Prevalence of heartworm in dogs and cats of Madrid, Spain

RESEARCH Open Access P e alence o hea wo m in dogs and ca s o Mad id, Spain José Albe o Mon oya-Alonso 1 , Rod igo Mo chón 2 , Yaiza Falcón-Co dón 1 , So aya Falcón-Co dón 1 , Fe nando Simón 2 and Elena Ca e ón 1* Abs ac Backg ound: Di o ila ia immi is causes hea wo m disease, a ch onic and po en ially a al ca diopulmona y disease which mainly a ec s dogs and ca s. I is p esen in mos o Spain, due o a ou able clima ic ac o s. Mad id, loca ed in he cen e o he Ibe ian Peninsula, is he mos highly popula ed ci y in he coun y. The e is a lack o cu en da a on canine hea wo m and he e a e no published epidemiological da a ega ding eline hea wo m in his egion, he e o e he aim o his s udy was o assess he p e alence and cu en dis ibu ion o canine and eline di o ila iosis in hep o inceo Mad id. Me hods: Se um samples om 1716 dogs and 531 ca s, om animals li ing in he me opoli an a ea o Mad id and adjacen a eas, we e s udied. All he samples, ei he om ca s and dogs, we e es ed o ci cula ing D. immi is an igens using a comme cial immunoch oma og aphic es ki . Fu he mo e, o es ablish he se op e alence o hea wo m in ec ion in ca s, se ological echniques o an i-D. immi is and an i-Wolbachia an ibody de ec ion we e used. Resul s: P e alence o D. immi is in he canine popula ion o Mad id was 3%, showing an inc ease in compa ison o p e ious da a. The p esence o hea wo m in he ci y cen e could be in luenced by he p esence o U ban Hea Islands, while he posi i e dogs om me opoli an and adjacen a eas we e mainly loca ed unde he in luence o i e s. Rega ding ca s, 0.2% we e posi i e o he an igens es and 7.3% we e se oposi i e o bo h an i-D. immi is and Wolbachia su ace p o ein an ibodies, which demons a e he p esence o eline hea wo m in Mad id. Se oposi i e ca s we e p esen in he same a eas whe e posi i e dogs we e ound. Indoo /ou doo ca s showed he highes se op e alence whe eas he lowes co esponded o indoo ca s, demons a ing ha p ophylac ic ea men s should be ca ied ou ega dless o li es yle. In ec ion was ound in 2.2% o dogs and 6.7% o he ca s < 1 yea - old, which indica es ha ea ly p e en i e campaigns in puppies and ki ens should be implemen ed. Conclusions: The esul s poin o he need o adequa e p ophylac ic measu es h ough he adminis a ion o mac ocyclic lac ones in animals li ing in Mad id. Ve e ina ians should be awa e o he p esence o his disease and include hea wo m in he di e en ial diagnosis when a pe p esen s wi h symp oms compa ible wi h D. immi is. Keywo ds: Feline, Canine hea wo m, Di o ila ia immi is, Vec o -bo ne diseases, P e alence, Epidemiology, Spain * Co espondence: [email p o ec ed] 1 In e nal Medicine, Facul y o Ve e ina y Medicine, Resea ch Ins i u e o Biomedical and Heal h Sciences (IUIBS), Uni e si y o Las Palmas de G an Cana ia, Las Palmas de G an Cana ia, Spain Full lis o au ho in o ma ion is a ailable a he end o he a icle © The Au ho (s). 2017 Open Access This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed. Mon oya-Alonso e al. Pa asi es & Vec o s (2017) 10:354 DOI 10.1186/s13071-017-2299-x Backg ound Hea wo m disease is caused by Di o ila ia immi is and is a ch onic, p og essi e and po en ially a al disease o he in ec ed animals. The main hos s a e dogs and ca s, al hough in ec ion has been epo ed in o he ca ni o es, including wild canines as well as domes ic and wild e- lids. Fu he mo e, humans can be in ec ed since i is a zoono ic disease [1]. The ansmission occu s h ough he bi e o he species o culicid mosqui oes belonging o he gene a Culex,Aedes and Anopheles, among o he s. Thus, p e