VECTOR-BORNE AND ZOONOTIC DISEASES Volume 14, Number 10, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/vbz.2014.1648
Molecular Epizootiology and Diagnosis of Porcine Babesiosis in Sardinia, Italy Rosanna Zobba,1 Anna Maria Nuvoli,1 Francesca Sotgiu,1 Roberta Lecis,1 Antonio Spezzigu,1 Gian Mario Dore,1 Marco Antonio Masia,1 Carla Cacciotto,1 Maria Luisa Pinna Parpaglia,1 Daniele Dessı`,2 Marco Pittau,1 and Alberto Alberti1
Abstract
The recent characterization of the 18S ribosomal RNA (rRNA) of a pathogenic Babesia species in a domestic sow paved the way for establishing diagnostic and epidemiological tools for porcine babesiosis. Here, we developed the first specific Babesia sp. Suis PCR, and we applied this test to a panel of samples collected from animals living in a typical Mediterranean environment (Sardinia, Italy), including domestic pigs, wild boars, and ticks. In domestic pigs, PCR coupled with sequencing revealed an estimated Babesia infection frequency of 26.2% and the presence of distinct 18S sequence types. The different distribution of sequence types in symptomatic and asymptomatic subjects might suggest the existence of phylogenetically closely related strains with variable pathogenicity in pigs. Moreover, molecular identification of tick species indicated Rhipicephalus sanguineus and Rhipicephalus bursa as candidate vectors potentially involved in the transmission of this pathogen. Collectively, the data reveal the suitability of 18S rRNA PCR/sequencing for molecular diagnosis of porcine babesiosis and for large-scale investigations on the presence and geographical distribution of Babesia sp. Suis genetic variants. Key Words:
Piroplasmida—Ticks—Tick-borne diseases—Protozoan—Anaplasmosis.
Introduction
B
abesiosis is a disease caused by protozoa of the genus Babesia, which invade and destroy erythrocytes. Babesia spp. are typically transmitted by Ixodidae ticks and are considered by some authors the second most common parasitic organisms in the blood of mammals, after trypanosomes (Schnittger et al. 2012). Babesiosis is raising veterinary and public health concerns for its great economic and medical impact worldwide (Setty et al. 2003, Colwell et al. 2011, Schnittger et al. 2012), and it is being increasingly acknowledged as an important emerging zoonosis (Kjemtrup and Conrad 2000, Lobo et al. 2013). In general, Babesia infections can cause a variety of nonspecific symptoms in immunocompetent patients. Common manifestations of babesiosis include mild to high fever, chills, anorexia, nausea, vomiting, anemia, and abortion. Host age, immunological status, and genetic factors may predispose the patient to more severe disease. In animals, co-infection with species of the Borrelia burgdorferi sensu lato (s.l.) complex, and/ or concurrent anaplasmosis can also dramatically complicate the clinical presentation of babesiosis (Homer et al. 2000, Lobo et al. 2013). 1
Babesia species infect a wide variety of both domestic and wild ungulates (Penzhorn 2006), usually occurring in closely related hosts. Examples of these are Babesia trautmanni, which is able to infect domestic pig and wild boar (Sus scrofa), but also the African bushpig Potamochoerus porcus and the warthog Phacochoerus aethiopicus. Furthermore, Babesia caballi and Theileria equi can infect horse and various zebra species (Penzhorn 2006). Porcine babesiosis has been reported in the former Soviet Union, southern Europe, Africa, and China (Yin et al. 1997, Penzhorn 2006, Uilenberg 2006), often associated with serious economical losses and significant mortality rates. Despite its importance, it still remains an overlooked disease, with the etiological agents still poorly investigated (Penzhorn 2006, Zobba et al. 2011). Literature on Babesia species affecting pigs is extremely scarce, and no specific molecular tools have been developed for these pathogens detection (Criado-Fornelio 2012). On the basis of morphological diagnosis, two Babesia spp. have been reported in pigs: B. trautmanni, transmitted by Rhipicephalus ticks and characterized by large piroplasms, and the smaller Babesia perroncitoi, whose vector has not been
Dipartimento di Medicina Veterinaria and 2Dipartimento di Scienze Biomediche, Universita` degli Studi di Sassari, Sassari, Italy.
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identified yet (Uilenberg 2006). As mentioned above, babesiosis outbreaks in worldwide pig populations are seldom investigated (Penzhorn 2006). On the other hand, cases of porcine babesiosis are systematically reported in Sardinia, Italy, although only at the local level (i.e., by veterinary practitioners) and sporadically in the scientific literature (Ligios and Scala 1993), on the basis of clinical diagnosis and microscopy. In 2011, a Babesia species was identified in a symptomatic sow, and its 18S rRNA was partially characterized (Zobba et al. 2011). The new isolate was found to be morphologically similar to B. trautmanni and genetically related to Ungulibabesids, However, because previous reports of B. trautmanni and B. perroncitoi lacked strain genetic characterization, this isolate could not be unambiguously assigned to one of these two species, and it was designated Babesia sp. Suis. Molecular data presented by Zobba and co-workers (2011) provided basic genetic information for the development of specific diagnostic tools for molecular epidemiological studies on porcine Babesia spp. In this work, we generated a highly sensitive and specific PCR test for Babesia sp. Suis that we applied to a panel of samples collected from animals and ticks in Sardinia, chosen as a representative of a Mediterranean subtropical area. Babesia sp. Suis strains were phylogenetically characterized, and host ticks species potentially involved in their transmission were also identified. Materials and Methods Animals, samples, and DNA extraction
Between 2011 and 2013, EDTA blood samples were obtained from 61 domestic swine traditionally farmed in small semifree herds in northern Sardinia. Herds were selected in areas where clinical cases had been previously reported. All domestic pigs were subjected to clinical examination and did not present tick infestation at the time of sampling. A total of 52 wild boar hunted in the same area were sampled and included in the analysis. Additionally, 64 engorged ticks were collected from domestic and wild animals farmed and/or free ranging in the same northern areas of the island (Table 1). EDTA blood was sampled from two bovines positive to Theileria buffeli and Babesia major, respectively, from a sheep naturally infected with T. ovis, from a horse naturally infected with T. equi, and from a sow infected with Babesia sp. Suis (Zobba et al. 2011). Positivity to T. buffeli, T. ovis, T. equi, and B. major was established by PCR (CriadoFornelio et al. 2003) and sequencing (unpublished). B. bovis, B. caballi, and T. equi fluorescence assay (FA) substrate slides were from Fuller Laboratories (Fullerton, CA). The presence of antigens in FA slides was confirmed by PCR (Criado-Fornelio et al. 2003). DNA was extracted from blood samples and from the FA slides using the DNeasy Blood and Tissue Kit (Qiagen, Italy), following the manufacturer’s recommendations. DNA was quantified with a BioPhotometer plus (Eppendorf, Italy).
