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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 Theileria and Babesia species are tick-borne haemoprotozoan parasites of vertebrates that have a major impact on livestock production, mainly cattle and small ruminants, in tropical and subtropical areas (Mehlhorn and Schein, 1984). Theileria annulata and Theileria parva cause lympho-proliferative disease with high morbidity and mortality, whereas Theileria buffeli/

§

Nucleotide sequence data reported in this paper are available in GenBank, EMBL and DDBJ databases under accession numbers from EU622821 to EU622825. * Corresponding author. Tel.: +90 424 237 0000; fax: +90 424 238 8173. E-mail address: [email protected] (M. Aktas). 0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.09.025

orientalis causes mild or asymptomatic disease in cattle. Bovine babesiosis is caused by Babesia bigemina, Babesia bovis, Babesia divergens and Babesia major. Babesia species have the potential for wide distribution wherever their tick vectors are encountered. Two species, B. bovis and B. bigemina, have a considerable impact on cattle health and productivity in tropical and subtropical countries (Uilenberg, 1995). Techniques for detection of these haemoparasites have been developed separately for use in each species. The traditional method of identifying the agents in infected animals is by microscopic examination of blood smears stained with Giemsa. This technique is usually adequate for detection of acute infections, but not for detection of carrier animals, where parasitaemias may be

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low (Friedhoff and Bose, 1994). Serological methods are employed in diagnosing subclinical infections in epidemiological studies, but false-positive and falsenegative results, due to cross-reactions or weakening of specific immune responses, are common (Passos et al., 1998). Therefore, a sensitive and highly specific method for the diagnosis of piroplasms is required. Recently, species-specific polymerase chain reaction (PCR) and PCR-based reverse line blot (RLB) hybridization methods have been developed for the detection and identification of Theileria and Babesia species (Figueroa et al., 1992; Calder et al., 1996; Gubbels et al., 1999; Georges et al., 2001; Aktas et al., 2005; Garcı´aSanmartı´n et al., 2006; Altay et al., 2007a; M’ghirbi et al., 2008). The main tick-borne haemoparasitic diseases occurring in cattle throughout Turkey are theileriosis and babesiosis. Bovine theileriosis has been investigated using molecular techniques, and the presence of T. annulata and T. buffeli/orientalis has been reported in the some part of the country (Aktas et al., 2002, 2006; Dumanli et al., 2005). B. bovis, B. bigemina and B. divergens have been detected by microscopy and serological tests (Aktas et al., 2001). However, these methods are less sensitive and specific in the detection of carrier animals and do not generally distinguish between current infections and previous exposures. Identification of carrier animals is important for the assessment of infection risk. They serve as reservoirs of infection for ticks and cause natural transmission of the disease (Calder et al., 1996). PCR-based techniques provide an alternative method for the direct detection of piroplasms in carrier animals. In the present study, a molecular survey of Theileria and Babesia species, based on PCR amplification and RLB hybridization,

was conducted in cattle in the East Black Sea Region of Turkey. The results of RLB were compared to those of examination of thin blood smears. 2. Materials and methods 2.1. Study area and collection of samples The study was conducted on cattle in the provinces of Tokat, Amasya, Gumushane, Giresun, Trabzon and Rize located in the East Black Sea Region of Turkey (Fig. 1). This area has varying weather conditions. Unlike the dry and hot East and Central Anatolia regions, the East Black Sea region covers two different climatic zones: an Atlantic climate in the coastal region with frequent rainfall and mild temperatures. The main tick species here are Ixodes ricinus, Haemaphysalis punctata, Haemaphysalis sulcata, Dermacentor marginatus, Rhipicephalus bursa and a Continental Mediterranean climate in the interior regions, with warmer summers and colder winters. The main tick species here are Hyalomma marginatum marginatum, Boophilus annulatus, Rhipicephalus spp. Blood samples from 389 cattle randomly selected from 74 farms, also randomly selected, were taken in EDTA containing tubes. A thin blood smear from each sample was prepared and numbered in the field by the same person. The blood samples were stored at 20 8C until DNA extraction. The age of animals ranged between 1 and 7 years, and all were clinically healthy. 2.2. Microscopic examination In the laboratory, the blood smears were fixed in 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

stain diluted with 5% buffer. Slides were examined for intra-erythrocytic forms of Theileria and Babesia spp. piroplasms at 100 objective magnification. Approximately 20 000 erythrocytes per slide were examined for the calculation of percentage of infected erythrocytes. The smears were recorded as negative for piroplasms if no parasites were detected in 50 oil-immersion fields. 2.3. DNA extraction DNA extraction was performed as described by d’Oliveira et al. (1995). Briefly, 125 ml of blood was added to 250 ml of lysis solution (0.32 M sucrose, 0.01 M Tris, 0.005 M MgCl2, 1% Triton X-100, pH 7.5). The mixture was centrifuged at 11 600  g for 1 min. The pellet was washed three times by centrifugation with 250 ml lysis buffer. The supernatants were discarded, and the final pellets were resuspended in 100 ml of PCR buffer (50 mM KCl, 10 mM Tris–HCl (pH 8), 0.1% Triton X-100, pH 8.3). Proteinase K (50 mg/ml) was added to the pellet suspension, and the mixture was incubated at 56 8C for 1 h. Finally, the samples were heated at 100 8C for 10 min. 2.4. 18S rRNA gene amplification and RLB hybridization

