I f M K G J a b D c J d a A R R A A K D H P W W 1 b b a v r s e T v U h 1 Ticks and Tick-borne Diseases 8 (2017) 470–476 Contents lists available at ScienceDirect Ticks and Tick-borne Diseases j ourna l ho me pa ge: www.elsev ier .com/ locate / t tbd is s the free-ranging jaguar (Panthera onca) a reservoir for Cytauxzoon elis in Brazil? ariana Malzoni Furtadoa,b,∗, Sueli Akemi Taniwakib, Betina Metzgerc, arina dos Santos Paduanc, Helena Lucia O’Dwyerc, Anah Tereza de Almeida Jácomoa, rasiela E.O. Porfírioa, Leandro Silveiraa, Rahel Sollmanna,1, Natália Mundim Tôrresa,d, osé Soares Ferreira Netob Jaguar Conservation Fund/Instituto Onç a-Pintada, Caixa Postal 193, 75830-000, Mineiros, GO, Brazil Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. r. Orlando Marques de Paiva, 87, 05508-000, São Paulo, SP, Brazil Instituto de Biociências de Botucatu, UNESP—Universidade Estadual Paulista, Campus de Botucatu, Departamento de Parasitologia, Distrito de Rubião únior, 18618-000, Botucatu, SP, Brazil Instituto de Biologia, Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Rua Ceará, s/n, 38400-902, Uberlândia, MG, Brazil r t i c l e i n f o rticle history: eceived 27 September 2016 eceived in revised form 3 February 2017 ccepted 5 February 2017 vailable online 6 February 2017 eywords: omestic cat a b s t r a c t This study investigated the occurrence of Cytauxzoon felis and Babesia spp. in free-ranging jaguars (Pan- thera onca), domestic dogs (Canis lupus familiaris) and domestic cats (Felis catus) from the Cerrado, Amazon and Pantanal biomes of Brazil. Blood samples were collected from 30 jaguars, 129 dogs and 22 cats for detection of the 18S rRNA genes of piroplasmids. All of the jaguars from the Pantanal (n = 22) and Cerrado (n = 4) and three of four jaguars from the Amazon were positive for C. felis, but no dogs or cats were posi- tive for the agent. All of the jaguars and domestic cats were negative for Babesia spp., while dogs from the Cerrado (7.9%; 5/63) and Amazon (10.6%; 5/47) biomes tested positive for the hemoparasite. Cytauxzoon emoparasite iroplasm ildlife ild felids nucleotide sequences detected were closely related to C. felis; and Babesia nucleotide sequences showed 100% of identity with Babesia vogeli. Although the pathogenicity of Cytauxzoon spp. genotypes that circu- late in Brazil is still unknown, free-ranging jaguars probably play an important role in the maintenance of C. felis in nature. In addition, even though there is no evidence of the circulation of Babesia spp. between jaguars and dogs, the presence of this hemoparasite should be monitored in jaguar populations. © 2017 Elsevier GmbH. All rights reserved. . Introduction Cytauxzoon felis and Babesia spp. are protozoan hemoparasites elonging to the Piroplasmida Order that are transmitted by tick ites and infect a wide range of mammals worldwide (Meinkoth nd Kocan, 2005; Penzhorn, 2006). C. felis has historically been iewed as highly fatal for domestic cats (Felis catus) while wild felids emain asymptomatic; however, recent studies have reported cats urviving infections as well as wild cats with fatal infections (Brown t al., 2010; Meinkoth et al., 2000; Nietfeld and Pollock, 2002; Rizzi ∗ Corresponding author at: Av. Dr. Silva Melo 520, Ed. Mamoré apt. 708, Jd. aquaral, São Paulo, SP, Brazil. E-mail address: marianafurtado@jaguar.org.br (M.M. Furtado). 