ORIGINAL PAPER
Molecular identification of
Theileria and Babesia in ticks collected
from sheep and goats in the Black Sea region of Turkey
Mehmet Fatih Aydin&Munir Aktas&Nazir Dumanli
Received: 19 July 2014 / Accepted: 23 September 2014 / Published online: 28 September 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract A molecular survey was undertaken in the Black Sea region of Turkey to determine the presence of Theileria and Babesia species of medical and veterinary importance. The ticks were removed from sheep and goats, pooled accord-ing to species and locations, and analyzed by PCR-based reverse line blot (RLB) and sequencing. A total of 2241 ixodid ticks belonging to 5 genus and 12 species were collected and divided into 310 pools. Infection rates were calculated as the maximum likelihood estimation (MLE) with 95 % confidence intervals (CI). Of the 310 pools tested, 46 (14.83 %) were found to be infected with Theileria or Babesia species, and the overall MLE of the infection rate was calculated as 2.27 % (CI 1.67–2.99). The MLE of the infection rates were calculated as 0.691 % (CI 0.171–1.78) in Haemaphysalis parva, 1.47 % (CI 0.081–6.37) in Rhipicephalus sanguineus, 1.84 % (CI 0.101– 7.87) in Ixodes ricinus, 2.86 % (CI 1.68–4.48) in Rhipicephalus turanicus, 5.57 % (CI 0.941–16.3) in Hyalomma marginatum, and 6.2 % (CI 4.02–9.02) in Rhipicephalus bursa. Pathogens identified in ticks included Theileria ovis, Babesia ovis, Babesia bigemina, and Babesia microti. Most tick pools were infected with a single pathogen. However, five pools displayed mixed infections with T. ovis and B. ovis. This study provides the first molecular evidence for the presence of B. microti in ticks in Turkey.
Keywords Babesia . RLB . Theileria . Tick
Introduction
Ticks are important vectors of a wide variety of protozoa, bacteria, fungi, and viruses that infect domestic livestock and wild animals in most regions of the world, causing dis-eases of zoonotic and veterinary importance (de la Fuente
et al. 2008; Ozdarendeli et al. 2008, 2010). Hyalomma
marginatum, Hyalomma anatolicum, Ixodes ricinus, Dermacentor marginatus, Haemaphysalis sulcata, Rhipicephalus turanicus, Rhipicephalus bursa, and Rhipicephalus sanguineus are the main tick species occurring
in Turkey (Aktas et al.2006,2009,2012,2013; Aktas2014;
Altay et al.2008a; Aydin et al.2012).
Piroplasmosis caused by Theileria and Babesia species leads to clinical infections in domestic and wild animals as
well as in humans (Friedhoff 1997; Aktas et al. 2007;
Heidarpour Bami et al.2009; Iqbal et al.2011; Altay et al.
2012; Vannier and Krause2012).
Epidemiologic studies concerning tick-borne diseases pro-vide information about endemic instability of these diseases
(Gachohi et al. 2010; Ekici et al. 2012). Varied diagnostic
methods were used to detect Theileria and Babesia species in ticks. Molecular methods provide improved sensitivity and specification compared to serological and microscopy
tech-niques (Altay et al.2008c; Aydin et al.2013; Berggoetz et al.
2014). Conventional PCR methods or their modified versions,
such as nested PCR, were used to detect pathogens in ticks
(Aktas et al.2006; Altay et al.2008a; Razmi and Yaghfoori
2013) with high sensitivity and specification but hampered by
their limited ability in the detection of multiple agents. Although there have been a number of studies of the prevalence of tick-borne pathogens in tick vectors in Turkey
(Aktas et al. 2009, 2010, 2012, 2013; Aktas 2014), little
Nucleotide sequence data reported in this paper are available in GenBank, EMBL 21, and DDBJ databases under accession numbers from KF737169 to KF737171.
