Introduction

Theileriosis and babesiosis caused by Theileria and Babesia species of the apicomplexa phylum, are transmitted by ixodid and argasid ticks and considered to be two of the most prevalent and economically important tick-borne haemoparasitic diseases (TBHDs) in mammalians [1,2,3,4]. Theileria and Babesia species are endemic in tropical and subtropical climatic regions along with the distribution of the vector tick species. Economic losses directly attributed to diseases caused by Theileria and Babesia species include mortality, production losses with the costs of veterinary diagnostic/treatment and tick control [5]. These losses are associated with a long-term carrier status that is being developed in survivor animals from the acute diseases [1, 2, 5, 6].

Several Theileria and Babesia species have been detected in cattle and small ruminants up to now. Theileria annulata, T. parva, T. mutans, T. sergenti/buffeli/orientalis, T. taurotragi, T. velifera, T. sinensis, B. bigemina, B. bovis, B. divergens, B. major, B. ovata, B. occultans, B. jakimovi and B. beliceri were observed in cattle [7,8,9]. In sheep and goats, the presence of T. ovis, T. lestoquardi (= T. hirci), T. recondita, T. separata, T. luwenshuni (= T. sp. China 1), T. uilenbergi (= T. sp. China 2), T. sp. OT1, T. sp. OT3, T. sp. MK, B. ovis, B. motasi, B. crassa, B. foliata, B. taylori, B. sp. Xinjiang and B. sp. BQ1 were shown [10,11,12,13,14]. Among Theileria and Babesia species observed in cattle and small ruminants; T. annulata, T. parva, T. lestoquardi, T. luwenshuni, T. uilenbergi, B. bovis, B. bigemina, B. divergens, B. ovis and B. motasi are considered to be pathogenic [1, 3, 15,16,17]. Although Theileria and Babesia species are considered to be host-specific parasites [1,2,3,4], some studies have reported that these parasites can be detected in other hosts apart from their known hosts [16, 18,19,20,21].

In endemic regions, conventional methods such as microscopical examination of Giemsa-stained blood smears are widely used to detect Theileria and Babesia species in animals with clinical outcome. However, following recovery from primary infection with Theileria and Babesia species, animals become persistent carriers of the parasites for an extended period of time [22, 23], which is characterised by the presence of very low numbers of parasites circulating within the blood. This situation makes it difficult and unreliable to obtain a positive diagnosis by examination of stained blood smears [24]. Moreover, it is extremely difficult to conclusively discriminate pathogenic species from non-pathogenic ones by morphology, especially when they simultaneously occur within the same host [6, 11, 25, 26]. Molecular based diagnosis techniques such as reverse line blot (RLB) hybridisation assay have been frequently used to detect and discriminate Theileria and Babesia species in ruminants especially over the last decade [11, 27,28,29,30]. Molecular methods provide much more sensitive and specific results in terms of detecting carrier animals compared to microscopic diagnosis [6, 11, 26, 31].

In the present study, the prevalence of Theileria and Babesia species in sheep, goat and cattle was determined using RLB hybridisation technique in Burdur province of Turkey. Additionally, possible cross infections between small ruminants and cattle that are using the same grazing area were also investigated.

Materials and Methods

Sample Collection and DNA Extraction

The present study was carried out in five districts of Burdur province (Fig. 1). Collection of 964 total blood samples were made from herds of cattle, sheep and goat grazing in the same or neighbouring pastures. The number of samples and sampled herds according to animal species and districts are given in Table 1. According to this, an average of 5–6 animals were sampled per herd. Blood samples were collected in EDTA blood collection tubes from vena jugularis or v. coccygea of randomly selected animals in herds. DNA was extracted via Promega Wizard genomic DNA extraction kit (Madison, WI, USA) following the manufactural protocol. Isolates of T. lestoquardi (Lahr) and B. crassa from Iran, isolates of T. uilenbergi (Longde), T. luwenshuni (Lintan) and B. motasi (Lintan) from China and isolates of T. ovis (Kayseri), T. sp. MK (Kayseri), B. ovis (Kayseri) and T. annulata (Aydin) from Turkey were used as positive control DNA samples.