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Table 1. Origin of Pig, Wild Boar, and Tick Samples Analyzed in This Study and Results of the 18S rRNA PCR (No. Positive/Total) Host species
Geographic location
Pig
Ala` dei Sardi Bortigiadas Chiaramonti Laerru Nule Perfugas Putifigari Sassari Tuili
Wild boar
Putifigari Villanova Monteleone
Ticks
Asinara (birds) Badesi (bovine) Bonassai (wild boar) Nuoro (dog) Nurra (horse, dog) Nurri (dog) Padru (dog) Perfugas (ruminants) Piedmont (dog) Putifigari (goat) Oristano (dog) Sassari (dog) Siniscola (dog) Monastir (deer)
No. of sampled animals
Babesia sp. Suis 18S rRNA PCR
16 3 10 3 4 10 6 6 3 61 47 5 52 6 1 1 4 4 1 1 21 15 1 6 2 1 1 65
1/16 1/3 0/10 2/3 0/4 6/10 0/6 6/6 0/3 16/61 0/47 0/5 0/52 0/6 0/1 0/1 0/4 1/4 0/1 0/1 4/21 0/15 0/1 0/6 0/2 0/1 0/1 5/65
primers were designed. The two primers were combined with the already published primer Bab/ThelF (Criado-Fornelio et al. 2003) to generate a specific heminested PCR test to detect Babesia sp. Suis in clinical samples. In the first PCR round, primers Bab/ThelF (5¢-GACACAGGGAGGTAGTG ACAAG-3¢) and Bsu/18S/1258R (5¢-CGTAGCGAAACC GAGTAACAC-3¢) were used to target an 860-bp fragment. Briefly, 100–150 ng of DNA were used in a 50-lL PCR reaction containing 200 lM deoxynucleotide triphosphates (dNTPs), 0.3 lM of each of the two primers, and 1.25 U of Taq DNA polymerase (Qiagen, Italy). One microliter of the first PCR product was used as a template in the second PCR in a similar reaction mix with the internal primer Bsu/18S/580F (5¢-ATTTCAGCCTTTTGCGATTG-3¢) combined to primer Bsu/18S/1258R, to amplify a product of approximately 680 bp. Both the first and second PCR amplifications were performed with an initial denaturation at 94C for 3 min, 30 cycles of: Denaturation at 94C (30 sec), annealing at 50C, and 55C in the first and second PCR, respectively (30 sec), extension at 72C (1 min), and final extension at 72C for 10 min. In total, 177 DNA samples obtained from 61 pigs, 52 wild boars, and 64 ticks were tested by 18S heminested PCR.
Babesia sp. Suis 18S heminested PCR
On the basis of the 18S rRNA sequence alignment of Babesia sp. Suis (accession no. HQ437690) with the corresponding region of different Babesia and Theileria species available in the GenBank database, two highly specific
Babesia sp. Suis 18S heminested PCR sensitivity and specificity
To test sensitivity of the 18S rRNA heminested PCR, plasmid pCR4/18s/Bspsuis was generated by cloning a
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first-round PCR product into the pCR4-TOPO vector (Life Technologies, Italy). According to the method used by Alberti and Sparagano (2006), pCR4/18s/Bspsuis was quantified and brought to a concentration of 100,000 copies/lL, after which serial two-fold dilutions of the plasmid were obtained until the concentration of one copy/lL was reached. One microliter of each dilution was coupled to 100 ng of calf thymus DNA (Sigma, Poole, UK) and tested with the Babesia sp. Suis 18S heminested PCR. Three independent serial dilutions were obtained and tested using PCR. Dilutions were considered PCR-positive when resulting in the desired band at least in two out of three serial dilutions replicates. To assess PCR specificity, a DNA sample extracted from blood obtained from a Babesia sp. Suis–positive sow (Zobba et al. 2011) was tested together with a panel of DNA samples representative of different Babesia species. In particular, the following species were tested by 18S heminested PCR: T. buffeli, B. major, T. ovis, T. equi, B. caballi, and B. bovis. Sequencing, 18S rRNA variability, and phylogenetic analyses
Positive PCR products were cloned into the plasmid pCR4TOPO vector (Life Technologies, CA) and used to transform One Shot TOP10 Chemically Competent Escherichia coli (Invitrogen, Italy). Plasmid DNA was extracted from positive colonies with PureLink Quick Plasmid MiniPrep Kit (Invitrogen, Italy) and automatically sequenced (BMR Genomics, Italy). At least five positive E. coli colonies were selected for each transformation. The generated sequences were edited with Chromas 2.2 (Technelysium, Australia) and aligned with ClustalW (Thompson et al. 1994) to assign them to unique sequence types. Unique sequence types, named after the host species and sequentially ordered, were checked against the GenBank database with nucleotide blast BLASTN (Altschul et al. 1990). To identify nucleotide signatures of porcine isolates, the 18S rRNA
sequence types obtained in this study were aligned with two sequences (accession nos. AY726557 and EU376017) representing the 18S rRNA gene variability of Ungulibabesids (Criado-Fornelio et al. 2003, Dawood et al. 2013). Phylogenetic analyses were performed aligning the porcine 18S rRNA sequence types with a set of 35 sequences belonging to the six Babesiadae clades identified by Schnittger et al. (2012). The set was composed by: Four clade-1 sequences (B. felis AY452707, B. leo AF244911, B. microti U09833, B. rodhaini M87565); three clade-2 sequences (B. lengau KC790443, B. conradae AF158702, B. duncani HQ289870); three clade-3 sequences (Theileria youngi AF245279, Cytauxzoon felis AY979105, T. bicornis AF499604); two clade-4 sequences (B. equi AY150064, B. bicornis AF419313); seven clade-5 sequences (T. parva L02366, T. taurotragi L19082, T. annulata M64243, T. buffeli DQ104611, T. velifera AF097993, T. mutans AF078815, T. separata AY260175); and 16 sequences belonging to clade-6 (B. orientalis AY596279, B. bovis AY150059; B. bennetti DQ402155; B. ovis AY150058; B. motasi AY533147; B. bigemina X59605; B. ovate AY603400; B. caballi Z15104; B. major GU194290; B. crassa AY260176; B. gibsoni DQ184507; B. divergens FJ944826; B. canis rossi DQ111760, B. canis canis AY072926, B. canis vogeli AY371198, B. poelea DQ200887). Additionally, Cardiosporidium cionae (EU052685) was included as outgroup in the phylogenetic analysis. Pairwise/multiple sequence alignments and sequences similarities were calculated using the ClustalW and the identity matrix options of Bioedit (Hall 1999), respectively. The evolutionary history of Babesia sp. Suis and of the 36 species (35 Babesia species and the outgroup species) considered in the analysis was inferred by using the maximum likelihood method based on the Kimura 2-parameters method (Tamura et al. 2004) identified as the best-suited evolutionary model for our data. Initial tree(s) for the heuristic search were obtained automatically by applying neighbor-joining (NJ)
Table 2. Designation, Sequence Type, Geographical Origin, and BLASTN Results of the 18S Sequence Types Identified in this Study (Maximum Identity) Sequence type Swinetick1
Swine1 Swine2 Swine3 Swine4 Swine5 Swine6 Tick1 Tick2 Tick3
Host
Geographical location
GenBank no.
BLAST analysis
Sus scrofa 01 Sus scrofa Bar02 Sus scrofa 223 Sus scrofa 224 Sus scrofa 292 Sus scrofa T01 Sus scrofa T02 Sus scrofa 14743 Sus scrofa S05 Sus Scrofa Gio1 Tick 73A Tick Candy12 Sus scrofa 280 Sus scrofa 220 Sus scrofa 14740 Sus scrofa 14742 Sus scrofa 14737 Sus scrofa S06 Tick 57 Tick 60B Tick 64A
Laerru (NW Sardinia) Laerru (NW Sardinia) Perfugas (NW Sardinia) Perfugas (NW Sardinia) Perfugas (NW Sardinia) Sassari (NW Sardinia) Sassari (NW Sardinia) Sassari (NW Sardinia) Perfugas (NW Sardinia) Perfugas (NW Sardinia) Perfugas (NW Sardinia) Nurra (NW Sardinia) Ala` dei Sardi (NE Sardinia) Bortigiadas (N Sardinia) Sassari (NW Sardinia) Sassari (NW Sardinia) Sassari (NW Sardinia) Perfugas (NW Sardinia) Perfugas (N Sardinia) Perfugas (N Sardinia) Perfugas (N Sardinia)
KJ801525 KJ801526 KJ801527 KJ801528 KJ801529 KJ801530 KJ801531 KJ801532 KJ801533 KJ801534 KJ801535 KJ801536 KJ801537 KJ801538 KJ801539 KJ801540 KJ801541 KJ801542 KJ801543 KJ801544 KJ801545
100% Babesia sp. Anglona/AA-2011
99% 99% 99% 99% 99% 99% 99% 99% 99%
Babesia Babesia Babesia Babesia Babesia Babesia Babesia Babesia Babesia
sp. sp. sp. sp. sp. sp. sp. sp. sp.