M. F. Aydin (*)
Research Laboratory, Higher Health School, University of Karamanoğlu Mehmetbey, 70100 Karaman, Turkey e-mail: veterinermfa@gmail.com
M. Aktas
:
N. DumanliDepartment of Parasitology, Faculty of Veterinary Medicine, University of Firat, 23119 Elazig, Turkey
information is available about the frequency of ixodid tick species and the prevalence of tick-borne diseases in most areas of the country.
This study aimed for the molecular detection and charac-terization of Theileria and Babesia species in ixodid ticks collected from sheep and goats in the Black Sea region of Turkey, which provides a new molecular parasitological data.
Materials and methods Tick samples
The material of this study was engorged ticks collected from small ruminants within 3 years in Bolu, Kastamonu, Çorum, Samsun, Tokat, Giresun, and Bayburt provinces of the Black Sea region of Turkey. Detailed information regarding the ticks used in this study was presented in the previous published
article (Aydin et al.2012).
Establishment of tick pools and DNA extraction
A total of 2241 ticks belonging to five genus and 12 species were analyzed for the presence of Theileria and Babesia species, which have veterinary and public importance. Ticks were pooled according to their species, province, host, sex, and blood sucking level. Ticks were disinfected in 70 % ethanol for 10 min, rinsed with sterilized distilled water for three times, and dried on a filter paper. Then ticks were transferred to a clean Eppendorf tube of 1.5 ml volume and
stored at−80 C to increase fragility at least overnight until
DNA extraction. For DNA extraction, frozen ticks were crushed using a disposable and sterile pestle in Eppendorf tubes of 1.5 ml volume by adding liquid nitrogen. DNA extraction was performed using a commercial DNA isolation kit (QIAamp DNA Mini Kit, 51306) following the manufac-turer’s instructions. Genomic DNAs were preserved at +4 C until use.
Polymerase chain reaction and agarose gel electrophoresis For the amplification of Theileria and Babesia species in ticks, a touchdown PCR was performed. For the amplification of an approximately 360–430-bp fragment of the hypervariable V4 region of the 18S rRNA gene, one set of primers [(RLB)-R2 (Biotin-5′-CTAAGAATTTCACCTCTGACAGT-3′) and
RLB-F2 (5′-GACACAGGGAGGTAGTGACAAG-3′)] was
used (Georges et al.2001).
The PCR was performed in a total reaction volume of 25μl
containing PCR buffer [750 mM Tris-HCl (pH 8.8), 200 mM
(NH4)2SO4, 0.1 % Tween 20], 5 mM MgCl2, 125 μM
deoxynucleotide triphosphates, 1.25 U Taq DNA polymerase,
primers (20 pmol/μl), and template DNA. Five microliters of
PCR product was visualized by UV transillumination in a 1.5 % agarose gel after electrophoresis and stained with ethidium bromide.
Reverse line blotting (RLB)
Probes of Theileria/Babesia catchall, Theileria spp.: T. ovis, T. separate, T. lestoquardi, T. uilenbergi, T. luwenshuni, Theileria sp. OT1, Theileria sp. OT3, Theileria sp. MK, T. annulata, T. buffeli/orientalis, T. mutans, T. taurotragi, T. velifera, T. equi; Babesia spp.: B. ovis, B. motasi, B. crassa, Babesia (Lintan) China, Babesia sp. Kashi, B. divergens, B. bovis, B. bigemina, B. odocoilei, B. major, B. canis rossi, B. canis vogeli, B. canis canis, B. gibsoni, B. microti, and B. caballi, were used with a range of 200–900 pmol/150 μl c o n c e n t r a t i o n a n d c o n t a i n N t e r m i n a l N --(trifluoracetamidohexyl-cyanoethyl,N,N-diisopropyl phosphoramidite [TFA])-C6 amino linker in the study. Bind-ing of probes on the Biodyne C membrane, reverse line blotting, and removing PCR products from the membrane were as previously described. We evaluated the black spots occurring in rows by ChemiDoc™ MP system (Bio-Rad, UK) having chemiluminescence detection feature (Aydin et al.
