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Molecular characterization and phylogenetic analysis of Babesia and Theileria spp. in ticks from domestic and wild hosts in Sardinia
Acta Tropica 196 (2019) 60–65
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Molecular characterization and phylogenetic analysis of Babesia and Theileria spp. in ticks from domestic and wild hosts in Sardinia Valentina Chisua,
⁎,1
T
, Alberto Albertib,c,1, Rosanna Zobbab, Cipriano Foxia, Giovanna Masalaa
a
Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Italy Dipartimento di Medicina Veterinaria, Università degli studi di Sassari, Sassari, Italy c Mediterranean Center for Disease Control (MCDC), University of Sassari, Sassari, Italy b
A R T I C LE I N FO
A B S T R A C T
Keywords: Ticks Tick-borne diseases Piroplasmids Phylogeny
Piroplasmoses are tick-borne protozoan diseases caused by hemoprotozoan parasites with considerable economic, veterinary and medical impact worldwide. Here, the presence and diversity of piroplasmids was investigated in ticks collected from domestic and wild hosts in a typical subtropical environment of Sardinia island by standard PCR, sequencing, and phylogenetic analyses. We demonstrate the presence of strains closely related to the Theileria buffeli/sergentii/orientalis complex in Rhipicephalus sanguineus s.l., Rh. bursa, Rh. annulatus, Hyalomma marginatum, Dermacentor marginatus and Haemaphysalis punctata ticks. A strain detected in two Rh. sanguineus s.l. ticks collected from dogs grouped with T. equi, the agent of equine piroplasmosis. T. ovis, the main etiological agent of ovine theileriosis, was detected in one Rh. bursa tick from a mouflon. Babesia bigemina, the causative agent of bovine babesiosis, was detected in two Rh. sanguineus s.l. ticks from dogs. Our findings expand the knowledge on the repertoire of tick-borne pathogens present in Mediterranean ticks. Further analyses are needed to determine the role of ticks in the biological or mechanical transmission of piroplasmoses in this area.
1. Introduction Ticks (Acari: Ixodida) are important vectors of microorganisms infecting both human and animal hosts (Estrada-Peña et al., 2014). Ticks feed on a large range of vertebrate host species, and are considered second to mosquitoes in pathogen transmission worldwide (de la Fuente et al., 2008). Among tick-borne diseases, piroplasmoses are caused by hemoprotozoan parasites of the order Piroplasmida (phylum Apicomplexa) and include four genera Babesia, Theileria, Cytauxzoon and Rangelia (Votýpka et al., 2017; Yabsley and Shock, 2013). The protozoans of this order play an important role in the world economy and Babesia represent the second most common blood parasites of vertebrates after trypanosomes (Alvarado-Rybak et al., 2016; Schnittger et al., 2012). Piroplasmids infections have been detected in livestock, dogs, cats, wild mammals and also, to a lesser extent, in humans (Baneth, 2014). The primary route of piroplasmid transmission is supposed to be through tick bites, but other manners of infection such as the direct dog-to-dog or the dog transplacental transmissions have been described for some piroplasmid species (Alvarado-Rybak et al., 2016). Blood transfusion is also considered an alternative route of direct transmission in human babesiosis (Young et al., 2012).
Once the sporozoites are released from tick salivary glands into the vertebrate host blood system (Alvarado-Rybak et al., 2016; Birkenheuer, 2012; Chauvin et al., 2009; Penzhorn, 2006), they can invade the erythrocytes (Babesia spp.), lymphocytes (Theileria spp.) (Mans et al., 2015; Chauvin et al., 2009) or macrophages, histiocytes, or reticuloendothelial cells of vertebrate hosts (Cytauxzoon spp. and R. vitalii) (Eiras et al., 2014; Kier et al., 1987). In Sardinia, the distribution of pyroplasmids in ticks has not been documented and only one study reported the molecular characterization, sequencing and phylogenetic analyses of a Babesia spp. from a symptomatic sow, showing that the detected piroplasm was phylogenetically closely related to the Ungulibabesids (Zobba et al., 2011). The aim of this study was to: a) assess the presence of piroplasmids of veterinary significance in ixodid ticks collected from domestic and wild animals; b) identify the tick species that carry these pathogens; c) identify piroplasmid 18S rRNA sequence types; c) reconstruct phylogeny of newly identified piroplasmid 18SrRNA sequence types isolated from different tick species infesting livestock and wildlife in Sardinia, Italy.
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Corresponding author. E-mail address: [email protected] (V. Chisu). 1 Authors contributed equally to this manuscript https://doi.org/10.1016/j.actatropica.2019.05.013 Received 9 April 2019; Received in revised form 13 May 2019; Accepted 13 May 2019 Available online 14 May 2019 0001-706X/ © 2019 Elsevier B.V. All rights reserved.