Anglona/AA-2011 Anglona/AA-2011 Anglona/AA-2011 Anglona/AA-2011 Anglona/AA-2011 Anglona/AA-2011 Anglona/AA-2011 Anglona/AA-2011 Anglona/AA-2011
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and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories [ + G, parameter = 0.5581]). The rate variation model allowed for some sites to be evolutionarily invariable ([ + I], 59.8244% sites). A total of 580 positions were included in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura et al. 2011). Statistical support for internal branches of the trees was evaluated by bootstrapping with 1000 iterations (Felsenstein 1985). NJ (Saitou and Nei 1987) trees and consensus values were generated using the same software. Trees were edited using NJplot (Perrie`re and Gouy 1996) and Treeview v. 1.5.2 (Page 1996). To reduce systematic errors, to investigate more deeply into taxa evolutionary relationships, and to test robustness of the phylogenetic analyses, sequence clusters were also detected by the analysis of phylogenetic networks inferred from uncorrected p-distances with the Neighbor-Net method in SplitsTree4 (Huson and Bryant 2006), not considering invariable sites. MEGA5 was also used to investigate the variability of the nucleotide positions in the 18S RNA sequences alignment. Babesia sp. Suis nucleotide sequence data reported in this paper are available in GenBank under the accession numbers reported in Table 2. Molecular identification of tick species and additional PCR tests
To identify tick species, a PCR amplifying a 460-bp fragment of mitochondrial 16S rRNA was performed on Babesia sp. Suis–positive ticks using primers 16S + 1/16S - 1, as in Black and Piesman (1994). PCR products were directly sequenced and checked against the GenBank database with BLAST. Tick 16S RNA sequences were submitted to GenBank (accession nos. KJ814006, KJ814007, KJ814008, KJ814009, and KJ814010). Animals positive to Babesia sp. Suis were also screened for the presence of Anaplasma spp. as previously described (Zobba et al. 2014). Results
On general physical examination, 10 out of 61 domestic pigs showed severe clinical signs indicative of babesiosis, such as high fever, chills, anorexia–dysorexia, nausea, vomiting, anemia, lateral recumbency, lameness, hematuria, and abortion. Clinical signs did not significantly differ among the subjects examined. Sensitivity of the Babesia sp. Suis 18S heminested PCR (Fig. 1A, B), evaluated by testing two-fold dilutions of plasmid pCR4/18s/Bspsuis, was 500 copies in the primary PCR and one copy in the heminested PCR. Both the newly developed primary and heminested PCRs were highly specific for Babesia sp. Suis (Fig. 1C, D), as only DNA samples obtained from Babesia sp. Suis–positive sows (Zobba et al. 2011) were PCR positive when tested together with a panel of DNA samples representative of different Babesia species. All DNA samples obtained from the 10 symptomatic pigs were positive to Babesia sp. Suis when tested by 18S rRNA heminested PCR and generated the predicted band of 680 bp on agarose electrophoresis (100% prevalence in symptomatic
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FIG. 1. Detection limit and specificity of the 18S rRNA Babesia sp. Suis–specific PCRs, developed during this study, using Taq DNA polymerase (Qiagen, Italy). (A and B) Results obtained by the first-round (A) and second-round PCRs (B) from two-fold dilutions of plasmid generated by cloning a first-round PCR product. Lanes A, 100,000 copies; lanes B, 50,000 copies; lanes C, 25,000 copies; lanes D, 10,000 copies; lanes E, 5000 copies; lanes F, 2500 copies; lanes G, 1000 copies; lanes H, 500 copies; lanes I, 100 copies; lanes L, 50 copies; lanes M, 10 copies; lanes N, one copy. (C and D) Results obtained with the first (C) and second (D) round of the 18S rRNA Babesia sp. Suis– specific PCR using DNA from a sow infected by Babesia sp. Suis (lanes 1); DNA from B. bovis (lanes 2), B. caballi (lanes 3), and T. equi (lanes 4); DNA from bovines positive to T. buffeli (lanes 5) and B. major (lanes 6); DNA from a sheep infected with T. ovis (lanes 7) and DNA from a horse infected with T. equi (lanes 8). Lanes ‘‘-’’ and ‘‘M’’ are the negative PCR controls (distilled water) and the 100-bp DNA ladder (Life Technologies, Italy), respectively. The same DNA ladder was used in A–D.
pigs). Additionally, six asymptomatic pigs were PCR positive (12% prevalence in asymptomatic pigs). Considering symptomatic and asymptomatic pigs together, Babesia sp. Suis infection frequency was therefore 26.2%. Babesia sp. Suis 18S rRNA was never identified in DNA samples extracted from the 52 wild boars sampled in geographical areas partially overlapping the domestic pigs distribution. Five engorged ticks, collected, respectively, from a horse and four goats, also tested positive for Babesia sp. Suis. On the basis of sequencing results, a total of 10 different 18S rRNA sequence types were identified (Table 2). Sequence type Swinetick1 was identified in 10 symptomatic pigs belonging to three geographically distant herds (Perfugas, Laerru, and Sassari). Also, two ticks collected from a horse and a goat in northern Sardinia tested positive for the same sequence type. A number of sequence types (Swine1–6) were found in asymptomatic pigs coming from two herds where symptomatic subjects were also present (Perfugas and Sassari), and from a herd (Bortigiadas) characterized by a clinically silent infection. Sequence types Tick1–3 were found exclusively in three ticks collected from three sheep coming from one herd (Perfugas).
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Table 3. Nucleotide Variable Positions and Signatures of the 10 Porcine Babesia sp. Suis Sequence Types Identified in This Study 18S rRNA variable positionsa 0 0 3 SwineTick1 T Swine1 Swine2 Swine3 Swine4 Swine5 Swine6 Tick1 Tick2 Tick3 Babesia sp. Kashi B. occultans C
0 0 5 G T
0 0 8 A G
0 1 1 G C
0 1 2 T C
0 1 5 G C
0 1 6 G T
0 1 9 A T
0 2 0 T C C
0 2 2 G C
0 2 7 A T
0 3 0 T C
0 3 2 G A
0 9 3 A G A
1 3 3 T
1 3 4 A T A
1 3 7 T C T
2 3 8 A G A
2 5 3 A G C A
3 4 3 A G A
3 6 0 A G A
3 8 2 T C T
4 2 6 A G A
4 6 4 C T C
5 6 3 T C T
6 0 6 T C T
6 1 2 A G A
6 3 8 A T
a Sites are numbered according to the alignment, starting from the first nucleotide position after primer sense. Variable positions conserved among porcine isolates are shaded in grey.