2013).
Sequencing analysis
The polymerase chain reaction products were purified from the agarose gel using a commercial PCR Clean up System (MinElute PCR Purification Kit, 28004) and directly se-quenced. Sequencing results were compared with the other
sequences available in NCBI database (http://www.ncbi.nlm.
nih.gov/nuccore) and submitted to GenBank. Calculation of infection rates in tick pools
The maximum likelihood estimation (MLE) with 95 % con-fidence intervals (CI) of infection rates of pooled samples per 100 ticks was calculated using MLE-IR software designed by
Dr. Weidong Gu (Gu et al.2003).
Results
Distribution of collected ticks and infection rates for Theileria
and Babesia species are presented in Table1. In this study, a
total of 2241 ticks, D. marginatus (n=367), Haemaphysalis concinna (n = 11), Haemaphysalis parva (n = 458), Haemaphysalis punctata (n = 55), H. sulcata (n = 26), Hyalomma excavatum (n=24), Hyalomma detritum (n=9), Hy. marginatum (n=38), I. ricinus (n=58), R. bursa (n= 468), R. sanguineus (n=77), and R. turanicus (n=650), were
analyzed using PCR and RLB in terms of the presence of Theileria and Babesia which have a potential to infect both animals and humans. Forty sixth of 310 pools were found to be positive in terms of Theileria and/or Babesia spp. with 2.27 % MLE (CI 1.67–2.99).
Infection rates of the examined tick pools varied among tick species and ranged from 0.691 % (CI 0.171–1.78) in H. parva to 6.2 % (CI 4.02–9.02) in R. bursa. No Theileria or Babesia species were detected in D. marginatus, H. concinna, Hy. detritum, Hy. excavatum, H. punctata, and H. sulcata.
T. ovis was found with the highest prevalence in 29/310 of pool at 1.38 % (CI 0.941–1.95) followed by B. ovis in 20/310 of pool at 0.921 % (CI 0.571–1.39), B. bigemina in 1/310 of pool at 0.041 % (CI 0.001–0.201), and B. microti in 1/310 of pool at 0.041 % (CI 0.001–0.201). Additionally, T. ovis + B. ovis were found together in 5/310 of pool at 0.221 % (CI 0.071–0.491). B. ovis was found in Hy. marginatum (2/14) at
5.57 % (CI 0.941–16.3), in H. parva (3/47) at 0.691 % (CI
0.171–1.78), in R. bursa (8/49) at 1.8 % (CI 0.821–3.33), and in R. turanicus (2/70) at 0.471 % (CI 0.111–1.22).
T. ovis was detected in 10/49 of R. bursa pools at 2.37 % (CI 1.19–4.13), in 1/6 of R. sanguineus pools at 1.47 % (CI 0.081–6.37), and in 13/70 of R. turanicus pools at 2.27 % (CI
1.25–3.72). Besides both R. bursa and R. turanicus were
found to be infected by B. ovis + T. ovis at 0.891 % (CI 0.271–2.05) and at 0.151 % (CI 0.001–0.681), respectively. B. bigemina was also detected in one R. bursa pool with the rate of 0.211 % (CI 0.011–0.951) and B. microti in a I. ricinus pool with the rate of 1.84 % (CI 0.101–7.87).
Discussion
Ticks and tick-borne diseases have a great importance throughout the world, and they affect animal and human health by sucking blood and also by transmitting protozoan, bacterial, rickettsial, spirochethal, and viral agents. Theilerosis, babesiosis, anaplasmosis, ehrlichiosis, and hepatozoonosis which are important tick-borne diseases infect a great variety of domestic and wild animals (Jongejan and
Uilenberg2004).
Several studies were conducted about tick-borne agents in ticks using different methods in some regions of Turkey
(Aktas et al.2004,2006; Tonbak et al.2006; Ica et al.2007;
Altay et al.2008a;2009; Aktas et al.2010,2012; Aktas2014).