alences a e in luenced by he p esence and abundance o ec o s, depending on clima ic ac o s such as empe a u e and humidi y [1, 2]. Animal di o ila iosis has been ex ensi ely s udied in Eu ope. The geog aphical loca ion o Spain, in sou he n Eu ope, si ua es he Ibe ian Peninsula in he endemic a ea o di o ila iosis on he con inen [3, 4]. In Spain, he in- ec ion is p esen in mos o he e i o y, he highes p e alences being ound in he sou hwes , Medi e anean coas and i iga ed a eas o inland Spain and in he Cana y Islands [5–7]. The p o ince o Mad id is in he cen e o he Ibe ian Peninsula. The al i ude a ies om 550 o 700 m in mos o he e i o y, excep in he no he nmos pa o he p o ince, whe e he mean heigh mainly a ies om 1000 o 1700 m, eaching abo e 2000 m a some poin s. Many i e basins c oss he e i o y om no h o sou h, ca ying he wa e low o he Ri e Tajo, which c osses he p o ince in he sou he n pa o he egion. The p o ince p esen s cold win e s and mild summe s in he no he n pa o he e i o y, while he es o he e i o y is cha ac e ized by cold win e s and ho sum- me s; hese a e he d ies seasons, while ains a e mo e equen in au umn and sp ing. The ci y o Mad id is he capi al o he p o ince, and is also he capi al o Spain. I is he mos highly popu- la ed ci y o he coun y, as only he cen e o Mad id has a census o o e 3 million people, 275,000 dogs and 65,000 ca s [8, 9]. The ci y is included wi hin he me o- poli an a ea, which su ounds he ci y ep esen ing he mos highly popula ed me opoli an a ea o Spain, wi h an es ima ed popula ion abo e 7 million and he i h mos popula ed me opoli an a ea o Eu ope. Published s udies epo ed canine di o ila iosis o 1.1 and 1.9%, in he p o ince o Mad id, be ween 1987 and 1990 [10, 11]. No s udies ollowed hese da a un il 2013, when a s udy epo ed p e alences o 2.3% in a wide a ea o cen al Spain which enclosed Mad id and Toledo [12]. The e a e no epidemiological da a published e- ga ding ca s. The e is a lack o cu en epidemiological da a o ani- mal hea wo m ocusing on he p o ince o Mad id. The e o e, he aim o his s udy was o assess he p e a- lence and cu en dis ibu ion o canine and eline di o ila iosis in he me opoli an a ea o Mad id and ad- jacen a eas. Me hods The p esen s udy included 1716 dogs and 531 ca s p e- sen ed o e e ina y clinics be ween Ma ch 2015 and Sep embe 2016. The samples we e collec ed a 20 e e - ina y cen es loca ed in he s udied a ea om animals li ing in he me opoli an a ea o Mad id and adjacen a eas, including 98 municipali ies in 218 di e en pos al codes (Fig. 1). The inclusion c i e ia o animals we e: being o e 6 mon hs o age; no ha ing a elled ou side he a ea o in e es o he s udy; ne e ha ing ecei ed ea men o hea wo m disease; and no p e ious his o y o hea - wo m in ec ion. A comple e eco d was kep o each animal, including iden i ica ion (age, sex and b eed), clinical his o y, and demog aphic da a. O he dogs included, 46.7% we e emales and 53.3% we e males. By b eed, he mos highly ep esen ed we e mong el dogs (26.3%), ollowed by Yo kshi e Te ie s (8.6%), Golden Re ie e s (6.9%), Cocke Spaniels (5.7%), Boxe (5.3%), F ench Bulldog (4.8%), Ge man Shephe d (4.1%) and 63 o he b eeds (38.3%). Rega ding he ca s s udied, 51.4% we e emales and 48.4% we e males; by b eed, he Eu opean sho hai was he mos highly ep- esen ed (65.3%), ollowed by Pe sian ca s (14.7%), Siam- ese ca s (12.2%) and o he 15 b eeds (7.8%). The ca s we e u he classi ied as indoo (57.6%), ou doo (12.6%) o indoo /ou doo (a leas 1–50% o he ime spen ou doo s) (29.8%). The age anges o dogs and ca s a e desc ibed in Table 1. The s udy included 845 dogs and 312 ca s