BLAST analyses conducted on the sequence types obtained in this study revealed a variable degree of nucleotide similarity with a Babesia sp. Suis strain (GenBank accession number HQ437690) already reported in the same geographical area and recovered from a symptomatic sow (Zobba et al. 2011). In particular, sequence type Swinetick1, found in symptomatic pigs and in ticks, showed 100% identity with HQ437690, whereas the other sequence types (Swine1–6, Tick1–3), found in asymptomatic subjects and in ticks were 99% similar to the same sequence. Alignments of the 10 18S sequence types together with sequences representative of Ungulibabesids (Criado-Fornelio et al. 2003, Dawood et al. 2013) allowed identification of 14 variable positions conserved among porcine isolates and therefore representing nucleotide signatures of this group of sequences (Table 3). Phylogenetic results obtained in this study were consistent with previous observations (Criado-Fornelio et al. 2003, Zobba et al. 2011, Schnittger et al. 2012, Dawood et al. 2013). Maximum likelihood trees generated with MEGA5 allowed the identification of six major Babesiadae clades (Fig. 2). The 10 Babesia sp. Suis sequence types rescued in this study grouped in the statistically supported clade 6 (Schnittger et al. 2012), together with the other Ungulibabesids. Network analyses (Fig. 3) confirmed maximum likelihood observations and identified the same Babesia groups. Tick DNA samples positive to Babesia sp. Suis generated a band of 460 bp when analyzed with the mitochondrial 16S rRNA diagnostic PCR (see Materials and Methods). Sequencing and BLAST analyses allowed identifying four ticks as Rhipicephalus bursa (nucleotide identity 99%) and one as R. sanguineus (nucleotide identity 96%). All DNA samples were negative to Anaplasma spp. Discussion
For their pathogenic role in animals and as zoonotic agents, Babesia species have been increasingly studied in the last decades. Although the major economic impact of babesiosis in animals is on the cattle industry, the relevance of infections in other domestic species, such as small ruminants, horse, and dogs, varies throughout the world. In
pigs, babesiosis is responsible for serious losses and produces antemortem clinical signs that may be similar to those of bovine babesiosis. Knowledge regarding porcine babesiosis is especially limited by the lack of population genetics and molecular epidemiology data, which hamper diagnostics and strain typing. For these reasons, porcine babesiosis remains an overlooked disease, scarcely reported in international literature (Zobba et al. 2011, Criado-Fornelio 2012). In the Mediterranean area, porcine babesiosis has been associated with two Babesia species, namely B. trautmanni and B. perroncitoi, classified on the basis of different size of piroplasmas (Kauffman 1996). Recently, Babesia sp. Suis has been characterized molecularly and suspected to represent a strain of B. trautmanni based on the size of piroplasms (Zobba et al. 2011). In this study we developed a molecular approach (based on specific PCR and sequencing) that has an inbuilt potential for improving molecular knowledge and specific diagnostics and represents a prerequisite for the study and management of porcine babesiosis. Indeed, 18S rRNA PCR is highly specific, as demonstrated by the selective amplification of Babesia sp. Suis when tested together with a panel of Babesia species (Fig. 1C, D). Furthermore, it is a very sensitive test able to detect up to one copy of DNA target in 100 ng of calf thymus DNA in sensitivity assays (Fig.1A, B). When applied to a panel of pig, wild boar, and tick samples, this PCR approach allowed estimation of the infection frequency of Babesia sp. Suis in the island of Sardinia, chosen as a typical Mediterranean environment. For its geographical location in the middle of the Mediterranean Sea, the island of Sardinia represents a natural laboratory for the study of tick-borne diseases. Its subtropical climate, favoring severe tick seasonal infestations caused by at least six tick genera (Rhipicephalus, Haemaphysalis, Hyalomma, Boophilus, Dermacentor, and Ixodes) that are present in the island at different relative abundances (Di Todaro et al. 1999, Alberti et al. 2005, Zobba et al. 2014), makes Sardinia a hot spot for tick-transmitted diseases. Sequencing of the PCR products resulted in 10 different 18S rRNA sequence types distributed in geographically separated populations of domestic pigs in northern Sardinia
PORCINE BABESIOSIS IN THE MEDITERRANEAN AREA
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FIG. 2. Evolutionary history of Babesia porcine strains and 34 different Piroplasmida species. Molecular phylogenetic analyses were conducted by the maximum likelihood method. Cardiosporidium cionae was included as outgroup. The tree with the highest log likelihood ( - 2357.3181) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying neighbor-joining (NJ) and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories [ + G, parameter = 0.1804]). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 45 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 580 positions in the final dataset. and in engorged ticks. Although no positivity was found in wild boars sampled in the same areas, a more geographically structured sampling including a greater number of wild boar populations is needed to investigate the role of this species as reservoir/host for Babesia sp. Suis. Notably all symptomatic pigs, belonging to three geographically separated herds (Table 1), were positive to 18S
rRNA PCR. Sequencing of PCR products obtained from symptomatic pigs constantly generated the same sequence (type Swinetick1), which was also rescued from two ticks collected from a horse and a goat, both asymptomatic and living in the same areas. On the other hand, other nine sequence types were found in ticks and in asymptomatic pigs (Table 2). The distribution of sequence types in
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FIG. 3. Network analyses of Babesia porcine strains and 34 different Piroplasmida species. Structure of the network overlaps the trees obtained with the maximum likelihood method. Babesia porcine strains group together in a cluster most closely related to B. orientalis.
asymptomatic/symptomatic pigs might indicate the existence of various Babesia sp. Suis strains associated with different degrees of pathogenicity in pigs. This suggests the potential use of our molecular approach for strain typing in large-scale epidemiological studies. However, more sequencing data from geographically distant populations and from symptomatic animals are needed to confirm this hypothesis. It should be considered that the existence of nonpathogenic strains should always be taken into account when planning a diagnostic investigation and subsequent treatment of a pig herd, especially when, due to intensive farming, a significant economical investment is necessary for therapy. Molecular phylogeny allowed the classification of piroplasmids and contributed to revealing evolutionary lineages and relations among species (Schnittger et al. 2012). Basing piroplasmids classification on molecular data instead of traditionally used phenotypic characters allows unraveling relationships that reflect a robust taxonomy (Reichard et al. 2005, Morrison 2009). In this study, both maximum likelihood trees and network analysis placed the porcine Babesia sequences obtained in a distinct branch of Ungulibabesids, which was strongly supported by statistical analysis. Porcine strains identified in this
study are actually monophyletic and most closely related to B. orientalis and B. occultans, respectively, isolated from water buffalos and cattle. In conclusion, the presence of distinct Babesia porcine strains in Rhipicephalus ticks and in symptomatic pigs suggests that intensive surveillance programs should be undertaken in Sardinia and generally in Mediterranean areas for effective monitoring of this tickborne disease. This would be essential to implement management of pig populations and herds where both ticks and vectored piroplasmids occur. Acknowledgments
Roberta Lecis and Rosanna Zobba were supported by the Fondazione Banco di Sardegna (project: Epidemiologia molecolare di agenti infettivi emergenti nella fauna selvatica sarda, importanti come agenti zoonotici o associati a patologie trasversali), and by Regione Autonoma della Sardegna, Italy, respectively. Author Disclosure Statement
No competing financial interests exist.
PORCINE BABESIOSIS IN THE MEDITERRANEAN AREA References
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Penzhorn BL. Babesiosis of wild carnivores and ungulates. Vet Parasitol 2006; 138:11–21. Perrie`re G, Gouy M. www-Query: An on-line retrieval system for biological sequence banks. Biochimie 1996; 78:364–369. Reichard MV, Van Den Bussche RA, Meinkoth JH, Hoover JP, et al. A new species of Cytauxzoon from Pallas’ cat caught in Mongolia and comments on the systematic and taxonomy of piroplasmids. J Parasitol 2005; 91:420–426. Saitou N, Nei M, The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 1987; 4:406–425. Schnittger L, Rodriguez AE, Florin-Christensen M, Morrison DA. Babesia: A world emerging. Infect Genet Evol 2012; 12:1788–1809. Setty S, Khalil Z, Schori P, Azar M, et al. Babesiosis. Two atypical cases from Minnesota and a review. Am J Clin Pathol 2003; 120:554–559. Tamura K, Nei M, Kumar S. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci USA 2004; 101:11030–11035. Tamura K, Peterson D, Peterson N, Stecher G, et al. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011; 28:2731–2739. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994; 22:4673–4680. Uilenberg G. Babesia—a historical overview. Vet Parasitol 2006; 138:3–10. Yin H, Lu W, Luo J. Babesiosis in China. Trop Anim Health Prod 1997; 29:11–15. Zobba R, Pinna Parpaglia ML, Spezzigu A, Pittau M, et al. First molecular identification and phylogeny of a Babesia sp. from a symptomatic sow (Sus scrofa Linnaeus 1758). J Clin Microbiol 2011; 49:2321–2324. Zobba R, Anfossi AG, Pinna Parpaglia ML, Dore GM, et al. Molecular investigation and phylogeny of Anaplasma spp. in Mediterranean ruminants reveal the presence of neutrophiltropic strains closely related to A. platys. Appl Environ Microbiol 2014; 80:271–280.
