and
Unhocomozinho (U&U, N11.31°, W16.40°, Fig. 1). A he
PNBA, he mean annual sea su ace empe a u e is 20.3 °C
( anging om 17.8 °C o 22.7 °C). A U&U, sea su ace
empe a u e a e age is 27.3 °C ( anging om 25.1 °C o
29.5 °C).
Cap u e Me hods
A he PNBA, wo ield ips we e held, in 8 May 2018 and
4–8 Ma ch 2019, and e o was 1.4 ne se s.day
-1
. Upon
obse ing u les su acing o b ea he, he pu se seine
me hod was used o cap u e hem, using h ee joined
en angling ne s (420 m long, 4 m deep, 32 cm mesh size),
wi h he assis ance o wo sailing boa s ope a ed by local
Im aguen ishe men. Cap u ed u les we e p ocessed on-
boa d he ishing essels. Th ee sepa a e missions we e held
a U&U on 18–22 Ma ch 2018, 21–25 Oc obe 2018 and
25–28 Ma ch 2019; e o was 1 ne se .day
-1
. Tu les we e
caugh using one en anglemen ne (800 m long, 4 m deep,
20 m mesh size), deployed om a pi ogue, ope a ed by
local Bijago
´s ishe s, enclosing o aging si es o pe iods o
1 h, unde cons an moni o ing o en angled u les.
Cap u ed u les we e b ough on-boa d a logis ic essel
(ancho ed nex o he ne ) o p ocessing.
Sample Collec ion and Tumou Sco ing
Fo each animal, an iden i ica ion pho og aph was aken o
he head and pos -o bi al scales, and he cu ed ca apace
leng h (CCL) was measu ed wi h a lexible measu ing ape,
o he nea es 0.1 cm. Tu les we e classi ied as ju enile
(<65 cm CCL), subadul (65–83 cm CCL) o adul
(83 cm CCL). The adul size-class was es ablished using
he minimum size o nes ing emales om he nea by g een
u le ooke y a Poila
˜o Island, Bijago
´s. Remaining size-
classes ollowed he c i e ia published by Pa ı
´cio e al.
(2016), as he e is no in o ma ion on he soma ic g ow h a
hese agg ega ions. Fo he lowe age classes, he gende was
no de e mined, gi en he absence o ob ious sexual
Figu e 1. Loca ion o g een u le o aging g ounds, in he coas o Wes A ica, assessed in he p esen s udy. aCap u e loca ions (black
diamond) wi hin he Na ional Pa k o he Banc d’A guin (PNBA), Mau i ania (polygon shows pa k limi s); bUnhocomo and Unhocomozinho
(U&U, black diamond) and limi s o he Bolama-Bijago
´s Biosphe e Rese e (polygon). In each panel, he pie cha s indica e he p e alence o
ib opapilloma osis (FP) and p e alence o chelonid he pes i us 5 (ChAHV5), linked o FP disease.
Fib opapilloma osis and he Chelonid Alphahe pes i us 5
dimo phism be o e he adul li e-s age. All u les we e
obse ed o he p esence o ex e nal ib opapillomas,
h oughou he su ace o he body and in he ocula and
o al egions. Fo a subse o 76 u les, biopsies we e col-
lec ed o gene ic de ec ion o he ChAHV5. (We did no
ha e biopsies o he 32 u les cap u ed du ing he las ield
season a he PNBA.) Fo each o hese u les, a sample o
appa en ly ‘no mal’ skin issue (i.e. issue wi h no isible
signs o FP) was collec ed om he igh shoulde , using a
s e ile biopsy punch (6 mm diame e ), and, i lesions wi h
he appea ance o ib opapillomas—p o uding peduncu-
la ed/ e ucous and/o la ge ex e nal g ow hs, anging in
colou om pale o pink, o pink g eyish and black–we e
p esen , samples we e collec ed om he mos se e e-
looking lesion, om he ex e nal su ace o he umou
punching app oxima ely 0.5 cm deep wi h a s e ile biopsy
punch (6 mm diame e ). Fo h ee indi iduals om U& U
p esen ing ocula lesions compa ible wi h ib opapillomas,
sampling was no pe o med o a oid inju y o he co nea.