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Table 1 Tick species collected on vertebrate hosts and vegetation from several collection sites in Sardinia and summary of PCR results. Tick species (total number of tested specimens)
Host/vegetation (tick number)
Coordinates
Collection site
Ecosystem type
18S rRNA PCR positive
Rhipicephalus bursa (22)
human (3)
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N
Urzulei Aritzo Talana S. Nicolò Gerrei Perdasdefogu Asinara Santadi Uta Baunei Urzulei Barisardo Cardedu Urzulei Triei Borutta Ozieri Sassari Alghero Sassari Tortolì Dorgali Pattada Alghero S. Anna Arresi Uta Orgosolo Alghero Asinara Uta Talana Tortolì Oschiri Santadi Oschiri
Mountain/Forest
– – 1 – – 1 1 1 2 – – – 1 – 4 4 3 1 – – 1
fox (3) goat (13)
Rhipicephalus sanguineus s.l. (97)
Ornitodorus marittimus (1) Rhipicephalus annulatus (13) Hyalomma marginatum (18)
Haemaphisalis punctate (6)
Dermacentor marginatus (9) Total 166
mouflon (1) Corsican red deer (1) cattle (1) goat (2) European pine marten (5) dog (83)
cat (1) vegetation (2) mouflon (3) Corsican red deer (1) human (1) cattle (13) human (1) wildboar (4) whinchat (8) cattle (4) mouflon(1) crow (1) cattle (4) Corsican red deer (5) cattle (4) 166
40° 05′; E 9° 30′ 39° 57'; E 09° 11' 40° 02′; E 9° 29′ 39° 29′; E 9° 18′ 39° 40'; E 09° 26' 41°03; E 8°16’ 39° 05′; E 8° 42′ 39° 17'; E 08° 57' 39°51'; E 09° 35' 40°05′; E 9°30′ 39° 51'; E 09° 35' 39° 47'; E 09° 37' 40° 05′; E 9° 30′ 40° 02′; E 9° 38′ 40° 31′; E 8°44′ 40° 35'; E 09°00' 40° 43’; E 8° 33′ 40° 33’; E 8° 19′ 40° 43′; E 8° 33′ 39° 55′; E 9° 39′ 40° 17′; E 9° 35′ 40° 34′; E 9° 06′ 40° 33; E 8° 19’ 39° 00′; E 8° 38′ 39° 17'; E 08° 57' 40° 12′; E 9° 21′ 40° 33′; E 8° 19′ 41° 03′; E 8° 16′ 39° 17'; E 08° 57' 40° 02′; E 9° 29′ 39° 55′; E 9° 39′ 40° 43′; E 9°06′ 39° 05′; E 8° 42′ 40° 43′; E 9° 06′
Hill/Farmland Hill/Pasture Hill/Mediterranean scrub Plain/ Farmland Mountain/Mediterranean scrub Mountain/Grazing land Mountain/Forest Plain/ Farmland Mountain/Forest Hill/ Grazing land Hill/Farmland Flat land/ Farmland Hill/Farmland Plain/ Farmland Hill/ Grazing land Mountain/ Grazing land Flat land/ Farmland Plain/ Farmland Mountain/Mediterranean scrub Mountain/Forest Plain/ Farmland Hill/Mediterranean scrub Mountain/Mediterranean scrub Mountain/Forest Plain/ Farmland Hill/ Grazing land Plain/ Farmland Hill/ Grazing land
– 1 – – – 2 1 – – 4 3 3 34 (20.5%)
2. Material and methods
2.2. Tick collection and morphological identification
2.1. Study area
A total of 166 ticks were collected from wild (three foxes, five muflon, four wild boars, seven Corsican red deer, five European pine marten, eight whinchat, and one crow) and domestic animals (83 dogs, one cat, 26 cattle, 15 goats). In addition, four ticks were collected from humans and two ticks from vegetation (Table 1). The wild animals sampled in this study were dead when brought to the Zooprofilattic Institute of the Sardinia (Sassari) for necropsy analyses. Ticks were removed from the hosts by tweezers, preserved in vials with 70% ethanol and stored at room temperature until morphological analyses and DNA extraction. Ticks from vegetation were collected by visual search methods, directly hand-collected and transported into vials with 70% ethanol to our labs for species identification. Ticks were then identified morphologically according to Manilla (1998).
The study was conducted from April to October 2017 in Sardinia, Italy. Sardinia is the second-largest island of the Mediterranean Sea with an area of 23,821 km2. The temperate Mediterranean climate of the island enables the survival of ticks throughout the year (Chisu et al., 2018). Sardinia environment provides a variety of ecosystems and ideal natural conditions to study disease ecology. Ticks were collected by veterinary practitioners from 22 collection sites (Table 1). Seven collection sites (Oschiri; Sassari, Alghero, Borutta; Ozieri; Pattada, Asinara), belonging to Northwest Sardinia, comprise a variety of ecosystems including plains, hills and promontories. Two sites, located in the center of Sardinia (Orgosolo and Dorgali) are morphologically complex and include sandy coasts, cliffs and mountains with forests of oak, juniper and yew. Ten collection sites (Urzulei, Aritzo, Talana, Baunei, Barisardo, Cardedu, Triei, Tortolì, Perdasdefogu and S. Nicolò Gerrei) are located in Southwest Sardinia where mountains, forests and green valleys are covered by the typical Mediterranean maquis. The landscape is also characterized by cultivated coastal plains, watercourses and rocky sheer coasts. The three collection sites of South-West Sardinia (Santadi, Uta and Sant’Anna Arresi) are located in a territory that present diversified and complex morphological features with the presence of plains, plateaus and reliefs at different height. Part of the territory presents karst cavities of morphological and mineralogical interest.
2.3. Tick processing and DNA extraction Ticks were individually immersed in distilled water for 10 min, dried on sterile filter paper, cut and crushed into small pieces with a sterile scalpel in microtubes (Eppendorf, Hilden, Germany) for DNA to be extracted efficiently. Sterile instruments were used for each individual tick cut. DNA was extracted from each tick using DNeasy® Blood & Tissue Kit (QIAGEN, Chatsworth, CA, USA), by following the manufacturer's instructions. 2.4. PCR amplification, purification and sequencing DNA extracts from all 166 ticks were screened for the presence of piroplasms by a conventional PCR that amplifies an approximately 450 61
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3. Results
bp fragment of the 18S ribosomal RNA gene of Babesia/Theileria species. The reaction was made up to a final volume of 25 μl containing 12.5 μL μl of Amplitaq Gold master mix (Quantitect Probe PCR Master Mix, Qiagen, Hilden, Germany), 1 μl of 25 μM of primer BJ1 (5′-GTCT TGTAATTGGAATGATGG-3′) and BN2 (5′-TAGTTTATGGTTAGGACT ACG-3′) (Casati et al., 2006), 9.5 μL of RNase nuclease-free water and 1 μl of genomic DNA. A negative control using RNase nuclease-free water (Qiagen, Hilden, Germany) and a DNA positive control of B. bovis were included in each PCR test. The reaction mixtures were subjected to an initial denaturation step of 15 min at 95 °C, followed by 40 cycles of denaturation at 94 °C for 1 min, annealing at 60 °C for 30 s., and elongation at 72 °C for 1 min. Amplification was completed by a further 5 min step at 72 °C. Negative and positive controls were included in each amplification assay. The PCR products were separated electrophoretically in 1.5% agarose gel under standard conditions. DNA Molecular Weight Marker VIII (Roche,) was used for DNA sizing. The products were treated with nontoxic SYBR® Green DNA Gel Stain (Invitrogen, Carlsbad, CA, USA), and visualized using standard UV transillumination. Positive PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol and then directly sequenced in both directions by using an ABI Prism BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA, USA).
