Introduction

The tick-borne diseases of livestock constitute a complex of several diseases with different etiological agents, such as protozoa, rickettsia, bacteria and viruses. The only common feature between these diseases is that they can all be transmitted by ticks. Theileriosis and Babesiosis belong to this complex and cause diseases in the livestock with high morbidity and mortality thereby resulting in high economical losses worldwide (Barnett 1974a, 1974b; Mehlhorn and Schein 1984; Mehlhorn et al. 1994; Ahmed et al. 2002).

For the long time it was assumed that Theileria lestoquardi is the only pathogenic parasite in small ruminants (Luo and Yin 1997). Recently, however, a previously unidentified parasite has been described as a species of Theileria, which is pathogenic for small ruminants as well, causing fatal diseases of small ruminants so read over North China (Luo and Yin 1997; Schnittger et al. 2000b, 2000c; Bai et al. 2002).

Interestingly, reviews of tick-borne diseases have made people increasingly aware of this public health problem. Babesia Divergens, the main agent of the bovine babesiosis in Europe is not only a cause of significant loss to the cattle industry, but can also infect immunocompromised humans, causing medical emergencies characterized by rapid fulmination and parasitemia that may exceed 70% (Zintle et al. 2003). Recently, a Babesia-like organism (WA1) has been detected and characterised as morphologically identical to Babesia microti, but biologically and genetically distinct, and more closely related to a known canine pathogen (Babesia gibsoni) and to the Theileria species than to some members of the genus Babesia (Thomford et al. 1994; Persing et al. 1995).

The Giemsa stained of blood smear is the common method for the identification and characterisation of these piroplasms in Iran, which accompanied with some technical problems cause false morphological diagnosis, and in some cases needs special diagnostic knowledge. In certain cases, serological methods such as the immune fluorescence antibody test (IFAT) or immunoperoxidase test have also been applied (Jianxus and Hung 1997; Leemans et al. 1997; Shayan et al. 1999).

One problem discussed in protozoan infection is the determination and characterization of the transmitter agent. Since many analyses were performed with the salivary gland smear using Methyl-green-puronin-staining method, or Feulgen-staining method, the transfer vector remains unanswered in some cases. Uilenberg (1997) stated that T. lestoquardi transmit by Hyalomma anatolicum anatolicum, while some other investigators believe that it is transmitted by Repicephalus bursa or probably also by Repicephalus sanguinius (Dschunkowsky and Urodschevich 1924; Ramzi et al. 2003). Considering the complicated preparation of the samples and their transport to specialized laboratories, we describe an easy method for the preparation of samples involving minimal space, and not requiring special cold storage. Furthermore, we show that the same sample can be used first for Giemsa-staining and than as a source for the extraction of DNA for further genetical analysis. In addition, we show that this material is suitable for the simultaneous detection and differentiation of genera Theileria and Babesia.

Materials and methods

Materials

We obtained 30 peripheral blood samples from sheep with suspicion of theileriosis or babesiosis. Ten of them were prepared with EDTA and ten samples were fixed with ethanol (1 ml blood/3 ml absolute ethanol), and ten unstained or Giemsa stained blood smears. All tissues had been obtained with consent given according to institutional guidelines.

DNA extraction

DNA extraction using phenol/chloroform

In the case of more than 200 μl blood, erythrocytes were first lysed in 0.155 M NH4Cl, 0.01 M KHCO3 and 0.1 mM EDTA for 10 min washed twice with PBS at 1000 g and the pellet was resolved in 200 μl of 10 mM NaCl, 20 mM Tris–HCl pH 8.0 and 1 mM EDTA. Then 20 μl proteinase K (10 mg/ml) was added and the sample incubated for 10 min at 55°C to digest the proteins. After addition of equal volume of Tris–HCl pH 8.0 saturated phenol, samples were gently vortexed and centrifuged at 12,000 rpm for 15 min. Upper liquid phase was transferred to the clear tube. The last step was repeated once with phenol/chloroform/Isoamylalcohol (25/24/1) to eliminate proteins and once with chloroform/Isoamylalcohol (24/1) to remove rest phenol in the solution. Finally, 6 M Na-acetat (1/10 volume of the sample) and ethanol 96% (2.5 volume of the sample) were added and the samples were incubated for 20 min at −70°C or overnight at −20°C. DNA was than precipitated at 12,000 rpm and, after washing with 70% ethanol, the pellet was dissolved in TE-buffer (10 mM Tris–HCl, 0.1 mM EDTA pH 8.0).

