First Molecular Evidence of Babesia Caballi and Theileria Equi Infections in Horses in Cuba

First molecular evidence of Babesia caballi and Theileria equi infections in horses in Cuba

Adrian Alberto Díaz-Sánchez, Marcus Sandes Pires, Carlos Yrurzun Estrada, Ernesto Vega Cañizares, Sergio Luis del Castillo Domínguez, et al.

Parasitology Research Founded as Zeitschrift für Parasitenkunde

ISSN 0932-0113

Parasitol Res DOI 10.1007/s00436-018-6005-5

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Parasitology Research https://doi.org/10.1007/s00436-018-6005-5

ORIGINAL PAPER

First molecular evidence of Babesia caballi and Theileria equi infections in horses in Cuba

Adrian Alberto Díaz-Sánchez1 & Marcus Sandes Pires2 & Carlos Yrurzun Estrada3 & Ernesto Vega Cañizares1 & Sergio Luis del Castillo Domínguez1 & Alejandro Cabezas-Cruz4 & Evelyn Lobo Rivero1 & Adivaldo Henrique da Fonseca2 & Carlos Luiz Massard2 & Belkis Corona-González1,5

Received: 3 April 2018 /Accepted: 4 July 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract Equine piroplasmosis is a disease of Equidae, including horses, donkeys, mules, and zebras, caused by either Theileria equi or Babesia caballi. This disease represents a serious problem for the horse industry and its control is critical for the international trade of horses. The objective of the present study was to detect B. caballi and T. equi infections in horses reared in western Cuba. Blood samples from 100 horses were tested for the presence of piroplasms by using Giemsa-stained blood smears and nested PCR (nPCR) assays targeting merozoite antigen genes of B. caballi (bc48) and T. equi (ema-1). All animals were inspected for the detection of tick infestation and tick specimens were collected for species identification. Erythrocyte inclusions were observed in 13 (13%) of the analyzed samples. nPCR analysis showed that 25 (25%) samples were positive for B. caballi, 73 (73%) for T. equi, and 20 (20%) showed dual infections. Only one tick species was found infesting horses, Dermacentor nitens.Inaddition, three nearly full-length sequences of T. equi 18S rRNA gene were obtained and subjected to phylogenetic analyses. This study reports a high prevalence of T. equi and B. caballi single and coinfections in horses in western Cuba. Molecular analysis of the 18S rRNA gene of T. equi suggested that different genotypes of this hemoparasite circulate in Cuba. To the best of our knowledge, this is the first report describing the molecular detection of B. caballi and T. equi in horses in Cuba.

Keywords Equine piroplasmosis . Babesia caballi . Theileria equi . nPCR . 18S rRNA

Section Editor: Neil Bruce Chilton Introduction

* Belkis Corona-González Equine piroplasmosis is a vector-borne disease affecting hors- [email protected] es and other equids (e.g., donkeys, mules, and zebras). This Adrian Alberto Díaz-Sánchez disease is caused by two obligate intraerythrocytic [email protected] hemoprotozoans, Theileria equi and Babesia caballi (Apicomplexa: Piroplasmida), which belong to the families 1 National Center for Animal and Plant Health (CENSA), Carretera de Babesiidae and Theileriidae, respectively (Mehlhorn and Tapaste y Autopista Nacional, km 22 1/2, 32700 San José de las Schein 1998). These parasites are widespread in tropical, sub- Lajas, Mayabeque, Cuba tropical, and some temperate regions, where tick vectors are 2 Department of Epidemiology and Public Health, Federal Rural present. Due to its morbidity and mortality, this disease has a University of Rio de Janeiro (UFRRJ), BR 465, km 7, Seropédica, Rio de Janeiro 23890-000, Brazil negative impact on the equid industry (Knowles Jr. 1996). The distribution of equine piroplasmosis is associated with the 3 Department of Animal Prevention, Veterinary Medicine College, Agrarian University of Habana (UNAH), Carretera de Tapaste y presence of competent vectors, mainly ixodid ticks of the gen- Autopista Nacional, km 23 1/2, San José de las Lajas, Mayabeque, era Dermacentor, Rhipicephalus, Hyalomma, Amblyomma, Cuba and Haemaphysalis (Scoles and Ueti 2015). Currently, 12 4 UMR BIPAR, INRA, ANSES, Ecole Nationale Vétérinaire d’Alfort, and 11 tick species are recognized as vectors of T. equi and Université Paris-Est, 94700 Maisons-Alfort, France B. caballi, respectively. However, the disease can be also 5 Animal Health Division, National Center for Animal and Plant transmitted through infected blood transfusion, as well as con- Health (CENSA), Carretera de Tapaste y Autopista Nacional, km 22 taminated syringes and surgical instruments (Short et al. 1/2, 32700 San José de las Lajas, Mayabeque, Cuba Author's personal copy

