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Genetic diversity of Hepatozoon spp. in Hydrochoerus hydrochaeris and Pecari tajacu from eastern Amazon
Ticks and Tick-borne Diseases xxx (xxxx) xxx–xxx
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Original article
Genetic diversity of Hepatozoon spp. in Hydrochoerus hydrochaeris and Pecari tajacu from eastern Amazon ⁎
Laise de Azevedo Gomesa, , Leopoldo Augusto Moraesa, Délia Cristina Figueira Aguiara, Hilma Lúcia Tavares Diasa, Ana Silvia Sardinha Ribeirob, Henrique Piram do Couto Rochab, ⁎ Márcio Roberto Teixeira Nunesc, Evonnildo Costa Gonçalvesa, a
Laboratório de Tecnologia Biomolecular, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, PA, Brazil Grupo de Estudos de Animais Selvagens, Universidade Federal Rural da Amazônia, Belém, PA, Brazil c Centro de Inovações Tecnológicas, Instituto Evandro Chagas, Levilândia, Ananindeua, PA, Brazil b
A R T I C L E I N F O
A B S T R A C T
Keywords: Hepatozoon canis Hepatozoon cuestensis Mammals Northern Brazil
This study aimed to identify and characterize genetically species of the genus Hepatozoon detected in Hydrochoerus hydrochaeris (capybaras) and Pecari tajacu (collared peccaries) from two localities from the Eastern Amazon. Blood samples from 196 free-living H. hydrochaeris from Marajó Island and 109 P. tajacu kept in captivity in Belém, Pará, were collected and analyzed for the presence of Hepatozoon spp. Partial sequences of the 18S rRNA gene were obtained and analyzed in comparison to others available in the NCBI database. Our results demonstrated a high prevalence of Hepatozoon canis in both mammals and the existence of four haplotypes of Hepatozoon spp., three of Hepatozoon canis and one of Hepatozoon cuestensis, found only in H. hydrochaeris. In addition, these data increase the genetic diversity of H. canis from the Eastern Amazon, as well as reporting, for the first time, the infection of mammals by H. cuestensis and P. tajacu by H. canis.
1. Introduction
Unlike most of the pathogens that are transmitted by an arthropod vector through the salivary glands during the blood repast, the main route of infection of the intermediate host by Hepatozoon spp. is oral ingestion of the definitive host containing mature polysporocyst oocysts (Baneth et al., 2007; Baneth, 2011; Desser, 1993). Thus, geographical distribution of the final host and the existence of reservoirs, such as some wild animals, can determine the dispersion patterns of Hepatozoon spp. (Dantas-Torres, 2010; Najm et al., 2014). In fact, foxes and golden jackals appear to have great importance in the distribution of H. canis (Duscher et al., 2013; Farkas et al., 2014; Imre et al., 2015; Najm et al., 2014). The worldwide occurrence of H. canis, including in places where there is no report of the existence of the tick vector Rhipicephalus sanguineus sensu lato, has reinforced the concept that other species of ticks have vectorial competence for H. canis (Majláthová et al., 2007; Mitková et al., 2014; Mitková et al.,2016). In Brazil, despite the high abundance of R. sanguineus sensu lato (Araújo et al., 2015; DantasTorres et al., 2009; Soares et al., 2006), studies carried out in an attempt to detect H. canis infection did not find any evidence of the presence of this protozoan (Demoner et al., 2013; Forlano et al., 2005, Gomes et al., 2010).
Hydrochoerus hydrochaeris (capybaras) and Pecari tajacu (collared peccaries) are widely distributed in South America and are among the Brazilian wild mammals that are of great economic importance due to the appreciation of their meat and to the interest of the international leather industry (Mayor et al., 2010; Ojasti, 1991). In nature, these animals appear to play an important role in maintaining the life cycle of apicomplexan protozoa such as Toxoplasma gondii (Abreu et al., 2016; Thois et al., 2003), Eimeria spp. (Wilber et al., 1996) and Hepatozoon canis (Criado-Fornelio et al., 2009). In wild mammals from Brazil, H. canis has been detected in Pseudalopex vetulus (hoary fox), Cerdocyon brachyurus (maned wolf) (André et al., 2010), Dusicyon thous (crab-eating fox), Pseudalopex gymnocercus (pampas fox) (Criado-Fornelio et al., 2006), Hydrochoerus hydrochaeris (capybara) (Criado-Fornelio et al., 2009) and Leopardus pardalis (ocelot) (Braz and Umeda, 2015). Considering that the genus Hepatozoon belongs to the group of hemogregarines, one of the characteristics of species of this genus is its heteroxenous life cycle involving an intermediate vertebrate host and a definitive invertebrate hematophagous host (Baneth and Shkap, 2003).
⁎ Corresponding authors at: Laboratório de Tecnologia Biomolecular, Instituto de Ciências Biológicas, Universidade Federal do Pará, Cidade Universitária, Av. Augusto Corrêa no. 1 Bairro Guamá, Belém, Pará, 66075-110, Brazil. E-mail addresses: [email protected] (L. de Azevedo Gomes), [email protected] (E. Costa Gonçalves).
https://doi.org/10.1016/j.ttbdis.2017.11.005 Received 24 April 2017; Received in revised form 28 October 2017; Accepted 9 November 2017 1877-959X/ © 2017 Elsevier GmbH. All rights reserved.
Please cite this article as: Gomes, L.d.A., Ticks and Tick-borne Diseases (2017), https://doi.org/10.1016/j.ttbdis.2017.11.005
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Table 1 Hepatozoon spp. 18S rRNA sequences used for phylogenetic analysis plus additional information retrieved from the GenBank database. Strain
GenBank accession No.