1 Current address: Department of Wildlife, Fish, and Conservation Biology, Uni- ersity of California Davis, 1088 Academic Surge, One Shields Ave, Davis, CA 95616, SA. ttp://dx.doi.org/10.1016/j.ttbdis.2017.02.005 877-959X/© 2017 Elsevier GmbH. All rights reserved. et al., 2015). C. felis appears to be observed primarily in the south- central regions of the United States of America (USA), where the disease has been extensively studied and the bob cat (Lynx rufus) has been identified as the natural reservoir of the parasite (Brown et al., 2010; Shock et al., 2013). Among the other felids, C. felis has also been diagnosed in lions (Panthera leo), tigers (Panthera tigris), pumas (Puma concolor), cheetahs (Acinonyx jubatus), ocelots (Leop- ardus pardalis), tigrinas (Leopardus tigrinus) and jaguars (Panthera onca) (André et al., 2009; Filoni et al., 2012; Lewis et al., 2012; Peixoto et al., 2007; Rotstein et al., 1999; Soares, 2001; Yabsley et al., 2006; Zinkl et al., 1981). Babesia spp. are responsible for causing major tick-borne diseases in the world, namely, babesiosis. Feline babesiosis is most frequently reported in wild and domestic cats from Africa (Penzhorn et al., 2004; Penzhorn, 2006). High parasite loads of Babesia were associated with the death of lions during the canine distemper epidemic in the Serengeti in 1994, where the climatic conditions probably favored coinfections with Babesia spp. and dx.doi.org/10.1016/j.ttbdis.2017.02.005 http://www.sciencedirect.com/science/journal/1877959X http://www.elsevier.com/locate/ttbdis http://crossmark.crossref.org/dialog/?doi=10.1016/j.ttbdis.2017.02.005&domain=pdf mailto:marianafurtado@jaguar.org.br dx.doi.org/10.1016/j.ttbdis.2017.02.005 M.M. Furtado et al. / Ticks and Tick-borne Diseases 8 (2017) 470–476 471 reas ( a s ( A G P r ( i T a t A 2 2 a i − c ( t ( l b t t a Fig. 1. Location of study a ltered the host-parasite relationship (Munson et al., 2008). Babesia pp. have been reported in leopards (Panthera pardus), pampas cat Leopardus colocolo), pumas, cheetahs, and lions (André et al., 2011; verbeck et al., 1990; Ayoob et al., 2010; Bosman et al., 2007; ithaka et al., 2012; Kelly et al., 2014; Lopez-Rebollar et al., 1999; enzhorn et al., 2001; Williams et al., 2014; Yabsley et al., 2006). The jaguar is the largest feline in the Americas and the only epresentative of the genus Panthera on the American continent Seymour, 1989). The jaguar is a top predator and as such plays an mportant role in balancing ecosystems (Soulé and Terborgh, 1999). his study investigated the presence of C. felis and Babesia spp. nd verified possible transmission between jaguars and domes- ic animals in three Brazilian biomes—the Cerrado, Pantanal and mazon. . Materials and methods .1. Study area The current study was conducted in Brazil. Specifically, jaguars nd domestic animals were sampled in the Cerrado biome, n the region of Emas National Park (ENP) (−18,061146 S; 52,941067 W); in the Pantanal biome, on the Caiman Ecologi- al Refuge (−19,80319 S; −56,27373 W) and Barranco Alto Ranch −19,57643 S; −56,16144 W); and in the transitional area between he Cerrado and Amazon biomes, in Cantão State Park (CSP) −9,64503 S; −50,13065 W) (Fig. 1). The Cerrado is the second largest Brazilian biome and an eco- ogical hotspot, with approximately 80% of its area being degraded y human activities (Cavalcanti and Joly, 2002). The ENP is one of he largest protected areas of the Cerrado grasslands and one of he last refuges for the native fauna, including the jaguar (Klink nd Moreira, 2002). The native vegetation surrounding the park dots) in Brazilian Biomes. has been converted to large-scale crop plantations, mainly soybean, corn and, more recently, sugar cane, as well as, to a lesser extent, extensive livestock ranching. The Pantanal is one of the largest contiguous habitats for jaguars outside the Amazon forest (Sanderson et al., 2002) and the largest wetland in the world. Rural properties in this area mainly engage in extensive cattle ranching and, in some cases, ecotourism (Harris et al., 2005). The CSP is located in the transitional area between the Amazon forest and the Cerrado grasslands. The main economic activity of the surrounding rural properties is extensive livestock ranching, and indigenous lands are found nearby (Morton et al., 2006). 