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2012). Transplacental transmission was also reported in increasingly important and plays a critical role in maintaining T. equi (Santetal.2016). the international market open to the horse industry. Per-acute, acute, sub-acute, and chronic forms of equine Although, the presence of equine piroplasmosis within the piroplasmosis have been described. The clinical signs of the Caribbean region is still controversial, the disease has been disease are diverse including anorexia, pyrexia, edema, hemo- reported on most islands, including Cuba (Asgarali et al. globinuria, anemia, intravascular hemolysis, jaundice, re- 2007;Oliveretal.1985; Zhang et al. 2015). However, limited duced work efficiency, weight loss, abortion in mares, and information is available on the status of this disease in equine death can occur in the acute phase of the infection (Zobba et herds of Cuba. The study on equine piroplasmosis in Cuba al. 2008). In general, infection with T. equi exhibits a more conducted by Salabarria et al. (1982) was largely based on severe clinical disease than B. caballi, although the signs and conventional blood smear examination and serological tests, severity can vary significantly from one region to another which as previously mentioned, are not the most suitable tools (Wise et al. 2013). Infection recovery is possible, but recov- for epidemiological assessments. Therefore, the present study ered animals may become carriers for long periods. The horses was aimed at detecting B. caballi and T. equi infections in that recover from the infection with T. equi may remain life- horses reared in western Cuba using microscopic and molec- long carriers, whereas infection by B. caballi is generally self- ular methods. limited up to 4 years (Rothschild 2013). B. caballi and T. equi infections can be diagnosed by different methods including conventional parasitological tech- Materials and methods niques (i.e., examination of stained thin blood smears) and serological and molecular techniques (Wise et al. 2013). The Study area utility of the microscopic examination of stained blood smears is limited because of its poor sensitivity, particularly in carrier The present study was performed on local horses of four prov- animals during chronic infection due to low parasitemia inces of the western region of Cuba. The island climate is (Malekifard et al. 2014). Several serological methods, such tropical and humid with two distinct climatic periods, a dry as the complement fixation technique (CFT), the indirect fluo- season from November to April and a warmer and rainy sea- rescence antibody test (IFAT), and the competitive inhibition son from May to October. In the dry season, the average tem- enzyme-linked immunosorbent assay (cELISA), are also perature varies between 15 and 26 °C, while temperatures available for the detection of parasite-specific antibodies in typically range between 22 and 32 °C in the wet season. the infected horse sera (Asgarali et al. 2007). Currently, the The horses studied were from six municipalities of the cELISA is considered by the World Organization for Animal provinces Mayabeque (San José de las Lajas, Jaruco, and Health (OIE) as the preferred test for international horse trade Madruga), Artemisa (Artemisa), Havana (Boyeros), and (OIE 2014). However, these serological assays are time con- Matanzas (Unión de Reyes) (Fig. 1). suming and labor intensive and cannot differentiate between current and previous infections due to persistence of antibodies after the infections were resolved (Wise et al. 2013). In Sample collection contrast, for the diagnosis of active infection, molecular methods are more sensitive than other diagnostic techniques One hundred blood samples were randomly taken from horses to detect carrier animals. Polymerase chain reaction (PCR) of different herds from September 2014 to August 2015. assays based on ribosomal 18S rRNA, B. caballi 48-kDa mer- Approximately 3–4 ml of blood was collected from each an- ozoite rhoptry-associated protein 1 (bc48), and T. equi mero- imal by puncturing the jugular vein using vacutainer zoite antigen 1 (ema-1) genes are routinely used to detect collecting tubes containing EDTA (BD Vacutainer®), and piroplasmosis in horses, and have proven to be sensitive, spe- transported to the laboratory. After preparing thin blood cific, and highly efficient (Alhassan et al. 2005;Battsetseget smears, blood samples collected in EDTA-coated tubes were al. 2002;Bhooraetal.2010). stored at −20 °C until DNA extraction. The socioeconomic impact of the disease and the restric- The horses were inspected visually for identification of tions to trade infected animals led the OIE Animal Health animals infested with ticks, using a systematic approach Code to categorize equine piroplasmosis as a notifiable dis- that began at the head and neck regions (including from ease (OIE 2014). Disease occurrence represents an obstacle to within the ears) and continued to the pectoral, axillary, international movement of horses; for this reason, many coun- and inguinal regions, concluding at the tail of each animal. tries have introduced stringent animal movement restrictions Adult ticks were collected and stored in polypropylene to prevent the introduction of B. caballi and T. equi into tubes containing isopropyl alcohol for identification to the disease-free areas (Knowles Jr. 1996). Therefore, the control species level using an appropriate published taxonomic key of equine piroplasmosis in endemic countries is becoming (Barros-Battesti et al. 2006). Author's personal copy

