Molecular Detection of Theileria and Babesia Infections in Cattle§ Kursat Altay, M

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Veterinary Parasitology 158 (2008) 295–301 www.elsevier.com/locate/vetpar

Molecular detection of Theileria and Babesia infections in cattle§ Kursat Altay, M. Fatih Aydin, Nazir Dumanli, Munir Aktas * Department of Parasitology, Faculty of Veterinary Medicine, University of Firat, 23119 Elazig, Turkey Received 31 May 2008; received in revised form 27 August 2008; accepted 15 September 2008

Abstract This study was carried out to determine the presence and distribution of tick-borne haemoprotozoan parasites (Theileria and Babesia) in apparently healthy cattle in the East Black Sea Region of Turkey. A total of 389 blood samples were collected from the animals of various ages in six provinces in the region. Prevalence of infection was determined by reverse line blot (RLB) assay. The hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene was amplified with a set of primers for members of the genera Theileria and Babesia. Amplified PCR products were hybridized onto a membrane to which generic- and species-specific oligonucleotide probes were covalently linked. RLB hybridization identified infection in 16.19% of the samples. Blood smears were also examined microscopically for Theileria and/or Babesia spp. and 5.14% were positive. All samples shown to be positive by microscopy also tested positive with RLB assay. Two Theileria (T. annulata and T. buffeli/orientalis) and three Babesia (B. bigemina, B. major and Babesia sp.) species or genotypes were identified in the region. Babesia sp. genotype shared 99% similarity with the previously reported sequences of Babesia sp. Kashi 1, Babesia sp. Kashi 2 and Babesia sp. Kayseri 1. The most frequently found species was T. buffeli/orientalis, present in 11.56% of the samples. T. annulata was identified in five samples (1.28%). Babesia infections were less frequently detected: B. bigemina was found in three samples (0.77%), B. major in two samples (0.51%) and Babesia sp. in five samples (1.28%). A single animal infected with T. buffeli/orientalis was also infected with B. bigemina. # 2008 Elsevier B.V. All rights reserved.

Keywords: Theileria; Babesia; PCR; Reverse line blot; Cattle; Turkey

1. Introduction orientalis causes mild or asymptomatic disease in cattle. Bovine babesiosis is caused by Babesia Theileria and Babesia species are tick-borne bigemina, Babesia bovis, Babesia divergens and haemoprotozoan parasites of vertebrates that have a Babesia major. Babesia species have the potential for major impact on livestock production, mainly cattle and wide distribution wherever their tick vectors are small ruminants, in tropical and subtropical areas encountered. Two species, B. bovis and B. bigemina, (Mehlhorn and Schein, 1984). Theileria annulata and have a considerable impact on cattle health and Theileria parva cause lympho-proliferative disease with productivity in tropical and subtropical countries high morbidity and mortality, whereas Theileria buffeli/ (Uilenberg, 1995). Techniques for detection of these haemoparasites have been developed separately for use in each species. § Nucleotide sequence data reported in this paper are available in The traditional method of identifying the agents in GenBank, EMBL and DDBJ databases under accession numbers from infected animals is by microscopic examination of blood EU622821 to EU622825. * Corresponding author. Tel.: +90 424 237 0000; smears stained with Giemsa. This technique is usually fax: +90 424 238 8173. adequate for detection of acute infections, but not for E-mail address: maktas@ﬁrat.edu.tr (M. Aktas). detection of carrier animals, where parasitaemias may be