2.2. Animals and biological samples collection Between February 2000 and May 2009, 29 free-ranging jaguars were captured as part of the Jaguar Long Term Monitoring Program conducted by the Brazilian Non-Governmental Organization Jaguar Conservation Fund. Two techniques to capture the animals were used: trained hounds and metal cage traps (Furtado et al., 2008). In addition, one juvenile jaguar, raised on an indigenous land near CSP, was also part of this sampling effort. The jaguars were anesthetized intramuscularly with a combi- nation of tiletamine-zolazepam (Zoletil ® or Telazol ® ) at an average dose of 9.7 mg/kg. Immediately after the animal was completely immobilized, physiologic parameters (heart rate, respiratory rate and rectal temperature) were evaluated and monitored at 10 min intervals. Blood samples were collected by internal femoral vein puncture in vacuum tubes with an anticoagulant (EDTA) and phys- ical examinations were conducted. All adult jaguars were fitted with a radiocollar. The animals were placed in a shaded and quiet area to recover from anesthesia. Recap- tures of five individuals were performed at ≥60 day-intervals. After 472 M.M. Furtado et al. / Ticks and Tick-borne Diseases 8 (2017) 470–476 Table 1 Piroplasms species and GenBank accession numbers used for design of the primers that amplify the 18S rRNA gene region. Species GenBank Amplicon (bp) B. canis AY272047; AY072926; AY649326 341 B. rossia AB935165; DQ111760; JQ613105 340 B. vogeli AY072925; AY371197; AY371198; DQ439545; EF636702 340 B. gibsoni AF175300; AF271082; AF396748 339 B. felis AF244912; AY452707 372 B. leo KC790444; AF244911; KC790441 369 B. rodhaini M87565 364 B. capreoli KX839234 341 B. microti AB071177 374 Babesia spp. AF244914; KC790442; JQ861965; JQ861971; AF244913 340–374 C. felisb AF399930; AY531524; AY679105; L19080; GU903911 366 Cytauxzoon spp. EF094470; EU622908; AY309956; KT361081; KT361080 365 T. annulata DQ287944 354 T. ovis FJ603460 358 T. equi KJ573374 353 Theileria spp. DQ866842; AF036336; KP410271; AF078816; JQ923446 349–361 b r T u 2 c p s t i a l ( r o a p t s 1 d C 2 c w ( o P i 2 g o 3 r C a 2 missmatches in foward primer. b 1 missmatch in foward primer. eing fitted with radiocollar, jaguars from the Cerrado and Pantanal egions were monitored through radiotelemetry and camera traps. he monitoring period comprises the interval between an individ- al’s capture day and the last location obtained until November 008, except one animal that was monitored until February 2010. Between May 2008 and January 2010, blood samples were also ollected from 129 domestic dogs and 22 domestic cats from rural roperties surrounding the protected areas. Domestic dogs were ampled in 28 rural properties from the Cerrado, six properties from he Pantanal and 13 from the Amazon. Domestic cats were sampled n six rural properties from the Cerrado, seven from the Pantanal nd three from the Amazon. Blood samples were collected by jugu- ar or cephalic vein puncture in vacuum tubes with anticoagulant EDTA). All blood samples were transported to the research base, sepa- ated into aliquots with or without the addition of an equal volume f saturated saline (100 mM Tris, 100 mM EDTA, 2% SDS), and frozen t −20 ◦C. Handling procedures were consistent with the Ethical Princi- les in Animal Research adopted by the Bioethics Commission of he School of Veterinary Medicine and Animal Science of Univer- ity of São Paulo (FMVZ-USP) and were approved by permit number 471/2008. Instituto Chico Mendes de Conservaç ão da Biodiversi- ade (ICMBio) granted field permits to work in ENP, Pantanal and SP (Permits number 14637, 11214 and 11628, respectively). .3. DNA extraction Biological samples were transported, on average, for 12 h, in oolers with ice packs to São Paulo-SP and stored at −20 ◦C. DNA extraction was performed by pre-incubating the blood ith proteinase K for 4 h at 56 ◦C and then for 15 min at 70 ◦C Rubini et al., 2005) under constant agitation. From 200 �l aliquots f the blood, DNA was isolated using