Fo all he biopsies, he egion o be sampled was p e i-
ously disin ec ed wi h a dilu ed po idone-iodine solu ion
and new glo es we e used o a oid c oss-con amina ion. All
samples we e s o ed in 90% e hanol, in 2-ml sc ew cap
mic ocen i uge ubes wi h unique labels.
Each indi idual umou was classi ied depending on
he app oxima e diame e : Class A – <1 cm; Class B–1 o
4 cm; Class C–4 o 10 cm; Class D – >10 cm. Tu les
we e assigned a umou sco e, om 0 o 3, based on he
numbe o umou s in each class, ollowing he classi ica-
ion by Wo k and Balazs (1999). The umou sco ing sys-
em e lec s he spec um o se e i y o g oss FP lesions in
g een u les; a sco e o 0 is a ibu ed o non-a lic ed
u les; 1 means ligh ly a ec ed; 2, mode a ely; and 3
hea ily a lic ed (Wo k and Balazs 1999).
Quan i a i e Polyme ase Chain Reac ion (qPCR)
and Valida ion o ChAHV5 De ec ion
Ex ac ion o o al DNA was ca ied ou wi h he DNeasy
Blood & Tissue ki (Qiagen, Hilden, Ge many), ollowing
manu ac u e s’ p o ocol o animal issues. DNA quan-
i ica ion was pe o med using he NanoD op
TM
2000
spec opho ome e (The mo Fishe Scien i ic, Wilming on,
USA).
Sc eening o he samples o ChAHV5 DNA p esence
was accomplished by a quan i a i e PCR (qPCR), a ge ing
an 86-bp highly conse ed genomic egion wi hin he DNA
polyme ase gene (UL30) (Quackenbush e al. 2001), using
he AccuS a II PCR Supe Mix (2x) (QUANTABio).
Reac ions we e un in a CFX96TM Op ical Reac ion
Module (Bio-Rad). The cycling condi ions we e as ollows:
ini ial dena u a ion a 95 °C o 10 min, 45 cycles o 90 °C
o 15 s and 60 °C o 1 min. A nega i e blank con ol was
included in all PCR uns. The h eshold cycle (C ) alue
was egis e ed.
To es ima e he numbe o i al copies in each sample,
he qPCR p o ocol desc ibed by Quackenbush e al. (2001)
was implemen ed and alida ed o cons uc a s anda d
cu e. Fi s , a plasmid DNA con aining a 486-base pai
long agmen om he ChAHV5 DNA polyme ase gene
was gene a ed by he con en ional PCR sys em desc ibed
by VanDe an e e al. (1996) and cloned in o he pGEM-T
Easy Vec o (P omega), and he ecombinan plasmid was
sequenced o con i m he inse was he desi ed pa ial
ChAHV5 gene sequence. T iplica es o en old se ial dilu-
ions o he ecombinan DNA anging om 10
–1
o 10
–11
we e p epa ed, quan i ied using he NanoD op
TM
2000
spec opho ome e , and es ed. The s anda d cu e was
gene a ed using he CFX Manage So wa e (Bio-Rad,
USA).
The sensi i i y o he qPCR me hod, exp essed as he
limi o de ec ion (LOD), was de e mined by using he
se ial dilu ions o he ecombinan DNA (10
–1
o 10
–11
).
The las dilu ion whe e all h ee eplica es ga e a posi i e
and speci ic ampli ica ion was conside ed as he LOD.
The ep oducibili y (in e -assay a iabili y) o he
me hod was es ed by epea ing he qPCR o he dilu ion
se ies in h ee independen uns. The epea abili y (in a-
assay a iabili y) o he me hod was es ed by p epa ing
h ee independen dilu ion se ies om 10
–2
un il 10
–6
and
subjec ing o independen uns qPCR. The ampli ica ions
used he same condi ions wi h he CFX96
TM
Op ical
Reac ion Module (Bio-Rad).
