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Development of a polymerase chain reaction method for diagnosis of Babesia ovis infection in sheep and goats
Veterinary Parasitology 133 (2005) 277–281 www.elsevier.com/locate/vetpar
Development of a polymerase chain reaction method for diagnosis of Babesia ovis infection in sheep and goats M. Aktas¸ *, K. Altay, N. Dumanlı Department of Parasitology, Faculty of Veterinary Medicine, University of Firat, 23119 Elazig, Turkey Received 3 May 2005; received in revised form 6 May 2005; accepted 27 May 2005
Abstract In this study, a pair of oligonucleotide primers were designed according to the nucleotide sequence of the small subunit ribosomal RNA (ssu rRNA) gene of Babesia ovis isolated from sheep in eastern Turkey. The primers were used to detect parasite DNA from blood samples of B. ovis-infected sheep and goats by polymerase chain reaction (PCR). A 549-bp DNA fragment was specifically amplified from blood samples from sheep and goats, naturally infected with B. ovis. No PCR products resulted from Babesia motasi, T. ovis, Theileria sp. OT1, Theileria sp. OT3, T. lestoquardi, B. canis, B. microti, T. annulata or normal sheep leucocytes DNA using these specific primers. B. ovis-infected erythrocytes with 1% parasitemia were subjected to 10-fold serial dilutions (from 101 to 109) using an uninfected sheep erythrocytes, and DNA was extracted from each diluted sample for testing the sensitivity of the PCR. The PCR was sensitive enough to detect parasite DNA from the dilution of 105 with 0.00001% parasitemia. This is more sensitive than examining 200 fields under light microscopy. In addition, 98 field samples collected from small ruminanats in eastern Turkey were tested for B. ovis infection. Four samples were positive Babesia spp. in blood smears, 21 samples were positive for B. ovis DNA by PCR. These results indicate that the PCR provides a useful diagnostic tool for the detection of B. ovis infection in sheep and goats. # 2005 Elsevier B.V. All rights reserved. Keywords: Babesia ovis; Sheep and Goats; PCR; Microscopy; Specific primers
1. Introduction Ovine babesiosis is the most important hemoparasitic tick-borne disease of small ruminants caused by Babesia ovis, Babesia motasi and Babesia crassa. These parasites are widespread in tropical and subtropical areas of the world (Uilenberg, 2001). * Corresponding author. Tel.: +90 424 237 0000; fax: +90 424 238 8173. E-mail address: [email protected] (M. Aktas¸).
B. ovis is highly pathogenic especially in sheep and causes severe infections which is charecterised by fever, anemia, icterus and hemoglobinuria. Mortality rates in susceptible hosts range from 30 to 50% in field infections. The pathogenecity of B. motasi is not high and appears to be moderately virulent. In contrast, B. crassa is considered as being non-pathogenic to small ruminants (Friedhoff, 1997; Hashemi-Fesharki, 1997). Microscopic examination of Giemsa stained blood smears remains the most appropriate for the diagnosis of acute babesiosis, but the low sensitivity of the
0304-4017/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2005.05.057
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method does not permit its use in epidemiological investigations (Almeria et al., 2001). Serological methods are frequently employed in determining subclinical infections in epidemiological studies. However, these methods lack specificity due to cross reactivity with other Babesia species (Passos et al., 1998). Furthermore, false positive and negative results are commonly observed in these tests. Therefore, development of a highly specific and sensitive method for the diagnosis of Babesia infections is required. Recently, molecular techniques have become the preferred methods for diagnosis of babesiosis and theileriosis, because these techniques are more sensitive and specific than other conventional methods (Nagore et al., 2004; Schnittger et al., 2004; Alhassan et al., 2005; Altay et al., 2005). In this study, the use of PCR for the specific detection of B. ovis in sheep and goats infected erythrocytes using oligonucleotides designed from the sequence of the small subunit ribosomal RNA (ssu rRNA) gene of B. ovis isolated from sheep in eastern Turkey is described.