Species
Host
Reference
Brazil Iran Turkey Croatia Brazil Brazil Hungray Croatia Czech Republic Brazil Brazil USA Brazil Japan Brazil Italy Bosnia and Herzegovina Bosnia and Herzegovina Croatia Croatia Ghana Canada Morocco Morocco Brazil Brazil Brazil Brazil Brazil Brazil Australia Australia China Brazil Chile
KU729738 KT736298 KX588232 HM212626 KU729737 EF622096 KJ572976 FJ497022 KU597242 KC342527 KC342524 AF176836 KU729739 AB771547 KY684005 KY649445 KX757031 KX757032 KT274177 KT274178 EF157822 AF130361 KU680464 KU680466 AY461377 KC127679 KY684007 KJ413113 KY684006 KY684004 AY252110 AY252105 KF939622 KU667308 FJ719813
H. canis H. canis H. canis H. canis H. canis H. canis H. canis H. canis H. canis H. cuestensis H. cuestensis H. americanum H. americanum H. felis H. felis H. silvestris H. silvestris H. silvestris H. ayorgbor H. ayorgbor H. ayorgbor H. catesbianae Hepatozoon sp. Hepatozoon sp. Hepatozoon sp. Hepatozoon sp. Hepatozoon sp. Hepatozoon sp. Hepatozoon sp. Hepatozoon sp. Hepatozoon sp. Hepatozoon sp. Hepatozoon sp. Hepatozoon sp. Hepatozoon sp.
Dog Dog Dog Fox Dog Capybara Golden Jackal Dog Tick Rattlesnake Rattlesnake Dog Dog Iriomote cat Ocelot Domestic cat European wild cat European wild cat Wood mouse Yellow-necked fieldmouse Ball python No data available Desert wall gecko Moorish gecko Fox Fox Caiman Caiman Turtle Paca Slaty grey snake Water python King rat snake Wild rodent Monito del monte
Gomes et al. (2016) Unpublished Unpublished Dezdek et al. (2010) Gomes et al. (2016) Criado-Fornelio et al. (2009) Farkas et al. (2014) Vojta et al. (2009) Hamšíková et al. (2016) O’Dwyer et al. (2013) O’Dwyer et al. (2013) Mathew et al. (2000) Gomes et al. (2016) Unpublished Soares et al. (2017) Giannelli et al. (2017) Hodžić et al. (2017) Hodžić et al. (2017) Unpublished Unpublished Sloboda et al. (2007) Carreno et al. (1999) Tomé et al. (2016) Tomé et al. (2016) Criado-Fornelio et al. (2006) Almeida et al. (2013) Soares et al. (2017) Unpublished Soares et al. (2017) Soares et al. (2017) Ujvari et al. (2004) Ujvari et al. (2004) Unpublished Demoner et al. (2016) Merino et al. (2009)
each sample was extracted from a 300μL aliquot of the blood by using a standard phenol-chloroform procedure, as described by Sambrook et al. (1989). DNA quality was checked by electrophoresis in an agarose gel, and the DNA was then quantified using the Qubit 2.0 fluorometer (Thermo Fisher Scientific).
In addition, there is no consensus so far about which tick species is in fact eco-epidemiologically important in the transmission of H. canis in Brazil. While O’Dwyer et al. (2001) indicated Amblyomma cajennense sensu lato as a potential vector, Demoner et al. (2013) refuted this possibility and suggested the existence of more than one strain of Amblyomma ovale capable of becoming infected by H. canis. The detection of mature oocysts of H. canis in the haemocoel of the cattle tick, Rhipicephalus microplus, collected from a dog and identification of DNA of the protozoan in this tick suggest that R. microplus can be a possible vector of H. canis (Miranda et al., 2011), however the experimental infestation of H. canis-infected dogs with R. microplus was not successful (Demoner et al., 2013). Most of our understanding of the disease caused by some species of the genus Hepatozoon comes from studies with domestic dogs (Aydin et al., 2015; Eiras et al., 2007; Ewing et al., 2000; Harvey et al., 2016; Li et al., 2008). In fact, the infection of wild animals by Hepatozoon spp. has been poorly studied in Brazil, principally in some regions, such as the Amazon. Thus, the objective of this study was to investigate the occurrence and genetically characterize the species of Hepatozoon found in H. hydrochaeris of extensive livestock farming and P. tajacu kept in captivity in the State of Pará, Brazil.
2.2. PCR amplification and DNA sequencing Molecular diagnosis of Hepatozoon spp. was undertaken according to Gomes et al. (2016) which is based on the partial amplification through nested PCR of the 18S rRNA gene of Hepatozoon spp. Briefly, we used HepF and HepR primers (Inokuma et al., 2002) in the first round of PCR followed by a second round of PCR with HepNF and HepNR primers (Gomes et al., 2016) whose amplification produced a fragment of approximately 300bp. In order to properly identify the Hepatozoon species, positive samples identified after molecular diagnosis, according to Gomes et al. (2016), were submitted to another nested PCR assay to amplify a larger fragment (∼670bp) of the 18S rRNA than the one amplified in the molecular diagnosis protocol. In this second assay, the first round of amplification included: NBA1 (5′GGTTGATCCTGCCAGTAGT3′) (Criado-Fornelio et al., 2003) and HPF2 (5′GACTTCTCCTTCGTCT AAG3′) (Criado-Fornelio et al., 2006) primers, while the second round PCR included HepF and HepR primers. First round PCR was carried out in 25μL reactions with 10–20 ng of the DNA template, 2.5mM MgCl2, 0.125mM each of deoxyribonucleotide triphosphates (dNTPs), 10mM Tris-HCl, 50mM KCl, 0.2μM of each primer, and 1 U Taq DNA polymerase (Invitrogen). The amplification reaction consisted of 40 cycles of 30 s at 95 °C, 30 s at 58 °C, and 2 min at 72 °C, preceded by 10 min at 95 °C and followed by 10 min at 72 °C. Second-round PCR was carried out under the same conditions of the first round PCR used here to the
2. Materials and methods 2.1. Samples and DNA extraction A total of 196 blood samples from H. hydrochaeris raised by extensive livestock farming on Marajó Island (State of Pará, Brazil) and 109 from P. tajacu maintained in captivity in the Brazilian Agricultural Research Cooperation (EMBRAPA Western Amazon) were collected into tubes containing ethylenediaminetetraacetic acid (EDTA). Total DNA of 2
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gene Hepatozoon spp. evidenced two clades well supported by posterior probability value (Fig. 1). CPV02 and CPV03 haplotypes, both derived from H. hydrochaeris samples and CPV01/CTU01 haplotype were included in the most derived clade that consisted of H. canis sequences, whose hosts were mostly mammals (dogs, fox, capybara and golden jackal). CPV04 haplotype grouped within the most ancestral clade represented by sequences H. cuestensis and Hepatozoon sp. obtained from biological samples of reptiles. This haplotype shared 99% nucleotide identity with H. cuestensis sequences (KC342527, KC342524, KU680464, KU680466) obtained from reptiles. Pairwise comparison of 40 sequences showed p distance ranging from 0 to 31.49% (Supplementary Table 1). While the genetic distances between H. canis strains was estimated at 2.04% (CPV02 x H. canis EF622096), the highest p distance between H. cuestensis strains was 2.55%.