However, our study is the first survey which investigates a large number of piroplasm species. We used probes for 30 different Theileria/Babesia species or genotypes having a potential to infect both animals and humans.
Among 12 tick species examined in terms of Theileria and Babesia species, six (R. bursa, R. turanicus, R. sanguineus, H. parva, Hy. marginatum, and I. ricinus) were found to be
Ta b le 1 Dis tributio n o f ixodid ticks collected fro m sheep and goats and infectio n rates for Th ei le ri a and Babesia spec ies T ick spe cie s N CT /N A T /N AP NP P M LE (%) 9 5 % CI B. ovis T . ovis T . ovis + B. ov is B. m icr oti B . b ig em ina R. bursa 51 1/468/49 23 6.2 4 .02 –9.02 8/49 (1.8 %) a CI 0.8 2 1– 3.33 10/49 (2 .37 % ) C I 1 .19 –4.13 4/49 (0.891 % ) C I 0.271 –2.05 – 1/49 (0.21 1 %) CI 0.01 1– 0 .951 R. turanicus 801/650/70 16 2.86 1.68 –4.48 2/70 (0.31 1 %) CI 0.051 –0.971 13/70 (2 .27 % ) C I 1 .25 –3.72 1/70 (0.151 % ) C I 0.001 –0.681 –– R. sanguineus 93/77/6 1 1.47 0.081 –6.37 – 1/6 (1.47 %) CI 0 .081 –6.37 –– – H. sul cat a 39/26/7 –– – – – – – – H. punctata 66/55/13 –– – – – – – – H. parva 632/458/47 3 0 .691 0.171 –1.78 3/47 (0.691 %) CI 0.171 –1.78 –– – – H. concinna 15/1 1 /4 –– – – – – – – Hy . m ar ginatum 62/38/14 2 5 .57 0 .941 –16.3 2 /14 (5.57 %) CI 0.941 –16.3 –– – – Hy . excavatum 33/24/7 –– – – – – – – H y. d et ritum 13/9/6 –– – – – – – – D. mar g inatus 463/367/73 –– – – – – – – I. ricinus 69/58/14 1 1 .84 0 .101 –7.87 –– – 1/14 (1.84 % ) CI 0.10 1– 7.87 – NC T number o f collected tick, NA T number o f analyzed tick, NA P number o f analyzed pool, NPP number o f p ositive pool, MLE ma ximu m like lih ood es ti mat ion, CI conf idenc e int erv als aT h er at e( … % ) shows the MLE result
infected by one or more species. Among the pathogens de-tected in ticks, T. ovis, B. ovis, B. bigemina, and B. microti were identified.
Since the Black Sea region of Turkey has suitable climate and geographical structure, there are a wide variety of tick species and tick-transmitted pathogens in the region (Aydin
et al.2012; Aktas et al.2010,2012; Altay et al.2008b). In a
previous study (Aydin et al.2013) carried out by our research
group, T. ovis, B. ovis, Theileria sp. OT3, and Theileria sp. MK were detected in sheep and goats in the same provinces of the region. Out of 1128 small ruminants, 327 of them were found to be infected with T. ovis, 23 of them with Theileria sp. OT3, 7 of them with Theileria sp. MK, and 5 of them with B. ovis. T. ovis was found with the highest prevalence (29/310 of tick pool at 1.38 %) with this study as in our previous study, and it was followed by B. ovis (20/310 of pool at 0.921 %). Additionally, T. ovis + B. ovis were found together in 5/310 of pool at 0.221 %. Theileria sp. OT3 and Theileria sp. MK were not detected in tick samples. It is thought that further studies are need to determine Theileria sp. OT3 and Theileria sp. MK in tick samples.
Although B. bovis, B. divergens, and Anaplasma phagocytophilum exist in Turkey, tick-borne infections affect-ing humans are not well documented, and there is a paucity of information about the presence of B. microti in the country. B. divergens and B. microti cause human babesiosis in Europe
and the USA, respectively (Vannier and Krause2012).