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Fig. 1 Map of the study area. A Island of Cuba. B Location of the municipalities in which the samples collections were taken in the provinces of Artemisa (Artemisa), Havana (Boyeros), Mayabeque (San José de las Lajas, Jaruco and Madruga), and Matanzas (Unión de Reyes). Scale bar = 40 km

Microscopic examination and purity of the DNA from all blood samples were determined by Colibri Microvolume Spectrophotometer (Titertek- The blood smears were fixed in methanol (Uni-Chem) and Berthold, Pforzheim, Germany). DNA was stored at − 20 °C stained in 10% Giemsa solution in phosphate buffered saline until further use. (PBS), pH 7.2. For the evaluation of intracellular parasites, the All DNA samples were subjected to nested PCR (nPCR) slides were examined using light microscopy (Carl Zeiss assays based on the procedures previously published by Microscopy GmbH, Jena, Germany), with an oil immersion Battsetseg et al. (2002), using primers (Table 1)totargetthe lens at a final magnification of × 1000. Approximately 100 48-kDa merozoite antigen (bc48) gene specific to B. caballi fields were evaluated per animal. The morphological and bio- and merozoite antigen-1 (ema-1) gene specific to T. equi.The metrical parameters including the shape, site location, and size PCRs were performed in a total volume of 25 μlcontaining of parasite in any infected erythrocyte were considered. In 1X of GoTaq® Green Master Mix (Promega®, Madison, WI, microscopic examination, B. caballi was identified as large USA), and 400 nM of each primer. Primary reactions used paired pyriform parasites, while the small T. equi parasites 2 μl of purified DNA as template, and nested amplifications were identified as paired pyriform, rounded, and tetrad or used 1 μl of the primary PCR product as template. The Maltese cross arrangement of merozoites (OIE 2014). thermocycling conditions used for either T. equi or B. caballi amplification were those described by Battsetseg et al. (2002) DNA extraction and nested PCR assays with modifications: 96 °C for 4 min, followed by 40 cycles, consisting of denaturation at 94 °C for 1 min, annealing at Genomic DNA was purified from 100 μl of the blood sample 56 °C for 1 min and extension at 72 °C for 1 min; with a final from each horse using the DNAeasy Blood and Tissue DNA extension cycle at 72 °C for 5 min. Nested cycling conditions Purification Kit (QIAGEN®, Valencia, CA, USA) according were those described above for the primary amplification, to the manufacturer’s recommendations. The concentration except that 30 cycles were used. All PCR amplifications were Author's personal copy

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Table 1 Nested polymerase chain reaction (nPCR) and sequencing primer pairs used in the assays for the detection of each pathogen

Species Primer Sequence [5′–3′] Target gene [size (bp)] Reference

B. caballi BC48F1 ACG AAT TCC CAC AAC AGC CGT GTT bc48 (530) Ikadai et al. (1999) BC48R3 ACG AAT TCG TAA AGC GTG GCC ATG BC48F11 GGG CGA CGT GAC TAA GAC CTT ATT bc48 (454) Battsetseg et al. (2002) BC48R31 GTT CTC AAT GTC AGT AGC ATC CGC T. equi EMA5 TCG ACT TCC AGT TGG AGT CC ema-1 (268) Battsetseg et al. (2002) EMA6 AGC TCG ACC CAC TTA TCA C EMA7 ATT GAC CAC GTC ACC ATC GA ema-1 (218) Battsetseg et al. (2002) EMA8 GTC CTT CTT GAG AAC GAG GT 18S-F1 AAC CTG GTT GAT CCT GCC AGT AGT CAT 18S rRNA (~ 1750) Liu et al. (2005) 18S-R1 GAT CCT TCT GCA GGT TCA CCT AC