Fig. 1
figure 1

Villages of Burdur province where the sampled herds located. Locations (villages) of the sampled herds are as follows; A1: Yesilbaskoy, A2: Kiprit, A3: Canakli, A4: Hisar, A5: Yumrutas, A6: Asagiyumrutas, A7: Camlidere, B1: Seydikoy, B2: Beskonak, B3: Cobanpinari, B4: Kizilli, B5: Kizilseki, B6: Karaot, B7: Bogazkoy, B8: Gundogdu, B9: Kuyubasi, B10: Urkutlu, B11: Yuregil, C1: Yakakoy, C2: Gokcebag, C3: Halicilar, C4: Cine, C5: Beskavak, C6: Aksu, C7: Kapakli, C8: Bozlar, C9: Karacaoren, C10: Callica, C11: Yassigume, C12: Basmakci, G1: Yamadi, G2: Uylupinar, G3: Hisarardi, G4: Kargali, G5: Karapinar, G6: Asmali, G7: Yesildere, G8: Camkoy, G9: Sorkun, Y1: Kayadibi, Y2: Caltepe, Y3: Bedirli, Y4: Bayirbasi, Y5: Baskuyu, Y6: Harmanli, Y7: Yarisli, Y8: Niyazlar, Y9: Horozkoy, Y10: Doganbaba, Y11: Yukarikirli, Y12: Guney

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Table 1 Number of collected samples and herds according to districts and animal species
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RLB Hybridization Assay for Detection of Theileria and Babesia Species

RLB hybridisation assay was used to detect the prevalence of Theileria and Babesia species in collected samples. Initially: 460–540 bp fragment of the V4 variable region of 18S small subunit ribosomal RNA gene of all Theileria and Babesia species was amplified by PCR, using genus specific primers of RLB-F (5'-GAC ACA GGG AGG TAG TGA CAA G -3') and RLB- R (5' biotin-CTA AGA ATT TCA CCT CTG ACA GT -3') [6]. Final volume of each PCR reaction mix were 50 μL and consisted of; 1 × PCR buffer, 1.5 mM MgCl2, 200 mM of each dNTP, 2.5 U of hotstart Taq polymerase (HOT FİREpol-Solis Biodyne), 25 pmol of each primer RLB-F and RLB-R (5′ biotin-labelled) and 2 μL of template DNA. Reaction conditions were as follows: an initial denaturation step at 94 °C for 10 min was followed by a touchdown programme including two cycles at each temperature, i.e. 20 s at 94 °C, 30 s at an annealing temperature of 67 °C, 30 s at 72 °C, with the annealing temperature being decreased from 67 to 57 °C in steps of 2 °C. Then, 40 cycles of 20 s at 94 °C, 30 s at 57 °C and 30 s at 72 °C were performed and followed by 72 °C for 10 min as a final extension.

Oligonucleotide probes used to detect all known Theileria/Babesia species both at species and genus level are listed in Table 2 and exemplar pictures of the RLB showing the reaction of the various positive controls with the probes can be seen in Supplementary Figs. 1 and 2. Probes were immobilised on a Biodyne-C nitrocellulose membrane (Pall Biosupport), via N-terminal N-trifluoracctamidohexyl-cyanocthyl, N, N-diisopropyl phosphoramidite [TFA]-C6 amino linker as described previously [11]. Then, 20 µl of biotine-labelled PCR products were diluted in 2 × SSPE/0.1% SDS solution to a total volume of 150 µl and screened by RLB hybridisation assay as previously described [11]. Each membrane was reused up to 12 times following post-hybridization and documentation of the membrane [11, 27, 29].

Table 2 Oligonucleotide probes, sequences, specificity and references
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Statistical Analysis

Statistical analysis was performed using Pearson chi-square test with help of MiniTab16 Statistics program. The Chi-square test was used to compare the positivity rates of the detected species and different districts. Observed differences were considered to be statistically significant when P < 0.05.

Results

The number of blood samples infected at least with one Theileria or Babesia species according to districts are given in Table 3. The overall positivity rate in sheep, goat and cattle samples were 80.6%, 16.33%, 7.18%, respectively. The positivity rate (266/330) of sheep was significantly higher (P < 0.05) compared to cattle and goat.