2. Materials and methods Blood samples were obtained from two sheep with clinical babesiosis in Elazig province, Turkey. DNAs extracted from the samples were revealed to be Babesia ovis by ssu rRNA gene sequence analysis. The nucleotide sequences of ssu rRNA gene of B. ovis have been submitted to the GenBank data base under accession no. AY998123 and AY998124. In this study, DNAs from these samples were used as positive controls for B. ovis specific PCR. Plasmid DNAs of Babesia motasi, Theileria sp. OT1 and Theileria sp. OT3 were kindly provided by Dr. Ana Hurtado (Department of Animal Health, Instituto Vasco de Investigacion y Desarrollo Agrario (NEIKER) Berreaga 1, 48160 Derio, Bizkaia, Spain). Genomic DNA of T. lestoquardi was kindly provided by Prof. Dr. J.S. Ahmed (Department of Immunology and Cell biology, Research Center, Borstel, Germany) and Babesia canis and Babesia microti were kindly provided by Dr. Bernard Carcy (Faculte´ de Pharmacie, Laboratoire de Biologie Cellulaire et Mole´culaire, Universite Montpellier, France). T. annulata and T. ovis were isolated from a cow and sheep at our laboratory (Department of
Parasitology, Faculty of Veterinary Medicine, University of Firat, Elazig, Turkey). Venous blood sample, taken from a healthy sheep without contact with ticks, was used as the negative control. Extraction of DNA was performed according to the method described by Clausen et al. (1999) with minor modifications. Briefly, 125 ml of blood was added to 250 ml of lysis mixture (0.32 M sucrose, 0.01 M Tris, 0.005 M MgCl2, 1% Triton X-100, pH 7.5) and 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 then incubated at 56 8C for 1 h. Lastly, the sample was boiled at 95 8C for 10 min. Two oligonucleotide primer targets for the 549-bp fragment were designed based on the ssu rRNA gene sequence of B. ovis isolated from sheep in eastern Turkey (GenBank accession numbers AY998123 and AY998124). Forward strand primer Bbo-F 50 TGGGCAGGACCTTGGTTCTTCT-30 , and reverse strand primer Bbo-R 50 -CCGCGTAGCGCCGGCTAAATA-30 were used in the PCR amplification. The PCR was performed in a touchdown thermocycler in a total reaction volume of 50 ml containing 5 ml of 10X PCR buffer [750 mM Tris–HCl (pH 8.8), 200 mM (NH4)2SO4, 0.1% Tween 20], 2 mM MgCl2, 250 mM of each of the four deoxynucleotide triphosphates, 1.25 U Taq DNA polymerase (Fermentas) and 50 pmol of each primer and 5 ml of template DNA. The reaction mixture was overlaid with 100 ml mineral oil and amplification was carried out in a no hot-lid minicycler (MJ Research, US). The reaction was repeated for 35 cycles under the following conditions: 1 min at 94 8C, 1 min at 62 8C and 1 min at 72 8C. PCR products were visualized by UV transillumination in a 1.5% agarose gel following electrophoresis and staining with ethidium bromide. DNAs from Babesia motasi, T. ovis, Theileria sp. OT1, Theileria sp. OT3, T. lestoquardi, B. canis, B. microti, T. annulata, and blood from a normal sheep with no exposure to ticks were tested in the PCR along with B. ovis DNA using the procedure described above. Primers RLB-F and -R (50 -GAGGTAGTGACAAGAAATAACAATA-30 and 50 -TCTTCGATCCC-
M. Aktas¸ et al. / Veterinary Parasitology 133 (2005) 277–281
CTAACTTTC-30 , described as primers for specific amplification (460–520-bp) of Babesia and Theileria species 18S ssu rRNA genes (Gubbels et al., 1999), were used in the PCR assay as a positive quality control for the Babesia and Theileria species. To determine the detection limit of the PCR assay, B. ovis-infected erythrocytes with 1% parasitemia calculated according to Oura et al. (2004) were subjected to 10-fold serial dilutions (from 101 to 109) using an uninfected sheep erythrocytes, and DNA was extracted from each diluted sample and processed for PCR as described above. A field study was conducted in Elazig province of eastern Turkey, to assess the suitability of the PCR for use with field samples. Blood samples were collected in tubes with EDTA from 98 small ruminants (68 sheep, 30 goats). These samples were used to prepare blood smears for microscopic examination and to extract DNA for PCR analysis. The blood smears were prepared immediately after taking the blood samples. The blood smears were fixed with methanol for 5 min, stained with Giemsa at a dilution of 5% in buffer solution for 30 min, and then examined at 1000 magnification for the presence of Babesia piroplasms by microscopy. The blood smears were recorded as negative for Babesia spp., if no piroplasms were observed in 200 oil-immersion fields.