a GFX ® Genomic Blood DNA urification Kit (GE Healthcare) according to the manufacturer’s nstructions. Each DNA sample was eluted in 100 �l of TE buffer. .4. Standardization of PCR Primers that amplify fragments of 339–374 bp of the 18S rRNA ene region were designed based on the conserved DNA sequences f piroplasm species (Table 1): Piro F (5′-TGACACAGGGAGGTAGT- ′) and Piro R (5′-CAACAAAATAGAACCAAAG-3′). The amplification eaction was performed in a total of 25 �l containing 1× Go Taq olorless Mastermix (Promega ® ), 10 pmol of each primer and 5 �l of extracted DNA. Ultrapure water was used as negative control and DNA from domestic dogs that tested positive for Babesia vogeli and DNA from a puma that tested positive for C. felis were used as positive controls. PCR reactions were performed using the fol- lowing thermal cycles: 95 ◦C for 5 min, 52 ◦C for 1 min, 72 ◦C for 2 min and 35 repetitive cycles of 94 ◦C for 30 s, 52 ◦C for 20 s and 72 ◦C for 20 s, followed by a final extension at 72 ◦C for 7 min. The purified amplicon of B. vogeli and C. felis, previously sequenced, were quantified using the Nanodrop 2000 (Thermo Fisher Scientific, Wilmington, DE) and the copy number was calculated. The sensi- tivity of PCR was performed with ten-fold dilutions (105–100) of amplicons using a canine negative DNA pool as a matrix (Helps et al., 2001). The specificity of the assay was tested with positive sam- ples of Babesia bovis, ‘Candidatus Mycoplasma haemominutum’, ‘Candidatus Mycoplasma turicensis’, Erlichia canis, Hepatozoon spp., Mycoplasma haemofelis, Rickettsia sp. strain NOD and Rangelia vitalli. Moreover, the primers were aligned with 72 sequences available in GenBank, including species of the genera Babesia, Cytauxzoon, Rangelia and Theileria, to analyze the nucleotide identity (Supple- mentary material). To confirm the agent and identify possible co-infections, two specific PCR protocols that separately amplified Babesia spp. and C. felis were performed. For C. felis detection, a 284 bp fragment of the 18S rRNA gene was amplified using the primers and amplification conditions described by Birkenheuer et al. (2006). For Babesia spp. detection, a 340 bp fragment of the 18S rRNA gene was amplified by PCR using the primers 455–479 F and 793-772 R as described by Birkenheuer et al. (2003). The amplification reactions were per- formed in a total of 25 �l containing 1× GoTaq Colorless Mastermix (Promega ® ), 10 pmol of each primer and 5 �l of extracted DNA. For C. felis, DNA from a puma that tested positive for the agent was used as the positive control; and for Babesia spp., DNA of an isolate of B. vogeli from a domestic dog was used as the positive control. Ultrapure water was used as the negative control. Aliquots of 5 �l of each of the amplified products were ana- lyzed in 1.5 or 2% agarose gel with gel Red (0.05 �l/ml; Uniscience ® ) by electrophoresis at 80 V for 30 min in TAE buffer and visualized under a UV transluminator. PCR fragments were estimated by com- parison with known amounts of eletrophoretic standards, using a 100 bp ladder (Invitrogen ® ). Diagnoses were presented according to the species and study area sampled. An animal that had at least one positive result during capture or recapture was considered to be positive (Patterson and Lello, 2003). ick-borne Diseases 8 (2017) 470–476 473 2 g i P M S G D t f r 1 i t U w l 2 p l j 1 z a m t i w t o q a T o ( ( g o u ( l 7 h n s t t a 3 c O w u t p Table 2 Results of molecular tests by study areas and species sampled between February 2000 and January 2010. Biome Species Examined Positive for C. felis (%) Babesia spp. (%) Cerrado Jaguar 4 4 (100) 0 Domestic dog 63 0 5 (7.9) Domestic cat 7 0 0 Pantanal Jaguar 22 22 (100) 0 Domestic dog 19 0 0 Domestic cat 10 0 0 Amazon Jaguar 4 3 (75) 0 M.M. Furtado et al. / Ticks and T .5. DNA integrity All samples that were PCR negative were tested for the