0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.09.025 296 K. Altay et al. / Veterinary Parasitology 158 (2008) 295–301 low (Friedhoff and Bose, 1994). Serological methods are was conducted in cattle in the East Black Sea Region of employed in diagnosing subclinical infections in Turkey. The results of RLB were compared to those of epidemiological studies, but false-positive and false- examination of thin blood smears. negative results, due to cross-reactions or weakening of specific immune responses, are common (Passos et al., 2. Materials and methods 1998). Therefore, a sensitive and highly specific method for the diagnosis of piroplasms is required. Recently, 2.1. Study area and collection of samples species-specific polymerase chain reaction (PCR) and PCR-based reverse line blot (RLB) hybridization The study was conducted on cattle in the provinces methods have been developed for the detection and of Tokat, Amasya, Gumushane, Giresun, Trabzon and identification of Theileria and Babesia species (Figueroa Rize located in the East Black Sea Region of Turkey et al., 1992; Calder et al., 1996; Gubbels et al., 1999; (Fig. 1). This area has varying weather conditions. Georges et al., 2001; Aktas et al., 2005; Garcıá- Unlike the dry and hot East and Central Anatolia Sanmartıń et al., 2006; Altay et al., 2007a; M’ghirbi regions, the East Black Sea region covers two different et al., 2008). climatic zones: an Atlantic climate in the coastal region The main tick-borne haemoparasitic diseases occur- with frequent rainfall and mild temperatures. The main ring in cattle throughout Turkey are theileriosis and tick species here are Ixodes ricinus, Haemaphysalis babesiosis. Bovine theileriosis has been investigated punctata, Haemaphysalis sulcata, Dermacentor mar- using molecular techniques, and the presence of T. ginatus, Rhipicephalus bursa and a Continental annulata and T. buffeli/orientalis has been reported in Mediterranean climate in the interior regions, with the some part of the country (Aktas et al., 2002, 2006; warmer summers and colder winters. The main tick Dumanli et al., 2005). B. bovis, B. bigemina and B. species here are Hyalomma marginatum marginatum, divergens have been detected by microscopy and Boophilus annulatus, Rhipicephalus spp. serological tests (Aktas et al., 2001). However, these Blood samples from 389 cattle randomly selected methods are less sensitive and specific in the detection from 74 farms, also randomly selected, were taken in of carrier animals and do not generally distinguish EDTA containing tubes. A thin blood smear from each between current infections and previous exposures. sample was prepared and numbered in the field by the Identification of carrier animals is important for the same person. The blood samples were stored at 20 8C assessment of infection risk. They serve as reservoirs of until DNA extraction. The age of animals ranged infection for ticks and cause natural transmission of the between 1 and 7 years, and all were clinically healthy. disease (Calder et al., 1996). PCR-based techniques provide an alternative method for the direct detection of 2.2. Microscopic examination piroplasms in carrier animals. In the present study, a molecular survey of Theileria and Babesia species, In the laboratory, the blood smears were fixed in based on PCR amplification and RLB hybridization, methanol for 5 min and stained for 30 min in Giemsa

Fig. 1. Map of the Turkish provinces, showing the locations surveyed in the current study. K. Altay et al. / Veterinary Parasitology 158 (2008) 295–301 297 stain diluted with 5% buffer. Slides were examined for agarose gel using a commercial kit (Wizard SV gel intra-erythrocytic forms of Theileria and Babesia spp. and PCR clean-up system, Promega, Madison, WI, piroplasms at 100 objective magnification. Approxi- USA). The purified PCR products were sequenced and mately 20 000 erythrocytes per slide were examined for submitted to GenBank. Each construct was sequenced the calculation of percentage of infected erythrocytes. at least three times and subjected to BLAST similarity The smears were recorded as negative for piroplasms if searches. A phylogenetic tree was created from the no parasites were detected in 50 oil-immersion fields. sequences of the 18S rRNA genes of cattle Babesia species identified in this study and those available from 2.3. DNA extraction GenBank, using the neighbour-joining method in MEGA version 3.1 (Kumar et al., 2004). The nucleotide DNA extraction was performed as described by sequences used in this study are available in GenBank d’Oliveira et al. (1995). Briefly, 125 ml of blood was under the following accession numbers: AY726556 for added to 250 ml of lysis solution (0.32 M sucrose, 0.01 M Babesia sp. Kashi 1; EF434786 for Babesia sp. Kayseri Tris, 0.005 M MgCl2, 1% Triton X-100, pH 7.5). The 1; AY726557 for Babesia sp. Kashi 2; AY596729 for B. mixture was centrifuged at 11 600 g for 1 min. The orientalis; AY603400, AY603401 and AY081192 for B. pellet was washed three times by centrifugation with ovata; DQ785311 and EF612434 for B. bigemina; 250 ml lysis buffer. The supernatants were discarded, and AY648886 and AY603339 for B. major; AY789076 and the final pellets were resuspended in 100 ml of PCR AY572456 for B. divergens; AF316893 for Plasmodium buffer (50 mM KCl, 10 mM Tris–HCl (pH 8), 0.1% vivax. Triton X-100, pH 8.3). Proteinase K (50 mg/ml) was added to the pellet suspension, and the mixture was 2.6. Statistical analysis incubated at 56 8C for 1 h. Finally, the samples were heated at 100 8C for 10 min. A x2-squared test was used to evaluate the differences among various parameters. P < 0.05 was 2.4. 18S rRNA gene amplification and RLB accepted to be statistically significant. hybridization 3. Results For the amplification of Theileria and Babesia species, one set of primers was used to amplify an 3.1. Specificity of the RLB assay approximately 390–430 bp fragment of the hypervariable V4 region of the 18S rRNA gene. The forward Primers RLB-F2 and RLB-R2 amplified bands of [RLB-F2 (50-GACACAGGGAGGTAGTGACAAG-30)] 390 and 430 bp corresponding to the hypervariable and the reverse [RLB-R2 (Biotin-50-CTAAGAATTT- V4 region of the 18S rRNA gene of Theileria and CACCTCTGACAGT-30)] primers were as described by Babesia species. PCR performed on uninfected control Georges et al. (2001). The PCR volume and reaction cattle DNA and a water control did not yield detectable conditions applied were similar to those described by product on agarose gel (data not shown). All PCR Altay et al. (2007a). The primers and oligonucleotide positive samples showed positive reactions with their probes (catchall Theileria/Babesia, Theileria spp., T. corresponding specific probes. However, some samples buffeli/orientalis, T. annulata, Babesia spp., B. bige- gave positive signals to catchall and Babesia genera- mina, B. bovis, B. divergens, B. major), containing an specific probes, but did not show any signal to the N-trifluoroacetamidohexyl-cyanoethyl, N,N-diisopro- species-specific probes tested (Fig. 2). This situation pyl phosphoramidite (TFA)-C6 aminolinker were indicated the presence of a novel Babesia genotype. synthesised by Isogen, Maarssen, Netherlands. Pre- paration, hybridization and stripping of RLB membrane 3.2. Sequencing and phylogenetic analysis were performed as described by Altay et al. (2007a). Two Theileria and three Babesia sequences were 2.5. Sequencing and phylogenetic analysis identified. The partial sequences of the 18S rRNA genes for T. buffeli/orientalis, B. bigemina, T. annulata, To confirm RLB results, representative PCR pro- Babesia sp. CS58 and B. major were deposited in the ducts were chosen randomly for sequencing. Generated EMBL/GenBank databases under accession numbers DNA fragment of approximately 390 and 430 bp of from EU622821 to EU622825, respectively. Theileria Theileria and Babesia were extracted from 1.5% sequences shared 99% identity with the recently 298 K. Altay et al. / Veterinary Parasitology 158 (2008) 295–301