3. Results The specificity of the primer pair was tested with DNA from B. ovis, B. motasi, B. canis, B. microti, Theileria sp. OT1, Theileria sp. OT3, T. lestoquardi, T. ovis and T. annulata. The expected 549-bp fragment was generated from B. ovis DNA, but was not generated from the other Babesia and Theileria DNAs (Fig. 1B). The Babesia and Theileria-specific primers RLB-F/-R were used as positive control and amplified a 460–520-bp fragment from the Babesia and Theileria species examined (Fig. 1A). The results of the detection limit of the assay are shown in Fig. 2. The blood dilutions between 1 and 105% are clearly positive with a diminishing signal as the target DNA concentration decreases. The lowest concentration of piroplasm detected was 0.00001% parasitemia in PCR. The PCR results for B. ovis showed that one infected cell out of 107 sheep erythrocytes or that
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Fig. 1. Specificity of the PCR method. Ethidium bromide stained agarose gel electrophoresis of amplication products from Babesia and Theileria species using Babesia and Theileria-specific primers or B. ovis-specific primers. (A) PCR with Babesia- and Theileriaspecific primer pair RLB-F/-R. (B) PCR with B. ovis-specific primer pair Bbo-F and -R. Lane M, 100-bp ladder DNA marker; lane 1, uninfected sheep blood (negative control); lane 2, B. ovis; lane 3, B. motasi; lane 4, T. ovis; lane 5, Theileria sp. OT1; lane 6, Theileria sp. OT3; lane 7, T. lestoquardi; lane 8, B. canis; lane 9, B. microti; lane 10, T. annulata.
parasites in blood with an equivalent parasitemia of 0.00001% could be detected. Thin blood smear examination of 98 small ruminants (68 sheep, 30 goats) from eastern Turkey showed that four animals (three sheep, one goat) were positive for Babesia spp. piroplasms. Using PCR, 21 of 98 (21.42%) animals (19 sheep, two goats) were positive. All of the positive samples by thin blood smears were also determined to be positive by PCR, whereas no piroplasms were seen by light microscopy in 17 of the 21 PCR positive animals.
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Fig. 2. Sensitivity of the PCR method. Ethidium bromide stained agarose gel electrophoresis of amplication products from 10-fold serial diluted samples (from 101 to 109%). Lane M, 100-bp ladder DNA marker; lane 1, undiluted blood with 1% parasitemia; lane 2, dilution of 101 with 0.1% parasitemia; lane 3, dilution of 102 with 0.01% parasitemia; lane 4, dilution of 103 with 0.001% parasitemia; lane 5, dilution of 104 with 0.0001% parasitemia; lane 6, dilution of 105 with 0.00001% parasitemia; lane 7, dilution of 106 with 0.000001% parasitemia; lane 8, dilution of 107 with 0.0000001% parasitemia; lane 9, dilution of 108 with 0.00000001% parasitemia; lane 10, dilution of 109 with 0.000000001% parasitemia; lane 11, positive control; lane 12, uninfected sheep blood (negative control).
4. Discussion The diagnosis of piroplasm infections in vertebrate hosts has been mainly carried out by microscopic examination of blood smear. However, the method requires expertise because these parasites have similar morphological features and therefore may confuse when mixed infections occur. Serological tests have also been used, but there are some difficulties with specificity and sensitivity (Passos et al., 1998). An exact differentiation between these parasites is crucial to understand their epidemiology. The detection of Babesia infection in carrier animals by DNA amplification was a powerful tool for epidemiological investigation, since these animals represent an important source of alimentary infection of Ixodid ticks. It is more sensitive and specific than detection of parasite by conventional methods (d’Oliveira et al., 1995; Aktas et al., 2002). Amplification of parasite DNA using specific PCR has been applied to various Babesia and Theileria species (Kirvar et al., 1998, 2000; Schnittger et al., 2000; Fukumoto et al., 2001; Oliveira-Sequira et al., 2005; Alhassan et al., 2005; Altay et al., 2005), but PCR specific for B. ovis
has not been available until now. In this study, a highly specific and sensitive PCR assay was developed for the detection of B. ovis in sheep and goats using a pair of oligonucleotide primers which target the 549-bp fragment in the ssu rRNA gene of the parasite. The specificity of the primers used in this study for B. ovis was determined by successful amplification of a 549-bp product from positive control and collected field samples. In contrast, no PCR products were seen with B. motasi, B. canis, B. microti, Theileria sp. OT1, Theileria sp. OT3, T. lestoquardi, T. ovis and T. annulata DNAs. The successful amplification of only B. ovis products indicates that the designed primers are specific for B. ovis DNA. The Babesia and Theileriaspecific primers RLB-F/-R, described by Gubbels et al. (1999), generated the expected a 460–520-bp fragment of Babesia and Theileria species examined, including B. ovis, confirming the integrity of the DNAs used to validate the specificity of the assay. We found that the detection limit of the test was 0.00001% parasitemia. This is equal to a parasitemia at the level of 105% and is over 100 times greater than can be attained by microscopic examination of blood smears. These results demonsrate that PCR using B. ovis-specific primers is capable of detecting B. ovis parasites present at extremely low parasitemias in sheep and goats and can be applied to samples collected under field conditions. Similar sensitivities in Theileria and Babesia PCR assays have been reported by d’Oliveira et al. (1995) for T. annulata, Kirvar et al. (1998, 2000) for T. lestoquardi and T. annulata, Fukumoto et al. (2001) for B. gibsoni, Oliveira-Sequira et al. (2005) for B. bigemina and B. bovis, Alhassan et al. (2005) for Babesia caballi and Babesia equi, Altay et al. (2005) for T. ovis. The results obtained from field samples indicated that the frequency of B. ovis detected by DNA amplification method was significantly higher than detection by light microscopy, because the latter method does not detect positive animals in the early phase of infection and the long-term carrier status, when the parasitemia is very low. The result agrees with previous report about the B. bovis (Calder et al., 1996). In conclusion, the primer pair described here could be useful tools for clarifiying the epidemiology of ovine babesiosis caused by B. ovis and for exact discrimination between B ovis and B. motasi infections in sheep and goats.