molecular diagnosis, except that the DNA samples were changed by 1μL of the PCR product amplified with NBA1/HPF2 primers. For both analyses (i.e. molecular diagnosis and taxonomic diversity), DNA of H. canis was used as the positive control, while sterile bi-distilled water was used as the negative control. A 100bp molecular marker (Invitrogen DNA ladder) was used to estimate the size of each amplified fragment Amplicons of the second round PCR (∼670bp) and, in some cases, those obtained by HepNF and HepNR primers (∼300bp) were enzymatically purified with Illustra ExoProStar (GE Healthcare). Nucleotide sequencing was performed in an ABI 3500 xL Genetic Analyzer (Thermo Fisher Scientific), according to the manufacturer’s specifications. BioEdit software (Hall, 1999) was used to align forward and reverse sequences. 2.3. Data analysis
4. Discussion In addition to sequences obtained here, the phylogenetic analysis was performed including thirty-five Hepatozoon spp. 18S rRNA sequences retrieved from the GenBank database (Table 1) and one Babesia sp. 18S rRNA sequence which was used as the outgroup. MrModeltest 2.3 (Nylander, 2004) was used to determine the appropriate model. Bayesian Inference (BI) was conducted based on Markov Chain Monte Carlo (MCMC) tree searches as implemented in MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). We conducted two parallel runs of four simultaneous MCMC searches for 5 million generations each, sampling one tree every 500 generations, and discarding the results of the first 1000 trees as burn-in. The remaining trees were used by MrBayes to estimate the posterior probability of each node in our phylogenetic reconstruction. Tracer v1.4.1 (Rambaut and Drummond, 2008) was used to check the stationarity of all parameters sampled by the chains. The p distances between pairwise sequences were performed using PAUP 4.0b10 (Swofford, 2002).
A notable result of this research, corroborating Gomes et al. (2016), is that molecular detection based only on the HepF and HepR primers (Inokuma et al., 2002) has low efficiency, probably due to the lower sensitivity of single PCR compared to the nested PCR. In addition, most of the positive samples were obtained through the protocol of Gomes et al. (2016), different from that achieved using the nested PCR protocol with the NBA1/HPF2 primers followed by amplification with the HepF/HepR primers. Thus, it is feasible to infer that the latter nested PCR protocol is less efficient than the nested PCR protocol described by Gomes et al. (2016). Sequencing of twenty-eight samples failed probably due to lack of success in the purification process. Based on sequencing, the high prevalence of H. canis in H. hydrochaeris (64/80, thus 80%) and P. tajacu (32/49, thus 65.3%) corroborates other studies, which have shown that presence of this pathogen seems to be common in certain wild animals, for example, 40.7% of foxes from Bosnia (Hodžić et al., 2015), 60% of golden jackals from Hungary (Farkas et al., 2014) and 64% of Brazilian capybara analyzed by Criado-Fornelio et al. (2009). Despite the lack of data on the development of disease due to infection by Hepatozoon spp. in wild animals, the high prevalence of infection as observed here suggests that H. hydrochaeris and P. tajacu may be reservoirs of these protozoa, but other studies such as transmission experiments need to be carried out. This is relevant because there is no evidence of host-specific parasitism with Hepatozoon spp. In fact, our detection of H. cuestensis (CPV04 haplotype) in H. hydrochaeris reinforces this hypothesis, since, up to present study, this parasite had been detected only in reptiles (O’Dwyer et al., 2013; Tomé et al.,2016). The largest value of genetic distance between sequences of this parasite was similar to that which we obtained for the H. canis sequences, proving that CPV04 haplotype belongs to the clade of H. cuestensis sequences found in reptiles. Infection of snakes by Hepatozoon spp. can occur through the ingestion of intermediate hosts containing cystic forms of these protozoa (Smith, 1996). In fact, the Brazilian snake Crotalus durissus terrificus, for which the species H. cuestensis (KC342527) was described, feeds on rodents, indicating that infection occurs if the prey is infected or if it has infected ticks or mites (O’Dwyer et al., 2013). On the other hand, H. hydrochaeris may accidentally ingest small insects infected with Hepatozoon spp. when feeding on grasses. To the best of our knowledge this study provided the first record of the causative agent of Old World canine hepatozoonosis in P. tajacu, whose haplotype (CPV01/CTU01) seems to be widely distributed in State of Pará, since it was also detected in H. hydrochaeris from Marajó Island and in dogs from the Belém metropolitan area (KU729738) by Gomes et al. (2016). In addition to the Belém 01 (KU729737) and Belém 02 (KU729738) haplotypes of H. canis identified by Gomes et al. (2016) in dogs from northern Brazil, we found two new haplotypes (CPV02 and CPV03) of H. canis in H. hydrochaeris from the same region,