Address correspondence to: Alberto Alberti Dipartimento di Medicina Veterinaria Universita` degli studi di Sassari Via Vienna, 2 07100 Sassari Italy E-mail: [email protected]
Molecular Epizootiology and Diagnosis of Porcine Babesiosis in Sardinia, Italy Rosanna Zobba,1 Anna Maria Nuvoli,1 Francesca Sotgiu,1 Roberta Lecis,1 Antonio Spezzigu,1 Gian Mario Dore,1 Marco Antonio Masia,1 Carla Cacciotto,1 Maria Luisa Pinna Parpaglia,1 Daniele Dessı`,2 Marco Pittau,1 and Alberto Alberti1
Abstract
The recent characterization of the 18S ribosomal RNA (rRNA) of a pathogenic Babesia species in a domestic sow paved the way for establishing diagnostic and epidemiological tools for porcine babesiosis. Here, we developed the first specific Babesia sp. Suis PCR, and we applied this test to a panel of samples collected from animals living in a typical Mediterranean environment (Sardinia, Italy), including domestic pigs, wild boars, and ticks. In domestic pigs, PCR coupled with sequencing revealed an estimated Babesia infection frequency of 26.2% and the presence of distinct 18S sequence types. The different distribution of sequence types in symptomatic and asymptomatic subjects might suggest the existence of phylogenetically closely related strains with variable pathogenicity in pigs. Moreover, molecular identification of tick species indicated Rhipicephalus sanguineus and Rhipicephalus bursa as candidate vectors potentially involved in the transmission of this pathogen. Collectively, the data reveal the suitability of 18S rRNA PCR/sequencing for molecular diagnosis of porcine babesiosis and for large-scale investigations on the presence and geographical distribution of Babesia sp. Suis genetic variants. Key Words:
Piroplasmida—Ticks—Tick-borne diseases—Protozoan—Anaplasmosis.
Introduction
B
abesiosis is a disease caused by protozoa of the genus Babesia, which invade and destroy erythrocytes. Babesia spp. are typically transmitted by Ixodidae ticks and are considered by some authors the second most common parasitic organisms in the blood of mammals, after trypanosomes (Schnittger et al. 2012). Babesiosis is raising veterinary and public health concerns for its great economic and medical impact worldwide (Setty et al. 2003, Colwell et al. 2011, Schnittger et al. 2012), and it is being increasingly acknowledged as an important emerging zoonosis (Kjemtrup and Conrad 2000, Lobo et al. 2013). In general, Babesia infections can cause a variety of nonspecific symptoms in immunocompetent patients. Common manifestations of babesiosis include mild to high fever, chills, anorexia, nausea, vomiting, anemia, and abortion. Host age, immunological status, and genetic factors may predispose the patient to more severe disease. In animals, co-infection with species of the Borrelia burgdorferi sensu lato (s.l.) complex, and/ or concurrent anaplasmosis can also dramatically complicate the clinical presentation of babesiosis (Homer et al. 2000, Lobo et al. 2013). 1
Babesia species infect a wide variety of both domestic and wild ungulates (Penzhorn 2006), usually occurring in closely related hosts. Examples of these are Babesia trautmanni, which is able to infect domestic pig and wild boar (Sus scrofa), but also the African bushpig Potamochoerus porcus and the warthog Phacochoerus aethiopicus. Furthermore, Babesia caballi and Theileria equi can infect horse and various zebra species (Penzhorn 2006). Porcine babesiosis has been reported in the former Soviet Union, southern Europe, Africa, and China (Yin et al. 1997, Penzhorn 2006, Uilenberg 2006), often associated with serious economical losses and significant mortality rates. Despite its importance, it still remains an overlooked disease, with the etiological agents still poorly investigated (Penzhorn 2006, Zobba et al. 2011). Literature on Babesia species affecting pigs is extremely scarce, and no specific molecular tools have been developed for these pathogens detection (Criado-Fornelio 2012). On the basis of morphological diagnosis, two Babesia spp. have been reported in pigs: B. trautmanni, transmitted by Rhipicephalus ticks and characterized by large piroplasms, and the smaller Babesia perroncitoi, whose vector has not been
Dipartimento di Medicina Veterinaria and 2Dipartimento di Scienze Biomediche, Universita` degli Studi di Sassari, Sassari, Italy.
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identified yet (Uilenberg 2006). As mentioned above, babesiosis outbreaks in worldwide pig populations are seldom investigated (Penzhorn 2006). On the other hand, cases of porcine babesiosis are systematically reported in Sardinia, Italy, although only at the local level (i.e., by veterinary practitioners) and sporadically in the scientific literature (Ligios and Scala 1993), on the basis of clinical diagnosis and microscopy. In 2011, a Babesia species was identified in a symptomatic sow, and its 18S rRNA was partially characterized (Zobba et al. 2011). The new isolate was found to be morphologically similar to B. trautmanni and genetically related to Ungulibabesids, However, because previous reports of B. trautmanni and B. perroncitoi lacked strain genetic characterization, this isolate could not be unambiguously assigned to one of these two species, and it was designated Babesia sp. Suis. Molecular data presented by Zobba and co-workers (2011) provided basic genetic information for the development of specific diagnostic tools for molecular epidemiological studies on porcine Babesia spp. In this work, we generated a highly sensitive and specific PCR test for Babesia sp. Suis that we applied to a panel of samples collected from animals and ticks in Sardinia, chosen as a representative of a Mediterranean subtropical area. Babesia sp. Suis strains were phylogenetically characterized, and host ticks species potentially involved in their transmission were also identified. Materials and Methods Animals, samples, and DNA extraction
Between 2011 and 2013, EDTA blood samples were obtained from 61 domestic swine traditionally farmed in small semifree herds in northern Sardinia. Herds were selected in areas where clinical cases had been previously reported. All domestic pigs were subjected to clinical examination and did not present tick infestation at the time of sampling. A total of 52 wild boar hunted in the same area were sampled and included in the analysis. Additionally, 64 engorged ticks were collected from domestic and wild animals farmed and/or free ranging in the same northern areas of the island (Table 1). EDTA blood was sampled from two bovines positive to Theileria buffeli and Babesia major, respectively, from a sheep naturally infected with T. ovis, from a horse naturally infected with T. equi, and from a sow infected with Babesia sp. Suis (Zobba et al. 2011). Positivity to T. buffeli, T. ovis, T. equi, and B. major was established by PCR (CriadoFornelio et al. 2003) and sequencing (unpublished). B. bovis, B. caballi, and T. equi fluorescence assay (FA) substrate slides were from Fuller Laboratories (Fullerton, CA). The presence of antigens in FA slides was confirmed by PCR (Criado-Fornelio et al. 2003). DNA was extracted from blood samples and from the FA slides using the DNeasy Blood and Tissue Kit (Qiagen, Italy), following the manufacturer’s recommendations. DNA was quantified with a BioPhotometer plus (Eppendorf, Italy).