3.1. Morphological detection of ticks The ticks were morphologically identified as follows: Rh. bursa (n = 22); Rh. sanguineus s.l. (n = 97); Rh. annulatus (n = 13); Hy. marginatum (n = 18); Hae. punctata (n = 6); D. marginatus (n = 9); Ornithodoros maritimus (n = 1) as shown in Table 1. 3.2. Molecular detection of piroplasmids Piroplasmid DNAs were detected in 34 out of 166 (21%) ticks removed from different wild and domestic hosts that showed no apparent clinical signs or symptoms at the sampling time (Table 1). More specifically, infection rates were 18% (4/22), 16.5% (16/97), 8% (1/13), 17% (3/18), 67% (4/6) and 67% (6/9) in Rh. bursa, Rh. sanguineus s.l., Rh. annulatus, Hy. marginatum, Hae. punctata, and D. marginatus ticks, respectively. All the 34 amplicons were sequenced and 19 good quality sequences were obtained. Fifteen poor quality sequences were discarded from further analysis. The 18S rRNA sequences obtained in this study were 99–100% identical to T. buffeli, T. equi, T. ovis and Babesia bigemina sequences (Table 2). Upon sequencing and ClustalX alignment, 18S rRNA sequences were assigned to nine different sequence types. TSard2018_1 (identified in one Rh. bursa, five Rh. sanguineus s.l., one Rh. annulatus, one Hy. marginatum, one Hae. punctata, and one D. marginatus collected from fox, cattle, goat, dogs and whinchat, PSard2018_2, PSard2018_3 and PSard2018_4 (include three sequences derived from three Rh. sanguineus s.l. ticks collected from dogs); PSard2018_5 (derived from a Rh. sanguineus s.l. collected from a mouflon); PSard2018_6 (from a Rh. bursa tick collected from a mouflon), PSard2018_7 and PSard2018_9 (include two sequences derived from two Rh. sanguineus s.l. ticks from dogs); TSard2018_8 (derived from one D. marginatus from a cattle). TSard2018_1, TSard2018_3, TSard2018_5, TSard2018_7, and TSard2018_8 sequence types shared 99–100% identity with T. buffeli, T. sergenti, and T. orientalis genotypes isolated worldwide from ruminants and ticks and deposited in the GenBank. TSard2018_2 and TSard2018_4 showed 99–100% identity with T. equi genotypes isolated from horses in Cuba (KY111762), Brazil (KX722519), Australia (KJ801925), and USA (JX177672). TSard2018_6 sequence type showed 100% identity to T. ovis. The sequence type named PSard2018_9 detected in two Rh. sanguineus s.l. ticks sampled from a dog shared 99% similarity with B. bigemina sequences isolated worldwide. Phylogenetic analysis (Fig. 1) of the partial 18S rRNA gene was performed by aligning the nine sequence types obtained in this study with selected Babesia and Theileria sequences. Sequence types PSard2018_1, PSard2018_3, PSard2018_5, PSard2018_7 and PSard2018_8 grouped with reference strains representative of T. buffeli. PSard2018_2, and PSard2018_4 sequence types were assigned to the T. equi clade. PSard2018_6 was closely related to T. ovis strain. PSard2018_9 grouped in a strongly supported monophyletic clade with B. bigemina.
2.5. Sequence and phylogenetic analysis Chromatograms of forward and reverse sequences generated with 18S rRNA primers were edited with Chromas 2.2 (Technelysium, Helensvale, Australia), and then aligned with CLUSTALX (Larkin et al., 2007) in order to assign them to unique sequence types, and checked against the GenBank database with nucleotide blast BLASTN (Altschul et al., 1990). Pairwise/multiple sequence alignments and sequence similarities were calculated using the CLUSTALW (Thompson et al., 1997) and the identity matrix options of Bioedit (Hall, 1999), respectively. For phylogenetic analyses, the nine sequence types obtained in this study (TSard2018_1-9) were aligned with a set of 43 sequences representing the 18S rRNA variability of the different species belonging to the genus Theileria and Babesia. The reference sequences used in this study are shown in Fig. 1. Sequences were aligned with ClustalX (Thompson et al., 1997). Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013) by using the Maximum Likelihood method based on the Kimura 2parameter model (Kimura, 1980) identified as the best model with the same software. Initial tree for the heuristic search was obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood approach, and then selecting the topology with superior log likelihood value. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985).
4. Discussion Theileriosis and babesiosis are tick-borne diseases that cause major economic losses and affect many domestic animals, mainly cattle and sheep, in tropical and subtropical regions (Uilenberg, 1995). In this study DNA of piroplasmids was detected in different tick species collected from several vertebrate hosts from a typical subtropical Mediterranean region, the island of Sardinia. Results indicate the presence of Theileria and Babesia species closely related to T. buffeli, T. equi, T. ovis and B. bigemina in Rh. sanguineus, Rh. bursa, Rh. annulatus, Hae. punctata, Hy. marginatum and D. marginatus ticks. As ticks were engorged when removed from their hosts, we cannot state that these ticks are competent vectors for the detected
2.6. Sequence accession numbers Representative Theileria and Babesia sequences were submitted to the NCBI database using the National Center for Biotechnology Information (NCBI; Bethesda, MD) BankIt v3.0 submission tool (http:// www3.ncbi.nlm.nih.gov/BankIt/) under accession numbers MK587691-MK587709.