performed in an Eppendorf Mastercycler Gradient numbers: B. caballi bc48 (KY111763-65), T. equi ema-1 Thermocycler (Eppendorf, Hamburg, Germany). DNA from (KY111766), and T. equi 18S rRNA (KY111760-62). horses known to be infected with B. caballi and T. equi were used as positive controls, and nuclease-free water was used as the negative control. The amplification products were submit- Phylogenetic analysis ted to electrophoresis on 1.5% agarose gels. The gels were stained with ethidium bromide (0.5 mg/ml) and photographed For phylogenetic analysis, 18S rRNA and ema-1 (T. equi), and under ultraviolet (UV) light. A 100-bp DNA ladder bc48 (B. caballi) nucleotide sequences were collected from (Promega®, Madison, WI, USA) was used to initially deter- GenBank; several isolates of T. equi and B. caballi were in- mine the molecular mass of PCR products. cluded in the analysis as shown in Figs. 2 and 3. Sequences were aligned using ClustalW algorithm (Thompson et al. Sequence analysis 1994). After alignment, non-aligned regions were removed with Gblocks (v 0.91b) (Castresana 2000) implemented in Six PCR products with the expected size were randomly se- Phylogeny.fr (Dereeper et al. 2008). The final alignment lected for sequencing (three from bc48 gene and three from contained 1411 (18S rRNA), 218 (ema-1), and 360 (bc48) ema-1 gene) to confirm nPCR results. Furthermore, three pos- gap-free nucleotide positions. The best-fit model of the se- itive samples for T. equi were randomly selected and the near- quence evolution was selected based on Corrected Akaike ly full-length 18S rRNA gene (approximately 1750 bp) was Information Criterion (cAIC) and Bayesian Information amplified and sequenced using the 18S-F1 and 18S-R1 Criterion (BIC) implemented in MEGA 7. TN93 (18S primers (Table 1) as previously described by Liu et al. rRNA) and Kimura 2-parameter (ema-1 and bc48) models (2005). The PCR products were purified using the QIAquick (Tamura and Nei 1993), which had the lowest values of Spin PCR Purification kit (QIAGEN®, Valencia, CA, USA), cAIC and BIC, were chosen for subsequent phylogenetic anal- submitted to direct sequencing using an ABI Prism BigDye™ yses. Maximum likelihood (ML) method, implemented in Terminator Cycle Sequencing kit (Applied Biosystems®, MEGA, was used to obtain the best tree topology. Initial trees Waltham, MA, USA) and analyzed with a commercial se- for the heuristic search were obtained automatically by apply- quencer ABI 3730 DNA Analyzer (Applied Biosystems®, ing neighbor joining and BioNJ algorithms to a matrix of Perkin Elmer, CA, USA). The raw sequences data obtained pairwise distances estimated using the Maximum Composite were assembled, edited, and analyzed using Molecular Likelihood (MCL) approach, and then selecting the topology Evolutionary Genetics Analysis (MEGA) 7 software (Kumar with superior log likelihood value. A proportion of Gamma et al. 2016). The Basic Local Alignment Search Tool distributed sites (+G) was estimated in MEGA for ema-1 (BLASTn) (Altschul et al. 1990) at the National Center for (0.38). Reliability of internal branches was assessed using Biotechnology Information (NCBI) was used to compare the the bootstrapping method (1000 replicates). The 18S rRNA Cuban B. caballi and T. equi nucleotide sequences with rep- gene sequences of Babesia divergens (AY098643) and B. resentative sequences available in the GenBank database by bigemina (JQ437261) were used as outgroups. Graphical rep- nucleotide sequence homology. The sequences were deposit- resentation and editing of the phylogenetic tree were per- ed in the GenBank database under the following accession formed with MEGA. Author's personal copy

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Fig. 2 Phylogenetic analysis based on B. caballi bc48 (A) and T. equi supported the branch. GenBank accession numbers are shown in paren- ema-1 (B) gene sequences. The phylogenetic trees were constructed using theses, the B. caballi and T. equi isolates identified in this study (H07, the maximum likelihood (ML) method, and the numbers above the inter- H10, H11 and H18) are indicated with an asterisk nal nodes indicate the percentages of 1000 bootstrap replicates that