li ing in he ci y o Mad id while 871 dogs and 219 ca s we e li ing in he es o he s udied a ea (me opoli an and adjacen a ea). Blood samples we e collec ed om he cephalic o jugula ein, placed in 3 ml se um ubes and cen i uged. Se um was kep a -20 °C un il es s we e pe o med. All he samples, ei he om ca s and dogs, we e es ed o ci cula ing D. immi is an igens using a comme cial immunoch oma og aphic es ki (U ano es Di o ila ia®, U ano Ve SL, Ba celona, Spain) acco ding o he manu- ac u e ’s ins uc ions. Fu he mo e, o es ablish he se op e alence o hea - wo m in ec ion in ca s, se ological echniques o an i- D. immi is and an i-Wolbachia an ibody de ec ion we e also used, as desc ibed by Mo chón e al. [13] wi h some modi ica ions. In b ie , he pla es we e coa ed wi h 0.8 μgo D. immi is soma ic an igen and Wolbachia su - ace p o ein (WSP). Se um samples we e p epa ed a 1/ 100 o an i-D. immi is se um an ibodies and 1/40 o an i-WSP an ibody de ec ion. An i- eline IgG an ibody, ho se adish pe oxidase-labelled (Ki kegaa d and Pe y Labo a o ies, Gai he sbu g, MD, USA), was applied a 1/ Mon oya-Alonso e al. Pa asi es & Vec o s (2017) 10:354 Page 2 o 6 Fig. 1 Dis ibu ion o canine and eline hea wo m in he p o ince o Mad id (Spain). The da k g ey a ea co esponds o he ci y o Mad id, while he pale g ey a ea co esponds o he me opoli an and adjacen a ea included in he s udy. The main i e s a e illus a ed as hick black lines. The posi i e animals a e ma ked as ollows: ed ci cles (hea wo m posi i e dogs); g een squa es (an ibodies-se oposi i e ca s); blue as e isk (an igens- posi i e ca ). The municipali ies wi h highe p e alences and se op e alences a e indica ed as: 1, A anjuez; 2, Alcalá de Hena es; and 3, Majadahonda Table 1 Dogs (n) and ca s (n) s udied by age anges, as well as p e alence o hea wo m (%) by each g oup o age Age ange Dogs (n) Canine p e alence (%) a Ca s (n) Feline se op e alence (%) b < 1 yea 45 2.2 15 6.7 1–3 yea s 320 3.7 78 1.3 3–6 yea s 393 3.6 213 8.0 6–9 yea s 429 3.3 88 11.4 9–12 yea s 396 2.0 74 5.4 > 12 yea s 133 2.3 63 9.5 a Based on de ec ion o ci cula ing D. immi is an igens b Based on se ological echniques o an i-D. immi is and an i-Wolbachia an ibody de ec ion (conside ed se oposi i e when an i-D. immi is and an i-Wolbachia su ace p o eins an ibodies p esen ed join ly) Mon oya-Alonso e al. Pa asi es & Vec o s (2017) 10:354 Page 3 o 6 4000 dilu ion. The op ical densi ies we e measu ed in an Easy-Reade (Bio-Rad Labo a o ies, He cules, CA, USA) a 492 nm. Cu -o poin s o ELISA D. immi is 0.8 and ELISA WSP 0.6 we e ob ained as a i hme ic mean op- ical densi y ± 3 s anda d de ia ions o se um o cli- nically heal hy ca s. Ca s we e conside ed se oposi i e when an i-D. immi is and an i-WSP an ibodies p esen ed join ly [7, 14–16]. Da a we e analysed using SPSS Base 20.0 so wa e o Windows (SPSS Inc./IBM, Chicago, IL, USA). Desc ip i e analysis o he conside ed a iables was ca ied ou con- side ing he p opo ions o he quali a i e a iables. χ 2 o compa e p opo ions was pe o med. In all cases, he sig- ni icance le el was es ablished a P<0.05. Resul s The p e alence o D. immi is in he canine popula ion o he s udied a ea o Mad id was 3% (52/1716). The e we e no signi ican di e ences in he p e alences be- ween males (3.2%) and emales (2.9%) (χ 2 = 0.006, d =1, P= 0.939). By b eed, mong el dogs showed highe bu no signi ican ly di e en p e alences (4.2%) when com- pa ed wi h pu e-b eed dogs (2.6%) (χ 2 = 2.902, d =1, P= 0.088). By age anges, he highes p e alences we e ound in dogs om 1 o 6 yea s, and signi ican di e ences we e only obse ed be ween dogs be wee 9–12 