lyceraldehydes-3-phosphate (GAPDH) gene to confirm the ntegrity of nucleic acids and to discard the presence of CR inhibitors. PCR was carried out with 1× GoTaq Green Astermix (Promega ® ), 10 pmol of each primer GAPDH fel (5′- GCCATCAATGACCCCTTCAT-3′) and GAPDH fel AS (5′- CCGTGGAATTTGCCGT-3′) (Leutenegger et al., 1999), 2.5 �l of the NA sample and ultrapure water to a final volume of 25 �l. Reac- ion conditions were 94 ◦C for 3 min, 35 cycles of 94 ◦C for 20 s, 55 ◦C or 30 s and 72 ◦C for 30 s, and final extension at 72 ◦C for 5 min. The eaction was considered positive when an amplicon of 81 (dog) or 64 bp (cats and jaguar) was present. Aliquots of 5 �l of each of the amplified products were analyzed n 2% agarose gel with gel Red (0.05 �l/ml; Uniscience ® ) by elec- rophoresis at 80 V for 30 min in TAE buffer and visualized under a V transluminator. PCR fragments were estimated by comparison ith known amounts of eletrophoretic standards, using a 100 bp adder (Invitrogen ® ). .6. Sequencing and phylogenetic analysis Sequencing and phylogenetic analysis were performed with six ositive samples that were randomly selected and include the fol- owing: two domestic dogs, C. lupus familiaris 31 and 117, and one aguar, P. onca 156, from the Cerrado, two jaguars, P. onca 136 and 43, from the Pantanal and one jaguar, P. onca 149, from the Ama- on. The PCR products were purified with the GFXTM PCR DNA nd Gel Band Purification Kit ® (GE Healthcare) according to the anufacturer’s recommendations and quantified in comparison to he Low Mass DNA Ladder ® (Invitrogen). Bidirectional sequenc- ng was performed using the Sanger’s method (Sanger et al., 1977) ith BigDye Terminator 3.1 Cycle Sequencing Kit (Applied Biosys- ems) and ABI-3500 sequencer (Applied BiosystemsTM). The quality f the sequences was determined using Phred electropherogram uality analysis software (Togawa and Brigido, 2003), available t http://asparagin.cenargen.embrapa.br/phph/, last updated 2012. he final sequence was obtained with the Cap-Conting application f the Bioedit Sequence Alignment Editor version 7.2.5 program Hall, 1999), and subject to comparison with the GenBank database http://www.ncbi.nlm.nih.gov/BLAST). The nucleotide sequences enerated for this study were aligned with homologous sequences f C. felis and Babesia spp. retrieved from the GenBank database sing CLUSTAL/W method implemented in MEGA 6.06 program Tamura, 2013). The values of the nucleotide identity were calcu- ated using the Bioedit Sequence Alignment Editor program version .2.5 (Hall, 1999). The C. felis and Babesia nucleotide sequences of these data set ave been deposited in the GenBank database under the accession umbers: KY346853 to KY346855. A phylogenetic tree was con- tructed with the program MEGA 6.06 (Tamura et al., 2013) using he Maximum Likelihood and Neighbor-Joining methods based on he Kimura 2-parameter model with a discrete Gamma distribution nd 1000 bootstrap replicates. . Results Almost all captured jaguars (96.7%, n = 29) were in good physical ondition and showed no clinical signs of any apparent disease. nly one of the female jaguars captured in the Amazon presented ith low body weight and dehydration; it also had no incisors, no pper left canine and the lower canines were worn. Jaguars from he Cerrado and Pantanal regions were monitored, on average, for eriods of 21.7 months (range of 1–91 months). Domestic dog 47 0 5 (10.6) Domestic cat 5 0 0 All of the samples analyzed were positive for the endogenous control GAPDH in the DNA extractions. The sensitivity analysis of the designed primers (Piro F and Piro R) showed a limit of detec- tion of 2 copies per reaction for both B. vogeli (KY346855) and C. felis (KY346854), which was confirmed in quadruplicate performed in 2 distinct runs. The specificity assay demonstrated no amplification of ‘Ca. Mycoplasma haemominutum’, ‘Ca. Mycoplasma turicensis’, M. haemofelis, Hepatozoon