Results Brazil (KP998150), the USA (AF092736), Mongolia (AB703057), Philippines (KX900447), and Egypt Prevalence of B. caballi and T. equi in horses of Cuba (KJ094947). The identity among Cuban bc48 gene ranged from 98 to 99%, while the similarities among the deduced The microscopic examination of 100 blood smears revealed amino acid sequences were found from 95 to 100%. The erythrocyte inclusions compatible with Babesia/Theileria par- bc48 gene-based phylogenetic analysis divided the sequences asites in 13/100 (13%; 95% CI, 8.6–20.4%) horses. The spe- into four genotypes as previously designed Munkhjargal et al. cies diagnosed microscopically were B. caballi 4/100 (4%; (2013)(Fig.2(A)), and Cuban isolates fell into clusters 3 and 95% CI, 1.6–9.8%) and T. equi 9/100 (9%; 95% CI, 4.8– 4. The Cuban bc48 gene sequences H07 (KY111763) and 16.2%). The nPCR assays confirmed the infection in the 13 H10 (KY111764) fell into cluster 3 together with Brazilian samples where erythrocyte inclusions were found. In addition, isolates (JN217099, KP998147, KP998151). Whereas, the the nPCR assays revealed that 78/100 (78%; 95% CI, 68.5– Cuban isolate H18 (KY111765) fell into cluster 4 was closely 85.7%) horses were infected with at least one of the two related to those of Egypt (KR811096, KR811097), Mongolia agents, including 5/100 (5%; 95% CI, 2.2–11.2%) for B. (AB703057, AB731210, AB731213, AB731218), and caballi infection, 53/100 (53%; 95% CI, 43.3–62.5%) with Philippines (KX900447). T. equi infection, and 20/100 (20%; 95% CI, 13.4–28.9%) In addition, sequences of three 218-bp nPCR amplicons of showed mixed infections (Table 2). As expected, the nPCR T. equi obtained from Cuban horses demonstrated identical products of bc48 (B. caballi)andema-1 (T. equi)were454 sequences between them, and showed 85 to 100% identity and 218 bp, respectively. A total of 155 adult ticks (98 females with previous published sequences from India (KT443898), and 57 males) were collected parasitizing horses, and all of Jordan (KX533889), Italy (KU923605), the USA them were identified as Dermacentor (Anocentor) nitens spe- (CP001669), Brazil (DQ250541), Japan (AB015216), cies, which is known as the tropical horse tick in the Americas. Mongolia (AB713965), and South Africa (JQ782611). The Parasitism by at least one tick was observed in 33 (33%) ema-1 gene-based phylogenetic analysis divided the se- animals. quences into five genotypes as previously designed by Munkhjargal et al. (2013)(Fig.2(B)), and Cuban isolates fell Genetic diversity of B. caballi and T. equi in horses into cluster 1. The Cuban isolate H11 (KY111766) fell into the of Cuba cluster 1 and was closely related (above 98% identity) to those of the USA (L13784), Brazil (U97167), Thailand The bc48 and ema-1 nPCR amplicons were 454 and 218 bp in (LC230156), Russia (AB015211), Morocco (U97168), length as expected, and the sequence analysis of them dem- Mongolia (AB731201, AB731193), and Israel (KX533891). onstrated the presence of B. caballi and T. equi-infected horses Three of 73 samples which were found positive in nPCR in Cuba. The BLASTn analysis of the three 454-bp nPCR assay for T. equi were randomly chosen, and successfully amplicons of B. caballi showed 96 to 99% identity with sequenced for the phylogenetic analyses of their nearly full- known sequences available in the GenBank database from length 18S rRNA gene sequences. The identity between Author's personal copy

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Fig. 3 Phylogenetic analysis of T. equi isolates from horses based on the numbers are shown in parentheses, and the T. equi samples identified in 18S rRNA gene sequence comparison. The phylogenetic tree was this study (H01, H12, and H16) are indicated with an asterisk. The T. equi constructed using the maximum likelihood (ML) method, and the num- 18S rRNA gene sequences fell into five main T. equi genotypes, desig- bers above the internal nodes indicate the percentages of 1000 bootstrap nated as genotypes A, B, C, E, and D, as previously described ini Qablan replicates that supported the branch. Babesia divergens (AY098643) and et al. (2013) B. bigemina (JQ437261) were used as outgroup. GenBank accession Author's personal copy