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agarose gel using a commercial kit (Wizard SV gel and PCR clean-up system, Promega, Madison, WI, USA). The purified PCR products were sequenced and submitted to GenBank. Each construct was sequenced at least three times and subjected to BLAST similarity searches. A phylogenetic tree was created from the sequences of the 18S rRNA genes of cattle Babesia species identified in this study and those available from GenBank, using the neighbour-joining method in MEGA version 3.1 (Kumar et al., 2004). The nucleotide sequences used in this study are available in GenBank under the following accession numbers: AY726556 for Babesia sp. Kashi 1; EF434786 for Babesia sp. Kayseri 1; AY726557 for Babesia sp. Kashi 2; AY596729 for B. orientalis; AY603400, AY603401 and AY081192 for B. ovata; DQ785311 and EF612434 for B. bigemina; AY648886 and AY603339 for B. major; AY789076 and AY572456 for B. divergens; AF316893 for Plasmodium vivax. 2.6. Statistical analysis A x2-squared test was used to evaluate the differences among various parameters. P < 0.05 was accepted to be statistically significant. 3. Results

For the amplification of Theileria and Babesia species, one set of primers was used to amplify an approximately 390–430 bp fragment of the hypervariable V4 region of the 18S rRNA gene. The forward [RLB-F2 (50 -GACACAGGGAGGTAGTGACAAG-30 )] and the reverse [RLB-R2 (Biotin-50 -CTAAGAATTTCACCTCTGACAGT-30 )] primers were as described by Georges et al. (2001). The PCR volume and reaction conditions applied were similar to those described by Altay et al. (2007a). The primers and oligonucleotide probes (catchall Theileria/Babesia, Theileria spp., T. buffeli/orientalis, T. annulata, Babesia spp., B. bigemina, B. bovis, B. divergens, B. major), containing an N-trifluoroacetamidohexyl-cyanoethyl, N,N-diisopropyl phosphoramidite (TFA)-C6 aminolinker were synthesised by Isogen, Maarssen, Netherlands. Preparation, hybridization and stripping of RLB membrane were performed as described by Altay et al. (2007a). 2.5. Sequencing and phylogenetic analysis To confirm RLB results, representative PCR products were chosen randomly for sequencing. Generated DNA fragment of approximately 390 and 430 bp of Theileria and Babesia were extracted from 1.5%

3.1. Specificity of the RLB assay Primers RLB-F2 and RLB-R2 amplified bands of 390 and 430 bp corresponding to the hypervariable V4 region of the 18S rRNA gene of Theileria and Babesia species. PCR performed on uninfected control cattle DNA and a water control did not yield detectable product on agarose gel (data not shown). All PCR positive samples showed positive reactions with their corresponding specific probes. However, some samples gave positive signals to catchall and Babesia generaspecific probes, but did not show any signal to the species-specific probes tested (Fig. 2). This situation indicated the presence of a novel Babesia genotype. 3.2. Sequencing and phylogenetic analysis Two Theileria and three Babesia sequences were identified. The partial sequences of the 18S rRNA genes for T. buffeli/orientalis, B. bigemina, T. annulata, Babesia sp. CS58 and B. major were deposited in the EMBL/GenBank databases under accession numbers from EU622821 to EU622825, respectively. Theileria sequences shared 99% identity with the recently

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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/ orientalis (EU407247) and T. annulata (AY508473). From the three Babesia sequences, two of which were most closely related to the B. bigemina and B. major, and they shared 99% identity to recently reported sequences for B. bigemina (EF612434) and B. major (AY603339). The third sequence differed clearly from the all known Babesia species infective for cattle, but shared 99% similarity with the unnamed Babesia isolates (Babesia sp. Kashi 1, Babesia sp. Kashi 2 and Babesia sp. Kayseri 1) and B. orientalis. The constructed phylogenetic tree revealed that Babesia species infective to cattle split into five monophyletic clades. Babesia sp. CS58 identified in this study demonstrated a close relationship and was included in the clade with unnamed Babesia isolates (Babesia sp. Kashi 1, Babesia sp. Kashi 2 and Babesia sp. Kayseri 1) and B. orientalis (Fig. 3).

examination. In the RLB and microscopy analysis, the highest number of positive samples and the highest carriers of piroplasms were obtained from the province of Tokat with 31.08% and 9.45%. Prevalence of each piroplasm species identified in cattle is shown in Table 2. Theileria spp. prevalence was 12.85% (50/389), and prevalence of Babesia spp. was 2.57% (10/389). 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: Babesia sp. were found in five samples (1.28%); B. bigemina in three samples (0.77%) and B. major in two samples (0.51%), one animal infected with T. buffeli/ orientalis was also infected with B. bigemina (Fig. 2, lane 8).