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Fig. 1. Molecular phylogenetic tree by using Maximum Likelihood method showing the evolutionary relationship of the nine identified piroplasmids sequence types and other 44 taxa used in this study. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.2366)). The analysis involved 52 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 285 positions in the final dataset.
the genus Hyalomma with the Hy. lusitanicum and Hy. marginatum species have been also documented in the island. A small percentage of tick population is represented by Ixodes festai and Argas reflexus (Chisu et al., 2018). However, these tick genera are recognized vectors of pathogenic bacteria with medical and veterinary relevance (Chisu et al., 2018). Although bovine theileriosis caused by T. annulata and T. parva is considered one of the four most important tick-borne haemoparasitic diseases of livestock (Uilenberg, 1995), there are increasing records of the T. orientalis/T.buffeli/T. sergenti group causing economically significant infections, especially in immunocompromized or stressed
piroplasmids, since they might have got the infection feeding on infected bacteremic animals, or/and by co-feeding with other infected ticks at the same feeding site. Sardinia is the second largest island in the Mediterranean Sea, and its ecosystem allows the survival and maintenance of a rich fauna of ixodid ticks throughout the year (Satta et al., 2011). Ticks of the Rhipicephalus genus are the best represented on the island and include five species: Rh. sanguineus, Rh. pusillus, Rh. turanicus, Rh. bursa and Rh. (Boophilus) annulatus infesting wild and domestic vertebrates (Chisu et al., 2018). The genus Dermacentor, with the D. marginatus which is the only species occurring in the island; the genus Haemaphysalis (with Hae. punctata, and Hae. sulcata species) and
Table 2 Sequence type and BLASTN identities of the 18S rRNA gene sequence types identified in this study. Sequence Type
Tick species (no. of positive)
Tick vertebrate host
GenBank accession number
BLASTN identity
TSard2018_1
Rh. sanguineus s.l. (5) Hy. marginatum (1) D. marginatus (1) Rh. bursa (1) Hae. punctate (1) Rh. annulatus (1) Rh. sanguineus s.l. (1) Rh. sanguineus s.l. (1) Rh. sanguineus s.l. (1) Rh. sanguineus s.l. (1) Rh. bursa (1) Rh. sanguineus s.l. (1) D. marginatus (1) Rh. sanguineus s.l. (2)
Dog,Goat Whinchat Cattle Fox Cattle Cattle Dog Dog Dog Mouflon Mouflon Dog Cattle Dog
MK587691-MK587695 MK5876916 MK587697 MK587698 MK587699 MK587700 MK587701 MK587702 MK587703 MK587704 MK587705 MK587706 MK587707 MK587708, MK587709
99% T. buffeli/sergenti/orientalis
TSard2018_2 TSard2018_3 TSard2018_4 TSard2018_5 TSard2018_6 TSard2018_7 TSard2018_8 TSard2018_9
63
99% T. equi 100% T. buffeli/sergenti/orientalis 100% T. equi 99% T. buffeli/ sergenti/ orientalis 100% T. ovis 100% T. buffeli/ sergenti/ orientalis 99% T. buffeli/sergenti/orientalis 99% B. bigemina
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animals (Shimizu et al., 2000; Kim et al., 1998). These piroplasms are transtadially transmitted by different Haemaphysalis spp. in Asia, Australia and New Zealand (Kamau et al., 2011; Fuujisaki, 1992). However, the transmission is not limited to this tick genus which could explain their worldwide occurrence (McFadden et al., 2011). In this study, five Theileria strains closely related to T. buffeli/sergenti/orientalis group were detected in different tick species (see Table 2) that may suggest their possible role as competent vectors for Theileria species in Sardinia, but their role in the transmission of T. buffeli/sergenti/orientalis complex remains to be shown. Theileria equi is a small piroplasm infecting erythrocytes causing equine piroplasmosis (EP) which affects the horse industry worldwide (Scoles and Ueti, 2015). EP is also an important cause of horse morbidity in Sardinia where both Babesia caballi and T. equi have been detected (Zobba et al., 2011). Prevalence of the disease is related to distribution of vector ticks (de Waal, 1992). Species of the genera Dermacentor, Hyalomma and Rhipicephalus are the biological vectors of T. equi (de La Fuente et al., 2008). All potential vectors of T. equi have been recorded in Sardinia (Chisu et al., 2018), suggesting their role as vectors of this piroplasm. In this study, two sequences phylogenetically related to T. equi were detected in two Rh. sanguineus s.l. ticks collected from dogs. Presence of pathogenic T. equi in symptomatic and asymptomatic dogs in Croatia and Spain has been previously reported (Beck et al., 2009; Criado-Fornelio et al., 2003). As Rh. sanguineus s.l. are well represented in the island and are mainly collected from dogs, the relationship between the newly identified parasite strain, the Rh. sanguineus s.l. ticks, and infection in dogs deserves further investigation. One Rh. bursa tick collected from a mouflon was positive for T. ovis. The transmission of T. ovis by the two-host tick Rh. bursa has been emphasized by e.g. Aktas et al. (2006) and Altay et al. (2005). Theileria ovis occurs in the Mediterranean basin as well as in other areas where the tick vector is present (Aydin et al., 2015; Yeruham et al., 1995). Subclinical infection in small ruminants caused by T. ovis has been reported from sheep in different countries (Altay et al., 2005; Nagore et al., 2004; Schnittger et al., 2012). Rh. bursa is widespread in the island and is frequently found on both domestic and wild ruminants (Chisu et al., 2018). Further research is needed in order to determine the presence of T. ovis in Rh. bursa ticks and related hosts in the island. In this study, B. bigemina was also detected in two Rh. sanguineus s.l. adult ticks, in agreement with literature data where the parasite was found in Rhipicephalus ticks in Iran (Rajabi et al., 2017) and Central and Southern Italy (Toma et al., 2017). Considering that B. bigemina is a common cause of bovine babesiosis in Italy (Toma et al., 2017), the population of Rh. sanguineus s.l. should be monitored, and further studies are needed to fully understand the link between this tick species and B. bigemina in the island. The identified Theileria and Babesia species are not associated with human infection. In Sardinia, ruminants are an important source of income and are exploited for milk and meat production. Monitoring the circulation of tick population on the island, the tick borne pathogens and the risk of animals and humans exposure to tick-borne diseases could be useful for tick bite risk assessment and for widening of surveillance methods. Further studies are nevertheless needed to better characterize piroplasmid strains by analyzing more discriminative genes, and to identify the main vectors implicated in their transmission in Sardinia.
