RESEARCH Open Access
T ansmission o Calicopho on daubneyi and
Fasciola hepa ica in Galicia (Spain):
Tempo al ollow-up in he in e media e
and de ini i e hos s
Ja ie Iglesias-Piñei o
2
, Ma a González-Wa le a
1
, José An onio Cas o-He mida
1
, Ma ía Có doba
3
,
Camino González-Lanza
4
, Yolanda Manga-González
4
and Me cedes Mezo
1*
Abs ac
Backg ound: Pa amphis omosis caused by Calicopho on daubneyi and asciolosis caused by Fasciola hepa ica a e
common pa asi ic diseases o li es ock animals. T ansmission o he diseases depends on he p esence o in e media e
hos s, i.e. eshwa e gas opods such as lymnaeids. We ca ied ou a 2-yea -long s udy o he dynamics o he snail
popula ion ac ing as he in e media e hos o hese pa asi es, conside ing he popula ion s uc u e in e ms o size/
age and in ec ion s a us. In addi ion, we de e mined he kine ics o ema ode egg exc e ion in g azing cows.
Gene alized Addi i e Models (GAMs) we e used o analyze he associa ions be ween di e en esponse a iables and
snail size, sampling mon h and wea he - ela ed a iables.
Resul s: O he molluscan species examined, Galba unca ula,Radix pe eg a,Anisus (Anisus)leucos oma and Pisidium
case anum (n= 2802), only G. unca ula was in ec ed wi h C. daubneyi o F. hepa ica, a p e alence a es o 8.2% and 4.
4% espec i ely. The p obabili y o in ec ion wi h C. daubneyi o F. hepa ica was linea ly ela ed o snail size, al hough in
di e en ways (nega i e o C. daubneyi and posi i e o F. hepa ica). The o al snail popula ion inc eased in win e ,
when specimens o all size classes we e ound. In ec ed snails we e mo e abundan du ing sp ing-au umn. Ma u e
ce ca iae o bo h pa asi es we e ound in mos seasons. In he s a is ical models, he sampling mon h accoun ed o a
high pe cen age (71.9–78.2%) o he obse ed a iabili y in snail abundance. The inclusion o clima ic a iables in he
models mode a ely inc eased he pe cen age o de iance explained (77.7–91.9%). Exc e ion o C. daubneyi eggs in cow
aeces was always highe han ha o F. hepa ica eggs.
Conclusions: Pa icula ca e should be aken o p e en pas u es and he su ounding en i onmen being con amina ed
wi h pa asi e eggs du ing win e -sp ing, when he numbe o snails suscep ible o mi acidial in ec ions is
maximal. This is he e o e he op imal ime o ea ing g azing animals. Ne e heless, con ol o ema odosis
based only on chemo he apy is di icul in an a ea such as he s udy a ea, whe e en i onmen al ac o s a ou
he egula appea ance o snail popula ions ha bou ing ma u e ce ca iae.
Keywo ds: Calicopho on daubneyi,Fasciola hepa ica, Snail hos s, Galba unca ula, Seasonal ends, GAM
* Co espondence: [email p o ec ed]s
1
Labo a o io de Pa asi ología, Cen o de In es igaciones Ag a ias de
Mabegondo, Ins i u o Galego de Calidade Alimen a ia-Xun a de Galicia,
Ca e e a AC-542 de Be anzos a Mesón do Ven o, Km 7, 15318 Abegondo (A
Co uña), Spain
Full lis o au ho in o ma ion is a ailable a he end o he a icle
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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
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Iglesias-Piñei o e al. Pa asi es & Vec o s (2016) 9:610
DOI 10.1186/s13071-016-1892-8
Backg ound
Pa amphis omosis caused by Calicopho on daubneyi
(Dinnik, 1962) and asciolosis caused by Fasciola hepa ica
Linnaeus, 1758 a e among he mos common pa asi ic
in ec ions o li es ock animals. These in ec ions appea
concu en ly on many a ms. Fasciolosis has been ecog-
nized o many yea s as a se e e pa hological en i y wi h
de imen al e ec s on animal wel a e and p oduc ion
pe o mance. On he con a y, pa amphis omosis has
adi ionally been conside ed o no medical signi icance,
a leas when li es ock animals a e main ained in good
nu i ional and heal h s a us, as is usual in Eu ope. How-
e e , his si ua ion seems o be changing, since cases o
se ious illness caused by pa amphis omes ha e been
desc ibed in F ance [1] and he UK [2–4]. Inc eased
p e alence o bo h in ec ions has also been epo ed in
a ious Eu opean coun ies [5–8]. The highe a e o
p e alence may e lec an inc eased isk o in ec ion, pos-
sibly igge ed by changes in clima ic condi ions a ou ing
highe ansmission a es [9] and also a ailu e o he
con ol measu es applied, which a e o en limi ed o ea -
men o li es ock wi h an helmin ics. A sus ained educ ion
in in ec ion a es can only be achie ed i ea ly ein ec ion o
ea ed animals is p e en ed. This can only be done a e
iden i ica ion o he spa io- empo al luc ua ions in he isk
o ansmission o in ec ion.