DNA extraction using TriPure isolation reagent

DNA extraction was performed according to the manufacturer’s instruction (Roche, Germany). Briefly, 5–10 × 106 blood cells were homogenized with 1 ml TriPure Isolation reagent. After addition of 0.5 ml Isopropanol, the suspension was first incubated for 10 min at room temperature, then centrifuged for 10 min at 12,000 g at 2–8°C. A 300 μl 96% ethanol was added to the lower and intermediate phases. DNA was precipitated after incubation for 2–3 min at room temperature by centrifugation at 2000 g at 2–8°C. The pellet was then washed twice with 0.1 M sodium citrate in 10% ethanol and subsequently dissolved in TE-buffer.

DNA extraction using MBST-kit

In contrast to the above mentioned two methods, this method is based on the specific binding of the DNA to the carrier. Therefore, neither phenol/chloroform nor DNA precipitation was used. For > 200 μl blood, erythrocytes were first lysed in blood samples using Erys-Lysing-Buffer. DNA was extracted using a DNA isolation kit (MBST, Germany/Iran) according to the manufacturer’s instructions. Briefly, cells were first lysed in 180 μl lysis buffer and the proteins were degraded with 20 μl proteinase K for 10 min at 55°C. After addition of 360 μl Bindings buffer and incubation for 10 min at 70°C, 270 μl ethanol (100%) was added to the solution and after vortexing, the complete volume was transferred to the MBST-column. The MBST-column was first centrifuged, then washed twice with 500 μl washing-buffer. Finally, DNA was eluted from the carrier with Elution buffer.

DNA extraction from ethanol fixed blood

Ethanol fixed blood was first centrifuged for 20 min at 13,000 rpm at 8°C and then air dried by converting of tube. The pellet material was dissolved in lysis buffer (obtained from Phenol/chloroform method or from MBST kit) using proteinase K for various time intervals, until the solution was homogenized. Finally, DNA was extracted according to the protocol of phenol/chloroform method or the MBST manufacturer’s instructions.

DNA extraction from blood smears

Each blood smear was cleaned in separate vessels by a short passage in acetone and ethanol. Approximately, half the blood smear before and after Giemsa staining was dissolved in 100 μl saturated phenol or 100 μl TriPure reagent or 100 μl Lysis buffer of the MBST kit. DNA was extracted as described above and dissolved in 100 μl TE buffer.

Polymerase chain reaction and seminnested PCR

Approximately 100 to 500 ng DNA, or 5–10 μl DNA solution in the case of extraction from blood smear, was used for the PCR analysis. The PCR was performed on 100 μl total volume including one time PCR buffer, 2.5 U Taq Polymerase (Cina gene, Iran), 2 μl of each primer (20 mM, MWG, Germany), 200 μM of each dATP, dTTP, dCTP and dGTP (Fermenta) and 1.5 mM MgCl2 in automated Thermocycler (Eppendorf, Germany) with the following program: 5 min incubation at 95°C to denature double strand DNA, 35–38 cycles of 45 s at 54–58°C (annealing step), 45 s, at 72°C (extension step) and 45 s at 94°C (denaturing step). Finally, PCR was completed with the additional extention step for 10 min. The PCR products were analysed on 1.8% Agarose gel in 0.5 times TBE buffer and visualized using Ethidium bromide and UV-eluminator.

To control the specificity of the PCR products from the 18s rRNA, seminested PCR technique was used, in which the additional primer is designated within the hyper variable region of the V4 region of 18s rRNA gene. The primers are listed in the Table 1.