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McFadden, A., Rawdon, T., Meyer, J., Makin, J., Morley, C., Clough, R., Tham, K., Mullner, P., Geysen, D., 2011. An outbreak of haemolytic anaemia associated with infection of Theileria orientalis in naive cattle. N. Z. Vet. J. 59, 79–85. Nagore, D., García-Sanmartín, J., García-Pérez, A.L., Juste, R.A., Hurtado, A., 2004. Identification, genetic diversity and prevalence of Theileria and Babesia species in a sheep population from Northern Spain. Int. J. Parasitol. 34, 1059–1067. Penzhorn, B., 2006. Babesiosis of wild carnivores and ungulates. Vet. Parasitol. 138, 11–21. Rajabi, S., Esmaeilnejad, B., Tavassoli, M., 2017. A molecular study on Babesia spp. in cattle and ticks in West-Azerbaijan province. Iran. Vet. Res. Forum. 8, 299–306. Satta, G., Chisu, V., Cabras, P., Fois, F., Masala, G., 2011. Pathogens and symbionts in ticks: a survey on tick species distribution and presence of tick-transmitted microorganisms in Sardinia. Italy. J. Med. Microbiol. 60, 63–68. 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Molecular characterization and phylogenetic analysis of Babesia and Theileria spp. in ticks from domestic and wild hosts in Sardinia Valentina Chisua,
⁎,1
T
, Alberto Albertib,c,1, Rosanna Zobbab, Cipriano Foxia, Giovanna Masalaa
a
Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Italy Dipartimento di Medicina Veterinaria, Università degli studi di Sassari, Sassari, Italy c Mediterranean Center for Disease Control (MCDC), University of Sassari, Sassari, Italy b
A R T I C LE I N FO
A B S T R A C T
Keywords: Ticks Tick-borne diseases Piroplasmids Phylogeny
Piroplasmoses are tick-borne protozoan diseases caused by hemoprotozoan parasites with considerable economic, veterinary and medical impact worldwide. Here, the presence and diversity of piroplasmids was investigated in ticks collected from domestic and wild hosts in a typical subtropical environment of Sardinia island by standard PCR, sequencing, and phylogenetic analyses. We demonstrate the presence of strains closely related to the Theileria buffeli/sergentii/orientalis complex in Rhipicephalus sanguineus s.l., Rh. bursa, Rh. annulatus, Hyalomma marginatum, Dermacentor marginatus and Haemaphysalis punctata ticks. A strain detected in two Rh. sanguineus s.l. ticks collected from dogs grouped with T. equi, the agent of equine piroplasmosis. T. ovis, the main etiological agent of ovine theileriosis, was detected in one Rh. bursa tick from a mouflon. Babesia bigemina, the causative agent of bovine babesiosis, was detected in two Rh. sanguineus s.l. ticks from dogs. Our findings expand the knowledge on the repertoire of tick-borne pathogens present in Mediterranean ticks. Further analyses are needed to determine the role of ticks in the biological or mechanical transmission of piroplasmoses in this area.
1. Introduction Ticks (Acari: Ixodida) are important vectors of microorganisms infecting both human and animal hosts (Estrada-Peña et al., 2014). Ticks feed on a large range of vertebrate host species, and are considered second to mosquitoes in pathogen transmission worldwide (de la Fuente et al., 2008). Among tick-borne diseases, piroplasmoses are caused by hemoprotozoan parasites of the order Piroplasmida (phylum Apicomplexa) and include four genera Babesia, Theileria, Cytauxzoon and Rangelia (Votýpka et al., 2017; Yabsley and Shock, 2013). The protozoans of this order play an important role in the world economy and Babesia represent the second most common blood parasites of vertebrates after trypanosomes (Alvarado-Rybak et al., 2016; Schnittger et al., 2012). Piroplasmids infections have been detected in livestock, dogs, cats, wild mammals and also, to a lesser extent, in humans (Baneth, 2014). The primary route of piroplasmid transmission is supposed to be through tick bites, but other manners of infection such as the direct dog-to-dog or the dog transplacental transmissions have been described for some piroplasmid species (Alvarado-Rybak et al., 2016). Blood transfusion is also considered an alternative route of direct transmission in human babesiosis (Young et al., 2012).
Once the sporozoites are released from tick salivary glands into the vertebrate host blood system (Alvarado-Rybak et al., 2016; Birkenheuer, 2012; Chauvin et al., 2009; Penzhorn, 2006), they can invade the erythrocytes (Babesia spp.), lymphocytes (Theileria spp.) (Mans et al., 2015; Chauvin et al., 2009) or macrophages, histiocytes, or reticuloendothelial cells of vertebrate hosts (Cytauxzoon spp. and R. vitalii) (Eiras et al., 2014; Kier et al., 1987). In Sardinia, the distribution of pyroplasmids in ticks has not been documented and only one study reported the molecular characterization, sequencing and phylogenetic analyses of a Babesia spp. from a symptomatic sow, showing that the detected piroplasm was phylogenetically closely related to the Ungulibabesids (Zobba et al., 2011). The aim of this study was to: a) assess the presence of piroplasmids of veterinary significance in ixodid ticks collected from domestic and wild animals; b) identify the tick species that carry these pathogens; c) identify piroplasmid 18S rRNA sequence types; c) reconstruct phylogeny of newly identified piroplasmid 18SrRNA sequence types isolated from different tick species infesting livestock and wildlife in Sardinia, Italy.
⁎
Corresponding author. E-mail address: [email protected] (V. Chisu). 1 Authors contributed equally to this manuscript https://doi.org/10.1016/j.actatropica.2019.05.013 Received 9 April 2019; Received in revised form 13 May 2019; Accepted 13 May 2019 Available online 14 May 2019 0001-706X/ © 2019 Elsevier B.V. All rights reserved.