S a is ical Analysis
The CCL alues conside ing all size classes (Shapi o–Wilk
es ; W = 0.915, p= 8.388 910
–5
), he C alues o
appa en ly no mal skin (W = 0.940, p= 0.031) and he C
alues o umou s (W = 0.873, p= 0.011) did no ollow a
no mal dis ibu ion, so we used he nonpa ame ic Mann–
Whi ney U es o assess whe he he e we e signi ican
di e ences in CCL and he C alues (‘no mal’ skin and
umou s) be ween he wo si es, and he K uskal–Wallis
es o assess whe he he e we e signi ican di e ences in
C alues (‘no mal’ skin and umou s) be ween li e-s ages
J. Mon ei o e al.
(ju enile, subadul and adul ) and be ween umou sco es.
The CCL alues excluding he adul u les ollowed a
no mal dis ibu ion (Shapi o–Wilk es ; W = 0.97962, p-
alue = 0.113); hus, we used a pa ame ic T- es o assess
whe he he e we e signi ican di e ences in he CCL o
imma u e u les be ween si es. We explo ed he ela ion-
ship be ween FP p esence and CCL a each s udy si e, using
gene alized addi i e modelling (GAM) implemen ed wi h
he package mcg (Wood and Wood, 2015) in RS udio
(RS udioTeam 2018). GAMs a e semipa ame ic models
ha allow nonlinea ela ionships be ween he esponse
a iable (FP p esence) and explana o y a iables ( u le
size, which is a p oxy o age). An alpha le el o 0.05 was
used o all s a is ical es s.
RESULTS
Fib opapilloma osis P e alence and Tumou Sco e
In o al, 108 g een u les we e examined o he p esence
and se e i y o FP: 36 we e cap u ed in U&U, and 72 a he
PNBA. A bo h si es, mos u les belonged o he ju enile
size class (Fig. 2). A U&U, ou adul s we e cap u ed ( wo
males and wo emales), anging in CCL om 87.5 o
97 cm, while a he PNBA only one adul emale was cap-
u ed wi h 96 cm CCL. The e was no signi ican di e ence
in CCL be ween si es (W = 1101, p= 0.205) when con-
side ing all size-classes, and he mean CCL o indi iduals
om U&U was 58.24 ±15.3 cm (mean ±SD, ange: 37.5
o 97 cm), sligh ly lowe han he mean CCL a he PNBA,
which was 59.52 ±9.44 cm ( anging om 39.7 o 96 cm).
Conside ing only imma u e-sized indi iduals, he e was a
signi ican di e ence be ween si es ( = -2.5103, d =
52.775, p= 0.015), wi h mean CCL a U&U being smalle
(54.0 ±9.8 cm) han a he PNBA (59.0 ±8.4 cm). O he
108 cap u ed animals, 32 (30%) exhibi ed g oss lesions
compa ible wi h FP (Table 1). The mean CCL o indi id-
uals p esen ing g oss FP lesions was 55.9 ±11.13 cm
( anging om 49.0 o 93.0 cm) in U&U, and
52.5 ±7.1 cm ( anging om 39.7 o 61.6 cm) a he
PNBA. The minimal adequa e GAMs showed ha he CCL
was signi ican ly co ela ed wi h FP isk a bo h si es (U&U:
p= 0.023, PNBA: GAM, p= 0.0003), a U& U he p ob-
abili y o de eloping FP was lowe o smalle indi iduals
hen inc easing and peaking o u les wi h CCL be ween
50–55 cm, dec easing he ea e ( Fig. 3a). A he PNBA,
he smalle u les had highe FP p obabili y, bu he e was
also a small peak o mid-sized ju eniles, a ound 60 cm
CCL (Fig. 3b). The adul size-class was excluded om ou
analysis due o he educed sample size (n = 5), which
could bias esul s. O e all, mos FP u les we e only mildly
a lic ed, as only ou u les (12.5%) had a umou sco e o
3, bu e en hese seemed o p esen a good body condi ion
(i.e. no emacia ed), and he umou s did no seem o be
a ec ing locomo ion o any i al unc ion (Table 2).