How-ever, seropositivity of B. microti was also detected in humans
in Europe (Foppa et al. 2002; Hunfeld et al. 2002). It is
transmitted by the genus Ixodes (Gray et al.2002). Here, we
report for the first time the molecular occurrence of B. microti in Turkey. The pathogen was expected to exist because it had been previously reported in squirrels and humans via
micro-scopic (Çiçek et al.2009) and serological methods (Poyraz
and Güneş 2010). Our study provides the first molecular
evidence of B. microti in I. ricinus in Turkey. It is suggested that the human population in the studied area may be at risk of B. microti infection and also further studies should be per-formed in humans and in ticks to investigate the pathogen.
B. ovis is potentially one of the most pathogenic Babesia species to infect small ruminant hosts. Ovine babesiosis caused by B. ovis is characterized by fever, anemia, icterus,
and hemoglobinuria (Friedhoff1997; Guan et al.2009). It has
been reported that the parasite is transmitted by R. bursa,
R. turanicus, and Hy. excavatum (Friedhoff 1997; Altay
et al.2008a). Although R. bursa and R. turanicus are the most
common species found in small ruminants in the region
(Aydin et al.2012), it has been reported that B. ovis is not
common in the region and it was found only in 5/1128 small
ruminants (Aydin et al.2013). Our results showed that 8 out of
49 pools (1.8 %, CI 0.821–3.33) derived from R. bursa, 2 out of 70 pools (0.311 %, CI 0.051–0.971) from R. turanicus, 3
out of 47 pools (0.691 %, CI 0.171–1.78) from H. parva, and
2 out of 14 pools (5.57 %, CI 0.941–16.3) from Hy. marginatum were infected with B. ovis. The finding that showed the detection of B. ovis in R. bursa is consistent with
the previous studies (Parviz et al.2008; Altay et al.2008a).
However, this is the first time that B. ovis was observed in R. turanicus, H. parva, and Hy. marginatum in Turkey.
B. bigemina has a considerable impact on cattle’s health in
tropical and subtropical areas (Uilenberg 1995; Altay et al.
2008b). It is transmitted by Rhipicephalus (Boophilus) microplus, Rhipicephalus (Boophilus) decoloratus, and
Rhipicephalus evertsi evertsi (de Vos and Potgieter 1994).
Several studies showed that B. bigemina and B. bovis are
transmitted by R. bursa (Ravindran et al. 2006; Tavassoli
et al. 2013). Our results showed that 1 out of 49 pools
(0.211 %, CI 0.011–0.951) consisting of R. bursa was infected by B. bigemina.
In conclusion, prevalent tick-borne pathogens of sheep and goats such as T. ovis and B. ovis were detected with high prevalence in ticks. B. ovis was mostly detected in R. bursa and R. turanicus, but rarely in H. parva and Hy. marginatum pools. T. ovis was found in R. bursa, R. turanicus, and R. sanguineus. In addition, an I. ricinus and a R. bursa pool were found to be positive in terms of the presence of B. microti and B. bigemina, respectively. It is thought that the data obtained through this study will be helpful to define risk zones and to create control methods for ticks and tick-borne dis-eases. We suggest that further studies with large number of ticks sampled around the country are needed to determine the prevalence of tick-borne pathogens having medical and vet-erinary importance.
Acknowledgments This work was supported financially by grants (109 O 766) from the Scientific and Technical Research Council of Turkey (TUBITAK) and (45-M-12) Commission of Scientific Research Projects, Karamanoğlu Mehmetbey University. We thank all veterinar-ians, technicveterinar-ians, and also animal breeders in the region for their kind help during sample collection. We are also grateful to Weidong Gu (Health Statistician, PhD, Centers for Disease Control and Prevention, Atlanta, GA, USA) for MLE-IR program used for estimating infection rates of pooled samples.
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