Table 3 RLB positivity rates of animals according to districts
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The overall single infections in sheep, goat and bovine samples were 46.66%, 16.33% and 6.88%, respectively. Overall mixed infection rates were 33.93%, 0% and 0.29% in sheep, goat and cattle, respectively. The positivity rates of single (154/330) and mixed (112/330) infections in sheep were significantly higher (P < 0.05) compared to cattle and goat (Table 4).

Table 4 The prevalence of overall Theileria and Babesia species and single and mixed infection status according to animals
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Differences in positivity rates of each host between districts were statistically significant (P < 0.05). The highest prevalence of overall infections in sheep was detected in Golhisar (96%) and Yesilova (96%), followed by Center (76.47%), Bucak (76%) and Aglasun (61.42%). Infection rates in goat samples were 58%, 16.66%, 8.23% and 6% in Golhisar, Yesilova, Aglasun, Bucak, respectively and the differences between districts were statistically significant (P < 0.05). All goat samples from Center were negative. The highest positivity rate in cattle samples was detected in Yesilova (36.66%), followed by Aglasun (2%) while none of cattle samples were positive in Bucak, Golhisar and Center (P < 0.05) (Table 3).

Regardless of single or mixed infection status, the overall prevalences of Theileria spp. and Babesia spp. were significantly higher (P < 0.05) in sheep than those in goat and cattle samples (Table 4).

The prevalence between single or mixed infections in overall sheep samples was statistically significant (P < 0.05), and the most prevalent was T. ovis single infection (46.36%), followed by T. ovis + Babesia spp. mixed infection (32.12%) as seen in Table 5. Single T. ovis infection was also significantly higher between districts (P < 0.05) with rates of 94.7%, 65.9% and 52% in Yesilova, Center and Bucak, respectively. According to districts, the prevalence of mixed infections with T. ovis + Babesia spp. in Golhisar, Aglasun, Bucak and Center was also significantly different (P < 0.05) with rates of 92%, 55.71%, 24% and 10.58%, respectively. Also regardless of being single or mixed infection, T. ovis and Babesia spp. were the most prevalent in sheep samples with a rate of 79.69% and 32.42%, respectively. The overall prevalence of T. ovis was 41.9% (264/630) in small ruminants. While B. crassa was detected in four sheep samples (1.12%) as mixed infections, B. ovis was not detected in any of the sheep samples. The prevalence of unidentified Babesia positive sheep samples (i.e. positive at Babesia spp. or Bm3, but not bind any species-specific probe) was 33.03% (109/330).

Table 5 RLB detections of Theileria/Babesia species in sheep samples according to districts
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Bm3 (comprising B. motasi, B. sp. China and B. crassa) (11%) and Babesia spp. (3.33%) were found to be most prevalent (P < 0.05) in goats compared to other detected parasites (Table 6). The prevalence of Bm3 was significantly different (P < 0.05) among districts and was most commonly detected in Golhisar with a rate of 50%. Unclassified Theileria/Babesia were detected in three goat samples collected from Golhisar and Yesilova. The prevalence of B. ovis was 0.66% in goat and was detected in only two samples from Aglasun and Golhisar. The prevalence of unidentified Babesia or Theileria positive goat samples (i.e. positive at Babesia spp., Bm3 or T/B catch all, but not binding to any species-specific probe) was 15.33% (46/300).

Table 6 RLB detections of Theileria/Babesia species in goat samples according to districts
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A significantly higher (P < 0.05) number of positive samples was detected in cattle from Yesilova (36.66%) compared to Aglasun district (2%), while cattle samples collected from Bucak, Golhisar and Center districts were negative. The most prevalent Babesia species in Yesilova were B. bovis (18.33%) and Babesia spp. (18.33%) regardless of single or mixed infection status. Two cattle from Aglasun and Yesilova were infected with T. annulata as single infection and T. annulata + Babesia spp. as mixed infection, respectively (Table 7). The overall prevalence of T. annulata was 0.59%. The prevalence of unidentified Babesia positive bovine samples (i.e. positive at Babesia spp., but not binding to any species-specific probe) was 3.59% (12/334).