Table 1 The sequences for primers used in PCR from heat shock protein (hsp70), T. lestoquardi ms1-2 gene, Babesia rhoptry protein gene and hyper variable region V of 18S rRNA gene and primers for seminesdted PCR from Theileria spp. and Babesia spp.
Full size table

Seminested PCR was performed with the PCR product isolated from agarose gel using the MBST-Kit according to the manufacturer’s instructions. Briefly, the DNA bands were cut from the gel under UV control and dissolved in the binding buffer at 60°C. The dissolved agarose was transferred into the MBST-column. After washing, the bound DNA was eluted using 100 μl TE-buffer. A 1–5 μl of the eluted DNA was amplified with the primers P8/P9, P8/P10 and P8/P11 separately.

Results and discussion

The specific PCR products could be amplified corresponding by with the DNA extracted by the three methods.

DNA was extracted from different sources of blood samples from suspected sheep infected with Theileria or Babesia.using the phenol/chloroform method, Tripure reagent and MBST-Kit. DNA extraction using the Tripure reagent must be modified in some cases such as ethanol fixed blood samples due to the absence of some reagents (Lysis buffer and Proteinase K). While the cell count was more than 106, DNA could be extracted and analysed by PCR with all the methods used. DNA isolation from the cells under 106 was not effective with the phenol/chloroform method and Tripure reagent, but it was clearly detectable on agarose gel by MBST-Kit. The possible explanation for this is that DNA isolation by MBST kit requires neither the phenol and chloroform extraction step nor the ethanol precipitation step and is grounded on the selective binding of nucleic acids to a silica based membrane in the presence of chaotropic salts.

DNA was extracted from Giemsa stained and unstained blood smears from infected and uninfected animals. The extracted DNA was resolved in 100 μl TE buffer then 5 μl of isolated DNA was amplified either with the Primers derived from the heat shock protein of Theileria annulata (hsp70) or with T. lestoquardi ms1-2 gene or with Babesia rhoptry protein gene (Figs. 1, 2). As a positive control for PCR analysis, DNA from Theileria annulata, T. lestoquardi infected cell lines and from the erythrocytes of an experimental sheep infected with Babesia ovis was used. In some cases, for PCR analysis, the isolated DNA with the Phenol/Chloroform and Tripure methods must be extracted again with Chloroform/Isoamylalcohol (24/1) and/or precipitated with ethanol to avoid the phenol and/or high salt contamination.

Fig. 1
figure 1

DNA was extracted from T. lestoquardi infected blood smear and amplified in PCR using primer derived from heat shock protein 70 (hsp70) and T. lestoquardi ms1-2 gene (Insert upper right: the same blood smear after DNA isolation, Insert lower left: M 100 bp Marker, lane 1 positive control with heat shock protein specific primer, lane 2 negative control, lane 3 amplified DNA from blood smear with heat shock protein specific primer, lane 4 amplified DNA from the same blood smear with T. lestoquardi ms1-2 gene)

Full size image
Fig. 2
figure 2

Extracted DNA from B. ovis infected blood smear (right) and B. motasi left) analysed using primer drived from Rhoptry protein gene. Insert: M 100 bp marker, 1 positive control, 2 negative control, 3 amplified DNA from B. ovis infected blood smear with primers P5/P6, 4 amplified DNA from B. motasi infected blood smear with primers P5/P6