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Table 1 Tick species collected on vertebrate hosts and vegetation from several collection sites in Sardinia and summary of PCR results. Tick species (total number of tested specimens)
Host/vegetation (tick number)
Coordinates
Collection site
Ecosystem type
18S rRNA PCR positive
Rhipicephalus bursa (22)
human (3)
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N
Urzulei Aritzo Talana S. Nicolò Gerrei Perdasdefogu Asinara Santadi Uta Baunei Urzulei Barisardo Cardedu Urzulei Triei Borutta Ozieri Sassari Alghero Sassari Tortolì Dorgali Pattada Alghero S. Anna Arresi Uta Orgosolo Alghero Asinara Uta Talana Tortolì Oschiri Santadi Oschiri
Mountain/Forest
– – 1 – – 1 1 1 2 – – – 1 – 4 4 3 1 – – 1
fox (3) goat (13)
Rhipicephalus sanguineus s.l. (97)
Ornitodorus marittimus (1) Rhipicephalus annulatus (13) Hyalomma marginatum (18)
Haemaphisalis punctate (6)
Dermacentor marginatus (9) Total 166
mouflon (1) Corsican red deer (1) cattle (1) goat (2) European pine marten (5) dog (83)
cat (1) vegetation (2) mouflon (3) Corsican red deer (1) human (1) cattle (13) human (1) wildboar (4) whinchat (8) cattle (4) mouflon(1) crow (1) cattle (4) Corsican red deer (5) cattle (4) 166
40° 05′; E 9° 30′ 39° 57'; E 09° 11' 40° 02′; E 9° 29′ 39° 29′; E 9° 18′ 39° 40'; E 09° 26' 41°03; E 8°16’ 39° 05′; E 8° 42′ 39° 17'; E 08° 57' 39°51'; E 09° 35' 40°05′; E 9°30′ 39° 51'; E 09° 35' 39° 47'; E 09° 37' 40° 05′; E 9° 30′ 40° 02′; E 9° 38′ 40° 31′; E 8°44′ 40° 35'; E 09°00' 40° 43’; E 8° 33′ 40° 33’; E 8° 19′ 40° 43′; E 8° 33′ 39° 55′; E 9° 39′ 40° 17′; E 9° 35′ 40° 34′; E 9° 06′ 40° 33; E 8° 19’ 39° 00′; E 8° 38′ 39° 17'; E 08° 57' 40° 12′; E 9° 21′ 40° 33′; E 8° 19′ 41° 03′; E 8° 16′ 39° 17'; E 08° 57' 40° 02′; E 9° 29′ 39° 55′; E 9° 39′ 40° 43′; E 9°06′ 39° 05′; E 8° 42′ 40° 43′; E 9° 06′
Hill/Farmland Hill/Pasture Hill/Mediterranean scrub Plain/ Farmland Mountain/Mediterranean scrub Mountain/Grazing land Mountain/Forest Plain/ Farmland Mountain/Forest Hill/ Grazing land Hill/Farmland Flat land/ Farmland Hill/Farmland Plain/ Farmland Hill/ Grazing land Mountain/ Grazing land Flat land/ Farmland Plain/ Farmland Mountain/Mediterranean scrub Mountain/Forest Plain/ Farmland Hill/Mediterranean scrub Mountain/Mediterranean scrub Mountain/Forest Plain/ Farmland Hill/ Grazing land Plain/ Farmland Hill/ Grazing land
– 1 – – – 2 1 – – 4 3 3 34 (20.5%)
2. Material and methods
2.2. Tick collection and morphological identification
2.1. Study area
A total of 166 ticks were collected from wild (three foxes, five muflon, four wild boars, seven Corsican red deer, five European pine marten, eight whinchat, and one crow) and domestic animals (83 dogs, one cat, 26 cattle, 15 goats). In addition, four ticks were collected from humans and two ticks from vegetation (Table 1). The wild animals sampled in this study were dead when brought to the Zooprofilattic Institute of the Sardinia (Sassari) for necropsy analyses. Ticks were removed from the hosts by tweezers, preserved in vials with 70% ethanol and stored at room temperature until morphological analyses and DNA extraction. Ticks from vegetation were collected by visual search methods, directly hand-collected and transported into vials with 70% ethanol to our labs for species identification. Ticks were then identified morphologically according to Manilla (1998).
The study was conducted from April to October 2017 in Sardinia, Italy. Sardinia is the second-largest island of the Mediterranean Sea with an area of 23,821 km2. The temperate Mediterranean climate of the island enables the survival of ticks throughout the year (Chisu et al., 2018). Sardinia environment provides a variety of ecosystems and ideal natural conditions to study disease ecology. Ticks were collected by veterinary practitioners from 22 collection sites (Table 1). Seven collection sites (Oschiri; Sassari, Alghero, Borutta; Ozieri; Pattada, Asinara), belonging to Northwest Sardinia, comprise a variety of ecosystems including plains, hills and promontories. Two sites, located in the center of Sardinia (Orgosolo and Dorgali) are morphologically complex and include sandy coasts, cliffs and mountains with forests of oak, juniper and yew. Ten collection sites (Urzulei, Aritzo, Talana, Baunei, Barisardo, Cardedu, Triei, Tortolì, Perdasdefogu and S. Nicolò Gerrei) are located in Southwest Sardinia where mountains, forests and green valleys are covered by the typical Mediterranean maquis. The landscape is also characterized by cultivated coastal plains, watercourses and rocky sheer coasts. The three collection sites of South-West Sardinia (Santadi, Uta and Sant’Anna Arresi) are located in a territory that present diversified and complex morphological features with the presence of plains, plateaus and reliefs at different height. Part of the territory presents karst cavities of morphological and mineralogical interest.
2.3. Tick processing and DNA extraction Ticks were individually immersed in distilled water for 10 min, dried on sterile filter paper, cut and crushed into small pieces with a sterile scalpel in microtubes (Eppendorf, Hilden, Germany) for DNA to be extracted efficiently. Sterile instruments were used for each individual tick cut. DNA was extracted from each tick using DNeasy® Blood & Tissue Kit (QIAGEN, Chatsworth, CA, USA), by following the manufacturer's instructions. 2.4. PCR amplification, purification and sequencing DNA extracts from all 166 ticks were screened for the presence of piroplasms by a conventional PCR that amplifies an approximately 450 61
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3. Results
bp fragment of the 18S ribosomal RNA gene of Babesia/Theileria species. The reaction was made up to a final volume of 25 μl containing 12.5 μL μl of Amplitaq Gold master mix (Quantitect Probe PCR Master Mix, Qiagen, Hilden, Germany), 1 μl of 25 μM of primer BJ1 (5′-GTCT TGTAATTGGAATGATGG-3′) and BN2 (5′-TAGTTTATGGTTAGGACT ACG-3′) (Casati et al., 2006), 9.5 μL of RNase nuclease-free water and 1 μl of genomic DNA. A negative control using RNase nuclease-free water (Qiagen, Hilden, Germany) and a DNA positive control of B. bovis were included in each PCR test. The reaction mixtures were subjected to an initial denaturation step of 15 min at 95 °C, followed by 40 cycles of denaturation at 94 °C for 1 min, annealing at 60 °C for 30 s., and elongation at 72 °C for 1 min. Amplification was completed by a further 5 min step at 72 °C. Negative and positive controls were included in each amplification assay. The PCR products were separated electrophoretically in 1.5% agarose gel under standard conditions. DNA Molecular Weight Marker VIII (Roche,) was used for DNA sizing. The products were treated with nontoxic SYBR® Green DNA Gel Stain (Invitrogen, Carlsbad, CA, USA), and visualized using standard UV transillumination. Positive PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol and then directly sequenced in both directions by using an ABI Prism BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA, USA).