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Table 1. Origin of Pig, Wild Boar, and Tick Samples Analyzed in This Study and Results of the 18S rRNA PCR (No. Positive/Total) Host species
Geographic location
Pig
Ala` dei Sardi Bortigiadas Chiaramonti Laerru Nule Perfugas Putifigari Sassari Tuili
Wild boar
Putifigari Villanova Monteleone
Ticks
Asinara (birds) Badesi (bovine) Bonassai (wild boar) Nuoro (dog) Nurra (horse, dog) Nurri (dog) Padru (dog) Perfugas (ruminants) Piedmont (dog) Putifigari (goat) Oristano (dog) Sassari (dog) Siniscola (dog) Monastir (deer)
No. of sampled animals
Babesia sp. Suis 18S rRNA PCR
16 3 10 3 4 10 6 6 3 61 47 5 52 6 1 1 4 4 1 1 21 15 1 6 2 1 1 65
1/16 1/3 0/10 2/3 0/4 6/10 0/6 6/6 0/3 16/61 0/47 0/5 0/52 0/6 0/1 0/1 0/4 1/4 0/1 0/1 4/21 0/15 0/1 0/6 0/2 0/1 0/1 5/65
primers were designed. The two primers were combined with the already published primer Bab/ThelF (Criado-Fornelio et al. 2003) to generate a specific heminested PCR test to detect Babesia sp. Suis in clinical samples. In the first PCR round, primers Bab/ThelF (5¢-GACACAGGGAGGTAGTG ACAAG-3¢) and Bsu/18S/1258R (5¢-CGTAGCGAAACC GAGTAACAC-3¢) were used to target an 860-bp fragment. Briefly, 100–150 ng of DNA were used in a 50-lL PCR reaction containing 200 lM deoxynucleotide triphosphates (dNTPs), 0.3 lM of each of the two primers, and 1.25 U of Taq DNA polymerase (Qiagen, Italy). One microliter of the first PCR product was used as a template in the second PCR in a similar reaction mix with the internal primer Bsu/18S/580F (5¢-ATTTCAGCCTTTTGCGATTG-3¢) combined to primer Bsu/18S/1258R, to amplify a product of approximately 680 bp. Both the first and second PCR amplifications were performed with an initial denaturation at 94C for 3 min, 30 cycles of: Denaturation at 94C (30 sec), annealing at 50C, and 55C in the first and second PCR, respectively (30 sec), extension at 72C (1 min), and final extension at 72C for 10 min. In total, 177 DNA samples obtained from 61 pigs, 52 wild boars, and 64 ticks were tested by 18S heminested PCR.
Babesia sp. Suis 18S heminested PCR
On the basis of the 18S rRNA sequence alignment of Babesia sp. Suis (accession no. HQ437690) with the corresponding region of different Babesia and Theileria species available in the GenBank database, two highly specific
Babesia sp. Suis 18S heminested PCR sensitivity and specificity
To test sensitivity of the 18S rRNA heminested PCR, plasmid pCR4/18s/Bspsuis was generated by cloning a
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first-round PCR product into the pCR4-TOPO vector (Life Technologies, Italy). According to the method used by Alberti and Sparagano (2006), pCR4/18s/Bspsuis was quantified and brought to a concentration of 100,000 copies/lL, after which serial two-fold dilutions of the plasmid were obtained until the concentration of one copy/lL was reached. One microliter of each dilution was coupled to 100 ng of calf thymus DNA (Sigma, Poole, UK) and tested with the Babesia sp. Suis 18S heminested PCR. Three independent serial dilutions were obtained and tested using PCR. Dilutions were considered PCR-positive when resulting in the desired band at least in two out of three serial dilutions replicates. To assess PCR specificity, a DNA sample extracted from blood obtained from a Babesia sp. Suis–positive sow (Zobba et al. 2011) was tested together with a panel of DNA samples representative of different Babesia species. In particular, the following species were tested by 18S heminested PCR: T. buffeli, B. major, T. ovis, T. equi, B. caballi, and B. bovis. Sequencing, 18S rRNA variability, and phylogenetic analyses
Positive PCR products were cloned into the plasmid pCR4TOPO vector (Life Technologies, CA) and used to transform One Shot TOP10 Chemically Competent Escherichia coli (Invitrogen, Italy). Plasmid DNA was extracted from positive colonies with PureLink Quick Plasmid MiniPrep Kit (Invitrogen, Italy) and automatically sequenced (BMR Genomics, Italy). At least five positive E. coli colonies were selected for each transformation. The generated sequences were edited with Chromas 2.2 (Technelysium, Australia) and aligned with ClustalW (Thompson et al. 1994) to assign them to unique sequence types. Unique sequence types, named after the host species and sequentially ordered, were checked against the GenBank database with nucleotide blast BLASTN (Altschul et al. 1990). To identify nucleotide signatures of porcine isolates, the 18S rRNA
sequence types obtained in this study were aligned with two sequences (accession nos. AY726557 and EU376017) representing the 18S rRNA gene variability of Ungulibabesids (Criado-Fornelio et al. 2003, Dawood et al. 2013). Phylogenetic analyses were performed aligning the porcine 18S rRNA sequence types with a set of 35 sequences belonging to the six Babesiadae clades identified by Schnittger et al. (2012). The set was composed by: Four clade-1 sequences (B. felis AY452707, B. leo AF244911, B. microti U09833, B. rodhaini M87565); three clade-2 sequences (B. lengau KC790443, B. conradae AF158702, B. duncani HQ289870); three clade-3 sequences (Theileria youngi AF245279, Cytauxzoon felis AY979105, T. bicornis AF499604); two clade-4 sequences (B. equi AY150064, B. bicornis AF419313); seven clade-5 sequences (T. parva L02366, T. taurotragi L19082, T. annulata M64243, T. buffeli DQ104611, T. velifera AF097993, T. mutans AF078815, T. separata AY260175); and 16 sequences belonging to clade-6 (B. orientalis AY596279, B. bovis AY150059; B. bennetti DQ402155; B. ovis AY150058; B. motasi AY533147; B. bigemina X59605; B. ovate AY603400; B. caballi Z15104; B. major GU194290; B. crassa AY260176; B. gibsoni DQ184507; B. divergens FJ944826; B. canis rossi DQ111760, B. canis canis AY072926, B. canis vogeli AY371198, B. poelea DQ200887). Additionally, Cardiosporidium cionae (EU052685) was included as outgroup in the phylogenetic analysis. Pairwise/multiple sequence alignments and sequences similarities were calculated using the ClustalW and the identity matrix options of Bioedit (Hall 1999), respectively. The evolutionary history of Babesia sp. Suis and of the 36 species (35 Babesia species and the outgroup species) considered in the analysis was inferred by using the maximum likelihood method based on the Kimura 2-parameters method (Tamura et al. 2004) identified as the best-suited evolutionary model for our data. Initial tree(s) for the heuristic search were obtained automatically by applying neighbor-joining (NJ)
Table 2. Designation, Sequence Type, Geographical Origin, and BLASTN Results of the 18S Sequence Types Identified in this Study (Maximum Identity) Sequence type Swinetick1
Swine1 Swine2 Swine3 Swine4 Swine5 Swine6 Tick1 Tick2 Tick3
Host
Geographical location
GenBank no.
