we collected samples of 49 dead wolves ( causes of death : legal hunting , road kill and 
 illegal killing ) from 2010 - 2013 in two regions of northwestern spain : asturias ( 4328n 
 527o ) and galicia ( 4317n 741o ) . carcasses were collected as part of a long - term 
 collection protocol of wolf samples approved by the regional governments of galicia and 
 asturias . this sample size represents about 2.5% of the wolves inhabiting the iberian 
 peninsula ( estimated in 2,000 individuals ) ( blanco & 
 corts 2012 ) . kidney samples were obtained and kept frozen until analyses . 
 once in the laboratory , samples were subjected to three types of analyses . direct 
 immunofluorescence ( if ) was carried out using rabbit igg against a pool of the studied 
 serovars of leptospira ( made in - house ) and goat anti - rabbit 
 igg - fluorescein isothiocyanate conjugate ( nordic immunological laboratories , 
 netherlands ) following the procedure of len - vizcano et 
 al . the serovars investigated were australis , autumnalis , ballum , 
 bataviae , bratislava , canicola , grippotyphosa , hardjo , hebdomadis , icterohaemorrhagiae , 
 pomona , saxkoebing , sejro and tarassovi . culture was carried out using 
 a standard ellinghausen - mccullough - johnson - harris ( emjh ) ( difco laboratories , madrid , 
 spain ) liquid medium following the procedures of baranton 
 and postic ( 1989 ) . kidney samples were homogenised in a 1:10 dilution with 
 emjh medium ( difco ) . homogenate samples were subjected to dna extraction , performed as 
 described by rojas et al . ( 2010 ) using a 
 commercial kit ( qiaamp dna mini kit ; qiagen iberia , spain ) . polymerase chain reaction ( pcr ) was performed as 
 described by moreno and agudelo - flrez ( 2010 ) 
 using primers lipl32/270f ( 5-cgctgaaatgggagttcgtatgatt-3 ) and lipl32/662r 
 ( 5-ccaacagatgcaacgaaagatccttt-3 ) as reported by levett 
 et al . pcr was performed 
 in a biometra t personal ( biometra , germany ) thermocycler and pcr products were 
 visualised through electrophoresis on a 1.5% agarose gel stained with ethidium bromide 
 and viewed under ultraviolet light . positive dna extractions were amplified by pcr in 
 order to differentiate leptospira serovars detecting the 
 oligonucleotide i - repl as a target of the amplification ( barocchi et al . ( 2010 ) using the primers rep1 ( 5- agcgggtatgactccgc-3 ) and irep1 
 ( 5-gcggactcatacccgct-3 ) ( barocchi et al . 2001 ) . 
 to detect antibodies against such serovars , kidney tissue exudates were analysed by the 
 indirect microscopic agglutination test following procedures outlined in faine ( 1982 ) , which is the standard method to detect 
 antibodies against leptospira serovars . differences in prevalence between seasons , regions and age 
 ( pups : younger than 1 year ; subadult : between 1 - 2 years ; adult : older than 2 years ) and 
 sex groups were tested with the  test or fisher s exact test using pasw 
 statistics 17.0 . ten wolves ( observed prevalence : 20.1% , 95% confidence interval = 11 - 33% ) showed 
 evidence of contact with leptospires ( table ) . eight wolves were actively infected as revealed by direct detection ( 16.3% ) ; of these , 
 three were positive by pcr and if and five only by pcr . overall , serovars detected were canicola ( n = 4 , 40% of the 
 positive cases ) , icterohaemorrhagiae ( n = 3 , 30% ) and sejro , ballum and grippotyphosa 
 ( n = 1 , 10% each ) . four of the seropositive wolves and three further wolves showed 
 titres against other serovars , including serovar australis , but below the cut - off level 
 ( table ) . no differences were found in 
 prevalence depending on the diverse factors studied ( see materials and methods ) . interestingly , a wolf pup had high antibody titres against serovar ballum , but was 
 apparently uninfected . tableserovars detected in 10 out of 49 wolves analysed for pathogenic 
 leptospires  direct detection in 
 kidney  serology in kidney 
 exudates 

 referenceregionage / sexserovarpositive by 
 serovar 
 ( titration)other titres below 
 the cut - off level 
 cl-012galiciasubadult / female--  canicola ( 1:400)ballum ( 1:10)cl-016galiciaadult / malecanicolapcr  canicola ( 1:100)-cl-017galiciapup / femalecanicolapcr  canicola ( 1:400)-cl-022asturiaspup / male--  ballum ( 1:800)canicola ( 1:20)cl-024galiciaadult / femalecanicolapcr , if  canicola ( 1:100)-cl-026asturiassubadult / femaleicterohaemorrhagiaepcr  icterohaemorrhagiae ( 1:100)-cl-034asturiassubadult / maleicterohaemorrhagiaepcr , if  -icterohaemorrhagiae ( 1:20)cl-035asturiassubadult / maleicterohaemorrhagiaepcr  icterohaemorrhagiae ( 1:200)australis ( 1:50)cl-063asturiassubadult / male 
 leptospira interrogans ( serovar nd)pcr  grippotyphosa ( 1:400)-cl-067asturiasadult / femalesejropcr , if  sejro ( 1:100)- 
 a : three further wolves showed titres below the cut - off 
 level : sejro ( 1:50 ) , sejro ( 1:10 ) and ballum ( 1:10 ) ; b : 
 all samples were negative by culture ; if : direct immunofluorescence ; nd : not 
 determined ; pcr : polymerase chain reaction . a : three further wolves showed titres below the cut - off 
 level : sejro ( 1:50 ) , sejro ( 1:10 ) and ballum ( 1:10 ) ; b : 
 all samples were negative by culture ; if : direct immunofluorescence ; nd : not 
 determined ; pcr : polymerase chain reaction . the prevalence detected in the present survey is higher than reported for wolf 
 populations elsewhere . very low seroprevalences were found in alaska ( zarnke et al . 2004 ) and scandinavia ( akerstedt et al . 2010 ) . in minnesota , about 10% of 
 wolves were seropositive ( khan et al . warmer winters in northwestern spain , as compared to these northern locations and 