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Table 2 Combined positivity by microscopy examination (Giemsa-stained blood smears) and nPCR assays for B. caballi and T. equi among horses from the western region of Cuba

Province Municipalities Samples Blood smear (%) nPCR (%)

B. caballi T. equi Coinf* B. caballi T. equi Coinf*

Artemisa Artemisa 9 ––––5(5) – Habana Boyeros 21 –––7 (7) 13 (13) 3 (3) Mayabeque Jaruco 8 ––––4(4) – Madruga 3 ––––1(1) – San José de las Lajas 50 4 (4) 8 (8) – 16 (16) 43 (43) 16 (16) Matanzas Unión de Reyes 9 – 1(1) – 2(2) 7(7) 1(1) Total 6 100 4 (4) 9 (9) – 25 (25) 73 (73) 20 (20)

*Coinfection

Cuban T. equi 18S rRNA gene sequences ranged from 97 to In the present study, occurrence of B. caballi and T. equi 99% each other, and were 1744 (KY111761, KY111763) and infections in horses in the western region of Cuba was inves- 1748 bp (KY111762) in length as expected. BLASTn analysis tigated by Giemsa-stained blood smear identification and revealed that the 18S rRNA gene sequences obtained share 95 nPCR assays. The positive rate of both hemoparasites detected to 99% identity with previous published sequences from the in our study on direct microscopic identification was very low USA (CP001669), Brazil (KY952237), Israel (KX227640), and was consistent with previous studies (Malekifard et al. Mexico (JQ390047), Spain (DQ287951), South Africa 2014). Although the visual examination of piroplasms by mi- (Z15105), and Switzerland (KM046920). In the current phy- croscopic identification is the simplest diagnostic test, with logenetic analysis, the five major T. equi genotypes 18S rRNA high specificity and the greatest accessibility, it lacks sensitiv- gene-based (A, B, C, D and E) were recovered using the ity, especially in diagnosis of subclinical infections when GenBank sequences previously described (n = 29) and the parasitemia becomes too low to detect positive cases (Zobba Cuban sequences described in this study (n = 3). The analyses et al. 2008). Considering the possible falsely diagnosed cases showed that the T. equi 18S rRNA gene sequence from the of equine piroplasmosis by microscopic examination, the de- isolates H12 (KY111761) and H16 (KY111762) belonged to finitive diagnosis of the disease is usually accomplished using genotype A together with sequences from USA the a combination testing by serological and PCR detection (JX177670-73), Israel (KX227640), Spain (AY150064), and methods (Wise et al. 2017). South Africa (Z15105); whereas the isolate H01 (KY111763) The percentage of positive animals determined by nPCR in fell into genotype C together with sequences from Brazil the studied population revealed the presence of B. caballi and (KU240066, KY464027), Mexico (JQ390047), Israel T. equi in 25 and 73% of the horses examined, respectively. (KX227630), and South Africa (EU642511) (Fig. 3). The high presence of hemoparasites detected in the horses by PCR is consistent with data reported in other Latin American countries: Costa Rica 20–46.2%, Venezuela 4.4–61.8%, Discussion Guatemala 48–52%, and Brazil 12.5–59.7% (Heim et al. 2007; Posada-Guzman et al. 2015; Rosales et al. 2013; Equine piroplasmosis is a disease of great economic impor- Teglas et al. 2005). In addition, the laboratory results sug- tance due to the constraints occasioned to the international gested both babesial agents could be endemic in the studied movement and trade of horses, especially for horses that travel areas, and showed piroplasms presence without causing seri- to equestrian sport events or are exported to a number of ous diseases in the horses. In this respect, 20% of the animals countries believed disease free. For these reasons, some coun- detected with mixed infections did not present clinical charac- tries maintain stringent restrictions that prevent import of teristics as well as those of healthy animals or with simple horses serologically positive for piroplasms species infections (Wise et al. 2013). Several studies based on molec- (Knowles Jr. 1996). As previously mentioned, piroplasmosis ular methods suggest T. equi to have a higher infection rate is an endemic tick-borne disease in most areas of the world (de than B. caballi; this difference in the prevalence between both Waal 1992), and the majority of infected equine appear clini- piroplasms is likely a consequence of the more efficient elim- cally normal, but they remain infected and develop into life- ination of B. caballi by the host immune system, in contrast to long carriers of the infection (Butler 2013). the lifelong persistence of T. equi (Rothschild 2013). In our Author's personal copy