T ansmission o he ema odes C. daubneyi and F.
hepa ica is associa ed wi h he p esence o eshwa e
gas opods ha ac as he in e media e hos s (IHs)
wi hin which he pa asi es mul iply. The ampli ying e i-
ciency o he in a-molluscan cycle de e mines he num-
be o me ace ca iae p oduced and he associa ed isk o
in ec ion in g azing animals. The pa asi es do no mul iply
wi hin he de ini i e hos s, and he magni ude o he bu -
den in li es ock, which in u n de e mines he mo bidi y
o in ec ion, depends di ec ly on he numbe o me ace -
ca iae inges ed. In o ma ion abou he gas opod popula-
ions ac ing as IHs is equi ed o a good unde s anding o
he epidemiology o pa amphis omosis and asciolosis.
Such in o ma ion would also help minimize ansmission
h ough ac ions aimed a dis up ing he li e-cycle o he
pa asi es.
Recen epo s on he spa ial dis ibu ion o pa amphis-
omosis and asciolosis explain he geog aphical he e o-
genei y in he p obabili y o in ec ion o li es ock as a
consequence o en i onmen al ac o s d i ing he li e-
cycle o bo h IHs and ema odes [10–13]. Howe e , as
en i onmen al (bo h abio ic and bio ic) ac o s a y
signi ican ly h oughou he yea , he isk o in ec ion also
a ies, and he empo a y luc ua ions in his pa ame e
mus he e o e also be aken in o accoun in epidemio-
logical s udies.
Many s udies ha e con i med ha Galba (Galba) unca-
ula (Mülle , 1774) is he main ansmi e o F. hepa ica
wi hin Eu ope [14–16], al hough some ecen da a show
ha o he lymnaeid snail species can pa icipa e in he
li e-cycle [17, 18]. Al hough he snail G. unca ula can
ac as an IH o C. daubneyi [18–20], he in ol emen o
o he gas opod species in he li e-cycle o his pa asi e
has sca cely been s udied [21]. In iew o he key ole o
lymnaeid snails in he li e-cycle o ema odes o medical
and e e ina y impo ance, p esen knowledge o he dy-
namics o snail popula ions and pa asi e-hos in e ac ions
is insu icien .
In his s udy, we add essed some key aspec s o he
ansmission o C. daubneyi and F. hepa ica in a egion
whe e he in ec ions caused by bo h ema odes a e en-
demic. We i s assessed whe he he di e en gas opod
species co-exis ing in he habi a ac as IHs o C. daub-
neyi and F. hepa ica. We hen s udied he dynamics o
he snail popula ion (G. unca ula) ac ing as IH o
bo h pa asi es, o e a pe iod o wo yea s, aking in o ac-
coun he popula ion s uc u e in e ms o size/age and
in ec ion s a us. We also de e mined he kine ics o
ema ode egg exc e ion in cows ha had access o he
s eam whe e he snails we e sampled. Finally, we used
Gene alized Addi i e Models (GAMs) o analyze he
associa ions be ween di e en ac o s (snail size, sam-
pling mon h and wea he - ela ed a iables) and he
esponse a iables s udied.
Me hods
Sampling
The s udy was ca ied ou on a dai y ca le a m (43°4′
N, 8°3′W) in Galicia (no h-wes Spain). The clima e in
he egion is classi ied as empe a e ma i ime, wi h an
a e age annual empe a u e o 11.6 °C and a e age
annual cumula i e ain all o 1065 mm [22]. In ec ions
caused by bo h F. hepa ica and C. daubneyi had p e i-
ously been diagnosed on he a m ia nec opsy o some
cows in he slaugh e house [13]. On ou i s isi o he
a m, we we e shown he ields whe e he cows we e
pas u ed. We obse ed some snails (possible in e medi-
a e hos s o bo h pa asi es) in a na ow (<1 m), shallow
(<50 cm), slow- lowing s eam whe e he animals had
ee access o d inking. The cows g azed all yea ound
and we e ne e ea ed wi h lukicides. The a m was
selec ed o s udy because o hese condi ions.
Th oughou wo consecu i e yea s (be ween July 2007
and June 2009), we made mon hly isi s o he a m o
simul aneous sampling o cows (de ini i e hos s) and
snails (po en ial in e media e hos s). Du ing each isi ,
all cows (n= 34) we e sampled indi idually and aeces
we e emo ed di ec ly om he ec um. Sys ema ic sam-
pling o snails was always ca ied ou by he same h ee
ained esea che s, a he same ime o day (be ween
11:00 h and 13:00 h) along he same s e ch o he
s eam (150 m long), including he s eam bed and s ips
Iglesias-Piñei o e al. Pa asi es & Vec o s (2016) 9:610 Page 2 o 14
o pas u e (50 cm wide) on bo h sides o he s eam. In-
o ma ion abou he p e alence o na u al in ec ions
caused by F. hepa ica in he in e media e hos s in Spain
is sca ce, and he e a e no da a a ailable abou na u al
in ec ions caused by C. daubneyi. Thus, assuming a
p e alence a e o in ec ion o 50% (e o ma gin 10%
and con idence in e al 95%), he minimum size o
sample equi ed was es ima ed o be 95 snails. Each
collec o sea ched 10 si es (abou 5 m apa om each
o he ) o 5 min pe si e and used so o ceps o pick
up all gas opods seen by he naked eye. When ewe
han 95 molluscs we e collec ed, he sampling was e-
pea ed. Wi h a iew o alida ing he esul s ob ained on
his a m, a la ge sample o eshwa e gas opods (n=
1097) was andomly collec ed on o he ca le a ms wi h
con i med cases o pa amphis omosis and asciolosis.