 relatively wet summers , probably enhance the survival of spirochetes in the environment . however , the difference observed with previous studies might only be a result of the 
 convenience sampling used in this study compared with samples obtained from live - trapped 
 animals in all other studies quoted ( khan et al . our study reveals , for the first time , active leptospira infection in 
 wolves . it is noteworthy that seroprevalence was only slightly higher than prevalence of 
 active infections , whereas previous studies in wild carnivores using both techniques 
 showed that this difference is usually marked ( milln et 
 al . the lack 
 of such differences in the present study may result from a poor performance of the 
 serological test due to the use of tissue exudates instead of serum . alternatively , 
 discrepancies may also indicate a short life of circulating antibodies in wolves or a 
 low degree of seroreactivity . in fact , antibody titres were low ( 1:100 ) in four wolves 
 that were concurrently infected . in addition , titres below the cut - off level were 
 detected in other cases , suggesting the existence of chronic infections in wolves that 
 could be acting as renal carriers ( table ) . dogs are 
 considered the natural host for serovar canicola ( andr - fontaine 2006 ) , though other species can be found infected by this 
 serovar ( garca et al . 2013 ) . given the genetic 
 proximity between wolves and dogs , it is not surprising that this was the most prevalent 
 serovar detected in the present survey . however , this was not the case in the study by 
 khan et al . ( 1991 ) in minnesota , where this 
 serovar was only the fourth most frequently detected . these differences may reflect the 
 fact that iberian wolves live in a more anthropised environment ( llaneza et al . 2012 ) and that wolves occurring in human - dominated 
 landscapes may be frequently exposed to canine pathogens . the main reservoirs for the serovars icterohaemorragiae , sejro and ballum are rodents 
 ( turk et al . no information is available about leptospiral 
 infections in rodents in the study areas . in a nearby region in western spain , the most 
 common serovar found was ballum ( garca et al . the abovementioned serovars were almost absent in wild ungulates in 
 asturias , as recently published ( esp et al . therefore , wolves may come into contact with these serovars after 
 consumption of rodents or perhaps lagomorphs , as suspected in other studies analysing 
 wild carnivores in southern europe ( milln et al . 2010 ) . however , the 
 proportion of these food items is very low or absent in the diets of wolves from the 
 study areas ( llaneza et al . . an alternative hypothesis 
 may be that the wolves became infected after drinking from rodent - contaminated water 
 sources or from consuming other preys or carrion of ungulates that can occasionally be 
 infected by these serovars ( slavica et al . serovar grippotyphosa , also detected in the present survey , was the most seroprevalent 
 in wolves from minnesota , representing one in four positive cases ( khan et al . 1991 ) . antibodies against this serovar were among the 
 most frequently detected in the survey of wild ungulates in asturias by esp et al . ( 2010 ) , especially in fallow deer 
 ( dama dama ) and wild boar ( sus scrofa ) . antibodies 
 against serovar grippotyphosa were also relatively prevalent in cattle farms in galicia 
 and asturias ( esp et al . thus , wolves probably come 
 into contact with this serovar after consumption of wild or domestic ungulates or their 
 products . lesions 
 ( chiefly chronic interstitial nephritis ) associated with leptospiral infection have been 
 recorded in other species of wild carnivores ( milln et 
 al . , leptospires can cause hepatic and renal failure and , 
 occasionally , death ( greene et al . 2012 ) . it is 
 generally assumed that serovars icterohaemorrhagiae and canicola are responsible for the 
 acute forms of canine leptospirosis . however , typical acute leptospirosis can also be 
 induced by strains belonging to other serogroups , including grippotyphosa ( andr - fontaine 2006 ) . in conclusion , our survey revealed infection and exposure in wolves with serovars 
 maintained by different reservoirs such as dogs , rodents and ungulates , confirming the 
 usefulness of the wolf and other large predators as an environmental sentinel for 
 leptospires and probably other environmental pathogens .
the iberian wolf ( canis lupus ) is the top predator in the iberian 
 environments in which it lives , feeding on a wide range of species , thus encountering 
 a wide range of disease agents . 
 therefore , the wolf can serve as sentinel of 
 environmental contamination with pathogens . 
 we investigated the exposure of 
 free - living wolves to 14 serovars of leptospira interrogans sensu 
 lato . 
 kidney samples from 49 wolves collected from 2010 - 2013 in 
 northwestern spain were analysed by culture , direct immunofluorescence and polymerase 
 chain reaction . 
 tissue fluids were analysed for antibodies by a microscopic 
 agglutination test . 
 ten wolves ( observed prevalence : 20% , 95% confidence interval = 
 11 - 33% ) showed evidence of contact with leptospires , eight through direct detection 
 and nine through serology ( 7 wolves were positive according to both techniques ) . 
 titres below the cut - off level were also detected in seven cases . 
 serovars confirmed 
 were canicola ( n = 4 ) , icterohaemorrhagiae ( n = 3 ) and sejro , ballum and 
 grippotyphosa ( n = 1 each ) , indicating that wolves were infected with serovars for 
 which dogs , rodents and ungulates , are the natural hosts and supporting the utility 
 of the wolf and other large predators as environmental sentinels for pathogens .