ChAHV5 P e alence
In o al, 76 indi iduals we e analysed o he p esence o
ChAHV5 DNA; 36 om U&U (12 o hese FP-a lic ed and
24 FP- ee), and 40 om he PNBA (15 FP a lic ed, 25 FP-
ee). A he PNBA, one umou sample was analysed om
each o he 15 FP-a lic ed u les, while a U&U we anal-
ysed one umou sample om each o nine FP-a lic ed
u les, as h ee u les we e no sampled o a oid inju y
(see me hods). O e all, a o al o 83.3% (20 ou o 24) o
he umou biopsies we e posi i e o he ChAHV5 DNA,
bu he e we e di e ences be ween si es: all o he umou s
om PNBA we e posi i e o i al de ec ion, compa ed o
app oxima ely hal o he umou s a U&U (Table 1). The
i al copy loads pe mg wi hin umou issues anged om
4.25 910
2
o 1.51 910
7
, wi h a mean o
3.99 910
6
±4.52 910
6
copies. The p esence o ChAHV5
Figu e 2. Numbe o g een u les cap u ed a o aging si es in
Unhocomo and Unhocomozinho, Guinea-Bissau (U&U), and in he
Na ional Pa k o he Banc d’A gin, Mau i ania (PNBA), dis ibu ed
by li e-s age (J: ju eniles, CCL <65 cm; SA: subadul s, 65 CCL
<83 cm; A: adul s, CCL 83 cm). CCL: cu ed ca apace leng h.
Tu les wi h ib opapilloma osis (FP) a e shown in da k g ey, u les
ee o FP lesions a e shown in ligh g ey.
Fib opapilloma osis and he Chelonid Alphahe pes i us 5

DNA was also de ec ed in 25.9% (7 ou o 27) o he
appa en ly no mal skin om FP-a lic ed u les and in
44.9% (22 ou o 49) o he no mal skin samples om
asymp oma ic u les, bu again his was highly a iable
be ween si es (Table 1). As expec ed, he numbe o i al
load (ChAHV5 DNA copies) was highe in umou samples
compa ed o samples o appa en ly no mal skin issue
(Fig. 4). The e was no s a is ically signi ican di e ence in
he C alues o umou samples be ween umou sco es
(
2
= 0.162, d = 1, p= 0.688), no be ween li e-s ages
(
2
= 1.3561, d = 2, p= 0.5076). Simila ly, we ound no
signi ican di e ences in he C alues o ‘no mal’ skin ei-
he be ween umou sco e (
2
= 1.414, d = 1, p= 0.234)
o be ween li e-s ages (
2
= 1.728, d = 2, p= 0.422). No
s a is ical associa ion was ound be ween he C ob ained
o he umou samples and he geog aphic loca ions
(W = 62, p= 0.205); howe e , he i al loads ound in he
no mal skin samples we e signi ican ly di e en be ween
Table 1. P e alence o Fib opapillomas (FP) and ChAHV5 DNA Among G een Tu les Fo aging a he Coas al Wa e s o Unhocomo
and Unhocomozinho Islands (U&U), in Guinea-Bissau, and he Banc d’A guin Na ional Pa k (PNBA), in Mau i ania, and P e alence o
ChAHV5 a Each Loca ion by Tissue Type.
Pa ame e U & U PNBA
FP p esence 12/36 (33.3%) 20/72 (28.0%)
ChAHV5 p esence 10/36 (28.0%) 32/40* (80.0%)
ChAHV5 in ’no mal’ skin o FP- ee u les 5/24 (20.8%) 17/25 (68.0%)
ChAHV5 in ’no mal’ skin o FP-a lic ed u les 1/12 (8.3%) 6/15 (40.0%)
ChAHV5 in FP umou s 5/9** (55.6%) 15/15 (100%)
*A he PNBA, biopsies o 40 ou o he 72 u les we e analysed o i al p esence (see me hods)
**A U&U h ee u les wi h umou s in he eyes we e no samples o a oid inju y (see me hods)
Figu e 3. G aphical summa y o gene alized addi i e models i ing he ela ionship be ween cu ed ca apace leng h (CCL, cm) and p esence o
ib opapilloma osis (FP) among imma u e g een u les o aginga wosi esinWes A ica:Unhocomo and Unhocomozinho, Guinea-Bissau
(U&U), and in he Na ional Pa k o he Banc d’A gin, Mau i ania (PNBA). Response a iable: p obabili y o FP. P edic o a iable: CCL.