Pa asi ological echniques
The aecal samples we e analyzed using a quan i a i e
sedimen a ion echnique. B ie ly, 10 g o aeces was added
o abou 100 ml o ap wa e and mixed ho oughly un il
ob aining a homogeneous suspension. Glass beads we e
added o he mix u e o help b eak up he ma e ial. The
esul ing suspension was hen passed h ough a 150 μm
sie e. The il e ed ma e ial was ans e ed o a 500 ml
conical lask o sedimen a ion (3 imes each o 20 min).
The inal sedimen was ans e ed o a ube and wa e
was added un il a olume o 10 ml. A e homogeniza ion,
a 1 ml aliquo (con aining 1 g o aeces) was emo ed and
examined unde he mic oscope (100×) o coun he Cali-
copho on and Fasciola eggs. The esul s we e exp essed as
numbe o eggs (o each ype) pe g am o aeces (epg).
The molluscs collec ed we e classi ied on he basis o
ex e nal mo phological cha ac e is ics. Some ep esen a-
i e specimens o each species we e p ese ed in 70%
e hanol o u he accu a e axonomic classi ica ion.
Helmin hological examina ions we e ca ied ou in i o.
Each snail was measu ed (shell heigh om he apex o
an e io ma gin) and he body was hen ex ac ed and dis-
sec ed unde a s e eomic oscope o sea ch o signs o
ema ode in ec ion. When ema ode la ae we e de-
ec ed, samples we e subsequen ly examined unde he
mic oscope, a highe magni ica ion, in o de o con i m
he species and de e mine he deg ee o de elopmen . The
ollowing 7 la al s ages we e eco ded: (1) spo ocys s
wi h ge minal masses; (2) spo ocys s con aining ediae; (3)
imma u e ediae o wi h ge minal masses; (4) ediae wi h
daugh e ediae; (5) ediae wi h imma u e ce ca iae; (6)
ediae wi h ma u e ce ca iae; and (7) ce ca iae jus shed
by he snails. Ne e heless, in o de o acili a e in e p e -
a ion o he esul s, 3 ca ego ies o in ec ion s a us we e
es ablished acco ding o he la al s ages obse ed: (i) spo-
ocys s and/o ediae wi h ge minal masses and/o daugh-
e ediae (s ages 1–4); (ii) ediae wi h imma u e ce ca iae
(s age 5); and (iii) ediae wi h ma u e ce ca iae and/o ee
ce ca iae (s ages 6 and 7). The e a e no desc ip ions o he
la al mo phology o C. daubneyi a ailable in he li e a-
u e. We he e o e iden i ied he la al s ages on he basis
o compa ison wi h he di e en s ages we ha e p e iously
obse ed in expe imen al in ec ions wi h mi acidia o his
pa asi e (da a no shown).
When spo ocys -like la al s ages we e obse ed by mi-
c oscopy, samples we e conse ed in liquid ni ogen o
subsequen molecula de e mina ion o he species using
he echnique de eloped by Ma ínez-Ibeas e al. [20].
Me eo ological da a
Me eo ological da a we e eco ded a he wea he
s a ion nea es o he a m unde s udy, i.e. he Olas
wea he s a ion, which belongs o he o icial me-
eo ological se ice o he Galician Go e nmen [22].
The s a ion is loca ed a a dis ance o 15 km om
he a m and a a simila al i ude. The ollowing
me eo ological a iables we e conside ed in he
s udy: daily mean ai empe a u e (°C), daily mean
ela i e humidi y (%), daily global sola adia ion
(10 kJ/m
2
day) and daily ain all (mm). Fo hese a -
iables, he a e age o accumula ed ( ain all) alues
co esponding o he in e -sampling pe iods we e
used. The in e -sampling pe iods included he days
be ween any sampling da e and he p e ious one.
Fo he i s sampling da e, he da a co esponding
o he p e ious 30 days we e conside ed.
S a is ical analysis
Gene alized Addi i e Models (GAMs) [23, 24] enable
assessmen o associa ions wi h independen a iables
whose e ec s a e no expec ed o emain cons an along
hei en i e ange o alues. We he e o e used GAMs o
model he associa ion be ween di e en ac o s (snail
size, sampling mon h and clima ic a iables) and he ol-
lowing esponse a iables: (i) p obabili y o in ec ion
(p esence o C. daubneyi o F. hepa ica); (ii) numbe o
indi iduals collec ed (conside ed as a p oxy o snail
popula ion size); and (iii) numbe o indi iduals in ec ed
wi h ei he C. daubneyi o F. hepa ica.
In he p obabili y models, we used a binomial dis ibu ion
wi h he complemen a y log-log link unc ion (clog-
log), η
i
=log (-log (1-(π
i
)) whe e η
i
= link, and π
i
=
p obabili y o p esence, which is mo e app op ia e o
bina y esponses wi h la ge numbe s o ze os. Fo he
models based on coun s (numbe s o snails), we used
a Poisson dis ibu ion wi h he loga i hmic link. Fo
he models o he numbe s o snails in ec ed wi h
each pa asi e, we included an o se o ake in o
accoun he numbe o snails examined on each
sampling da e.