yea -old compa ed wi h dogs be ween 1 and 6 yea -old (χ 2 =2.753, d =5,P=0.738)(Table1). An igens es we e posi i e in 0.2% (1/531) o he ca s, while 7.3% (39/531) o ca s we e se oposi i e o bo h an i-D. immi is and WSP an ibodies. The e we e no s a- is ically signi ican di e ences be ween emales (7.7%) and males (7.0%) (χ 2 = 0.172, d =2,P= 0.918). By b eed, he Eu opean sho hai p esen ed a se op e a- lence o 7.5% while he mean se op e alence in he o he b eeds was 7.1% (χ 2 = 0.036, d =1,P= 0.850). Indoo / ou doo ca s showed he highes se op e alence (11.4%), ollowed by ou doo ca s (10.4%) while he lowes co e- sponded o indoo ca s (4.6%) (χ 2 = 8.201, d =2, P= 0.017). By age anges, he highes se op e alence was ound in ca s be ween 6 and 9 yea -old; no signi i- can di e ences we e ound be ween all g oups o age (χ 2 = 7.265, d =5,P= 0.202) (Table 1). The p e alence o hea wo m in ec ion in he ci y o Mad id in he canine popula ion was 3.3%. By sex, bo h males and emales showed he same p e alence (3.3%). The e we e posi i e dogs in 15 o he 21 dis ic s o he ci y. The p e alence in he me opoli an and adjacen a eas was 2.7% (2.4% in emales and 3% in males); he dis ibu ion was no uni o m, he posi i e dogs being mainly loca ed in he a ea o in luence o he majo i- e s. By municipali ies, he highes p e alences we e ob- se ed in A anjuez (10.0%), Majadahonda (6.4%) and Alcalá de Hena es (5.9%), all o hem in luenced by i- e s (Fig. 1). When he ca s we e analysed, he se oposi i i y in he ci y o Mad id was 6.1%. By sex, he se op e alence was 7.6% in emales and 4.3% in males. The e we e se oposi- i e ca s p esen in 11 o he 21 dis ic s o he ci y. The se op e alence was highe in he me opoli an and adjacen a eas, his being 9.1% (7.7% in emales and 10.3% in males). Posi i e animals we e mainly ound in a eas close o i e s. By municipali ies, he highes se o- p e elances we e obse ed in Alcalá de Hena es (27.3%), A anjuez (20.8%) and Majadahonda (11.1%), all o hem unde he in luence o i e basins. The only ca posi i e o he an igens es co esponded o a emale Eu opean sho hai , li ing ou doo s in A anjuez (Fig. 1). Discussion The p esen s udy epo s he p esence o hea wo m in- ec ion in he pe popula ion o Mad id and es ablishes he cu en canine p e alence a 3%. P e ious epo ed da a on hea wo m in ec ion in dogs om Mad id be- ween 1987 and 1990 a ied om 1.1 o 1.9% [10, 11]. Mo e ecen da a epo ed a p e alence o 2.3% in 2013, al hough he p e alence was de ined o a wide a ea o cen al Spain which enclosed Mad id and Toledo wi h a ela i ely low sample size [12]. Conside ing p e ious da a, i can be conside ed ha he p e alence o canine hea - wo m in Mad id has inc eased. The e a e se e al clima ic condi ions ha could a ou he ansmission o di o ila iosis in he s udied a ea. The p o ince o Mad id is unde he in luence o se e al i e basins, which p o ides a humid en i onmen ap- p op ia e o he de elopmen o he mosqui oes. The esul s om he s udy show how cu en ly posi i e dogs ou side he ci y cen e a e mainly concen a ed in a eas unde he in luence o he i e s Tajo, Ja ama, Hena es and Guada ama. The p e alence de ec ed in A anjuez is especially s iking; he a ea is a small u banized muni- cipali y unde he in luence o he Ri e Tajo, whe e dogs li e ou doo s mo e equen ly han in u ban a eas. An inc ease in he p e alence om ha 6.8% epo ed in 1990 [10] o he cu en 10% was also obse ed. Fu he - mo e, he ci y o Mad id is also unde he in luence o he Ri e Manzana es, which c osses he ci y cen e om wes o eas . The high le els o u baniza ion o he ci y o Mad id and he closes me opoli an a ea a e unde he in luence o he