3.3. Prevalence of piroplasm infections in cattle

Cattle with subclinical theileriosis and babesiosis become chronic carriers of the piroplasm and, hence, sources of infection for tick vectors. Therefore, latent infections are important in the epidemiology of the diseases. The diagnoses of piroplasm infections are based on clinical findings and microscopic examination of Giemsa-stained blood smears. However, this method is not sensitive enough or sufficiently specific to detect chronic carriers, particularly when mixed infections occur. Molecular techniques enable sensitive and specific detection of the parasites. The RLB method is an effective and practical tool, since it is able to detect extremely low parasitaemia levels and simultaneously identify Theileria and Babesia species using specific oligonucleotide probes (Gubbels et al., 1999; Altay

Thin blood smears revealed parasitaemia in infected cattle ranging from 0.01% to 0.1%. Piroplasms, detected inside erythrocytes, were pleomorph and ringor pear-shaped. Prevalence of piroplasms detected by microscopy and RLB from samples at locations in the Black Sea Region of Turkey are presented in Table 1. Of the 389 blood samples examined, microscopy revealed 20 (5.14%) positive for piroplasms, whereas 63 (16.19%) of DNA amplified products hybridized with the probes for catchall, and genera- and species-specific probes. These results demonstrated that RLB showed a significantly higher rate of detection of Theileria and Babesia infections (P < 0.01) than did microscopic

4. Discussion

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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 PCR amplification and RLB hybridization was performed for detection of bovine Theileria and Babesia species. By this method, 63 of 389 (16.19%) samples examined showed a positive signal to one or more species-specific probes as well as to the corresponding genus-specific probes. It was reported that the oligonucleotide probes used in this study reacted with their corresponding species and did not cross-react, with the exception of the T. lestoquardi-specific probe which cross-reacted with T. annulata (Nagore et al., 2004; Altay et al., 2007a). T. lestoquardi infects sheep and goats and has not been reported in Turkey (Altay et al., 2007a,b). The survey identified different two Theileria (T. annulata, T. buffeli/orientalis) and three Babesia (B. bigemina, B. major, and a new Babesia genotype) species and genotypes infecting cattle. The survey revealed that the most frequently found species was T. buffeli/orientalis, present in 11.56% of the samples. We also found that T. annulata was present in the same area, but the prevalence of this species was lower (1.28%).

These results are not in agreement with previous studies carried out in Eastern Turkey (Dumanli et al., 2005; Aktas et al., 2006). The lower prevalence of T. annulata compared to T. buffeli/orientalis was related to the geographic distribution of the tick vectors associated Table 1 Light-microscopy examination of thin blood smears and RLB results (Bovine Theileria and Babesia) by locations in the East Black Sea Region of Turkey. Province

Trabzon Giresun Rize Amasya Gumushane Tokat Total

n

Test Lightmicroscopy

RLB

+

%

+

%

56 57 60 58 84 74

4 3 0 1 5 7

7.14 5.26 0 1.72 5.95 9.45

4 10 0 12 14 23

7.14 17.54 0 20.68 16.66 31.08

389

20

5.14

63

16.19

n, number of samples; +, positive samples.

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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 anatolicum anatolicum, the main vector tick of T. annulata in Turkey, was not found on cattle, whereas Haemaphysalis spp. were the dominant tick species (unpublished data). The prevalence of Babesia infection was lower than that of Theileria infection. In a previous serological study in the same region, serum antibodies against B. bigemina, B. bovis and B. divergens were detected in 62%, 44% and 75% of the samples, respectively (Dincer et al., 1991). In the present study, the lower prevalence of Babesia species detected among carrier cattle as compared to carriers of Theileria species could be explained by the fluctuations in parasitaemia that occur in the chronic phase of infection by Babesia species (Calder et al., 1996; Gubbels et al., 1999). This situation could also be explained by the low number of intraerythrocytic piroplasms circulating in the bloodstream of Babesia carriers (Homer et al., 2000). By sequencing the 18S rRNA gene of the Babesia isolates identified in this study, a phylogenetic tree was created (Fig. 2). It showed that the Babesia sp. CS58 isolate was in the clade with the unidentified Babesia isolates from China (Kashi 1 and Kashi 2) and Turkey (Kayseri 1) as well as with B. orientalis. Sequence comparisons (357 nucleotides) of Babesia sp. CS58 revealed that the isolate differed clearly from all known Babesia species infective for cattle but shared 99% similarity with the Babesia sp. Kashi 1 and Kashi 2 isolated from H. anatolicum anatolicum (Luo et al., 2005) and with Babesia sp. Kayseri 1, isolated from H. marginatum marginatum (Ica et al., 2007). These results demonstrated that B. bigemina, B. major, and unnamed Babesia sp. were present in cattle in Turkey. The presence of B. bigemina was expected, since the parasite has been reported previously (Dincer et al., 1991). However, B. major was detected for the first time in Turkey and therefore contributed greater insight into bovine piroplasm distribution and phylogenetic diver-

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