Iglesias-Piñei o e al. Pa asi es & Vec o s (2016) 9:610 Page 3 o 14
In addi ion, we used GAMs wi h a gamma dis ibu ion
and he iden i y link o explo e he ela ionships
be ween he sampling mon h and he mean numbe o
ema ode eggs pe g am o aeces exc e ed by cows.
The smoo hness selec ion c i e ia used (i.e. he ule o
he deg ee o lexibili y allowed) o he GAM models was
he Unbiased Risk Es ima o C i e ia (UBRE) [25]. Pena-
lised splines [26] we e used as smoo he s.
All analyses we e implemen ed using he mgc package
[24] in R so wa e [27]. The ela ionships be ween he
same explana o y a iable (e.g. sampling mon h) and
di e en esponse a iables a e included in he same
igu e (al hough analyzed by di e en models) o enable
di ec compa ison. Fo he sake o cla i y, he con idence
in e als a e no shown. The igu es co esponding o
each indi idual model and con idence in e als a e
supplied as supplemen a y ma e ial (Addi ional ile 1:
Figu es S1-S6).
Resul s
T ema ode in ec ions in in e media e hos s
A o al o 2802 specimens o eshwa e molluscs
belonging o he species G. unca ula (n= 1141), Radix
pe eg a (n= 1084), Anisus (Anisus)leucos oma (n= 476)
and Pisidium case anum (n= 101) we e collec ed om
he s udied a m. Only he lymnaeids we e ound o be
in ec ed wi h ema ode la ae. Speci ically, la ae o he
amily Plagio chiidae we e obse ed in a small pe cen -
age o R. pe eg a snails (1.9%), while coloniza ion by a
g ea e di e si y o ema odes was obse ed in G. un-
ca ula, i.e. No oco ylidae (0.5%), Plagio chiidae (3.4%), F.
hepa ica (4.4%) and C. daubneyi (8.2%). Mixed in ec-
ions we e ne e obse ed. Only da a on C. daubneyi
and F. hepa ica (which a e o e e ina y ele ance and
he a ge o his s udy) and hei in e media e hos , G.
unca ula, a e shown.
To de e mine he e ec o he snail size on he p obabil-
i y o in ec ion wi h C. daubneyi and F. hepa ica,sizeclass
dis ibu ions o he o al popula ion o snails and only in-
ec ed snails we e es ablished (Fig. 1). In he o al popula-
ion, mos indi iduals belonged o he 3.0–3.9 mm
(33.1%) and 4.0–4.9 mm classes (30.9%) (Fig. 1a). The dis-
ibu ion was almos iden ical in snails in ec ed wi h F.
hepa ica, wi h p opo ions o espec i ely 36.0 and 30.0%
in he co esponding size classes (Fig. 1b). Howe e , a
ma ked skew owa ds he lowe ange was obse ed in he
snails in ec ed wi h C. daubneyi, so ha 52.7% (49/93)
we e included wi hin he 3.0–3.9 mm class.
Mon hly da a o G. unca ula abundance we e g ouped,
o clea e p esen a ion o esul s, wi hin a seasonal
amewo k in which win e was conside ed o consis o
he mon hs Janua y, Feb ua y and Ma ch, sp ing o com-
p ise Ap il, May and June, summe o include July, Augus
and Sep embe , and au umn o comp ise Oc obe ,
No embe and Decembe . The numbe o snails was
ex emely low a he beginning o he s udy (summe -au-
umn o 2007) and hen inc eased sha ply (Fig. 2a). In all
yea s, he o al snail popula ion inc eased clea ly in win e ,
when specimens belonging o all size classes we e ound
(Tables 1 and 2), and hen dec eased, wi h he comple e
disappea ance o he la ges snails (≥6 mm) du ing sum-
me -au umn. A di e en seasonal pa e n was seen when
we ocusedonlyonin ec edsnails,whichwe emo eabun-
dan du ing sp ing-au umn o 2008, jus when he o al
popula ion dec eased. This seasonal a ia ion was mo e
ma ked in he case o C. daubneyi in ec ion (Fig. 2b).
De ailed mic oscopic examina ion o he in ec ed snails
enabled us o de e mine he di e en s ages o la al de-
elopmen al (see Me hods and Fig. 3). Tables 1 and 2
summa ize he in ec ion s a us o he snails in ela ion o
hei size and sampling season. All size classes o snails,
excep he > 7 mm class, ha bou ed la al s ages anging
Fig. 1 aSize dis ibu ion o he snail popula ion. bEnla ged g aph showing he numbe o in ec ed indi iduals in each size class. To al numbe
o snail collec ed (whi e ba ), numbe o snail ha bou ing C. daubneyi (ligh g ey ba ), numbe o snails ha bou ing F. hepa ica (da k g ey ba )
Iglesias-Piñei o e al. Pa asi es & Vec o s (2016) 9:610 Page 4 o 14
om ba ely e ol ed (indica i e o ecen o a es ed in-
ec ions) o ully de eloped. Among he di e en size
classes, he p opo ion o in ec ed snails ha bou ing
ma u e ce ca iae a ied widely [ anges o C. daubneyi:
8.7% (2/23) –33.3% (2/6); anges o F. hepa ica:20.0%
(3/15) –75.0% (3/4)]. Howe e , he absolu e numbe o
snails wi h ma u e ce ca iae wi hin each class was simila
( anges: 1–5 o C. daubneyi;3–5 o F. hepa ica). All size
classes should ha e a simila capaci y o con amina e pas-
u es. Conside ing he season, some di e ences we e ob-
se ed be ween bo h pa asi es, especially a e he win e
o 2008 when he snail popula ion inc eased signi ican ly.