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Acknowledgements The authors would like to thank all veterinarians and technicians for their kind help during sample collection for this study. For the gifts of genomic DNA from Jabbar Ahmed, Ana Hurtado and Bernard Carcy are gratefully acknowledged. This study was supported by a grant (104 O 393) from The Scientific and ´ TAK). ¨ BY Technical Research Council of Turkey (TU
References Aktas, M., Dumanli, N., Cetinkaya, B., Cakmak, A., 2002. Field evaluation of PCR in detecting Theileria annulata infection in cattle in the east of Turkey. Vet. Rec. 150, 548–549. Alhassan, A., Pumidonming, W., Okamura, M., Hirita, H., Battsetseg, B., Fujisaki, F., Yokoyama, N., Igarashi, I., 2005. Development of a single-round and multiplex PCR method for the simultaneous detection of Babesia caballi and Babesia equi in horse blood. Vet. Parasitol. 129, 43–49. Almeria, S., Castella`, J., Ferrer, D., Ortun˜o, A., Estrada-Pen˜a, A., Gutierrez, J.F., 2001. Bovine piroplasm in Minorca (Balaric Island Spain): a comparison of PCR-based and light microscopy detection. Vet. Parasitol. 99, 249–259. Altay, K., Dumanli, N., Holman, P.J., Aktas, M., 2005. Detection of Theileria ovis infected sheep by nested PCR. Vet. Parasitol. 127, 99–104. Calder, J.A.M., Reddy, G.R., Chieves, L., Courney, C.H., Littell, R., Livengood, J.R., Norval, R.A.I., Smith, G., Dame, J.B., 1996. Monitoring B. bovis in cattle using PCR-based test. J. Clin. Microbiol. 34, 2748–2755. Clausen, P.H., Wiemann, A., Patzelt, R., Kakaire, D., Po¨tzsch, C., Peregrine, A., Mehlitz, D., 1999. Use of a PCR assay for the specific and sensitive detection of Trypanosoma spp. in naturally infected dairy cattle in peri-urban Kampala, Uganda. Ann. NY Acad. Sci. 29, 21–31. d’Oliveira, C., Van der Weide, M., Habela, M.A., Jacquiet, P., Jongejan, F., 1995. Detection of Theileria annulata in blood samples of carrier cattle by PCR. J. Clin. Microbiol. 33, 2665– 2669. Friedhoff, K.T., 1997. Tick-borne diseases of sheep and goats caused by Babesia, Theileria or Anaplasma spp.. Parasitologia 39, 99– 109.