spp., Rickettsia sp. strain NOD, R. vitalli, E. canis and B. bovis. In silico analysis, with alignment of 72 sequences available in GenBank, showed 100% to 94% (1 mismatch) of iden- tity with the genera Babesia, Cytauxzoon and Theileria, except the species B. rossi, B. lengau and B. bovis (Supplementary material). Results of molecular tests are shown in Table 2. Five recaptured jaguars (one from the Cerrado and four from the Pantanal) pre- sented with the same diagnosis as their initial captures. Four rural properties from the Cerrado (14.3%) and two from the Amazon (15.4%) had dogs only infected with B. vogeli but no dogs or cats were infected with C. felis. The four Cytauxzoon nucleotide sequences (330 bp) had 99.6% of identity between them. As P. onca 136 and 143 had one degener- ate base in the same position, a comparison with other sequences from the GenBank database was not included. P. onca 153 and 156 showed 100% and 99.6% of identity, respectively, with C. felis from a domestic cat with a fatal infection from the USA (AY679105); 98.7% and 99% of identity from a captive ocelot (GU903911) and a tigrina (DQ382277) from Brazil; 91.2% and 90.9% of identity with Cytaux- zoon spp. from wildcat (Felis silvestris) (KT361081) and 90.9% and 90.6% of identity from lynx (Lynx lynx) (KT361080) from Europe. Babesia spp. nucleotide sequences (304 bp) from the domestic dogs were identical and showed 100% of identity with B. vogeli from dogs (KT333456 KU710803) and cat (EF636702) from Brazil. The phylogenetic analysis shows that P. onca 153 (KY346853) and 156 (KY346854) sequences segregated with C. felis, and C. lupus familiaris 31 (KY346855) segregated with B. vogeli sequences retrieved from GenBank (Fig. 2). 4. Discussion The C. felis infection in all but one jaguar suggests the high preva- lence of this pathogen in the study sites and the important role of jaguars in the maintenance of the parasite in the wild. This is the first report of C. felis in free-ranging jaguars from the Cerrado, Pantanal and Amazon biomes. In Brazil, C. felis had been detected in jaguars, ocelots, pumas and lions kept in captivity (André et al., 2009; Filoni et al., 2012; Peixoto et al., 2007). A higher incidence of C. felis in free-ranging felids is probably related to a higher exposure to vectors in the natural environment. The jaguar raised in the indigenous land was the only negative result for C. felis in our study, probably because this animal did not move through natural habitats and had less contact with ticks. http://asparagin.cenargen.embrapa.br/phph/ http://asparagin.cenargen.embrapa.br/phph/ http://asparagin.cenargen.embrapa.br/phph/ http://asparagin.cenargen.embrapa.br/phph/ http://asparagin.cenargen.embrapa.br/phph/ http://asparagin.cenargen.embrapa.br/phph/ http://asparagin.cenargen.embrapa.br/phph/ http://www.ncbi.nlm.nih.gov/BLAST http://www.ncbi.nlm.nih.gov/BLAST http://www.ncbi.nlm.nih.gov/BLAST http://www.ncbi.nlm.nih.gov/BLAST http://www.ncbi.nlm.nih.gov/BLAST http://www.ncbi.nlm.nih.gov/BLAST http://www.ncbi.nlm.nih.gov/BLAST 474 M.M. Furtado et al. / Ticks and Tick-bo Fig. 2. Phylogenetic tree of the 18S rRNA gene nucleotide sequences of C. felis from jaguars and B. vogeli from domestic dogs using Maximum Likelihood and Neighbor- Joining methods based on the Kimura 2-parameter model with a discrete Gamma distribution and 1000 bootstrap replicates. The circles indicate the sequences from this study. Bootstrap values superior of 70 are shown at the nodes. Bar represents substitutions number per site. rne Diseases 8 (2017) 470–476 In the USA, Dermacentor variabilis and Amblyomma americanum are responsible for transmitting C. felis (Blouin et al., 1984; Reichard et al., 2009; Reichard et al., 2010). However, these two tick species do not occur in South America, where other ixodid species are probably involved in the hemoparasite transmission (Peixoto et al., 