Table 2. Numbe o Fo aging G een Tu les Pa i ioned by
S udy Si e and Li e-S age, Wi hin Each Tumou Sco e (TS).
Si e TS Li e-s age
Ju enile Subadul Adul
U&U 0 18 4 2
18 0 1
20 0 0
31 1 1
PNBA 0 35 16 1
118 0 0
21 0 0
31 0 0
U&U: Unhocomo and Unhocomozinho islands, Bijago
´s, Guinea-Bissau.
PNBA: Banc d’A guin Na ional Pa k, Mau i ania. TS0 = FP absence,
TS1 = mild FP, TS2 = mode a e FP, TS3 = se e e FP (Wo k and Balazs
1999). FP: ib opapilloma osis
J. Mon ei o e al.
si es (W = 262, p= 0.005), wi h highe C alues, co e-
sponding o lowe i al loads obse ed in samples om
U&U (Fig. 4).
Quan i a i e Polyme ase Chain Reac ion (qPCR)
and Valida ion o ChAHV5 De ec ion
The DNA concen a ion yielded om bo h no mal and
umou skin samples a ied be ween 6.9 and 51.7 ng/ll.
Fo u les whe e bo h hese samples we e collec ed, he
amoun o DNA ob ained om umou s was, in gene al,
highe han om no mal skin (mean ±SD = 38.11 ±
5.85 ng/mL and 18.34 ±19.48 ng/mL, espec i ely). The
mean DNA quan i y ob ained om no mal skin was
17.44 ng/mL, while om umou s was 38.11 ng/mL. The
s anda d cu e gene a ed had 100% ampli ica ion e iciency
(slope = 3.322) and a high co ela ion coe icien
(
2
= 0.995). The LOD o he qPCR me hod was i e copies
o i al DNA (1.8 910
-17
g) deduc ed om he s anda d
cu e.
DISCUSSION
Aside om he p esen , he e a e no s udies epo ing FP
p e alence among o aging g een u les in he egion o
Wes A ica. The p e alence o FP ound in his s udy was
highe han ha epo ed a wo g een u le o aging si es
in Cen al A ica: Loango Bay, Congo, and Co isco Bay,
Equa o ial Guinea, wi h mean FP p e alence alues o 10%
(2008–2014) and 16.98% (1998–2006), espec i ely (Gi a d
2016), and a one eeding si e in Eas A ica: he Ba en
Islands, Madagasca (Eas A ica) wi h FP a es o 9-13%
(2010–2012, Le oux 2010, Campillo 2011, 2012). Ou al-
ues we e mo e simila o he FP p e alence epo ed o
g een u les om P ı
´ncipe Island, Sa
˜o Tome
´and P incipe
(Cen al A ica), whe e 32% o ju eniles (n = 25) and 36%
o subadul s (n = 22) we e ound o ha e FP (2009,
Lou ei o and Ma os 2009). Compa ed o o he si es
wo ldwide, whe e an h opogenic impac is p esumably
highe , FP p e alence a ou s udy si es seems o be mod-
e a e, e.g. Indian Ri e Lagoon (Flo ida, USA) wi h
p e alence up o 70% (Eh ha e al. 2016), Pue o Mangla
Bay (Pue o Rico) wi h p e alence up o 75% (Pa ı
´cio
e al. 2016) and Mo e on Bay (Aus alia) wi h p e alence
up o 70% (Agui e e al. 2000). The si es whe e he u les
we e cap u ed o his s udy ha e low human p esence,
limi ed o a isanal ishe ies and ew local inhabi an s. The
PNBA is a ma ine-p o ec ed a ea wi h e y low popula ion
densi y, whe e only local Im aguen people a e allowed o
ish, and using only adi ional sailing boa s (wi h no en-
gine). The U&U islands ha e also low popula ion densi y,
concen a ed in h ee small illages. The main local ac i -
i ies a e small-scale ishe ies engaged by local illage s and
some mig an ishe s om nea by Wes A ican coun ies.