Table 7 RLB detections of Theileria/Babesia species in bovine samples according to districts
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According to single or multiple infection status, of the 330 sheep sampled from five districts; 154 (46.66%), 110 (33.33%) and two (0.6%) sheep were found to be infected with single, two and three species, respectively. Of 300 goats, 49 (16.33%) were infected with single species, while no mixed infections were detected. Twenty-three of 334 (6.88%) cattle were infected with one species and only one (0.29%) was infected with two species. No cross-infection was observed between small ruminants and cattle.

Discussion

Theileriosis and babesiosis are considered among the most important diseases responsible for major health problems and economic losses for small ruminant and cattle production, especially in countries located in tropic and subtropic climates [1, 3, 5, 32]. There are several Theileria and Babesia species including highly pathogenic species detected in sheep, goat and cattle causing theileriosis and babesiosis [1, 3, 16]. Conventional methods such as microscopic examination can be used to diagnose acute clinical forms of these diseases, however, these are inadequate to detect persistent carrier animals and differentiate species in mixed infections [16, 23, 33]. In contrast, RLB hybridisation assay, also used in the present study, is a molecular based method, and allows more specific, sensitive and simultaneous detection of small ruminant and bovine Theileria and Babesia species and possible cross-infections [6, 11, 33].

Theileria ovis, considered as non-pathogenic [12, 32], was the only Theileria species detected in small ruminants in Burdur province with an overall prevalence of 41.9%. A significant difference (P < 0.0001) was observed among sheep and goats for the presence of T. ovis and the prevalence rates were 79.69% and 0.3% in sheep and goats, respectively. Similarly, in studies conducted in the Eastern Black Sea Region [33] and in Nigde province [34] of Turkey, significant differences were observed between sheep and goats for prevalence of Theileria infections. In Sivas [17], and Kayseri [28, 35] significant differences were also observed in the prevalence of T. ovis among sheep and goats. Factors including seasonal activity of vector tick species and their infection rates together with, infestation rates of animals, different grazing behaviours of sheep and goat and parasite epidemiology were stated to be related with the observed differences among sheep and goats [28, 35].

In the present study, samples were collected from sheep and goat herds grazing in the same or neighbouring areas at similar days. This increased the possibility of being exposed to same tick species; however, the possible differences in infection and/or infestation rates among tick and hosts should not be ignored. Another factor effecting the infection rate is the difference in the immunological response among sheep and goats that needs to be further investigated. Theileria species considered as pathogenic were not detected in sheep and goats in this study. This indicated the presence of asymptomatic but carrier animals with very low parasitaemia of non-pathogenic Theileria species among sampled sheep.

Although T. ovis was the only Theileria species detected in small ruminants in the present study, the presence of pathogenic species T. uilenbergi and T. luwenshuni have been revealed in Burdur region in a previous study [29]. According to that; T. ovis (62.77%) and T. sp. OT1 (2.91%) were found by RLB and T. ovis (70.8%), T. uilenbergi (8.75%), T. luwenshuni (2.18%) and T. sp. MK (0.72%) were detected by species-specific PCR in sheep samples from Burdur province [29]. Therefore, local and/or general control measures such as controlled herd movements and anti-tick applications are still needed for effective prevention of epidemics.

The presence of ovine babesiosis in small ruminants caused by B. ovis (0.4–5.43%) [28, 29, 35, 36], B. motasi (0.1%) [29], B. crassa (4%) [29] and Babesia spp. (5.4%) [29] have been reported with similar prevalence rates in Turkey, also B. crassa and Babesia spp. were found in rates of 3.64% and 5.84% respectively in Burdur province [29]. In this study, B. ovis was only detected in two goat samples with a prevalence of 0.66% and no B. ovis was detected in sheep. Besides, B. crassa was detected as a part of mixed infection in sheep with a rate of 1.12% (4/330) but not in goat. The most prevalent Babesia infections in small ruminants were Babesia spp. (18.57%) and Bm3 group (5.87%) comprising B. motasi, B. sp. China and B. crassa. The significantly high prevalence of unclassified Babesia spp., detected in the present study, needs further investigation using species-specific PCR, sequence and phylogenetic analysis and other techniques to reveal the small ruminant babesiosis status in this region.