Parasitol Res study, the low number of positive samples of B. caballi ob- The Cuban isolates H12 and H16 belong to genotype A, served could be also associated to the low loads of parasites which predominantly contains sequences isolated from horses circulating in blood due to the erythrocytes infected are se- linked with equine piroplasmosis studies reported in the USA, questered in the spleen, which could not be detected with the Israel, Spain, and South Africa; whereas the isolate H01 fell babesial DNA by the PCR protocol used (Machado et al. into genotype C, which predominantly contains sequences 2012). This is the first report using PCR as a diagnostic tool isolated from horses in Brazil, Israel, and Mexico, and from for equine piroplasmosis in Cuba; therefore, it is not possible zebras in South Africa. Our results show that T. equi exhibits to perform a comparative analysis at the present time. an overlapping occurrence of different genotypes within the In our study, the only tick species identified infesting the same population of equids, which is in agreement with the horses was D. nitens, which is well known as a transovarial previous reports confirming the sequence heterogeneity in and intraestadial vector of B. caballi in the Americas, based on the rRNA genes of the causal parasites of equine experimental and epidemiological evidence (Kerber et al. piroplasmosis (Kizilarslan et al. 2015;Liuetal.2016). 2009; Schwint et al. 2008). Probably the single collection Considering that the information available about the T. equi period in our study contributed that it was the only tick species and B. caballi genotypes circulating between horses and other observed, and other transmission ways or other arthropods equids species in Cuba is scarce, future studies focused to could be involved in maintaining the high level of T. equi identify and characterize Theileria and Babesia genotypes infections observed in the studied region (Wise et al. 2013). would be necessary. Little is known concerning the presence of efficient tick vec- In conclusion, to our knowledge, this is the first molecular tors in the studied region; therefore, further studies are re- report on T. equi and B. caballi infecting horses reared in quired to ascertain which tick species would be involved as Cuba. Interestingly, we found a high number of horses posi- competent vectors for T. equi and B. caballi in the western tive to T. equi and B. caballi, suggesting that they could be a region of Cuba. reservoir of infection to other horses and tick vectors. The merozoite antigen genes of B. caballi (bc48) and T. Considering that equine piroplasmosis can cause great eco- equi (ema-1) are considered as important candidates for the nomic losses, additional epidemiological surveys are recom- development of effective diagnostic assays of equine mended with the aim of identifying all the genotypes of B. piroplasmosis caused by the respective parasite species caballi and T. equi present in the equine population in Cuba. (Xuan et al. 2001). In the present study, we performed phylo- The correlations with vector distribution and other factors in- genetic analyses based on the genes encoding both antigens to volved in susceptibility to infection, such as age, gender, determine their genetic diversity. The Cuban bc48 gene se- breed, and geographical location, should also be considered. quence analysis showed that B. caballi in Cuba include at least two different genotypes, and the Cuban ema-1 gene sequence Acknowledgements The authors would like to thank Dr. MVZ Osvaldo analysis found a unique genotype. However, at different pre- Fonseca Rodríguez for his kindness in preparing the map included in this study, as well as Nelson Albelo Chávez and Eleuterio Hernández vious studies conducted by Munkhjargal et al. (2013)in Hernández for their technical assistance, and Dr. nat. sc. Marina L. Meli Mongolia, between the Cuban bc48 and ema-1 gene se- at University of Zurich (UZH) for her very helpful comments on the quences was observed a high homology as was further con- manuscript. firmed by the findings of the phylogenetic trees. Therefore, further studies are needed based on analyses of large numbers Funding sources This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. of bc48 and ema-1 gene sequences from geographically different endemic areas that might shed an additional insight on Compliance with ethical standards the actual genetic variations between isolates. The present study also provided the first data on the Conflict of interests The authors declare that they have no conflict of molecular characterization and the genotypes of T. equi in interest. Cuban horse populations based on 18S rRNA gene sequences, which have been constantly used in the phylogenetic analyses and genotyping of the equine piroplasms References (Halletal.2013). The phylogenetic analyses based on the sequence heterogeneity in the 18S rRNA gene within T. 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