phenomenon called U ban Hea Island (UHI). As buildings and asphal e ain hea du ing he day which is e-emi ed owa ds he a mosphe e, he empe a u e inc eases se e al deg ees inside he ci y c ea ing mic ocli- ma es [17, 18]. In he case o Mad id, mul iple s udies on UHI ha e been ca ied ou du ing he las wo decades [19, 20]. Fu he mo e, human ac i i y inc eases he Mon oya-Alonso e al. Pa asi es & Vec o s (2017) 10:354 Page 4 o 6 densi y o mosqui oes and de elops a sui able en i on- men o i s p oli e a ion due o an inc ease in he p o ision o wa e sou ces and ege a ion which can be ound in he p esence o pa ks and g een u ban a eas. Since he mosqui oes can ep oduce in small con aine s wi h wa e ( lowe po s, ponds, oun ains), all hese ac o s a ou he clima ic condi ions o he de elopmen o hea wo m la ae in mosqui o ec o s du ing he cold mon hs o he yea . This phenomenon was also obse ed in Ba celona, ano he la ge ci y in Spain [16, 21]. In he p esen s udy, he p e alence obse ed in he ci y cen e was 3.3%. A p e ious s udy epo ed a p e alence o 0.6% in he u ban a ea o Mad id in 1990 [10], he e o e i can be concluded ha he p e alence in he ci y cen e has also inc eased. This could be due o he exace ba ion o he phenomenon o he UHI, a g ea e p esence o g een u ban a eas, a highe densi y o dogs in he ci y and a low incidence chemop ophylaxis. Dogs li ing in u ban a eas a e no sa e om hea wo m in ec ion and should ecei e adequa e p ophylaxis wi h mac ocyclic lac ones, as con- cluded in a simila s udy in Ba celona [21]. Geog aphical in o ma ion sys ems (GIS) can p edic he dis ibu ion and epidemiological beha iou o di o- ila iosis in di e en e i o ies [3, 21–23] based on cli- ma ic, ecological and many o he da a ela ed o he de eloping equi emen s o pa asi es and ec o s. A GIS esea ch on he gene al p edic ion o he dis ibu ion o di o ila iosis in Spain showed ha he a ea included in his s udy is a high isk o D. immi is in ec ion [23]; among o he isk ac o s, due o he exis ence o i i- ga ed c ops which inc eases he ansmission isk, p o- iding excellen habi a s o mosqui o b eeding. Rega ding ca s, he p esen esul s epo he i s epi- demiological da a on eline D. immi is and demons a e he p esence o eline hea wo m in Mad id. To da e, in Spain epidemiological s udies in hea wo m in ec ion in ca s ha e been p e iously epo ed only in he Cana y Islands and in Ba celona [7, 14, 16]. The esul s showed ha 0.2% o ca s we e posi i e o D. immi is an igens in compa ison wi h 7.3% se oposi i i y o an ibodies. This may be due o he ac ha he sensi i i y o an igen es- ing is ela i ely low in ca s, and because hese only de- ec adul and emale wo ms. The e o e, a nega i e esul does no ule ou an in ec ion om male wo ms, p e-adul wo ms o a single emale adul wo m, mos o which a e common in ca s [24, 25]. On he o he hand, an ibody es s de ec an ibodies p oduced by he hos in esponse o in ec ion, which he e o e can emain posi i e o a long ime a e he dea h o he pa asi eanddono di e en ia eacu en o apas in ec ion [24, 26]. Gi en he na u al esis ance o he eline hos o hea wo m in ec ion, i is es ima ed ha he eline di o ila iosis is 5–20% o ha o he canine popula ion in he same a ea [27, 28], and eline hea wo m epo ed in he p esen s udy is wi hin hese anges. The esul s showed ha ca s li ing indoo /ou doo and indoo p esen ed highe se op e alences, p esu- mably because hey a e mo e exposed o mosqui o bi es. Howe e , 4.6% o indoo ca s showed D. immi is an i- bodies demons a ing ha an indoo li es yle does no p o ec ca s om in ec ion [16, 29]. Ca s do no usually ecei e chemop ophylaxis and he e o e he isk o in- ec ion