This study revealed the presence of T. annulata in cattle populations in Burdur province with a low prevalence of 0.59%, while T. buffeli/orientalis were not detected in any samples. The presence of T. annulata, causative agent of tropical theileriosis, has been reported in bovine samples in Turkey with a range of 1.28–18.1% [37,38,39]. The data obtained in previous studies indicated that the prevalence of T. annulata is closely related with the endemic status of the sampling region. Regions with higher prevalence show signs of being endemic or having an endemic stability for tropical theileriosis, while some regions having lower prevalence seems to be sporadic for T. annulata. The lower prevalence of T. annulata detected in the present study clearly indicated that tropical theileriosis is not a major problem for farmers in Burdur region. These results resembled a sporadic situation rather than an endemic stability in regions where samples were collected for tropical theileriosis. This is supported by Yukarı and Umur [40], where the prevalence of vector tick species (Hyalomma marginatum) of tropical theileriosis was shown to be 0.8% in Burdur. However, it should be noted that uncontrolled cattle movement and neglected tick control programmes may potentially cause outbreaks especially in regions where host and vector ticks species overlap.

So far, the most important causative agents of bovine babesiosis B. bovis, B. bigemina, B. divergens and less pathogenic species B. major and B. occultans were detected in Turkey [15, 41]. In the present study, only B. bovis and Babesia spp. were detected in cattle with prevalence rates of 3.29% and 3.59% respectively. It should be noted that all B. bovis and all except one Babesia spp. positive samples were found in Yesilova, only one bovine sample from Aglasun was infected with Babesia spp. and all bovine samples from other districts were negative for Babesia species. Hence, prevalence of bovine babesiosis is significantly higher in Yesilova district with a rate of 36.66% (22/60). Animals that survive from acute bovine babesiosis develop persistent infection and are carriers of Babesia species, which causes more transmissions and persistence of parasites in endemic regions, where usually high prevalence of Babesia parasites could be detected but number of clinical bovine babesiosis is low because of endemic stability [41].

Another subject that needs to be discussed is possible cross-infections. In a study conducted in Vietnam [20]; B. bigemina was detected in a blood sample of a goat and this finding was also confirmed by sequence analysis. As a result of the AMA-1 (Apical Membrane Antigen) gene sequence analysis, it was interpreted that B. bigemina is in the same group with other isolates detected in cattle in Vietnam, but the genetic diversity in B. bigemina populations can be better determined with the identification of more genes and the development of markers [20]. In another similar study conducted in Egypt [16]; B. bovis and B. bigemina were detected in two sheep via species-specific PCR method. As a result of the sequence analysis; the genes encoding RAP-1 (Rhoptri Associated Protein) of B. bovis and AMA-1 protein of B. bigemina were preserved at high rates of 99.3–100% and 95.3–100%, respectively, in these isolates [16]. Elsify et al. [16] suggests that, although the pathobiological meaning behind the parasite's host change is unknown, it may be a survival strategy used by blood parasites. Furthermore, the unidentified isolates in the present study may have different genetic characteristics from the identified species, and may become more adaptive for the different hosts other than their own ones. Piroplasms can be transmitted to different host animal species other than their own ones due to the blood-sucking habits of vector ticks, hence it could be expected to detect DNA of piroplasms in blood samples of different hosts. Supporting Elsify et al. [16], we can consider that the piroplasm species may be adapting to a new similar or familiar hosts, however, phylogenetic analysis is particularly needed for evaluation and discussion.

In the present study, no cross-infection was observed between cattle, sheep and goat sharing the same grazing area. However, a cross infection among animal species may not be expected in herds where infection rates are at such low levels. On the other hand, samples positive at only genus level needs to be further identified. These isolates were attempted to be cloned for DNA sequence analysis for multiple times without success, so they are still unclassified (denoted) species of Theileria or Babesia.

More detailed studies covering investigation of tick species and their vector potential for this region are needed to improve our knowledge about prevalence, pathogenicity, host preference of these parasites and epidemiology and impacts of diseases to develop more effective control strategies.