BLAST analysis
Sus scrofa 01 Sus scrofa Bar02 Sus scrofa 223 Sus scrofa 224 Sus scrofa 292 Sus scrofa T01 Sus scrofa T02 Sus scrofa 14743 Sus scrofa S05 Sus Scrofa Gio1 Tick 73A Tick Candy12 Sus scrofa 280 Sus scrofa 220 Sus scrofa 14740 Sus scrofa 14742 Sus scrofa 14737 Sus scrofa S06 Tick 57 Tick 60B Tick 64A
Laerru (NW Sardinia) Laerru (NW Sardinia) Perfugas (NW Sardinia) Perfugas (NW Sardinia) Perfugas (NW Sardinia) Sassari (NW Sardinia) Sassari (NW Sardinia) Sassari (NW Sardinia) Perfugas (NW Sardinia) Perfugas (NW Sardinia) Perfugas (NW Sardinia) Nurra (NW Sardinia) Ala` dei Sardi (NE Sardinia) Bortigiadas (N Sardinia) Sassari (NW Sardinia) Sassari (NW Sardinia) Sassari (NW Sardinia) Perfugas (NW Sardinia) Perfugas (N Sardinia) Perfugas (N Sardinia) Perfugas (N Sardinia)
KJ801525 KJ801526 KJ801527 KJ801528 KJ801529 KJ801530 KJ801531 KJ801532 KJ801533 KJ801534 KJ801535 KJ801536 KJ801537 KJ801538 KJ801539 KJ801540 KJ801541 KJ801542 KJ801543 KJ801544 KJ801545
100% Babesia sp. Anglona/AA-2011
99% 99% 99% 99% 99% 99% 99% 99% 99%
Babesia Babesia Babesia Babesia Babesia Babesia Babesia Babesia Babesia
sp. sp. sp. sp. sp. sp. sp. sp. sp.
Anglona/AA-2011 Anglona/AA-2011 Anglona/AA-2011 Anglona/AA-2011 Anglona/AA-2011 Anglona/AA-2011 Anglona/AA-2011 Anglona/AA-2011 Anglona/AA-2011
PORCINE BABESIOSIS IN THE MEDITERRANEAN AREA
and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories [ + G, parameter = 0.5581]). The rate variation model allowed for some sites to be evolutionarily invariable ([ + I], 59.8244% sites). A total of 580 positions were included in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura et al. 2011). Statistical support for internal branches of the trees was evaluated by bootstrapping with 1000 iterations (Felsenstein 1985). NJ (Saitou and Nei 1987) trees and consensus values were generated using the same software. Trees were edited using NJplot (Perrie`re and Gouy 1996) and Treeview v. 1.5.2 (Page 1996). To reduce systematic errors, to investigate more deeply into taxa evolutionary relationships, and to test robustness of the phylogenetic analyses, sequence clusters were also detected by the analysis of phylogenetic networks inferred from uncorrected p-distances with the Neighbor-Net method in SplitsTree4 (Huson and Bryant 2006), not considering invariable sites. MEGA5 was also used to investigate the variability of the nucleotide positions in the 18S RNA sequences alignment. Babesia sp. Suis nucleotide sequence data reported in this paper are available in GenBank under the accession numbers reported in Table 2. Molecular identification of tick species and additional PCR tests
To identify tick species, a PCR amplifying a 460-bp fragment of mitochondrial 16S rRNA was performed on Babesia sp. Suis–positive ticks using primers 16S + 1/16S - 1, as in Black and Piesman (1994). PCR products were directly sequenced and checked against the GenBank database with BLAST. Tick 16S RNA sequences were submitted to GenBank (accession nos. KJ814006, KJ814007, KJ814008, KJ814009, and KJ814010). Animals positive to Babesia sp. Suis were also screened for the presence of Anaplasma spp. as previously described (Zobba et al. 2014). Results
On general physical examination, 10 out of 61 domestic pigs showed severe clinical signs indicative of babesiosis, such as high fever, chills, anorexia–dysorexia, nausea, vomiting, anemia, lateral recumbency, lameness, hematuria, and abortion. Clinical signs did not significantly differ among the subjects examined. Sensitivity of the Babesia sp. Suis 18S heminested PCR (Fig. 1A, B), evaluated by testing two-fold dilutions of plasmid pCR4/18s/Bspsuis, was 500 copies in the primary PCR and one copy in the heminested PCR. Both the newly developed primary and heminested PCRs were highly specific for Babesia sp. Suis (Fig. 1C, D), as only DNA samples obtained from Babesia sp. Suis–positive sows (Zobba et al. 2011) were PCR positive when tested together with a panel of DNA samples representative of different Babesia species. All DNA samples obtained from the 10 symptomatic pigs were positive to Babesia sp. Suis when tested by 18S rRNA heminested PCR and generated the predicted band of 680 bp on agarose electrophoresis (100% prevalence in symptomatic
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FIG. 1. Detection limit and specificity of the 18S rRNA Babesia sp. Suis–specific PCRs, developed during this study, using Taq DNA polymerase (Qiagen, Italy). (A and B) Results obtained by the first-round (A) and second-round PCRs (B) from two-fold dilutions of plasmid generated by cloning a first-round PCR product. Lanes A, 100,000 copies; lanes B, 50,000 copies; lanes C, 25,000 copies; lanes D, 10,000 copies; lanes E, 5000 copies; lanes F, 2500 copies; lanes G, 1000 copies; lanes H, 500 copies; lanes I, 100 copies; lanes L, 50 copies; lanes M, 10 copies; lanes N, one copy. (C and D) Results obtained with the first (C) and second (D) round of the 18S rRNA Babesia sp. Suis– specific PCR using DNA from a sow infected by Babesia sp. Suis (lanes 1); DNA from B. bovis (lanes 2), B. caballi (lanes 3), and T. equi (lanes 4); DNA from bovines positive to T. buffeli (lanes 5) and B. major (lanes 6); DNA from a sheep infected with T. ovis (lanes 7) and DNA from a horse infected with T. equi (lanes 8). Lanes ‘‘-’’ and ‘‘M’’ are the negative PCR controls (distilled water) and the 100-bp DNA ladder (Life Technologies, Italy), respectively. The same DNA ladder was used in A–D.
pigs). Additionally, six asymptomatic pigs were PCR positive (12% prevalence in asymptomatic pigs). Considering symptomatic and asymptomatic pigs together, Babesia sp. Suis infection frequency was therefore 26.2%. Babesia sp. Suis 18S rRNA was never identified in DNA samples extracted from the 52 wild boars sampled in geographical areas partially overlapping the domestic pigs distribution. Five engorged ticks, collected, respectively, from a horse and four goats, also tested positive for Babesia sp. Suis. On the basis of sequencing results, a total of 10 different 18S rRNA sequence types were identified (Table 2). Sequence type Swinetick1 was identified in 10 symptomatic pigs belonging to three geographically distant herds (Perfugas, Laerru, and Sassari). Also, two ticks collected from a horse and a goat in northern Sardinia tested positive for the same sequence type. A number of sequence types (Swine1–6) were found in asymptomatic pigs coming from two herds where symptomatic subjects were also present (Perfugas and Sassari), and from a herd (Bortigiadas) characterized by a clinically silent infection. Sequence types Tick1–3 were found exclusively in three ticks collected from three sheep coming from one herd (Perfugas).
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Table 3. Nucleotide Variable Positions and Signatures of the 10 Porcine Babesia sp. Suis Sequence Types Identified in This Study 18S rRNA variable positionsa 0 0 3 SwineTick1 T Swine1 Swine2 Swine3 Swine4 Swine5 Swine6 Tick1 Tick2 Tick3 Babesia sp. Kashi B. occultans C
0 0 5 G T
0 0 8 A G
0 1 1 G C
0 1 2 T C
0 1 5 G C
0 1 6 G T
0 1 9 A T
0 2 0 T C C
0 2 2 G C
0 2 7 A T
0 3 0 T C
0 3 2 G A
0 9 3 A G A
1 3 3 T
1 3 4 A T A
1 3 7 T C T
2 3 8 A G A
2 5 3 A G C A
3 4 3 A G A
3 6 0 A G A
3 8 2 T C T
4 2 6 A G A
4 6 4 C T C
5 6 3 T C T
6 0 6 T C T
6 1 2 A G A
6 3 8 A T
a Sites are numbered according to the alignment, starting from the first nucleotide position after primer sense. Variable positions conserved among porcine isolates are shaded in grey.