I is cu ious, he e o e, ha hese emo e a eas, wi h di -
e en habi a s and e y limi ed an h opogenic impac , s ill
hold *30% FP p e alence, posing he ques ion o wha
o he ac o s con ibu e o disease exp ession.
A U & U FP p obabili y peaked among indi iduals
wi h *50–60 cm in CCL, while a he PNBA, u les wi h
FP lesions we e smalle , bu a small peak was also obse ed
a *60 cm CCL. A bo h si es, a e *60-cm CCL FP
Figu e 4. C alues in di e en samples om o aging g een u les: aFP ( ib opapilloma) samples (26.32 ±4.26), ‘no mal’ skin ( ee o FP
lesions) om FP-a lic ed animals (34.70 ±SD) and ‘no mal’ skin om FP- ee u les (35.50 ±1.87)—s udy si es pooled; b‘no mal’ skin
samples om FP- ee g een u les om he Banc d’A guin Na ional Pa k, Mau i ania (PNBA, 34.01 ±1.75) and om Unhocomo and
Unhocomozinho islands, Guinea-Bissau (U&U, 36.21 ±3.73). Boxes indica e median, uppe and lowe qua iles, whiske s indica e highes and
lowes obse a ion. FP: ib opapilloma osis.
Fib opapilloma osis and he Chelonid Alphahe pes i us 5
p obabili y dec eased, simila o wha was ound a Loango
Bay, Congo (Gi a d 2016). This end o la ge ju eniles
and subadul s ha ing lowe p obabili y o exp essing u-
mou s is consis en o wha was ound in a long- e m
cap u e-ma k- ecap u e (CMR) s udy a a g een u le
o aging agg ega ion in Pue o Rico (Pa ı
´cio e al. 2016),
and in a long- e m s udy looking a s anded animals in
Flo ida, USA (Foley e al. 2005), al hough a leas one s udy
epo ed highe FP p e alence among la ge subadul s
(Bap is o e, 2007). Gene ally, he mean CCL o a lic ed
u les was in acco dance wi h p e ious s udies (Balazs
1991; dos San os e al. 2010; Foley e al. 2005; Pa ı
´cio e al.
2016). The lowe isk o lesions among la ge indi iduals
suppo s he hypo hesis ha g een u les may acqui e
immuni y wi h age (Pa ı
´cio e al. 2016). Howe e , despi e
a low isk o ha ing umou s being desc ibed o adul s
(Foley e . al, 2005), wo o he ou adul s cap u ed om
U&U p esen ed umou s. We cap u ed e y ew (PNBA:
n = 1; U&U: n = 3) ‘ ec ui ’ size indi iduals (i.e. u les
wi h CCL <40 cm, as pe Pa ı
´cio e al. 2014), bu we
know hey exis in hese loca ions, as we ha e ound se e al
s anded animals and old ca apaces wi hin he ‘ ec ui ’
size-class, a bo h si es. Po en ially, smalle u les managed
o escape he en anglemen ne s due o he la ge mesh sizes.
Thus, we canno in e i g een u les a i e a he o aging
si es ee o FP, o acqui e he disease upon ec ui men .
Fu u e su eys should adop a echnique o a ge his size-
class.
Mos FP-a lic ed u les (84.3%) we e only mildly
a ec ed by he disease, wi h 12.5% classi ied as hea ily a -
lic ed. The de elopmen o FP has been linked o o he co-
in ec ions and/o o immunological agili y, which may
explain why some animals de elop mo e se e e symp oms
(He bs and Klein 1995). The hea ily a lic ed indi iduals
may po en ially ha e o he inc eased isk ac o s, such as
poo e nu i ional condi ion o concomi an in ec ions/
diseases leading o a mo e se e e exp ession o he clinical
disease (Agui e and Balazs, 2000). All u les obse ed
appea ed o be in good body condi ion, ye we did no
assess blood pa ame e s, which would gi e a mo e com-
ple e pic u e o o e all heal h condi ion, po en ially
explaining he obse ed di e ences in FP exp ession
(Domiciano e al. 2019). The con inua ion o he in-wa e
CMR moni o ing will gi e mo e insigh o he pa hogenesis
o he disease, as his s udy ep esen s a snapsho in ime,
and he e may be empo al a ia ions in FP exp es-
sion/se e i y, o example, media ed by seasonali y in sea-
wa e empe a u es, which a e mo e a iable in Mau i ania,
as wa me seawa e empe a u es ha e been p oposed o be
linked wi h inc eases in umou g ow h (He bs e al. 1994,
He bs e al. 1995, Foley e al. 2004). Indeed, a his si e,
mo e g een u les a e ound s anded in he wa me
mon hs (June and July, PNBA unpublished da a), bu we
ha e no de ails on FP p esence o se e i y among hese
s anded animals.
