Title: Genetic Variation of the -tubulin of two Babesia caballi strains J Arthropod-Borne Dis, September 2017, 11(3): 344–353 MG Montes-Cortés et al.: Genetic Variation of … 344 http://jad.tums.ac.ir Published Online: September 08, 2017 Original Article Genetic Variation of the -tubulin Gene of Babesia caballi Strains María Guadalupe Montes-Cortés 1, *José Luis Fernández-García 2, Miguel Ángel Habela Martínez-Estéllez 1 1Parasitology and Parasitological Diseases, Veterinary Faculty, Universidad de Extremadura, Cáceres, Spain 2Genetics and Animal Breeding, Veterinary Faculty, Universidad de Extremadura, Cáceres, Spain (Received 23 Mar 2015; accepted 10 Dec 2016) Abstract Background: Equine piroplasmosis is caused by two haemoprotozoan parasites: Babesia caballi and Theileria equi. Negative economic impact on international trade has been associated to endemic sites. This is the reason why carrier detection requires reliable diagnostic methods. Various diagnostic modalities can be used alone or in combination including PCR. However, genetic variation of commonly used genes is still of debate. The aim of this research was to sequence the -tubulin gene of a B. caballi strain from Spain and to compare it with known -tubulin sequences. Methods: DNA was isolated from a cryopreserved strain from Spain and acute and chronic carrier horses. Firstly, degenerated primer pairs were designed based on GenBank sequences of different Babesia and Theileria species for sequencing. The primers were redesigned to amplify both parasites, simultaneously. Finally, a species-specific pri- mer pair for B. caballi was designed and a Restriction Fragment Length Polymorphism-PCR (PCR-RFLP) assay performed to know the difference of known B. caballi strains. Results: We provided new insights of the -tubulin gene and a good molecular coverage of this gene, contributing with a number of useful primers to amplify T. equi and B. caballi. Moreover, PCR-RFLP assays based on the exon II of this gene confirmed the causative B. caballi strain in Spanish horses. Conclusion: We reported useful primer pairs for diagnostic and a new sequence of the -tubulin gene of B. caballi, which will facilitate the development of future assays and the detection of infected horses, preventing thus the spread of this disease worldwide. Keywords: Babesia caballi, -tubulin gene, Equine piroplasmosis, PCR-RFLP marker Introduction Equine piroplasmosis is caused by two dif- ferent haemoprotozoan parasites: Babesia ca- balli (1) and Theileria equi (formerly Babesia equi Laveran 1901) (2). Both species are transmitted to horses by ticks found in tropi- cal and subtropical regions (3). These para- sites cause disease characterized by fever, anaemia, jaundice, weakness, loss of weight, blood urine, oedema, lymphadenopathy and hepatomegaly. Moreover, after infection, hors- es may remain life-long carriers when they are infected of T. equi, whereas horses may remain carriers of B. caballi from 1 to 4yr (4). In spite of this difference, both parasi- taemia have a serious negative impact on international trade (5), with southern Europe particularly affected as it is an endemic zone of this disease (5-6). Detection of chronic carriers of Equine piro- plasmosis represents a problem for the eq- uine industry, due to the limitations of the available diagnostic methods. Therefore, various diagnostic modalities can be used alone or in combination to diagnose infec- tions (7). These methods are indirect methods such as ELISA and the indirect fluorescent antibody test (IFAT) or direct diagnosis such as microscopic examination of blood smears or PCR (8-7, 9). *Corresponding author: Dr JL Fernández-García, E-mail: mahabela@unex.es http://jad.tums.ac.ir/ mailto:mahabela@unex.es J Arthropod-Borne Dis, September 2017, 11(3): 344–353 MG Montes-Cortés et al.: Genetic Variation of … 345 http://jad.tums.ac.ir Published Online: September 08, 2017 Classical single PCR (6), multiplex PCR (10-11), nested PCR (12, 13-14) or real-time PCR (15) are used to diagnose Equine piro- plasmosis. Oligonucleotides used in PCR were designed based on genes such us 18S rRNA (6-13-10- 15) or equi merozoite antigen gene -ema1- (12-16) to amplify T. equi genes. On the other hand, to diagnose B. caballi, genes such as 18S rRNA (6-10) or the rhoptry- associated protein gene -rap1- (17) are commonly used. The -tubulin gene is one of the genes used to diagnose Piroplasmids (18), although its use is not widespread