2007). In the present study, jaguars were mainly parasitized by dif- ferent species of the Amblyomma genus (data not shown), that could have acted as vector for C. felis. Nearly 100% of infected jaguars did not show clinical signs or alterations in their movement patterns during the monitor- ing period suggesting that they did not develop clinical disease and strengthening the hypothesis of the involvement of jaguars as potential carriers of C. felis in the study areas. The poor physical condition observed in a single jaguar from the Amazon was likely due to the animal’s advanced age and not related to the presence of C. felis. André et al. (2009) suggested that Brazilian wild felids could act as potential reservoirs of C. felis, similar to bobcats and pumas in the USA (Blouin et al., 1984; Glenn et al., 1983; Rotstein et al., 1999; Shock et al., 2011). Although not common, fatal infections caused by C. felis were reported in bobcats (Nietfeld and Pollock, 2002), tigers (Garner et al., 1996) and lions (Peixoto et al., 2007)—highlighting the importance of monitoring the agent in jaguars, and not discard- ing the potential threat to immunosuppressed animals or animals with low genetic variability. The PCR developed in the present study revealed high sensitivity and expected specificity when tested with the main hemoparasites of the studied species. It demonstrates the utility of this assay in screening studies since the primers match with different species of piroplasms. Comparing 298 bp from the Cytauxzoon nucleotide sequence from our jaguars with sequences from the other Brazil- ian jaguar described in the literature (EU376527), 100% of identity occurred with P. onca 153, and 99.6% of identity with P. onca 156 was found. Additionally, our sequences were closely related either to the domestic cat with fatal infection (AY679105) and the domes- tic cat with survival infection (AF399930). The identical sequences appear to support the hypothesis of a common source of C. felis infection to wild and domestic cats, but larger fragment sequencing and different target genes are still needed to confirm this hypoth- esis. The four jaguars that tested positive in the recapture events con- firmed the presence of the agent after the time intervals of 7–38 months, suggesting reinfection during that time or persistent infec- tions, as reported for bobcats (Brown et al., 2010; Meinkoth and Kocan, 2005). The absence of Cytauxzoon spp. in domestic dogs was expected since C. felis is a specific parasite of felines (Kier et al., 1982; Meinkoth and Kocan, 2005), but the agent was also absent in the domestic cats sampled. As the pathogenic potential of Cytauxzoon isolates in Brazil has not been utterly assessed, this absence cannot be related to the high fatality rate observed in the USA (Meinkoth and Kocan, 2005). Cats from our study were in good physical condi- tion and apparently had no clinical signs of the infectious disease. In domestic cats in Brazil, C. felis has already been reported in the state of Rio de Janeiro in a cat co-infected with ‘Ca. Mycoplasma haemominutum’, which died four days after the onset of symp- toms (Maia et al., 2013) and in the state of Minas Gerais (André et al., 2015). Conversely, cats from catteries in central-west Brazil (Miceli et al., 2013) and from a zoo environment in southeast Brazil (André et al., 2014) were PCR negative for the agent. The pathogenicity of Cytauxzoon spp. genotypes that circulate in domestic and wild cats in Brazil remains unknown. André et al. (2014) suggested that C. felis circulates more often in wild felids compared to cats in Brazil, a statement corroborated by our findings. Babesia spp. was not detected in jaguars or domestic cats from the three study areas, and only dogs from the Cerrado and the Ama- zon surroundings tested positive for B. vogeli. As in the present ick-bo s P e t w a R e d ( b t h ( r e c 2 t p