The de ec ion o he ChAHV5 DNA in mos umou
samples is consis en wi h he ole o his i us in he
ae iology o FP (Lu e al. 2000; Quackenbush e al. 1998)
and u he demons a es ha umou s a e he bes ma ix
o i al de ec ion. Howe e , no all samples we e posi i e,
which could be due o inhibi o ac o s, he s age o he
umou , ela i e low sensi i i y o he qPCR echnique used
(Al a o-Nu
´n
˜ez and Gilbe 2014), o quali y o he ex-
ac ed DNA. A hi d o he samples om appa en ly
no mal skin o FP-a lic ed u les we e posi i e o
ChAHV5 DNA. La ency, a known abili y o he pes i uses is
an impo an poin o add ess in ChAHV5 in es iga ions.
Resea ch has shown ha ChAHV5 has co-e ol ed wi h i s
hos o a leas 8.9 millions o yea s (Pa ı
´cio e al. 2012),
de eloping he abili y, a e es ablishing la ency, o minimal
i al exp ession o a oid de ec ion by he hos immune
sys em (Al a o-Nu
´n
˜ez e al. 2016). Du ing he ac i e i -
aemic phase, i al DNA may be de ec ed h oughou he
body, while in ch onic in ec ions i al ac i i y may be
limi ed o umou s (Al a o-Nu
´n
˜ez e al. 2014). This may
lead o an unequal i al dis ibu ion ac oss issue ypes o
body egions wi hin he same indi idual and explain why,
in mos cases, only he umou s had high i al loads. No-
ably, he e was no co ela ion be ween he umou sco e
and he C alue in umou samples. The numbe o i al
copies could be mo e likely associa ed wi h umou
de elopmen s age, han o he se e i y o FP. Fo example,
some o he umou s sampled om animals mildly a lic ed
(TS = 1) could be unde apid g ow h, wi h p oli e a ion
o hos cells in ec ed by ChAHV5, hus inc easing he
numbe o i al copies (Ye sko e al. 2020). Because ou
s udy ep esen s a snapsho in ime, we do no know,
howe e , which u les we e de eloping umou s, which
ones we e in mo e s able diseased condi ions o e en
unde going umou eg ession.
This s udy also e ealed a ele an p opo ion o
asymp oma ic indi iduals (PNBA: 68.0%, U&U: 20.8%)
in ec ed wi h ChAHV5, simila o wha has been ound in
o he s udies (Dua e e al. 2012, Page-Kajian e al. 2012,
Al a o-Nu
´n
˜ez e al. 2014). This phenomenon was highe in
u les om he PNBA, Mau i ania. The C alues o he
J. Mon ei o e al.
appa en ly no mal skin samples we e signi ican ly lowe in
he u les om Mau i ania, indica ing highe i al loads,
compa ed o Guinea-Bissau u les. This di e ence could
indica e a a ia ion in esis ance o in ec ion be ween he
wo o aging agg ega ions. Al e na i ely, FP ou b eaks and
p e alence may be mo e hea ily in luenced by en i on-
men al ac o s and/o hos immuni y han hey a e by i al
ci cula ion in he popula ions (He bs e al. 2008). The ac
ha FP- ee u les a e ca ie s o he i us shows ha hey
a e asymp oma ic ese oi s, which may be unde going
ei he ea ly o la en in ec ion, o al e na i ely, umou
eg ession. In he cases o la ency o ea ly in ec ion, hese
u les may de elop clinical disease in he u u e (Al a o-
Nu
´n
˜ez e al. 2014; Quackenbush e al. 2001). The high
p e alence o ChAHV5 in asymp oma ic animals sugges s
ha FP is enzoo ic a ou s udy si es (Page-Ka jian e al.