Fig. 2. Reverse line blot assay of the PCR products generated by amplification of genomic DNA from cattle samples infected with Theileria and Babesia species, and from negative and positive samples as template. Oligonucleotide probes are shown in rows, and samples are applied in columns. Samples bearing identified single and mixed infections and negative and positive controls are showed as follows: lane 1, T. buffeli/orientalis; lane 2, T. annulata; lane 3, B. bigemina (field sample); lane 4, B. bovis (positive template); lane 5, B. divergens (positive template); lane 6, B. major (field sample); lane 7, Babesia sp. (field sample); lane 8, T. buffeli/orientalis and B. bigemina (field sample); lane 9, negative control (genomic DNA of uninfected cattle); lane 10, negative PCR control; lane 11, T. buffeli/orientalis 18S rRNA gene sequence; lane 12, T. annulata 18S rRNA gene sequence; lane 13, B. bigemina 18S rRNA gene sequence; lane 14, B. major 18S rRNA gene sequence; lane 15, Babesia sp. CS58 18S rRNA gene sequence. reported sequences for the 18S rRNA gene of T. buffeli/ examination. In the RLB and microscopy analysis, the orientalis (EU407247) and T. annulata (AY508473). highest number of positive samples and the highest From the three Babesia sequences, two of which were carriers of piroplasms were obtained from the province most closely related to the B. bigemina and B. major, of Tokat with 31.08% and 9.45%. and they shared 99% identity to recently reported Prevalence of each piroplasm species identified in sequences for B. bigemina (EF612434) and B. major cattle is shown in Table 2. Theileria spp. prevalence was (AY603339). The third sequence differed clearly from 12.85% (50/389), and prevalence of Babesia spp. was the all known Babesia species infective for cattle, but 2.57% (10/389). The most frequently found species was shared 99% similarity with the unnamed Babesia T. buffeli/orientalis, present in 11.56% of the samples. T. isolates (Babesia sp. Kashi 1, Babesia sp. Kashi 2 and annulata was identified in five samples (1.28%). Babesia sp. Kayseri 1) and B. orientalis. Babesia infections were less frequently detected: The constructed phylogenetic tree revealed that Babesia sp. were found in five samples (1.28%); B. Babesia species infective to cattle split into five bigemina in three samples (0.77%) and B. major in two monophyletic clades. Babesia sp. CS58 identified in samples (0.51%), one animal infected with T. buffeli/ this study demonstrated a close relationship and was orientalis was also infected with B. bigemina (Fig. 2, included in the clade with unnamed Babesia isolates lane 8). (Babesia sp. Kashi 1, Babesia sp. Kashi 2 and Babesia sp. Kayseri 1) and B. orientalis (Fig. 3). 4. Discussion