3. Results Only 11 samples, all from H. hydrochaeris, were positive using the HepF and HepR primers (Inokuma et al., 2002), while through the protocol of Gomes et al. (2016) 80 (40.8%) H. hydrochaeris and 49 (44.9%) P. tajacu were positive. Of 129 (80 of H. hydrochaeris and 49 of P. tajacu) positive samples according to the protocol of Gomes et al. (2016), only 92 (60 of H. hydrochaeris and 32 of P. tajacu) were amplified with the nested PCR protocol based on the NBA1/HPF2 (CriadoFornelio et al., 2003, 2006) and HepF/HepR primers. For the diversity analysis we obtained 101 Hepatozoon spp. DNA sequences, 69 (60 of ∼660bp and 9 of ∼300bp) of H. hydrochaeris and 32 (∼660bp) of P. tajacu. Sequencing of twenty-eight samples (11 of H. hydrochaeris and 17 of P. tajacu) failed. Hepatozoon sequences of H. hydrochaeris resulted in four haplotypes, whose analysis with the NCBI BLAST tool indicated three of H. canis and one of Hepatozoon cuestensis: CPV01/CTU01 (GenBank accession number KY965141), CPV02 (GenBank accession number KY965142), CPV03 (GenBank accession number KY965143) e CPV04 (GenBank accession number KY965144). Hepatozoon sequences obtained from P. tajacu resulted in a single haplotype, CTU01, which was identical to the CPV01 haplotype found in H. hydrochaeris. CPV04 haplotype (H. cuestensis) presented the highest number of polymorphisms with eight transitions and four transversions in relation to the CPV01/CTU01 haplotype of H. canis (Table 2). The most frequent haplotype was CPV01/CTU01, while the least frequent was CPV02. Bayesian Inference assumed a GTR + G model of nucleotide substitution with estimated base frequencies (A = 0.3142, C = 0.1613, G = 0.2047, T = 0.3199), substitution model (A-C = 0.9128, AG = 3.3867, A-T = 1.6333, C-G = 0.1355, C-T = 3.3867, G-T = 1), and rates for variable sites following a gamma distribution (G = 0.6607). A topology tree using partial sequences of 18S rRNA 3
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Table 2 Polymorphic nucleotide sites between the haplotypes of the 18S rRNA partial gene of Hepatozoon spp. found in H. hydrochaeris and P. tajacu from Eastern Amazon. Haplotype
CPV01/CTU01 CPV02 CPV03 CPV04
Host
Capybara/Collared peccary Capybara Capybara Capybara
Number of samples
60/32 1 3 5
Sequence length (bp)
656 300 300 300
Nucleotide position 253
284
287
288
289
290
302
316
326
331
362
364
365
377
T . G G
C . . T
T . . C
T . . A
T . . A
G . . A
A . . G
A . . G
G . . A
G . A A
A G . .
C . . T
C . . A
A . G .
The symbol (.) indicates a conserved nucleotide.
Fig. 1. Phylogenetic relationship between the partial sequences of the Hepatozoon 18S rRNA gene of the species found in H. hydrochaeris and P. tajacu from the Eastern Amazon and species of the same genus. The numbers in the nodes indicate the value of the Bayesian posterior probability. The scale bar indicates an evolutionary distance of 0.05 nucleotides per position in the sequence. The sequences obtained in this study are in bold. One Babesia sp. sequence was used as outgroup.
Brazilian Agricultural Research Cooperation (EMBRAPA) for ceding the biological samples from P. tajacu.
indicating this parasite has high genetic diversity in northern Brazil. Among the H. canis haplotypes, CPV03 was the one with the most variable sites and high nucleotide identity (99%) shared with a sequence of H. canis (KU597242) isolated from an Ixodes ricinus tick found in the Czech Republic (Hamšíková et al., 2016), but this ectoparasite does not occur in Brazil (Diniz et al., 2016). In some municipalities in the State of Pará, R. sanguineus sensu lato ticks, the main ectoparasite responsible for spreading Old World canine hepatozoonosis (Baneth et al., 1998; Baneth et al., 2007), and A. cajennense sensu lato are highly prevalent (Serra-Freire, 2010), but the two species seem to have no vector competence for H. canis (Demoner et al., 2013; Forlano et al., 2005). In general, the present study indicates that the amount of H. canis genotypes that were found in animals from North of Brazil reveals only a small portion of the real genetic diversity of this parasite in this region. In addition, our results highlight the need to understand the ecological plasticity of H. cuestensis, for the first time identified in mammals, in infecting different hosts.