Full size image

The localization of heat shock protein 70 (hsp70) in the mitochondria of Theileia macroschizonts has been shown by Schnittger et al. (2000a). Amplification of mitochondrial hsp70 gene with the template DNA from infected blood smear revealed that not only the genomic DNA of parasites but also the DNA from other parasite organels could be detectable by MBST-Kit (Fig. 1). The DNA extracted from infected blood smear with T. lestoquardi, Theileria annulata, B. ovis and Babesia motasi could be amplified with the common primer pair derived from hyper variable region V4 of 18S rRNA (P7 and P8) (Table 1, Fig. 3). Interestingly, the Theilerial hypervariable region V4 of 18S rRNA consists of more nucleotides than in Babesia spp. (Schnittger et al. 2004). Therefore, the application of the mentioned primer pair (P7 and P8) could easily and simultaneously differentiate between Theileria spp. and Babesia spp. The PCR product of Theileria spp. and Babesia spp. is 426–430 and 389–402 bp, respectively. The difference of ca. 30 bp in the length of the PCR product is easily determinable in 1.8% agarose gel (Fig. 2). Additional primers (P9 for T. lestoquardi, P10 for B. ovis and P11 for B. motasi) (Schnittger et al. 2004) were designed from the variable region V4 as a sense primer to show that PCR products were species specific (Fig. 3). Seminested PCR of the PCR products has been done with primer P8 as an antisense primer and P9, P10 or P11 as a sense primer. The results showed that the PCR products with primers P7 and P8 were amplifiable with the above mentioned primers and produced the calculated DNA length derived from the data bank. Seminested PCR of the PCR products of B. ovis with the primers P8/P11 and B. motasi with the primers P8/P10 were amplifiable as well (Fig. 3). It is most probably due to the partial similarity between B. ovis and B. motasi hypervariable region V4 of 18S rRNA genes specially with the 3′-ends of the primers. Interestingly, additional repeatable smaller DNA band occurred by seminested PCR with the PCR product of B. motasi with the primers P8/P11, its source remaining unknown (Fig. 3).

Fig. 3
figure 3

Extracted DNA from infected blood smear with T. listoquardi, B. ovisand B. motasi analysed with the primers drived from hyper variable region V4 of 18S rRNA. a chematicaly demonstration of the partial gene from hyper variable region V4 of 18S rRNA and localization of the different primers for PCR and seminested PCR. b Amplification of DNA from blood smear infected with T. annulata (lane 1), T. lestoquardi (lane 2), B. ovis lane 3) and B. motasi lane 4) using primer P7/P8, M 100 bp marker. c Seminested PCR with the PCR products from B: Lane 1 T. lestoquardi PCR product amplified with primer P9/P8, lane 2 and 3 B. motasi PCR product amplified with primers P11/P8 and P10/P8, respectively, Lane 4 and 5 B. ovis PCR product amplified with primers P10/P8 and P11/P8, respectively

Full size image

Control and management of livestock health could be understood as the two sides of a gold coin for a successful and healthy economy in stock-farming. Here, the control of tick-borne diseases plays a prominent role. One of the most important diseases in small ruminant is the infection with protozoan parasites, Theileria and Babesia, which cause annually high economical losses in Iran. Furthermore, reviews of tick-borne diseases has been increasingly recognized worldwide as highlighting this public health problem. Babesia Divergens, the main agent of the bovine babesiosis in Europe is not only a cause of significant loss to the cattle industry, but can also infect immunocompromised humans, causing medical emergencies characterised by rapid fulmination and parasitemia that may exceed 70% (Zintle et al. 2003). B. ovis was also described as a pathogenic agent in humans (Rios et al. 2003). Taken together, these piroplasms are not only important in the animal health but also in public health. Taking a high number blood samples, cold storage and sending probes to the laboratory are time consuming costly and problematic.

Our results suggest that it is possible to develop new strategies on the basis of blood smear for the collection of samples and sending them to specialized laboratories without the problems of cooling and high transport cost. Furthermore, our results revealed that it is possible to extract the piroplasm’s DNA from the already stained blood sample and analyse the DNA using PCR. Therefore, in cases of doubt old registered blood smear can be reanalysed with the described method.

Schnittger et al. (2004) recently described a reverse line blotting method for the simultaneous detection and differentiation of Theileria and Babesia parasites infecting small ruminants. At present in Iran this method is unpracticable due to the high cost. But our results showed that a common primer derived from hyper variable region V4 of 18S rRNA can be used for simultaneous differentiation of Theileria from Babesia by PCR on the 1.8% agarose gel in the carrier animals.