in the detection of B. caballi because little is yet known about the genetic variability of this gene in this spe- cies, the proof of this is that there is only genetic information from one sequence in data bases (Acc. Nº: AJ289246) The prevalence of B. caballi in horses in our country ranges from 7.9% (García-Bo- canegra et al. 2013) to 21.3% (20). Whereby, the detection of chronic carriers is a prime goal to prevent the spread of this disease, which can be achieved by increasing the knowledge about genetic variation of the -tubulin gene with differential diagnostic purposes. The aim of this research was to sequence the -tubulin gene from one isolate of B. caballi from Spain by developing primers using - tubulin genes deposited in Gene Bank from different haemoprotozoan species, to com- pare it with one known sequence of this gene from B. caballi and to provide markers suita- ble for diagnosis and molecular epidemiology. Materials and Methods Blood samples All the blood samples used in this re- search were collected from the jugular vein into sterile vacuum tubes (vacutainer®) with and without anticoagulant (EDTA) and rou- tinely thin blood smears were stained and were examined under a microscope at 1500x magnification. Babesia caballi was detected in the blood sample from Málaga (Andalu- sia, Spain) which was sent to our laborato- ries in Cáceres in 2006. This parasite was cultured in equine erythrocytes (21) and cry- opreserved in liquid nitrogen, which still be- longs to our sample bank (named GM Malaga strain). This isolated was used to obtain tem- plate DNA for sequencing the -tubulin gene. Moreover, blood samples of acute in- fected and chronic carrier horses were col- lected from different regions of centre and southwest Spain in 2011 as follows: five samples from acute infected horses (Cáceres province) and samples from chronic carriers from Segovia (n=5), Cádiz (n=7), Badajoz (n=6) and Cáceres (n=2) (Fig. 1). All the acute cases were positive by microscopic examination but chronic carriers were nega- tive to this procedure. These last blood sam- ples were checked by IFAT (22) and by commercial cELISA test (kits from VMRD Inc. Pullman, WA, USA), being all of them B. caballi positive to both techniques (Fig. 1). DNA extraction Whole genomic DNA was obtained from the cryopreserved strain, five samples from acute infected horses and 20 blood samples collected from chronic carriers, tested by cELISA to assess them as carriers of B. ca- balli parasite. Forty L or 250L of culture fluid or red cell, respectively, were processed by a proteinase K/salting out procedure (23- 24) modified using Zymo-Spin IIC columns (Zymo Research Corporation, Irvine, CA 92614, USA) to purify nucleic acids. Ge- nomic DNA was semi quantitatively an- alysed using Lambda DNA Marker (λ DNA- HindIII Digest, New England Biolabsinc.) by visualization in 0.8% agarose gel electro- phoresis stained with ethidium bromide fol- lowing manufacturer recommendations. Primer design and PCR amplification Several available complete or partial se- quences of the -tubulin gene were down- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2017, 11(3): 344–353 MG Montes-Cortés et al.: Genetic Variation of … 346 http://jad.tums.ac.ir Published Online: September 08, 2017 loaded from the Gene Bank (Table 1). These sequences, aligned using MEGA4 software (25), aimed at finding a conserved segment where universal PCR primers could be de- signed to amplify the longest fragment of this gene by conventional PCR. All aligned stretches which expanded from nucleotide 845478 (ie, -2 nucleotide from start codon) to nucleotide 846287 regarding sequence CP001669 were selected for primers design. Because completely conserved seg- ments were not found at both endpoints, use- ful primers, which included degenerate nu- cleotides, were obtained from this alignment as follows: B-Tub F and reverse B-Tub R (Table 2). The fragment of -tubulin gene of B. ca- balli was obtained by PCR based on these degenerate primers (which included nucleo- tides ambiguities, Table 2). In the first step, DNA from in vitro cultured B. caballi strain was used. Duplicated PCR were carried out in 50L reaction volumes with 5L of DNA template and 45L mixture containing final concentration of 1X PCR buffer, 0.2mM dNTPs, 1pmol each primer (B-Tub R and F or B-Tub R2 and F2 depending on the de- sired reaction), 1.5MgCl2 and 1 unit of Taq DNA polymerase (Ecogen®) on a thermal cycler 2720 (Life Technologies®). To max- imize amplification success with these de- generate primers a modified-touchdown pro- gram was used as follows: (1) each of the first four cycles were run at different annealing temperatures ranging from 55 ºC to 51 ºC and (2) the remaining 37 cycles at 52 ºC. The gen- eral amplification conditions were: 96 ºC for 5min, then each cycle at 96º for 30s, primer annealing (see (1) above) for 30s and 72 ºC for 1min. extension, finally 72 ºC for 7min. Five L of products for both PCR and, later, PCR-RFLP were always run in 1.5% LM agarose gels (Low Melting agarose) buffered on 0.5% TBE with power supply limited to 120v for 35–45min according the expected fragment size and stained with ethidium bro- mide for checking. PCR products obtained from the Málaga isolated were further used for sequencing. After sequencing the PCR product and realigning it to those in Table 2, a less de- generated primer pair was designed but in such way that it was able to amplify both the DNA template from B. caballi and T. equi simultaneously, since both species can be found in infected horses. The new designed pri- mers were as follows: B-Tub F2 and B-Tub R2 (Table 2). The PCR conditions for this assay were: 94 ºC for 5min, 94 ºC 45s, pri- mer annealing for 52 ºC for 1min and 72 ºC for 1min extension with 37 cycles, finally ex- tension 72 ºC for 7min. Reaction volumes consisted of 1L of template and 19L mix- ture containing final concentration of 1X PCR buffer, 0.2mM dNTPs, 1pmol each pri- mer, 1.5MgCl2 and 1 unit of Taq DNA pol- ymerase (Ecogen®) on a thermal cycler 2720 (Life Technologies®). DNA sequencing and resequencing PCR product from the cultured isolate from Málaga was purified with ExoSAP-IT (GE Healthcare®). Sequencing and resequenc- ing was performed to determine the genomic variations by the BigDye Terminator v.3.1 Cycle Sequencing Kit in both directions us- ing B-Tub F2 and B-Tub R2, respectively (26). Species-specific primers for Babesia caballi Three primer pairs were designed to am- plify the -tubulin gene of B. caballi from genomic DNA of cryopreserved strain. But the primer pair GM B-Tub F and GM B-Tub R (Table 2), which expanded from the start of the first intron to partially the second exon, was designed based on both sequences (this research and Acc. N. AJ289246) to specifically amplify B. caballi. Its specificity was studied using template DNA from T. equi, which showed no amplification (data not showed). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2017, 11(3): 344–353 MG Montes-Cortés et al.: Genetic Variation of … 347 http://jad.tums.ac.ir Published Online: September 08, 2017 Restriction fragment length polymorphism analysis (PCR-RFLP) To test the B. caballi strain described here a PCR-RFLP analysis was used. The PCR- RFLP assays were designed to discriminate both known sequences using software simu- lation. Using PROPHET 5.0 software (BBN Systems and technologies) a restriction map- ping analysis was performed to predict re- striction enzymes useful to discriminate be- tween both known strains of B. caballi (the Spanish isolated Acc. N. KX358867 and the French one with Acc. N. AJ289246). Based on simulation of cleavage sites, two restriction enzymes, HaeIII and HinfI, were selected. The cutting prediction for each strain can be seen in Table 3. Results This research provided a new sequence of the -tubulin gene of B. caballi (GM Málaga strain). This DNA stretch was sufficiently long for a successful primer pairs design. As a result of comparing the -tubulin gene of the two most important piroplasms species that impair horses, T. equi and B. caballi, a first primer pair (B-TubF and B-TubR, Table 2) was designed in order to amplify that gene from a DNA template of both parasites. More- over, new and less degenerated primers pair could be designed (B-TubF2 and B-TubR2, Table 2) used for conventional PCR applica- tions. The resulted amplicons had a size of 718bp and 826bp for T. equi and B. caballi, respectively (Fig. 2). According to (27), the -tubulin gene se- quences from each of the two species dif- fered in length (100bp approx.). The se- quence of the -tubulin gene of B. caballi obtained with these primers provided more molecular information about the genetic var- iations of this conserved gene. Both sequences (AJ289246 and KX358867) were truncated to accommodate to the short- est molecule, i.e., 460bp. Thus, this fragment of the -tubulin gene of B. caballi partly