3.3. Prevalence of piroplasm infections in cattle Cattle with subclinical theileriosis and babesiosis become chronic carriers of the piroplasm and, hence, Thin blood smears revealed parasitaemia in infected sources of infection for tick vectors. Therefore, latent cattle ranging from 0.01% to 0.1%. Piroplasms, infections are important in the epidemiology of the detected inside erythrocytes, were pleomorph and ring- diseases. The diagnoses of piroplasm infections are or pear-shaped. Prevalence of piroplasms detected by based on clinical findings and microscopic examination microscopy and RLB from samples at locations in the of Giemsa-stained blood smears. However, this method Black Sea Region of Turkey are presented in Table 1.Of is not sensitive enough or sufficiently specific to detect the 389 blood samples examined, microscopy revealed chronic carriers, particularly when mixed infections 20 (5.14%) positive for piroplasms, whereas 63 occur. Molecular techniques enable sensitive and (16.19%) of DNA amplified products hybridized with specific detection of the parasites. The RLB method the probes for catchall, and genera- and species-specific is an effective and practical tool, since it is able to detect probes. These results demonstrated that RLB showed a extremely low parasitaemia levels and simultaneously significantly higher rate of detection of Theileria and identify Theileria and Babesia species using specific Babesia infections (P < 0.01) than did microscopic oligonucleotide probes (Gubbels et al., 1999; Altay K. Altay et al. / Veterinary Parasitology 158 (2008) 295–301 299

Fig. 3. Neighbour-joining analysis of the 18S rRNA gene of the bovine Theileria and Babesia identified in this study and those present in the GenBank database. Numbers above the branch demonstrate bootstrap support from 1000 replications. The tree was created using the MEGA 3.1 package. The origin (Country) and GenBank accession numbers are indicated in parentheses. Sequences described in this study are indicated in bold. Scale bar represents nucleotide substitutions per position. et al., 2007a). In this study, a molecular survey based on These results are not in agreement with previous studies PCR amplification and RLB hybridization was per- carried out in Eastern Turkey (Dumanli et al., 2005; formed for detection of bovine Theileria and Babesia Aktas et al., 2006). The lower prevalence of T. annulata species. By this method, 63 of 389 (16.19%) samples compared to T. buffeli/orientalis was related to the examined showed a positive signal to one or more geographic distribution of the tick vectors associated species-specific probes as well as to the corresponding genus-specific probes. It was reported that the Table 1 oligonucleotide probes used in this study reacted with Light-microscopy examination of thin blood smears and RLB results (Bovine Theileria and Babesia) by locations in the East Black Sea their corresponding species and did not cross-react, with Region of Turkey. the exception of the T. lestoquardi-specific probe which cross-reacted with T. annulata (Nagore et al., 2004; Province n Test Altay et al., 2007a). T. lestoquardi infects sheep and Light- RLB goats and has not been reported in Turkey (Altay et al., microscopy 2007a,b). +% +% The survey identified different two Theileria (T. Trabzon 56 4 7.14 4 7.14 annulata, T. buffeli/orientalis) and three Babesia (B. Giresun 57 3 5.26 10 17.54 bigemina, B. major, and a new Babesia genotype) Rize 60 0 0 0 0 species and genotypes infecting cattle. The survey Amasya 58 1 1.72 12 20.68 revealed that the most frequently found species was T. Gumushane 84 5 5.95 14 16.66 Tokat 74 7 9.45 23 31.08 buffeli/orientalis, present in 11.56% of the samples. We also found that T. annulata was present in the same area, Total 389 20 5.14 63 16.19 but the prevalence of this species was lower (1.28%). n, number of samples; +, positive samples. 300 K. Altay et al. / Veterinary Parasitology 158 (2008) 295–301