3.1. Morphological detection of ticks The ticks were morphologically identified as follows: Rh. bursa (n = 22); Rh. sanguineus s.l. (n = 97); Rh. annulatus (n = 13); Hy. marginatum (n = 18); Hae. punctata (n = 6); D. marginatus (n = 9); Ornithodoros maritimus (n = 1) as shown in Table 1. 3.2. Molecular detection of piroplasmids Piroplasmid DNAs were detected in 34 out of 166 (21%) ticks removed from different wild and domestic hosts that showed no apparent clinical signs or symptoms at the sampling time (Table 1). More specifically, infection rates were 18% (4/22), 16.5% (16/97), 8% (1/13), 17% (3/18), 67% (4/6) and 67% (6/9) in Rh. bursa, Rh. sanguineus s.l., Rh. annulatus, Hy. marginatum, Hae. punctata, and D. marginatus ticks, respectively. All the 34 amplicons were sequenced and 19 good quality sequences were obtained. Fifteen poor quality sequences were discarded from further analysis. The 18S rRNA sequences obtained in this study were 99–100% identical to T. buffeli, T. equi, T. ovis and Babesia bigemina sequences (Table 2). Upon sequencing and ClustalX alignment, 18S rRNA sequences were assigned to nine different sequence types. TSard2018_1 (identified in one Rh. bursa, five Rh. sanguineus s.l., one Rh. annulatus, one Hy. marginatum, one Hae. punctata, and one D. marginatus collected from fox, cattle, goat, dogs and whinchat, PSard2018_2, PSard2018_3 and PSard2018_4 (include three sequences derived from three Rh. sanguineus s.l. ticks collected from dogs); PSard2018_5 (derived from a Rh. sanguineus s.l. collected from a mouflon); PSard2018_6 (from a Rh. bursa tick collected from a mouflon), PSard2018_7 and PSard2018_9 (include two sequences derived from two Rh. sanguineus s.l. ticks from dogs); TSard2018_8 (derived from one D. marginatus from a cattle). TSard2018_1, TSard2018_3, TSard2018_5, TSard2018_7, and TSard2018_8 sequence types shared 99–100% identity with T. buffeli, T. sergenti, and T. orientalis genotypes isolated worldwide from ruminants and ticks and deposited in the GenBank. TSard2018_2 and TSard2018_4 showed 99–100% identity with T. equi genotypes isolated from horses in Cuba (KY111762), Brazil (KX722519), Australia (KJ801925), and USA (JX177672). TSard2018_6 sequence type showed 100% identity to T. ovis. The sequence type named PSard2018_9 detected in two Rh. sanguineus s.l. ticks sampled from a dog shared 99% similarity with B. bigemina sequences isolated worldwide. Phylogenetic analysis (Fig. 1) of the partial 18S rRNA gene was performed by aligning the nine sequence types obtained in this study with selected Babesia and Theileria sequences. Sequence types PSard2018_1, PSard2018_3, PSard2018_5, PSard2018_7 and PSard2018_8 grouped with reference strains representative of T. buffeli. PSard2018_2, and PSard2018_4 sequence types were assigned to the T. equi clade. PSard2018_6 was closely related to T. ovis strain. PSard2018_9 grouped in a strongly supported monophyletic clade with B. bigemina.
2.5. Sequence and phylogenetic analysis Chromatograms of forward and reverse sequences generated with 18S rRNA primers were edited with Chromas 2.2 (Technelysium, Helensvale, Australia), and then aligned with CLUSTALX (Larkin et al., 2007) in order to assign them to unique sequence types, and checked against the GenBank database with nucleotide blast BLASTN (Altschul et al., 1990). Pairwise/multiple sequence alignments and sequence similarities were calculated using the CLUSTALW (Thompson et al., 1997) and the identity matrix options of Bioedit (Hall, 1999), respectively. For phylogenetic analyses, the nine sequence types obtained in this study (TSard2018_1-9) were aligned with a set of 43 sequences representing the 18S rRNA variability of the different species belonging to the genus Theileria and Babesia. The reference sequences used in this study are shown in Fig. 1. Sequences were aligned with ClustalX (Thompson et al., 1997). Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013) by using the Maximum Likelihood method based on the Kimura 2parameter model (Kimura, 1980) identified as the best model with the same software. Initial tree for the heuristic search was obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood approach, and then selecting the topology with superior log likelihood value. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985).
4. Discussion Theileriosis and babesiosis are tick-borne diseases that cause major economic losses and affect many domestic animals, mainly cattle and sheep, in tropical and subtropical regions (Uilenberg, 1995). In this study DNA of piroplasmids was detected in different tick species collected from several vertebrate hosts from a typical subtropical Mediterranean region, the island of Sardinia. Results indicate the presence of Theileria and Babesia species closely related to T. buffeli, T. equi, T. ovis and B. bigemina in Rh. sanguineus, Rh. bursa, Rh. annulatus, Hae. punctata, Hy. marginatum and D. marginatus ticks. As ticks were engorged when removed from their hosts, we cannot state that these ticks are competent vectors for the detected
2.6. Sequence accession numbers Representative Theileria and Babesia sequences were submitted to the NCBI database using the National Center for Biotechnology Information (NCBI; Bethesda, MD) BankIt v3.0 submission tool (http:// www3.ncbi.nlm.nih.gov/BankIt/) under accession numbers MK587691-MK587709.