2020). E en hough he e idence o ese oi s does no
co ela e o he p e alence o FP, i is impo an e idence o
help unde s and he pa hogenesis o he disease and sup-
po s i s panzoo ic s a us (He bs and Klein 1995; He bs
1994).
Final Conside a ions
In ec ious disease ou b eaks in ma ine ecosys ems ha e
been inc easing, eac i a ing p e iously la en diseases and
in oducing new, po en ially a al ones (Cunningham e al.
2017). The implica ions o u u e clima e change on FP a e
unce ain, bu enhanced wa e empe a u es as a esul o
global wa ming, and unbalanced ecosys ems, ha e he
po en ial o g ea ly inc ease he p e alence and i ulence o
wildli e diseases (Agui e and Lu z 2004; Bu ge e al. 2014).
Guinea-Bissau, in Wes A ica, is home o one o he la ges
g een u le popula ions globally (Pa ı
´cio e al. 2019), wi h
s ong connec i i y o o aging g ounds in Mau i ania
(Godley e al. 2010). Al hough bo h he Guinea-Bissau and
he Mau i ania s udy loca ions seem o ha e e y limi ed
an h opogenic s esso s, hey had ele an FP p e alence
alues. Thus, o be e unde s and he FP dynamics o Wes
A ica g een u les, long- e m moni o ing is needed, as FP
p e alence, and possibly i s i ulence, may luc ua e
h ough ime. This would inc ease sample size and allow
he ecap u e o u les, key o assess disease p og ession o
eg ession. Thus, we ecommend iden i ying u les wi h
lippe ags, commonly used in ma ine u le moni o ing
p og ams (NOAA, 2008). This s udy epo s o he i s
ime he p esence o in ec ion by he ChAHV5 in Wes
A ica g een u les, and i es ablishes a baseline o he
p e alence o FP and o ChAHV5 in wo key a eas o his
species.
ACKNOWLEDGEMENTS
Resea ch pe mi s and logis ic suppo in Mau i ania we e
p o ided by he Pa c Na ional du Banc d’A guin (PNBA),
and in Guinea-Bissau by he Ins i u o da Biodi e sidade e das
A
´ eas P o egidas, D . Al edo Sima
˜o da Sil a (IBAP). We
would like o acknowledge P o . Paulo Ca y (MARE–ISPA),
o his key ole in he de elopmen o sea u le esea ch
p ojec s in bo h coun ies. Fieldwo k was conduc ed wi h he
collabo a ion o membe s om he local communi ies, o
whom we a e deeply hank ul. This s udy was unded by he
p ojec ‘‘Consolida ion o sea u le conse a ion a he
Bijago
´s A chipelago, Guinea-Bissau’’ awa ded o IBAP by he
MAVA ounda ion, and by he p ojec s ‘‘To ue d’A guin’’
and ‘‘Habi a To ue’’, awa ded o he PNBA by MAVA and
by he PRCM, espec i ely; molecula analyses we e sup-
po ed by he Cen o de In es igac¸a
˜o In e disciplina em
Sanidade Animal (CIISA), Faculdade de Medicina Ve e -
ina
´ ia, Uni e sidade de Lisboa (UIDP/CVT/00276/2020—
FCT); and by a g an om Fundac¸a
˜o pa a a Cie
ˆncia e a
Tecnologia, Po ugal (UIDB/04292/2020 and UIDP/04292/
2020) awa ded o MARE. Labo a o y wo k was conduc ed a
he Vi ology Labo a o y o Ins i u o Nacional de In es-
igac¸a
˜o Ag a
´ ia e Ve e ina
´ ia (INIAV). This manusc ip
bene i ed om he commen s o wo anonymous e iewe s.
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Fib opapilloma osis and he Chelonid Alphahe pes i us 5