w b a v s i t a o s a a u 5 j a p r s a A a E a f a R a C t d a w I L M.M. Furtado et al. / Ticks and T tudy, no free-ranging jaguars in Venezuela, French Guiana and the antanal were found to be positive for Babesia spp. (Criado-Fornelio t al., 2009; Thoisy et al., 2000). André et al. (2011) reported cap- ive jaguars seropositive for B. vogeli, however, the same individuals ere negative when performing molecular testing. The low exposure of dogs to Babesia spp. in the present study gree with the frequencies of 5.2% and 18.8% found in dogs from io de Janeiro (O’Dwyer et al., 2001) and from Minas Gerais (Maia t al., 2007) in Brazil. The identification of B. vogeli in two positive ogs agrees with the findings of Passos et al. (2005) and Maia et al. 2007) that this is the species responsible for most cases of canine abesiosis in Brazil. The absence of Babesia spp. in domestic cats is consistent with he literature (Dantas-Torres and Figueredo, 2006), stating that cats ave a lower predisposition to contract the infection than dogs Ayoob et al., 2010). Feline babesiosis was diagnosed and has been eported regularly in South Africa (Ayoob et al., 2010; Penzhorn t al., 2004). In Brazil, B. vogeli was recently detected in domestic ats from São Paulo (André et al., 2014), Mato Grosso (André et al., 015) and Rio Grande do Sul (Malheiros et al., 2016). Regarding he wild felids, Babesia closely related to B. leo was detected in a ampas cat kept in captivity (André et al., 2011). The vector of B. vogeli, the tick Rhipicephalus sanguineus s.1., as identified parasitizing domestic dogs in the three study areas ut was not found in jaguars (data not shown). This reinforces the bsence of Babesia in jaguars and the specificity of B. vogeli to this ector. Munson et al. (2008) highlighted the importance of Babesia spp. howing that coinfection with the canine distemper virus resulted n the death of lions in the outbreak of 1994 in Serengeti. Thus, he presence of Babesia spp. in domestic dogs from the Cerrado nd the Amazon surroundings should be monitored, as well as its ccurrence in jaguars. Babesia spp. possibly have specificity for host pecies and the infection by species usually diagnosed in domestic nimals could be fatal to wildlife, especially in stressful situations nd in animals that are immunosuppressed or in endangered pop- lations (Penzhorn, 2006). . Conclusions The results of this study suggest the participation of free-ranging aguars in the maintenance of C. felis in nature and the existence of sylvatic cycle of the agent in the study areas. Otherwise, jaguars robably are not the normal host for Babesia spp., but the occur- ence of this hemoparasite in dogs from surrounding properties hould be carefully monitored due to the history related to the gent. cknowledgments This work was supported by the FAPESP (Fundaç ão de Amparo Pesquisa do Estado de São Paulo), grant number 2007/50941-5, arthwatch Institute and the Memphis Zoo. MF received a schol- rship from FAPESP (Process no. 2007/50942-1). We thank ICMBio or granting us permission to work in ENP, Pantanal and CSP; ENP nd CSP managements, Caiman Ecological Refuge, Barranco Alto anch and Naturatins for logistical support; and dog/cat owners for llowing biological sample collection. We are indebted to Marcelo arvalho, Mario Ferraro, Natália Camargo and Earthwatch volun- eers for their invaluable help with biological sample collection of omestic animals and to Cyntia Kashivakura, Eduardo Ramos, Fabi- no Bortolini, James Bortolini and Tiago Boscarato for their help ith field work and jaguar capture. We are also grateful to Cassia kuta and Gisele Oliveira for their helpful support and Marcelo Baia abruna for the positive controls used in the specificity analysis. rne Diseases 8 (2017) 470–476 475 Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ttbdis.2017.02. 005. 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