Table 2 Distribution and frequency of bovine Theileria and Babesia species detected by RLB (n = 389). Positive Parasite species Theileria spp. T. annulata T. buffeli Babesia spp. Babesia sp. B. bigemina B. major 5+ + 44 + + 1+ ++ + 3 + + 2 + + 5 ++ 63 (16.19%) 50 (12.85%) 5 (1.28%) 45 (11.56%) 10 (2.57%) 5 (1.28%) 3 (0.772%) 2 (0.51%) with each species. In the present study, Hyalomma sity. B. bovis and B. divergens were not detected in the anatolicum anatolicum, the main vector tick of T. cattle examined, although I. ricinus, B. annulatus and R. annulata in Turkey, was not found on cattle, whereas bursa, the vector ticks of these species, were identified Haemaphysalis spp. were the dominant tick species among the tick collected (unpublished data). The (unpublished data). absence of these species in RLB can possibly be The prevalence of Babesia infection was lower than explained by fluctuations in low level parasitaemia. that of Theileria infection. In a previous serological In conclusion, this study has revealed two Theileria study in the same region, serum antibodies against B. (T. annulata and T. buffeli/orientalis) and three Babesia bigemina, B. bovis and B. divergens were detected in (B. bigemina, B. major and Babesia sp.) infecting cattle. 62%, 44% and 75% of the samples, respectively (Dincer The RLB performed has revealed a novel bovine et al., 1991). In the present study, the lower prevalence Babesia genotype. The assay provided more accurate of Babesia species detected among carrier cattle as data on prevalence of infection and allowed direct compared to carriers of Theileria species could be identification of species. explained by the fluctuations in parasitaemia that occur in the chronic phase of infection by Babesia species Acknowledgement (Calder et al., 1996; Gubbels et al., 1999). This situation could also be explained by the low number of intra- This work was supported financially by a grant (106 erythrocytic piroplasms circulating in the bloodstream of Babesia carriers (Homer et al., 2000). O 416) from the Scientific and Technical Research By sequencing the 18S rRNA gene of the Babesia Council of Turkey (TUBITAK). isolates identified in this study, a phylogenetic tree was created (Fig. 2). It showed that the Babesia sp. CS58 References isolate was in the clade with the unidentified Babesia isolates from China (Kashi 1 and Kashi 2) and Turkey Aktas, M., Dumanlı, N., Karaer, Z., Cakmak, A., Sevgili, M., 2001. (Kayseri 1) as well as with B. orientalis. Sequence Elazig, Malatya ve Tunceli illerinde Babesia tu¨rlerinin seropre- comparisons (357 nucleotides) of Babesia sp. CS58 valansi. Turk. J. Vet. Anim. Sci. 25, 447–451 (in Turkish). Aktas, M., Dumanli, N., Ç etinkaya, B., Cakmak, A., 2002. Field revealed that the isolate differed clearly from all known evaluation of PCR in detecting Theileria annulata infection in Babesia species infective for cattle but shared 99% cattle in the east of Turkey. Vet. Rec. 150, 548–549. similarity with the Babesia sp. Kashi 1 and Kashi 2 Aktas, M., Altay, K., Dumanli, N., 2005. Development of a poly- isolated from H. anatolicum anatolicum (Luo et al., merase chain reaction method for diagnosis of Babesia ovis 2005) and with Babesia sp. Kayseri 1, isolated from H. infection in sheep and goats. Vet. Parasitol. 133, 277–281. Aktas, M., Altay, K., Dumanli, N., 2006. A molecular survey of bovine marginatum marginatum (Ica et al., 2007). These results Theileria parasites among apparently healthy cattle and with a demonstrated that B. bigemina, B. major, and unnamed note on the distribution of ticks in eastern Turkey. Vet. Parasitol. Babesia sp. were present in cattle in Turkey. The 138, 179–185. presence of B. bigemina was expected, since the Altay, K., Aktas, M., Dumanli, N., 2007a. Molecular identification, parasite has been reported previously (Dincer et al., genetic diversity and distribution of Theileria and Babesia species infecting small ruminants. Vet. Parasitol. 147, 161–165. 1991). However, B. major was detected for the first time Altay, K., Aktas, M., Dumanli, N., 2007b. Theileria infections in small in Turkey and therefore contributed greater insight into ruminants in the East and Souteast Anatolia. Turkiye Parasitol. bovine piroplasm distribution and phylogenetic diver- Derg. 31, 268–271. K. Altay et al. / Veterinary Parasitology 158 (2008) 295–301 301

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