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Conflict of interest statement No competing financial or non-financial interests exist. Acknowledgements ECG is grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for individual support (grant 311686/2015-0). LAG acknowledges a doctoral scholarship from Conselho de Aperfeiçoamento de Pessoal (CAPES). All authors are grateful to the 4
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Giannelli, A., Latrofa, M.S., Nachum-Biala, Y., Hodžić, A., Greco, G., Attanasi, A., Annoscia, G., Otranto, D., Baneth, G., 2017. Three different Hepatozoon species in domestic cats from southern Italy. Ticks Tick-Borne Dis. 8, 721–724. Gomes, P.V., Mundim, M.J.S., Mundim, A.V., Ávila, D.F., Guimarães, E.C., Cury, M.C., 2010. Occurrence of Hepatozoon sp. in dogs in the urban area originating from a municipality in southeastern Brazil. Vet. Parasitol. 174, 155–161. Gomes, L.A., Moraes, P.H., do Nascimento, L.C., O'Dwyer, L.H., Nunes, M.R., Rossi, A.D., Aguiar, D.C., Gonçalves, E.C., 2016. Molecular analysis reveals the diversity of Hepatozoon species naturally infecting domestic dogs in a northern region of Brazil. Ticks Tick-Borne Dis. 7, 1061–1066. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95–98. Hamšíková, Z., Silaghi, C., Rudolf, I., Venclíková, K., Mahríková, L., Slovák, M., Mendel, J., Blažejová, H., Berthová, L., Kocianová, E., Hubálek, Z., Schnittger, L., Kazimírová, M., 2016. Molecular detection and phylogenetic analysis of Hepatozoon spp. in questing Ixodes ricinus ticks and rodents from Slovakia and Czech Republic. Parasitol. Res. 115, 3897–3904. Harvey, T.V., Guedes, P.E., Oliveira, T.N., Assunção, M.S., Carvalho, F.S., Albuquerque, G.R., Silva, F.L., Carlos, R.S., 2016. Canine hepatozoonosis in southeastern Bahia, Brazil. Genet. Mol. Res. 15 (gmr.15038623). Hodžić, A., Alić, A., Fuehrer, H., Harl, J., Wille-Piazzai, W., Duscher, G.G., 2015. A molecular survey of vector-borne pathogens in red foxes (Vulpes vulpes) from Bosnia and Herzegovina. Parasites Vectors 8, 88. Hodžić, A., Alić, A., Prašović, S., Otranto, D., Baneth, G., Duscher, G.G., 2017. 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Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Original article
Genetic diversity of Hepatozoon spp. in Hydrochoerus hydrochaeris and Pecari tajacu from eastern Amazon ⁎
Laise de Azevedo Gomesa, , Leopoldo Augusto Moraesa, Délia Cristina Figueira Aguiara, Hilma Lúcia Tavares Diasa, Ana Silvia Sardinha Ribeirob, Henrique Piram do Couto Rochab, ⁎ Márcio Roberto Teixeira Nunesc, Evonnildo Costa Gonçalvesa, a
Laboratório de Tecnologia Biomolecular, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, PA, Brazil Grupo de Estudos de Animais Selvagens, Universidade Federal Rural da Amazônia, Belém, PA, Brazil c Centro de Inovações Tecnológicas, Instituto Evandro Chagas, Levilândia, Ananindeua, PA, Brazil b
A R T I C L E I N F O
A B S T R A C T
Keywords: Hepatozoon canis Hepatozoon cuestensis Mammals Northern Brazil
This study aimed to identify and characterize genetically species of the genus Hepatozoon detected in Hydrochoerus hydrochaeris (capybaras) and Pecari tajacu (collared peccaries) from two localities from the Eastern Amazon. Blood samples from 196 free-living H. hydrochaeris from Marajó Island and 109 P. tajacu kept in captivity in Belém, Pará, were collected and analyzed for the presence of Hepatozoon spp. Partial sequences of the 18S rRNA gene were obtained and analyzed in comparison to others available in the NCBI database. Our results demonstrated a high prevalence of Hepatozoon canis in both mammals and the existence of four haplotypes of Hepatozoon spp., three of Hepatozoon canis and one of Hepatozoon cuestensis, found only in H. hydrochaeris. In addition, these data increase the genetic diversity of H. canis from the Eastern Amazon, as well as reporting, for the first time, the infection of mammals by H. cuestensis and P. tajacu by H. canis.
1. Introduction
Unlike most of the pathogens that are transmitted by an arthropod vector through the salivary glands during the blood repast, the main route of infection of the intermediate host by Hepatozoon spp. is oral ingestion of the definitive host containing mature polysporocyst oocysts (Baneth et al., 2007; Baneth, 2011; Desser, 1993). Thus, geographical distribution of the final host and the existence of reservoirs, such as some wild animals, can determine the dispersion patterns of Hepatozoon spp. (Dantas-Torres, 2010; Najm et al., 2014). In fact, foxes and golden jackals appear to have great importance in the distribution of H. canis (Duscher et al., 2013; Farkas et al., 2014; Imre et al., 2015; Najm et al., 2014). The worldwide occurrence of H. canis, including in places where there is no report of the existence of the tick vector Rhipicephalus sanguineus sensu lato, has reinforced the concept that other species of ticks have vectorial competence for H. canis (Majláthová et al., 2007; Mitková et al., 2014; Mitková et al.,2016). In Brazil, despite the high abundance of R. sanguineus sensu lato (Araújo et al., 2015; DantasTorres et al., 2009; Soares et al., 2006), studies carried out in an attempt to detect H. canis infection did not find any evidence of the presence of this protozoan (Demoner et al., 2013; Forlano et al., 2005, Gomes et al., 2010).
Hydrochoerus hydrochaeris (capybaras) and Pecari tajacu (collared peccaries) are widely distributed in South America and are among the Brazilian wild mammals that are of great economic importance due to the appreciation of their meat and to the interest of the international leather industry (Mayor et al., 2010; Ojasti, 1991). In nature, these animals appear to play an important role in maintaining the life cycle of apicomplexan protozoa such as Toxoplasma gondii (Abreu et al., 2016; Thois et al., 2003), Eimeria spp. (Wilber et al., 1996) and Hepatozoon canis (Criado-Fornelio et al., 2009). In wild mammals from Brazil, H. canis has been detected in Pseudalopex vetulus (hoary fox), Cerdocyon brachyurus (maned wolf) (André et al., 2010), Dusicyon thous (crab-eating fox), Pseudalopex gymnocercus (pampas fox) (Criado-Fornelio et al., 2006), Hydrochoerus hydrochaeris (capybara) (Criado-Fornelio et al., 2009) and Leopardus pardalis (ocelot) (Braz and Umeda, 2015). Considering that the genus Hepatozoon belongs to the group of hemogregarines, one of the characteristics of species of this genus is its heteroxenous life cycle involving an intermediate vertebrate host and a definitive invertebrate hematophagous host (Baneth and Shkap, 2003).