covered exon I (nucleotide 1 to 63), but com- pletely covered intron I (nucleotide 64 to 233) and exon II (nucleotide 234 to 460). Sixty- eight polymorphic sites were found between both sequences. The comparison of both se- quences using BLASTN 2.2.20 (28) yielded 81% nucleotides identities and 3% open gaps. Focusing on exon II, they shared 92% identities with 19 (19 of 76 triples) silent mutations (synonymous substitutions), as estimated using the DnaSP software version 4.0. This analysis reported an unexpected num- ber of polymorphisms between both strains at the coding region but without changing the primary structure of the protein, as should be in the case of strongly conserved genes such as the -tubulin gene (27). Moreo- ver, the first intron showed a minor size var- iation due to a 2bp deletion in the Spanish sequence. All these facts suggest the se- quence of -tubulin gene could be variable among and within species of Babesia para- sites. Twenty DNA samples from chronic carriers and 5 samples from acute infected horses were amplified using our species- specific primers for B. caballi. All samples were positive. These PCR were digested with both restriction enzymes (HaeIII and Hinf I) to determine the strain causing the infection. HinfI cut the PCR amplicons but HaeIII does not cut them as was predicted. As a result, this assay was able to confirm that the infecting strain of these horses would be the Spanish strain (Fig. 3). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2017, 11(3): 344–353 MG Montes-Cortés et al.: Genetic Variation of … 348 http://jad.tums.ac.ir Published Online: September 08, 2017 Table 1. Sequences used to design primers Accession number Species Gene Chromosome Nucleotide posi- tions or size Author CP001669 Babesia equi Complete genome 1 845478…846856 Kappmeyer et al. 2012 AAGK01000002 Theileria par- va Complete genome 2 923513…925358 Gardner et al. 2005. AJ289249 Babesia equi Partial -- 349bp Cacciò et al. 2000 XM-001611566 Babesia bovis Complete cds -- 1343bp Brayton et al. 2007 KC465972 Babesia odoi- coilei Partial cds -- 1275bp Zamoto-Niikura et al. 2014 AB083377 Babesia mi- croti Complete cds -- 1651bp Zamoto et al. 2004 AB860327 Babesia ovata Partial cds -- 457bp Damdinjav et al. un- published AP011947 Theileria ori- entalis Complete genome 2 933579…934685 Hayashida et al. 2012 AJ289246 Babesia ca- balli partial -- 460bp Cacciò et al. 2000 Table 2. Oligonucleotide primers used to amplify and sequence parasite -tubulin gene. Primer Pair Forward Sequence 5´-3´ (F) Reverse Sequence 5´-3´ (R) B. caballi Amplicon size T. equi Amplicon size B-Tub GAATGAGRGARATCGTW- CACA CARCTTYAGNGTNCKRAA- GCARA 826bp Not done B-Tub 2 GAATGAGGGARATCGTW- CACA CAGCTTTAGRGTTCKGAA- GCARAT 826bp 718 GM B-Tub ACCCGG- TAAGTCGTTAAACC AGTTGTCRGGYCTGAA- GAGT 345bp No amplifica- tion Table 3. Restriction mapping analysis for predicting restriction enzymes. (*excluding cuts within primers) B. caballi strain Size of PCR product Predicted fragment sizes HaeIII (GGCC) HinfI (GANTC) Cacciò et al. (2000) 347 249,88 * 299,48 This research 345 Uncut * 297,48 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2017, 11(3): 344–353 MG Montes-Cortés et al.: Genetic Variation of … 349 http://jad.tums.ac.ir Published Online: September 08, 2017 Fig. 1. Geographical distribution of collected samples Fig. 2. Amplicons of Theileria equi and Babesia caballi using primers pairs B-Tub 2. Lane M, 100bp size marker. Lane 1: Amplicon of T. equi (718bp) Lane 2: Amplicon of B. caballi (826bp) Fig. 3. Restriction enzymes assays. Lane M, 100bp size marker. Lane 1a: PCR product of DNA from cultivated isolate amplified with primers pairs GM B- Tub. Lane 1b: PCR product digested with HaeIII (uncut). Lane 1c: PCR product digested with HinfI. Lane 2a: PCR product of DNA from an acute infected horse with primers pairs GM B-Tub. Lane 2b: PCR product digested with HaeIII (uncut). Lane 2c: PCR product digested with HinfI Discussion This research has provided new insights into the -tubulin gene of B. caballi, the key factor being that there was a unique and par- tial sequence available in the Gene Bank for this piroplasm. This sequence belonged to a different B. caballi strain after comparison with the sequence from (27). Moreover, it can be suggested that genetic variation should be expected among B. caballi strains even for conserved genes. The occurrence of genetic variations in certain conserved genes like the -tubulin gene (described in this research), could indicate not only the exist- ence of different strains of B. caballi, but also suggests the study of possible higher taxonomic levels because in our study the shared identity was within the 92% for cod- ing segments. Similar results were recently reported for the β-tubulin gene sequence of B. ovata. This Babesia shared 94.8–100% identity within species but the shared se- quence identity of B. ovata with other spe- cies was 87.0–90.6%, 83.7–86.3%, and 82.7– 85.4% to B. bigemina, B. odocoilei and B. di- vergens, respectively (29). Based on genetic knowledge of 18S rRNA gene both for B. caballi and T. equi (30- 31) there were different genotypes in Spain for both species. On the one hand, (32) conducted phylogenetic analysis based on the same gene to report that there were two B. caballi clades. However, (33) established four B. caballi clades using the BC48 gene. On the other hand, (32) found three T. equi clades. However, (34) announced that there were not three, but four T. equi clades. Phylogenetic analysis by (33) using the ema-1 gene divid- ed the sequences into four T. equi clades. (35) using a phylogenetic study based on both the 18S rRNA gene and the -tubulin gene have shown T. annae and its synonyms are not Theileria parasites instead they are http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2017, 11(3): 344–353 MG Montes-Cortés et al.: Genetic Variation of … 350 http://jad.tums.ac.ir Published Online: September 08, 2017 Babesia species, being the -tubulin decisive to determine that reclassification. This information suggests that taxonomic classification of equine piroplasms is not yet defined completely as reported (3). Accord- ing to (36) and (37), a new diagnostic test on additional or new antigens is needed because nowadays there is not a gold standard test for detecting Equine piroplasmosis. Specifically, (37) showed there was lack of concordance between the B. caballi cELISA, IFAT, and nPCR. Hence, the importance of this study that contributes to the enrichment of current genetic information of an unknown -tubulin gene of B. caballi. Based on the PCR-RFLP analysis, it was possible to discriminate different piroplasms species as shown by (27) where cattle piro- plasms such as Theileria sergenti, T. annula- ta, B. bigemina, B. bovis and B.a major were efficiently discriminated using RsaI restriction enzyme. Another study also valued PCR- RFLP as a specific analysis to confirm spe- cies diagnosis as has been shown for B. bi- gemina with VspI digestion of the small subunit rRNA . Using BslI and HinfI with PCR amplicons from the 18S ribosomal RNA gene, discriminated subspecies of canine piro- plasms from other species such as T. equi, T. annae and B. conradae. PCR-RFLP assays may not be necessary to discriminate between B. caballi and T. equi species due to the different sizes of their intron I (27 and this study). However, this difference in size was as short as 2bp be- tween both B. caballi strains, which cannot be seen with agarose gels. This is the reason why PCR-RFLP assays were selected to dis- criminate the GM Málaga strain from the strain described by (27), so this study shows for the first time that PCR-RFLP assays were used in order to discriminate among different strains of B. caballi species assuming predictions based on the sequence deposited in the Gene Bank. Moreover, although PCR- RFLP compares a limited number of nucleo- tides, restriction enzyme digestion was in full accordance with the predicted target based on the sequence of the GM Málaga strain even when using DNA from naturally infected horses. Finally, this study has provided several new and useful primer pairs although they should be more deeply assessed for diagnostic pur- poses (acute infected horses and chronic B. caballi carriers) in future assays. Conclusion We reported here that the -tubulin gene of B. caballi shows high genetic variation, which will be useful to facilitate the devel- opment of more precise molecular assays for the detection of infected horses. This is equally relevant at local and international levels, help- ing to prevent the spread of this disease world- wide in the future. Acknowledgements The authors wish to acknowledge the col- laboration of the veterinarians and owners of the horse farms where the samples were col- lected. The authors declare that there is no conflict of interest. References 1. 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