is highe in his species han in dogs. Se opo- si i e ca s we e p esen in he same a eas whe e posi i e dogs we e ound, in luenced by he same clima ic condi- ions. The se op e alence is highe in ca s om me o- poli an and adjacen a eas when compa ed wi h he ci y cen e; p obably one o he easons may be because ca s li ing in hose municipali ies spend ime ou doo s mo e equen ly. The e o e, adequa e p ophylac ic ea men s wi h mac ocyclic lac ones should be ca ied ou in his species, ega dless o li es yle. Al hough no s a is ically signi ican , mong el dogs showed highe p e alences when compa ed wi h pu e- b eed dogs, as epo ed in o he s udies [21]. Rega ding ca s, no signi ican di e ences we e obse ed be ween Eu opean sho hai and all he o he b eeds. By age, he highes p e alence was ound in dogs om 1 o 3 yea s ollowed by dogs om 3 o 6 yea s; o he s udies showed he highes p e alences in olde dogs [14, 23]. In ca s, a g ea a iabili y in he p e alences by age anges can be obse ed when he esul s we e compa ed wi h o he s udies [7, 14–16, 29]. I is no ewo hy o men ion ha 2.2% o dogs < 1 yea -old we e in ec ed and 6.7% o he ca s we e se oposi i e, which indica es ha ea ly p e- en i e campaigns in puppies and ki ens wi h mac o- cyclic lac ones should be ca ied ou . Conclusions The esul s show he p esence o canine and eline hea wo m in he p o ince o Mad id, in he cen e, me opoli an and adjacen a eas and an inc ease in he p e alence compa ed o p e ious s udies. These esul s poin o he need o p ophylac ic measu es wi h he ad- minis a ion o mac ocyclic lac ones o con ol he dis- ease and o a oid zoono ic in ec ions. Ve e ina y clinicians should be awa e o he p esence o his disease and include hea wo m in he di e en ial diagnosis when a pe p esen s wi h espi a o y signs o symp oms compa ible wi h D. immi is. Abb e ia ions GIS: Geog aphical in o ma ion sys ems; UHI: U ban Hea Island; WSP: Wolbachia su ace p o ein Acknowledgmen s The au ho s would like o hank all he e e ina y clinics which kindly coope a ed in he p esen s udy; wi hou hem his s udy could no ha e been ealized. Mon oya-Alonso e al. Pa asi es & Vec o s (2017) 10:354 Page 5 o 6 Funding This esea ch was suppo ed by Baye Hispania S. A. A ailabili y o da a and ma e ials All da a gene a ed o analysed du ing his s udy a e included in his published a icle. Au ho s’con ibu ions JAMA and EC designed he s udy and w o e he manusc ip . RM, YFC, SFC and FS o ganized he ieldwo k, pa icipa ed in he da a collec ions and analysed he samples. All au ho s analysed he da a, ead and app o ed he inal manusc ip . E hics app o al and consen o pa icipa e All he owne s ga e hei consen o pa icipa ion in his s udy. The s udy was app o ed by he e hical commi ee o he Ve e ina y Medicine Se ice o he Uni e si y o Las Palmas de G an Cana ia and was ca ied ou in acco dance wi h he cu en Eu opean legisla ion on animal p o ec ion. Consen o publica ion No applicable. Compe ing in e es s The au ho s decla e ha hey ha e no compe ing in e es s. Publishe ’sNo e Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a ilia ions. Au ho de ails 1 In e nal Medicine, Facul y o Ve e ina y Medicine, Resea ch Ins i u e o Biomedical and Heal h Sciences (IUIBS), Uni e si y o Las Palmas de G an Cana ia, Las Palmas de G an Cana ia, Spain. 2 Labo a o y o Pa asi ology, Facul y o Pha macy, Ins i u e o Biomedical Resea ch o Salamanca (IBSAL), Uni e si y o Salamanca, Salamanca, Spain. Recei ed: 18 Ap il 2017 Accep ed: 18 July 2017 Re e ences 1. Simón F, Siles-Lucas M, Mo chón R, González-Miguel J, Mellado I, Ca e ón E, e al. 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