⁎ Corresponding authors at: Laboratório de Tecnologia Biomolecular, Instituto de Ciências Biológicas, Universidade Federal do Pará, Cidade Universitária, Av. Augusto Corrêa no. 1 Bairro Guamá, Belém, Pará, 66075-110, Brazil. E-mail addresses: [email protected] (L. de Azevedo Gomes), [email protected] (E. Costa Gonçalves).
https://doi.org/10.1016/j.ttbdis.2017.11.005 Received 24 April 2017; Received in revised form 28 October 2017; Accepted 9 November 2017 1877-959X/ © 2017 Elsevier GmbH. All rights reserved.
Please cite this article as: Gomes, L.d.A., Ticks and Tick-borne Diseases (2017), https://doi.org/10.1016/j.ttbdis.2017.11.005
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Table 1 Hepatozoon spp. 18S rRNA sequences used for phylogenetic analysis plus additional information retrieved from the GenBank database. Strain
GenBank accession No.
Species
Host
Reference
Brazil Iran Turkey Croatia Brazil Brazil Hungray Croatia Czech Republic Brazil Brazil USA Brazil Japan Brazil Italy Bosnia and Herzegovina Bosnia and Herzegovina Croatia Croatia Ghana Canada Morocco Morocco Brazil Brazil Brazil Brazil Brazil Brazil Australia Australia China Brazil Chile
KU729738 KT736298 KX588232 HM212626 KU729737 EF622096 KJ572976 FJ497022 KU597242 KC342527 KC342524 AF176836 KU729739 AB771547 KY684005 KY649445 KX757031 KX757032 KT274177 KT274178 EF157822 AF130361 KU680464 KU680466 AY461377 KC127679 KY684007 KJ413113 KY684006 KY684004 AY252110 AY252105 KF939622 KU667308 FJ719813
H. canis H. canis H. canis H. canis H. canis H. canis H. canis H. canis H. canis H. cuestensis H. cuestensis H. americanum H. americanum H. felis H. felis H. silvestris H. silvestris H. silvestris H. ayorgbor H. ayorgbor H. ayorgbor H. catesbianae Hepatozoon sp. Hepatozoon sp. Hepatozoon sp. Hepatozoon sp. Hepatozoon sp. Hepatozoon sp. Hepatozoon sp. Hepatozoon sp. Hepatozoon sp. Hepatozoon sp. Hepatozoon sp. Hepatozoon sp. Hepatozoon sp.
Dog Dog Dog Fox Dog Capybara Golden Jackal Dog Tick Rattlesnake Rattlesnake Dog Dog Iriomote cat Ocelot Domestic cat European wild cat European wild cat Wood mouse Yellow-necked fieldmouse Ball python No data available Desert wall gecko Moorish gecko Fox Fox Caiman Caiman Turtle Paca Slaty grey snake Water python King rat snake Wild rodent Monito del monte
Gomes et al. (2016) Unpublished Unpublished Dezdek et al. (2010) Gomes et al. (2016) Criado-Fornelio et al. (2009) Farkas et al. (2014) Vojta et al. (2009) Hamšíková et al. (2016) O’Dwyer et al. (2013) O’Dwyer et al. (2013) Mathew et al. (2000) Gomes et al. (2016) Unpublished Soares et al. (2017) Giannelli et al. (2017) Hodžić et al. (2017) Hodžić et al. (2017) Unpublished Unpublished Sloboda et al. (2007) Carreno et al. (1999) Tomé et al. (2016) Tomé et al. (2016) Criado-Fornelio et al. (2006) Almeida et al. (2013) Soares et al. (2017) Unpublished Soares et al. (2017) Soares et al. (2017) Ujvari et al. (2004) Ujvari et al. (2004) Unpublished Demoner et al. (2016) Merino et al. (2009)
each sample was extracted from a 300μL aliquot of the blood by using a standard phenol-chloroform procedure, as described by Sambrook et al. (1989). DNA quality was checked by electrophoresis in an agarose gel, and the DNA was then quantified using the Qubit 2.0 fluorometer (Thermo Fisher Scientific).
In addition, there is no consensus so far about which tick species is in fact eco-epidemiologically important in the transmission of H. canis in Brazil. While O’Dwyer et al. (2001) indicated Amblyomma cajennense sensu lato as a potential vector, Demoner et al. (2013) refuted this possibility and suggested the existence of more than one strain of Amblyomma ovale capable of becoming infected by H. canis. The detection of mature oocysts of H. canis in the haemocoel of the cattle tick, Rhipicephalus microplus, collected from a dog and identification of DNA of the protozoan in this tick suggest that R. microplus can be a possible vector of H. canis (Miranda et al., 2011), however the experimental infestation of H. canis-infected dogs with R. microplus was not successful (Demoner et al., 2013). Most of our understanding of the disease caused by some species of the genus Hepatozoon comes from studies with domestic dogs (Aydin et al., 2015; Eiras et al., 2007; Ewing et al., 2000; Harvey et al., 2016; Li et al., 2008). In fact, the infection of wild animals by Hepatozoon spp. has been poorly studied in Brazil, principally in some regions, such as the Amazon. Thus, the objective of this study was to investigate the occurrence and genetically characterize the species of Hepatozoon found in H. hydrochaeris of extensive livestock farming and P. tajacu kept in captivity in the State of Pará, Brazil.