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Fig. 1. Molecular phylogenetic tree by using Maximum Likelihood method showing the evolutionary relationship of the nine identified piroplasmids sequence types and other 44 taxa used in this study. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.2366)). The analysis involved 52 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 285 positions in the final dataset.
the genus Hyalomma with the Hy. lusitanicum and Hy. marginatum species have been also documented in the island. A small percentage of tick population is represented by Ixodes festai and Argas reflexus (Chisu et al., 2018). However, these tick genera are recognized vectors of pathogenic bacteria with medical and veterinary relevance (Chisu et al., 2018). Although bovine theileriosis caused by T. annulata and T. parva is considered one of the four most important tick-borne haemoparasitic diseases of livestock (Uilenberg, 1995), there are increasing records of the T. orientalis/T.buffeli/T. sergenti group causing economically significant infections, especially in immunocompromized or stressed
piroplasmids, since they might have got the infection feeding on infected bacteremic animals, or/and by co-feeding with other infected ticks at the same feeding site. Sardinia is the second largest island in the Mediterranean Sea, and its ecosystem allows the survival and maintenance of a rich fauna of ixodid ticks throughout the year (Satta et al., 2011). Ticks of the Rhipicephalus genus are the best represented on the island and include five species: Rh. sanguineus, Rh. pusillus, Rh. turanicus, Rh. bursa and Rh. (Boophilus) annulatus infesting wild and domestic vertebrates (Chisu et al., 2018). The genus Dermacentor, with the D. marginatus which is the only species occurring in the island; the genus Haemaphysalis (with Hae. punctata, and Hae. sulcata species) and
Table 2 Sequence type and BLASTN identities of the 18S rRNA gene sequence types identified in this study. Sequence Type
Tick species (no. of positive)
Tick vertebrate host
GenBank accession number
BLASTN identity
TSard2018_1
Rh. sanguineus s.l. (5) Hy. marginatum (1) D. marginatus (1) Rh. bursa (1) Hae. punctate (1) Rh. annulatus (1) Rh. sanguineus s.l. (1) Rh. sanguineus s.l. (1) Rh. sanguineus s.l. (1) Rh. sanguineus s.l. (1) Rh. bursa (1) Rh. sanguineus s.l. (1) D. marginatus (1) Rh. sanguineus s.l. (2)
Dog,Goat Whinchat Cattle Fox Cattle Cattle Dog Dog Dog Mouflon Mouflon Dog Cattle Dog
MK587691-MK587695 MK5876916 MK587697 MK587698 MK587699 MK587700 MK587701 MK587702 MK587703 MK587704 MK587705 MK587706 MK587707 MK587708, MK587709
99% T. buffeli/sergenti/orientalis
TSard2018_2 TSard2018_3 TSard2018_4 TSard2018_5 TSard2018_6 TSard2018_7 TSard2018_8 TSard2018_9
63
99% T. equi 100% T. buffeli/sergenti/orientalis 100% T. equi 99% T. buffeli/ sergenti/ orientalis 100% T. ovis 100% T. buffeli/ sergenti/ orientalis 99% T. buffeli/sergenti/orientalis 99% B. bigemina
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animals (Shimizu et al., 2000; Kim et al., 1998). These piroplasms are transtadially transmitted by different Haemaphysalis spp. in Asia, Australia and New Zealand (Kamau et al., 2011; Fuujisaki, 1992). However, the transmission is not limited to this tick genus which could explain their worldwide occurrence (McFadden et al., 2011). In this study, five Theileria strains closely related to T. buffeli/sergenti/orientalis group were detected in different tick species (see Table 2) that may suggest their possible role as competent vectors for Theileria species in Sardinia, but their role in the transmission of T. buffeli/sergenti/orientalis complex remains to be shown. Theileria equi is a small piroplasm infecting erythrocytes causing equine piroplasmosis (EP) which affects the horse industry worldwide (Scoles and Ueti, 2015). EP is also an important cause of horse morbidity in Sardinia where both Babesia caballi and T. equi have been detected (Zobba et al., 2011). Prevalence of the disease is related to distribution of vector ticks (de Waal, 1992). Species of the genera Dermacentor, Hyalomma and Rhipicephalus are the biological vectors of T. equi (de La Fuente et al., 2008). All potential vectors of T. equi have been recorded in Sardinia (Chisu et al., 2018), suggesting their role as vectors of this piroplasm. In this study, two sequences phylogenetically related to T. equi were detected in two Rh. sanguineus s.l. ticks collected from dogs. Presence of pathogenic T. equi in symptomatic and asymptomatic dogs in Croatia and Spain has been previously reported (Beck et al., 2009; Criado-Fornelio et al., 2003). As Rh. sanguineus s.l. are well represented in the island and are mainly collected from dogs, the relationship between the newly identified parasite strain, the Rh. sanguineus s.l. ticks, and infection in dogs deserves further investigation. One Rh. bursa tick collected from a mouflon was positive for T. ovis. The transmission of T. ovis by the two-host tick Rh. bursa has been emphasized by e.g. Aktas et al. (2006) and Altay et al. (2005). Theileria ovis occurs in the Mediterranean basin as well as in other areas where the tick vector is present (Aydin et al., 2015; Yeruham et al., 1995). Subclinical infection in small ruminants caused by T. ovis has been reported from sheep in different countries (Altay et al., 2005; Nagore et al., 2004; Schnittger et al., 2012). Rh. bursa is widespread in the island and is frequently found on both domestic and wild ruminants (Chisu et al., 2018). Further research is needed in order to determine the presence of T. ovis in Rh. bursa ticks and related hosts in the island. In this study, B. bigemina was also detected in two Rh. sanguineus s.l. adult ticks, in agreement with literature data where the parasite was found in Rhipicephalus ticks in Iran (Rajabi et al., 2017) and Central and Southern Italy (Toma et al., 2017). Considering that B. bigemina is a common cause of bovine babesiosis in Italy (Toma et al., 2017), the population of Rh. sanguineus s.l. should be monitored, and further studies are needed to fully understand the link between this tick species and B. bigemina in the island. The identified Theileria and Babesia species are not associated with human infection. In Sardinia, ruminants are an important source of income and are exploited for milk and meat production. Monitoring the circulation of tick population on the island, the tick borne pathogens and the risk of animals and humans exposure to tick-borne diseases could be useful for tick bite risk assessment and for widening of surveillance methods. Further studies are nevertheless needed to better characterize piroplasmid strains by analyzing more discriminative genes, and to identify the main vectors implicated in their transmission in Sardinia.
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