Fo C. daubneyi (Table 1), he h ee ca ego ies o in ec ion
s a us appea ed con inuously om sp ing 2008, while o
F. hepa ica (Table 2), some de elopmen al s ages we e ab-
sen in some seasons. Speci ically, ma u e ce ca iae we e
Fig. 2 aTo al numbe o G. unca ula collec ed pe season. bEnla ged g aph showing he numbe o pa asi ized snails. To al numbe o snails
collec ed (whi e ba ), numbe o snails ha bou ing C. daubneyi (ligh g ey ba ), numbe o snails ha bou ing F. hepa ica (da k g ey ba ).
Abb e ia ions: A, Au umn; S, Summe ; Sp, Sp ing; W, Win e
Table 1 Numbe s o Galba unca ula ha bou ing Calicopho on daubneyi acco ding o season and snail size
No. o snails in ec ed/ No. o snails collec ed
No. o snails ha bou ing di e en la al s ages o he pa asi e
Yea Season < 3 mm 3–3.9 mm 4–4.9 mm 5–5.9 mm 6–6.9 mm ≥7 mm To al
2007 Summe 0/1 1/9
1
c
1/6
1
c
1/3
1
c
3/19
3
c
Au umn 0/3 0/3
2008 Win e 1/13
1
b
0/33 4/67
2
a
2
b
3/75
3
a
1/41
1
b
0/5 9/234
5
a
4
b
Sp ing 5/11
5
b
17/39
4
a
13
b
4/23
4
b
1/12
1
b
3/24
1
a
1
b
1
c
1/12
1
b
31/121
5
a
25
b
1
c
Summe 1/8
1
a
10/53
7
a
2
b
1
c
2/23
2
a
0/3 13/87
10
a
2
b
1
c
Au umn 2/11
1
a
1
c
13/66
3
a
8
b
2
c
5/52
5
b
0/2 20/131
4
a
13
b
3
c
2009 Win e 0/46 3/105
3
a
3/123
2
a
1
b
1/75
1
c
0/23 0/7 7/379
5
a
1
b
1
c
Sp ing 0/21 5/70
3
a
1
b
1
c
4/58
2
a
1
b
1
c
0/14 1/4
1
a
10/167
6
a
2
b
2
c
TOTAL 9/111
2
a
6
b
1
c
49/378
20
a
24
b
5
c
23/352
8
a
13
b
2
c
6/184
3
a
1
b
2
c
5/92
2
a
2
b
1
c
1/24
1
b
93/1141
35
a
47
b
11
c
a
Spo ocys s and/o ediae wi h ge minal masses and/o daugh e ediae
b
Rediae wi h imma u e ce ca iae
c
Rediae wi h ma u e ce ca iae and/o ee ce ca iae
Iglesias-Piñei o e al. Pa asi es & Vec o s (2016) 9:610 Page 5 o 14
no ound in he summe o 2008, and ba ely e ol ed la -
al s ages we e no de ec ed in he au umn o 2008 o
sp ing o 2009.
Pa ency o C. daubneyi and F. hepa ica in ec ions in
de ini i e hos s
The seasonal a ia ion in exc e ion o eggs o bo h lukes
ia cow aeces, obse ed du ing he s udy, is shown in
Fig. 4. All cows we e posi i e o C. daubneyi eggs in all
seasons, while some a ia ions we e obse ed in he
pa ency o F. hepa ica in ec ion, p obably because he
numbe o eggs was close o he de ec ion limi o he
cop ological echnique on some sampling occasions.
The F. hepa ica egg coun s we e always e y low, wi h
mean alues anging om 4.2 ± 1.3 (± s anda d de i-
a ion, SD) epg, in sp ing 2008, o 9.5 ± 3.8 epg, in
au umn o he same yea . The C. daubneyi egg coun s
we e highe , wi h mean alues anging om 32.6 ± 7.8,
in summe 2007, o 191.9 ± 44.2, in au umn 2008. Mixed
in ec ions by bo h lukes we e de ec ed in all animals in
summe -au umn 2007 and in au umn 2008.
Modelling C. daubneyi and F. hepa ica in ec ions in
in e media e and de ini i e hos s
The esul s o GAMs o he p obabili y o in ec ion o
G. unca ula in ela ion o he snail size and collec ion
mon h a e shown in Table 3. The p obabili y o in ec ion
wi h C. daubneyi la ae was signi ican ly associa ed wi h
bo h a iables, while he e ec o he size was only ma -
ginally signi ican (P= 0.058) o in ec ion wi h F.
hepa ica. The p esence o la ae o bo h ema ode spe-
cies in he snails was linea ly ela ed o snail size, bu
wi h opposing ends (Fig. 5a). Thus, he p obabili y o
in ec ion wi h C. daubneyi dec eased and he p obabili y
o in ec ion wi h F. hepa ica inc eased as snail size
inc eased. Rega ding he empo al a ia ion in he p ob-
abili y o in ec ion o e he s udy pe iod, simila pa e ns
we e obse ed o bo h ema odes (Fig. 5b). Thus, he
p obabili y o in ec ion inc eased du ing sp ing-summe ,
eaching alues abo e 0.8, and hen dec eased in au umn o
minimal alues o < 0.4 in win e . In bo h models, he pe -
cen age o explained de iance was 19.0% o C. daubneyi
and 12.8% o F. hepa ica.