2.2. PCR amplification and DNA sequencing Molecular diagnosis of Hepatozoon spp. was undertaken according to Gomes et al. (2016) which is based on the partial amplification through nested PCR of the 18S rRNA gene of Hepatozoon spp. Briefly, we used HepF and HepR primers (Inokuma et al., 2002) in the first round of PCR followed by a second round of PCR with HepNF and HepNR primers (Gomes et al., 2016) whose amplification produced a fragment of approximately 300bp. In order to properly identify the Hepatozoon species, positive samples identified after molecular diagnosis, according to Gomes et al. (2016), were submitted to another nested PCR assay to amplify a larger fragment (∼670bp) of the 18S rRNA than the one amplified in the molecular diagnosis protocol. In this second assay, the first round of amplification included: NBA1 (5′GGTTGATCCTGCCAGTAGT3′) (Criado-Fornelio et al., 2003) and HPF2 (5′GACTTCTCCTTCGTCT AAG3′) (Criado-Fornelio et al., 2006) primers, while the second round PCR included HepF and HepR primers. First round PCR was carried out in 25μL reactions with 10–20 ng of the DNA template, 2.5mM MgCl2, 0.125mM each of deoxyribonucleotide triphosphates (dNTPs), 10mM Tris-HCl, 50mM KCl, 0.2μM of each primer, and 1 U Taq DNA polymerase (Invitrogen). The amplification reaction consisted of 40 cycles of 30 s at 95 °C, 30 s at 58 °C, and 2 min at 72 °C, preceded by 10 min at 95 °C and followed by 10 min at 72 °C. Second-round PCR was carried out under the same conditions of the first round PCR used here to the
2. Materials and methods 2.1. Samples and DNA extraction A total of 196 blood samples from H. hydrochaeris raised by extensive livestock farming on Marajó Island (State of Pará, Brazil) and 109 from P. tajacu maintained in captivity in the Brazilian Agricultural Research Cooperation (EMBRAPA Western Amazon) were collected into tubes containing ethylenediaminetetraacetic acid (EDTA). Total DNA of 2
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gene Hepatozoon spp. evidenced two clades well supported by posterior probability value (Fig. 1). CPV02 and CPV03 haplotypes, both derived from H. hydrochaeris samples and CPV01/CTU01 haplotype were included in the most derived clade that consisted of H. canis sequences, whose hosts were mostly mammals (dogs, fox, capybara and golden jackal). CPV04 haplotype grouped within the most ancestral clade represented by sequences H. cuestensis and Hepatozoon sp. obtained from biological samples of reptiles. This haplotype shared 99% nucleotide identity with H. cuestensis sequences (KC342527, KC342524, KU680464, KU680466) obtained from reptiles. Pairwise comparison of 40 sequences showed p distance ranging from 0 to 31.49% (Supplementary Table 1). While the genetic distances between H. canis strains was estimated at 2.04% (CPV02 x H. canis EF622096), the highest p distance between H. cuestensis strains was 2.55%.
molecular diagnosis, except that the DNA samples were changed by 1μL of the PCR product amplified with NBA1/HPF2 primers. For both analyses (i.e. molecular diagnosis and taxonomic diversity), DNA of H. canis was used as the positive control, while sterile bi-distilled water was used as the negative control. A 100bp molecular marker (Invitrogen DNA ladder) was used to estimate the size of each amplified fragment Amplicons of the second round PCR (∼670bp) and, in some cases, those obtained by HepNF and HepNR primers (∼300bp) were enzymatically purified with Illustra ExoProStar (GE Healthcare). Nucleotide sequencing was performed in an ABI 3500 xL Genetic Analyzer (Thermo Fisher Scientific), according to the manufacturer’s specifications. BioEdit software (Hall, 1999) was used to align forward and reverse sequences. 2.3. Data analysis
4. Discussion In addition to sequences obtained here, the phylogenetic analysis was performed including thirty-five Hepatozoon spp. 18S rRNA sequences retrieved from the GenBank database (Table 1) and one Babesia sp. 18S rRNA sequence which was used as the outgroup. MrModeltest 2.3 (Nylander, 2004) was used to determine the appropriate model. Bayesian Inference (BI) was conducted based on Markov Chain Monte Carlo (MCMC) tree searches as implemented in MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). We conducted two parallel runs of four simultaneous MCMC searches for 5 million generations each, sampling one tree every 500 generations, and discarding the results of the first 1000 trees as burn-in. The remaining trees were used by MrBayes to estimate the posterior probability of each node in our phylogenetic reconstruction. Tracer v1.4.1 (Rambaut and Drummond, 2008) was used to check the stationarity of all parameters sampled by the chains. The p distances between pairwise sequences were performed using PAUP 4.0b10 (Swofford, 2002).
A notable result of this research, corroborating Gomes et al. (2016), is that molecular detection based only on the HepF and HepR primers (Inokuma et al., 2002) has low efficiency, probably due to the lower sensitivity of single PCR compared to the nested PCR. In addition, most of the positive samples were obtained through the protocol of Gomes et al. (2016), different from that achieved using the nested PCR protocol with the NBA1/HPF2 primers followed by amplification with the HepF/HepR primers. Thus, it is feasible to infer that the latter nested PCR protocol is less efficient than the nested PCR protocol described by Gomes et al. (2016). Sequencing of twenty-eight samples failed probably due to lack of success in the purification process. Based on sequencing, the high prevalence of H. canis in H. hydrochaeris (64/80, thus 80%) and P. tajacu (32/49, thus 65.3%) corroborates other studies, which have shown that presence of this pathogen seems to be common in certain wild animals, for example, 40.7% of foxes from Bosnia (Hodžić et al., 2015), 60% of golden jackals from Hungary (Farkas et al., 2014) and 64% of Brazilian capybara analyzed by Criado-Fornelio et al. (2009). Despite the lack of data on the development of disease due to infection by Hepatozoon spp. in wild animals, the high prevalence of infection as observed here suggests that H. hydrochaeris and P. tajacu may be reservoirs of these protozoa, but other studies such as transmission experiments need to be carried out. This is relevant because there is no evidence of host-specific parasitism