BLAST analyses conducted on the sequence types obtained in this study revealed a variable degree of nucleotide similarity with a Babesia sp. Suis strain (GenBank accession number HQ437690) already reported in the same geographical area and recovered from a symptomatic sow (Zobba et al. 2011). In particular, sequence type Swinetick1, found in symptomatic pigs and in ticks, showed 100% identity with HQ437690, whereas the other sequence types (Swine1–6, Tick1–3), found in asymptomatic subjects and in ticks were 99% similar to the same sequence. Alignments of the 10 18S sequence types together with sequences representative of Ungulibabesids (Criado-Fornelio et al. 2003, Dawood et al. 2013) allowed identification of 14 variable positions conserved among porcine isolates and therefore representing nucleotide signatures of this group of sequences (Table 3). Phylogenetic results obtained in this study were consistent with previous observations (Criado-Fornelio et al. 2003, Zobba et al. 2011, Schnittger et al. 2012, Dawood et al. 2013). Maximum likelihood trees generated with MEGA5 allowed the identification of six major Babesiadae clades (Fig. 2). The 10 Babesia sp. Suis sequence types rescued in this study grouped in the statistically supported clade 6 (Schnittger et al. 2012), together with the other Ungulibabesids. Network analyses (Fig. 3) confirmed maximum likelihood observations and identified the same Babesia groups. Tick DNA samples positive to Babesia sp. Suis generated a band of 460 bp when analyzed with the mitochondrial 16S rRNA diagnostic PCR (see Materials and Methods). Sequencing and BLAST analyses allowed identifying four ticks as Rhipicephalus bursa (nucleotide identity 99%) and one as R. sanguineus (nucleotide identity 96%). All DNA samples were negative to Anaplasma spp. Discussion
For their pathogenic role in animals and as zoonotic agents, Babesia species have been increasingly studied in the last decades. Although the major economic impact of babesiosis in animals is on the cattle industry, the relevance of infections in other domestic species, such as small ruminants, horse, and dogs, varies throughout the world. In
pigs, babesiosis is responsible for serious losses and produces antemortem clinical signs that may be similar to those of bovine babesiosis. Knowledge regarding porcine babesiosis is especially limited by the lack of population genetics and molecular epidemiology data, which hamper diagnostics and strain typing. For these reasons, porcine babesiosis remains an overlooked disease, scarcely reported in international literature (Zobba et al. 2011, Criado-Fornelio 2012). In the Mediterranean area, porcine babesiosis has been associated with two Babesia species, namely B. trautmanni and B. perroncitoi, classified on the basis of different size of piroplasmas (Kauffman 1996). Recently, Babesia sp. Suis has been characterized molecularly and suspected to represent a strain of B. trautmanni based on the size of piroplasms (Zobba et al. 2011). In this study we developed a molecular approach (based on specific PCR and sequencing) that has an inbuilt potential for improving molecular knowledge and specific diagnostics and represents a prerequisite for the study and management of porcine babesiosis. Indeed, 18S rRNA PCR is highly specific, as demonstrated by the selective amplification of Babesia sp. Suis when tested together with a panel of Babesia species (Fig. 1C, D). Furthermore, it is a very sensitive test able to detect up to one copy of DNA target in 100 ng of calf thymus DNA in sensitivity assays (Fig.1A, B). When applied to a panel of pig, wild boar, and tick samples, this PCR approach allowed estimation of the infection frequency of Babesia sp. Suis in the island of Sardinia, chosen as a typical Mediterranean environment. For its geographical location in the middle of the Mediterranean Sea, the island of Sardinia represents a natural laboratory for the study of tick-borne diseases. Its subtropical climate, favoring severe tick seasonal infestations caused by at least six tick genera (Rhipicephalus, Haemaphysalis, Hyalomma, Boophilus, Dermacentor, and Ixodes) that are present in the island at different relative abundances (Di Todaro et al. 1999, Alberti et al. 2005, Zobba et al. 2014), makes Sardinia a hot spot for tick-transmitted diseases. Sequencing of the PCR products resulted in 10 different 18S rRNA sequence types distributed in geographically separated populations of domestic pigs in northern Sardinia
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FIG. 2. Evolutionary history of Babesia porcine strains and 34 different Piroplasmida species. Molecular phylogenetic analyses were conducted by the maximum likelihood method. Cardiosporidium cionae was included as outgroup. The tree with the highest log likelihood ( - 2357.3181) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying neighbor-joining (NJ) and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories [ + G, parameter = 0.1804]). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 45 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 580 positions in the final dataset. and in engorged ticks. Although no positivity was found in wild boars sampled in the same areas, a more geographically structured sampling including a greater number of wild boar populations is needed to investigate the role of this species as reservoir/host for Babesia sp. Suis. Notably all symptomatic pigs, belonging to three geographically separated herds (Table 1), were positive to 18S
rRNA PCR. Sequencing of PCR products obtained from symptomatic pigs constantly generated the same sequence (type Swinetick1), which was also rescued from two ticks collected from a horse and a goat, both asymptomatic and living in the same areas. On the other hand, other nine sequence types were found in ticks and in asymptomatic pigs (Table 2). The distribution of sequence types in
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FIG. 3. Network analyses of Babesia porcine strains and 34 different Piroplasmida species. Structure of the network overlaps the trees obtained with the maximum likelihood method. Babesia porcine strains group together in a cluster most closely related to B. orientalis.
asymptomatic/symptomatic pigs might indicate the existence of various Babesia sp. Suis strains associated with different degrees of pathogenicity in pigs. This suggests the potential use of our molecular approach for strain typing in large-scale epidemiological studies. However, more sequencing data from geographically distant populations and from symptomatic animals are needed to confirm this hypothesis. It should be considered that the existence of nonpathogenic strains should always be taken into account when planning a diagnostic investigation and subsequent treatment of a pig herd, especially when, due to intensive farming, a significant economical investment is necessary for therapy. Molecular phylogeny allowed the classification of piroplasmids and contributed to revealing evolutionary lineages and relations among species (Schnittger et al. 2012). Basing piroplasmids classification on molecular data instead of traditionally used phenotypic characters allows unraveling relationships that reflect a robust taxonomy (Reichard et al. 2005, Morrison 2009). In this study, both maximum likelihood trees and network analysis placed the porcine Babesia sequences obtained in a distinct branch of Ungulibabesids, which was strongly supported by statistical analysis. Porcine strains identified in this
study are actually monophyletic and most closely related to B. orientalis and B. occultans, respectively, isolated from water buffalos and cattle. In conclusion, the presence of distinct Babesia porcine strains in Rhipicephalus ticks and in symptomatic pigs suggests that intensive surveillance programs should be undertaken in Sardinia and generally in Mediterranean areas for effective monitoring of this tickborne disease. This would be essential to implement management of pig populations and herds where both ticks and vectored piroplasmids occur. Acknowledgments
Roberta Lecis and Rosanna Zobba were supported by the Fondazione Banco di Sardegna (project: Epidemiologia molecolare di agenti infettivi emergenti nella fauna selvatica sarda, importanti come agenti zoonotici o associati a patologie trasversali), and by Regione Autonoma della Sardegna, Italy, respectively. Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to: Alberto Alberti Dipartimento di Medicina Veterinaria Universita` degli studi di Sassari Via Vienna, 2 07100 Sassari Italy E-mail: [email protected]