The seasonal changes in he snail popula ion will de e -
mine he pe iod du ing which ema ode li e-cycles can
con inue. This will, in u n, de e mine he seasonal abun-
dance o in ec ed snails and ul ima ely he isk o in ec ion
o he de ini i e hos h oughou he yea . GAM analysis
was also used o model he abundance o snails ( o al
popula ion and numbe o specimens in ec ed wi h C.
daubneyi and F. hepa ica) in ela ion o ime o yea . The
sampling mon h p o ed o be a highly signi ican explana-
o y a iable o snail abundance, accoun ing o a high
pe cen age (71.9–78.2%) o he obse ed a iabili y in he
h ee popula ions s udied (Table 4).
The obse ed seasonali y in he abundance o snails
(Fig. 6) led us o explo e he e ec o he a ailable
clima ic a iables, by including hem in he models
(which o iginally only included ime as an independen
a iable). Because o concu i y in he clima ic da a,
Table 2 Numbe s o Galba unca ula ha bou ing Fasciola hepa ica acco ding o season and snail size
No. o snails in ec ed/ No. o snails collec ed
No. o snails ha bou ing di e en la al s ages o he pa asi e
Yea Season < 3 mm 3–3.9 mm 4–4.9 mm 5–5.9 mm 6–6.9 mm ≥7 mm To al
2007 Summe 0/1 3/9
2
b
1
c
1/6
1
b
2/3
2
c
6/19
3
b
3
c
Au umn 0/3 0/3
2008 Win e 1/13
1
a
0/33 1/67
1
a
0/75 3/41
1
b
2
c
0/5 5/234
2
a
1
b
2
c
Sp ing 2/11
2
c
3/39
1
a
2
b
1/23
1
b
2/12
2
c
1/24
1
c
0/12 9/121
1
a
3
b
5
c
Summe 0/8 7/53
6
a
1
b
5/23
4
a
1
b
0/3 12/87
10
a
2
b
Au umn 1/11
1
c
3/66
1
b
2
c
4/52
3
b
1
c
0/2 8/131
4
b
4
c
2009 Win e 0/43 2/105
1
b
1
c
2/123
1
a
1
c
2/75
1
b
1
c
2/23
1
a
1
c
0/7 8/379
2
a
2
b
4
c
Sp ing 0/21 0/70 1/58
1
c
1/14
1
b
0/4 2/167
1
b
1
c
To al 4/111
1
a
3
c
18/378
7
a
7
b
4
c
15/352
6
a
6
b
3
c
7/184
2
b
5
c
6/92
1
a
1
b
4
c
0/24 50/1141
15
a
16
b
19
c
a
Spo ocys s and/o ediae wi h ge minal masses and/o daugh e ediae
b
Rediae wi h imma u e ce ca iae
c
Rediae wi h ma u e ce ca iae and/o ee ce ca iae
Iglesias-Piñei o e al. Pa asi es & Vec o s (2016) 9:610 Page 6 o 14
each clima ic a iable was included sepa a ely in he
models. The o al snail numbe s we e signi ican ly
ela ed o all clima ic a iables, and he abundance o
snails in ec ed wi h each o he ema ode species was
also signi ican ly associa ed wi h all clima ic a iables,
excep accumula ed ain all (Table 5). Mo eo e , he in-
clusion o he clima ic a iables in he GAMs inc eased
he pe cen age o explained de iance in all models, ex-
cep in he GAMS o he numbe o C. daubneyi-in-
ec ed snails, which included he mean daily global sola
adia ion and he accumula ed ain all. Conside ing he
pe cen age o explained de iance as a i ing c i e ion,
he bes i s we e p o ided by he ollowing models: (i)
o he o al popula ion o snails, he model including
he global sola adia ion (explained de iance = 91.9%;
model 3); (ii) o he numbe o C. daubneyi-in ec ed
snails, he model including mean empe a u e (explained
de iance = 77.7%; model 1); and (iii) o he numbe o
F. hepa ica-in ec ed snails, he model i ed wi h he
mean ela i e humidi y (explained de iance = 83.2%;
model 2). The indi idual e ec s o he clima ic a iables
on he snail popula ions a e shown in Fig. 7. Those asso-
cia ed wi h empe a u e, ela i e humidi y and sola
adia ion dese e special a en ion due o he obse ed
signi ican e ec s on he h ee snail popula ions s udied.
The numbe o snails in ec ed wi h C. daubneyi was
Fig. 3 La al s ages o C. daubneyi and F. hepa ica.aSpo ocys o C. daubneyi.bSpo ocys o F. hepa ica con aining edia. cRedia o C. daubneyi
wi h de ail showing he an e io po ion (inse ). dRediae o F. hepa ica (di e en gene a ions wi hin he same snail). eMa u e ce ca ia o C.
daubneyi. Ma u e ce ca ia o F. hepa ica.gMe ace ca ia o C. daubneyi. hMe ace ca ia o F. hepa ica. La al s ages in aand bwe e con i med by
molecula echniques acco ding o Ma ínez-Ibeas e al. [20]. La al s ages in c-hwe e classi ied mo phologically by mic oscopic echniques.