with Hepatozoon spp. In fact, our detection of H. cuestensis (CPV04 haplotype) in H. hydrochaeris reinforces this hypothesis, since, up to present study, this parasite had been detected only in reptiles (O’Dwyer et al., 2013; Tomé et al.,2016). The largest value of genetic distance between sequences of this parasite was similar to that which we obtained for the H. canis sequences, proving that CPV04 haplotype belongs to the clade of H. cuestensis sequences found in reptiles. Infection of snakes by Hepatozoon spp. can occur through the ingestion of intermediate hosts containing cystic forms of these protozoa (Smith, 1996). In fact, the Brazilian snake Crotalus durissus terrificus, for which the species H. cuestensis (KC342527) was described, feeds on rodents, indicating that infection occurs if the prey is infected or if it has infected ticks or mites (O’Dwyer et al., 2013). On the other hand, H. hydrochaeris may accidentally ingest small insects infected with Hepatozoon spp. when feeding on grasses. To the best of our knowledge this study provided the first record of the causative agent of Old World canine hepatozoonosis in P. tajacu, whose haplotype (CPV01/CTU01) seems to be widely distributed in State of Pará, since it was also detected in H. hydrochaeris from Marajó Island and in dogs from the Belém metropolitan area (KU729738) by Gomes et al. (2016). In addition to the Belém 01 (KU729737) and Belém 02 (KU729738) haplotypes of H. canis identified by Gomes et al. (2016) in dogs from northern Brazil, we found two new haplotypes (CPV02 and CPV03) of H. canis in H. hydrochaeris from the same region,
3. Results Only 11 samples, all from H. hydrochaeris, were positive using the HepF and HepR primers (Inokuma et al., 2002), while through the protocol of Gomes et al. (2016) 80 (40.8%) H. hydrochaeris and 49 (44.9%) P. tajacu were positive. Of 129 (80 of H. hydrochaeris and 49 of P. tajacu) positive samples according to the protocol of Gomes et al. (2016), only 92 (60 of H. hydrochaeris and 32 of P. tajacu) were amplified with the nested PCR protocol based on the NBA1/HPF2 (CriadoFornelio et al., 2003, 2006) and HepF/HepR primers. For the diversity analysis we obtained 101 Hepatozoon spp. DNA sequences, 69 (60 of ∼660bp and 9 of ∼300bp) of H. hydrochaeris and 32 (∼660bp) of P. tajacu. Sequencing of twenty-eight samples (11 of H. hydrochaeris and 17 of P. tajacu) failed. Hepatozoon sequences of H. hydrochaeris resulted in four haplotypes, whose analysis with the NCBI BLAST tool indicated three of H. canis and one of Hepatozoon cuestensis: CPV01/CTU01 (GenBank accession number KY965141), CPV02 (GenBank accession number KY965142), CPV03 (GenBank accession number KY965143) e CPV04 (GenBank accession number KY965144). Hepatozoon sequences obtained from P. tajacu resulted in a single haplotype, CTU01, which was identical to the CPV01 haplotype found in H. hydrochaeris. CPV04 haplotype (H. cuestensis) presented the highest number of polymorphisms with eight transitions and four transversions in relation to the CPV01/CTU01 haplotype of H. canis (Table 2). The most frequent haplotype was CPV01/CTU01, while the least frequent was CPV02. Bayesian Inference assumed a GTR + G model of nucleotide substitution with estimated base frequencies (A = 0.3142, C = 0.1613, G = 0.2047, T = 0.3199), substitution model (A-C = 0.9128, AG = 3.3867, A-T = 1.6333, C-G = 0.1355, C-T = 3.3867, G-T = 1), and rates for variable sites following a gamma distribution (G = 0.6607). A topology tree using partial sequences of 18S rRNA 3
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Table 2 Polymorphic nucleotide sites between the haplotypes of the 18S rRNA partial gene of Hepatozoon spp. found in H. hydrochaeris and P. tajacu from Eastern Amazon. Haplotype
CPV01/CTU01 CPV02 CPV03 CPV04
Host
Capybara/Collared peccary Capybara Capybara Capybara
Number of samples
60/32 1 3 5
Sequence length (bp)
656 300 300 300
Nucleotide position 253
284
287
288
289
290
302
316
326
331
362
364
365
377
T . G G
C . . T
T . . C
T . . A
T . . A
G . . A
A . . G
A . . G
G . . A
G . A A
A G . .
C . . T
C . . A
A . G .
The symbol (.) indicates a conserved nucleotide.
Fig. 1. Phylogenetic relationship between the partial sequences of the Hepatozoon 18S rRNA gene of the species found in H. hydrochaeris and P. tajacu from the Eastern Amazon and species of the same genus. The numbers in the nodes indicate the value of the Bayesian posterior probability. The scale bar indicates an evolutionary distance of 0.05 nucleotides per position in the sequence. The sequences obtained in this study are in bold. One Babesia sp. sequence was used as outgroup.
Brazilian Agricultural Research Cooperation (EMBRAPA) for ceding the biological samples from P. tajacu.
indicating this parasite has high genetic diversity in northern Brazil. Among the H. canis haplotypes, CPV03 was the one with the most variable sites and high nucleotide identity (99%) shared with a sequence of H. canis (KU597242) isolated from an Ixodes ricinus tick found in the Czech Republic (Hamšíková et al., 2016), but this ectoparasite does not occur in Brazil (Diniz et al., 2016). In some municipalities in the State of Pará, R. sanguineus sensu lato ticks, the main ectoparasite responsible for spreading Old World canine hepatozoonosis (Baneth et al., 1998; Baneth et al., 2007), and A. cajennense sensu lato are highly prevalent (Serra-Freire, 2010), but the two species seem to have no vector competence for H. canis (Demoner et al., 2013; Forlano et al., 2005). In general, the present study indicates that the amount of H. canis genotypes that were found in animals from North of Brazil reveals only a small portion of the real genetic diversity of this parasite in this region. In addition, our results highlight the need to understand the ecological plasticity of H. cuestensis, for the first time identified in mammals, in infecting different hosts.
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Conflict of interest statement No competing financial or non-financial interests exist. Acknowledgements ECG is grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for individual support (grant 311686/2015-0). LAG acknowledges a doctoral scholarship from Conselho de Aperfeiçoamento de Pessoal (CAPES). All authors are grateful to the 4
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