Ma u e ce ca iae eleased du ing dissec ion o in ec ed snails encys ed on he slides o o m he me ace ca iae shown in g-h. All pho og aphs a e
o iginal wo k by Yolanda Manga-González
Iglesias-Piñei o e al. Pa asi es & Vec o s (2016) 9:610 Page 7 o 14
nega i ely and linea ly ela ed o empe a u e. By con-
as , he numbe o snails in ec ed wi h F. hepa ica and
he o al snail popula ion we e posi i ely ela ed o em-
pe a u es anging om 10–16 °C and om 8–14 °C,
espec i ely (Fig. 7a). The numbe o snails in ec ed wi h
each ema ode species was posi i ely associa ed wi h
he ela i e humidi y, whe eas he o al popula ion was
nega i ely ela ed o ela i e humidi y, mainly a
alues exceeding 80% (Fig. 7b). The size o he h ee
snail popula ions was posi i ely ela ed o sola adi-
a ion, al hough he e ec on he in ec ed popula ions
was weake (Fig. 7c).
In he de ini i e hos s, he mean numbe o luke eggs
in he aeces was signi ican ly associa ed wi h he
sampling mon h. The e ec was mo e ma ked o C.
daubneyi (ed =1.0; Re . d =1.0; F= 34.78; P< 0.001;
explained de iance = 57.1%) han o F. hepa ica (ed =
4.89; Re . d =5.94; F= 5.29; P< 0.01; explained de i-
ance = 56.8%). Fo bo h ema odes, egg exc e ion
ended o inc ease h oughou he s udy pe iod (Fig. 8).
Ne e heless, he ela ionship (linea ) was much mo e
cons an and s onge o C. daubneyi han o F. hep-
a ica (see shape o cu es and scale o y axes).
Discussion
Changes in he popula ions o eshwa e molluscs and
he na u al in ec ions associa ed wi h C. daubneyi and F.
hepa ica we e s udied h oughou 2 yea s on a ca le a m
whe e bo h in ec ions coexis ed. As bo h pa asi es and
molluscs sha ed he habi a and we e hus exposed o he
same en i onmen al condi ions (changes in wea he , phys-
icochemical a iables and/o biocoenosis), any di e ence
in he dynamics o hese in ec ions could be a ibu ed o
pa icula beha iou al ai s o he di e en molluscan and
ema ode species in ol ed. Al hough all species o mol-
luscs sampled we e exposed o he same isk o in ec ion
by mi acidia, G. unca ula was he only species in ol ed
in he ansmission o C. daubneyi and F. hepa ica. This
e eals he impo ance o his snail as an in e media e
hos o F. hepa ica, as is well known, and also o C.
daubneyi, a pa asi e whose epidemiology has sca cely
been s udied un il now. In addi ion, we analyzed 1097
eshwa e gas opods belonging o he species R. pe eg a,
P. case anum,Po amopy gus an ipoda ium,Anisus ani-
sus,Physa on inalis and Succinea pu is, collec ed om
o he a ms wi h ca le in ec ed by C. daubneyi and F.
hepa ica. We did no ind la al s ages o ei he pa asi e in
any o he specimens. The in ol emen o species o he
han G. unca ula in he ansmission o bo h ema odes
in his egion can he e o e be uled ou . In he UK, Jones
e al. [18] also ound ha C. daubneyi in ec ed G. unca-
ula bu no R. bal hica o P. an ipoda um collec ed om
he same habi a s. Ano he lymnaeid snail, Lymnaea gla-
b a, has also been epo ed o be a na u al hos o C.
daubneyi and F. hepa ica in F ance [21, 28]. Ne e heless,
Ab ous e al. [15] showed ha he ole o his snail species
as a ansmi e was e y limi ed (p e alence e y low o
nil) when G. unca ula was also p esen in he same habi-
a . Ou indings in ela ion o F. hepa ica do no appea
o be consis en wi h hose o Rel e al. [17] and Jones e
al. [18], who de ec ed pa asi e DNA in a high p opo ion
o R. pe eg a snails in I eland (66 ou o 167) and he UK
(13 ou o 52). Ne e heless, he p esence o li e F. hepa -
ica la ae in hese snails was no de ini ely demons a ed
(i.e. by mic oscopic isualiza ion), and he DNA-posi i e
eac ions may e lec unsuccess ul in ec ions in which he
mi acidia en e ed he snail bu we e no able o de elop a
all o only did so up o e y ea ly la al s ages. Indeed,
spo ocys encapsula ion has equen ly been obse ed in
R. pe eg a snails expe imen ally exposed o he mi acidia
o F. hepa ica [29]. Such mic oscopically unde ec able,
abo i e ea ly in ec ions canno be uled ou in he p esen
s udy. Howe e , we belie e ha he examina ion o such a
la ge numbe o R. pe eg a specimens allows us o s a e
Fig. 4 Seasonal a ia ion in exc e ion o C. daubneyi (ligh g ey)andF.
hepa ica (da k g ey) eggs in cow aeces. Pe cen age o posi i e cows
(lines) and mean coun s ± s anda d de ia ion, SD (ba s). Abb e ia ions:
A, Au umn; S, Summe ; Sp, Sp ing; W, Win e
Table 3 Es ima ed componen s o he GAMs o he
ela ionships be ween he p obabili y o in ec ion by ema ode
la ae and snail size and sampling mon h, wi h co esponding
e ec i e deg ees o eedom (ed ), Chi-squa e s a is ic, P- alues,
and pe cen age o de iance explained by he models (%DE)
Smoo hing
e ec (s) a iable
P obabili y o in ec ion
C. daubneyi F. hepa ica
ed χ
2
P%DE ed χ
2
P%DE
Snail size 1.0 4.6 0.035 19.0 1.0 3.6 0.058 12.8
Sampling mon h 6.8 92.8 < 0.001 5.4 49.9 < 0.001
Iglesias-Piñei o e al. Pa asi es & Vec o s (2016) 9:610 Page 8 o 14
ha his snail species does no ansmi F. hepa ica in
Galicia.
The a es o in ec ion wi h C. daubneyi and F. hepa ica
we e much lowe in he in e media e hos (8.1 and 4.4%,
espec i ely) han in he de ini i e hos (all cows shed eggs
o bo h pa asi es h oughou he s udy pe iod). Acco ding
o he published da a, simila o e en lowe p e alence
a es a e gene ally obse ed o in ec ions caused by a
g ea a ie y o ema ode species, including C. daubneyi
and F. hepa ica, in lymnaeid popula ions in na u al eco-
sys ems [14, 16, 18, 30]. In a e ospec i e s udy ca ied
ou in Cen al F ance o e a 12-yea pe iod, di e ences in
he p e alence o C. daubneyi in ec ion we e obse ed in
he de ini i e and in e media e hos s. Thus, he p e alence
inc eased s eadily in ca le ( om 5.23 o 44.72%), while i
luc ua ed be ween low alues in he snails (be ween 0.8
and 6.3%) [31]. This may be a ibu ed o di e en ac o s
depending on snails and pa asi es and hei complex ela-
ionships. Fo ins ance, in some snail- ema ode sys ems,
i has been demons a ed expe imen ally ha some non-
hos snail species ( esis an o in ec ion) can ac as a
decoy, hus in e e ing and consequen ly educing he a e
o in ec ion o he hos species wi h which he habi a is
sha ed [32]. Popula ions wi h pheno ypic di e ences in
suscep ibili y o in ec ion by ce ain ema ode species
ha e also been desc ibed wi hin he same species [33].
The suscep ibili y has been shown o be associa ed wi h
exp ession o ce ain s ess genes, in he case o he
snail Biomphala ia glab a a and he pa asi e ema ode
Schis osoma mansoni [34, 35]. Finally, he indings o
he p esen s udy con i m he a i y o na u al co-
in ec ions wi h di e en digenean species [20, 21, 36],
p obably as a esul o compe i ion, which would play
an impo an ole in he con igu a ion o ema ode in-
ec ion s a us o snail popula ions in each habi a . The
mechanisms unde lying compe i i e p ocesses emain
la gely unknown; howe e , such mechanisms may con-
s i u e a basis o de eloping s a egies o in e up he
li e-cycle o ema odes. Fu he esea ch is needed o
p o ide da a on which o base imp o emen s in he
con ol o pa amphis omosis and asciolosis, agains
which no accines o e ec i e ea men s a e cu en ly
a ailable.
The dynamics o na u al in ec ions by C. daubneyi and
F. hepa ica in hei in e media e snail hos s we e also
add essed wi h he ul ima e aim o cons uc ing s a is-
ical models o es ima e he isk o ansmission o ca le
in he a ea. Speci ically, wo in ec ion a iables we e
ollowed-up o e ime: he p obabili y ha an indi idual
is in ec ed and he numbe o indi iduals in ec ed wi hin
he whole popula ion. Fo he p obabili y o in ec ion,
associa ions we e obse ed wi h bo h snail size and
sampling mon h. Howe e , some di e ences be ween he
wo pa asi es we e obse ed, mainly in ela ion o snail
size, which was posi i ely associa ed wi h he p obabili y
o p esence o F. hepa ica, bu nega i ely associa ed wi h
Fig. 5 Rela ionship be ween he p obabili y o in ec ion o G. unca ula by luke la ae and snail size (a) and sampling mon h (b) es ima ed
h ough he smoo h e ec (s) yielded by he GAMs shown in Table 3. The dashed line ep esen s C. daubneyi da a and he do ed line, F. hepa ica
da a. The seasons, sepa a ed by e ical lines, a e indica ed a he op o g aph b.Abb e ia ions: A, Au umn; S, Summe ; Sp, Sp ing; W, Win e
Table 4 Es ima ed componen s o he GAMs o he ela ionships be ween snail abundance and sampling mon h, wi h
co esponding e ec i e deg ees o eedom (ed ), Chi-squa e s a is ic, P- alues, and pe cen age o de iance explained by he models
(%DE)
Smoo hing
e ec (s)
a iable
Numbe o snails
To al no. collec ed No. ha bou ing C. daubneyi No. ha bou ing F. hepa ica
ed χ
2
P%DE ed χ
2
P%DE ed χ
2
P%DE
Sampling mon h 8.9 438.9 < 0.001 78.2 6.7 76.1 < 0.001 71.9 4.1 37.5 < 0.001 72.2
Iglesias-Piñei o e al. Pa asi es & Vec o s (2016) 9:610 Page 9 o 14