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Fukumoto, S., Xuenan, Xuan, Shigeno, S., Kimbita, E., Igarashi, I., Nagasawa, H., Fujisaki, K., Mikami, T., 2001. Development of a polymerase chain reaction method for diagnosis Babesia gibsoni infection in dog. J. Vet. Med. Sci. 63, 977–981. Gubbels, J.M., de Vos, A.P., Van der Weide, M., Viseras, J., Schouls, L.M., de Vries, E., Jongejan, F., 1999. Simultaneous detection of Bovine Theileria and Babesia species by reverse line blot hybridisation. J. Clin. Microbiol. 37, 1782–1789. Hashemi-Fesharki, R., 1997. Tick-borne diseases of sheep and goats and their related vectors in Iran. Parasitologia 39, 115–117. Kirvar, E., Ilhan, T., Katzer, F., Wilkie, G., Hooshmand-Rad, Brown, C.G.D., 1998. Detection of Theileria lestoquardi (hirci) in ticks, sheep, goats using polymerase chain reaction. Ann. NY Acad. Sci. 849, 52–62. Kirvar, E., Ilhan, T., Katzer, F., Wilkie, G., Hooshmand-Rad, P., Zweygarth, E., Gestenberg, C., Phipps, P., Brown, C.G.D., 2000. Detection of Theileria annulata in cattle and vector ticks by PCR using the Tams1 gene sequences. Parasitology 120, 245– 254. Nagore, D., Garcı´a-Sanmartı´n, J., Garcı´a-Pe´rez, A.L., Juste, R.A., Hurtado, A., 2004. Identification, genetic diversity and prevalence of Theileria and Babesia species in a sheep population from Nortern Spain. Int. J. Parasitol. 34, 1059–1067. Oliveira-Sequira, T.C.G., Oliveira, M.C.S., Araujo, J.P., Amarante, A.F.T., 2005. PCR-based detection of Babesia bovis and Babesia bigemina in their natural host Boophilus microplus and cattle. Int. J. Parasitol. 35, 105–111. Oura, C.A.L., Bishop, R.P., Wampande, E.M., Lubega, G.W., Tait, A., 2004. Application of a reverse line blot assay to the study of haemoparasites in cattle in Uganda. Int. J. Parasitol. 34, 603– 613. Passos, L.M.F., Bell-Sakyi, L., Brown, C.G.D., 1998. Immunochemical charecterization of in vitro culture-derived antigens of Babesia bovis and Babesia bigemina. Vet. Parasitol. 76, 239– 249. Schnittger, L., Yin, H., Jianxun, L., Lugwing, W., Shayan, P., Rahbari, S., Voss-Holtmann, A., Ahmed, J.S., 2000. Ribosomal small-subunit RNA gene-sequence analysis of Theileria lestoquardi and a Theileria species highly pathogenic for small ruminants in China. Parasitol. Res. 86, 352–358. Schnittger, L., Yin, H., Qi, B., Gubbels, M.J., Beyer, D., Nieman, S., Jongejan, F., Ahmed, J.S., 2004. Simultaneous detection and differentiation of Theileria and Babesia parasites infecting small ruminants by reverse line blotting. Parasitol. Res. 92, 189–196. Uilenberg, G., 2001. Babesiosis. In: Servise, M.W. (Ed.), Encyclopedia of arthropod-transmitted infection of man and domestic animals. CABI Publishing, Wallinford, pp. 53–60.
Development of a polymerase chain reaction method for diagnosis of Babesia ovis infection in sheep and goats M. Aktas¸ *, K. Altay, N. Dumanlı Department of Parasitology, Faculty of Veterinary Medicine, University of Firat, 23119 Elazig, Turkey Received 3 May 2005; received in revised form 6 May 2005; accepted 27 May 2005
Abstract In this study, a pair of oligonucleotide primers were designed according to the nucleotide sequence of the small subunit ribosomal RNA (ssu rRNA) gene of Babesia ovis isolated from sheep in eastern Turkey. The primers were used to detect parasite DNA from blood samples of B. ovis-infected sheep and goats by polymerase chain reaction (PCR). A 549-bp DNA fragment was specifically amplified from blood samples from sheep and goats, naturally infected with B. ovis. No PCR products resulted from Babesia motasi, T. ovis, Theileria sp. OT1, Theileria sp. OT3, T. lestoquardi, B. canis, B. microti, T. annulata or normal sheep leucocytes DNA using these specific primers. B. ovis-infected erythrocytes with 1% parasitemia were subjected to 10-fold serial dilutions (from 101 to 109) using an uninfected sheep erythrocytes, and DNA was extracted from each diluted sample for testing the sensitivity of the PCR. The PCR was sensitive enough to detect parasite DNA from the dilution of 105 with 0.00001% parasitemia. This is more sensitive than examining 200 fields under light microscopy. In addition, 98 field samples collected from small ruminanats in eastern Turkey were tested for B. ovis infection. Four samples were positive Babesia spp. in blood smears, 21 samples were positive for B. ovis DNA by PCR. These results indicate that the PCR provides a useful diagnostic tool for the detection of B. ovis infection in sheep and goats. # 2005 Elsevier B.V. All rights reserved. Keywords: Babesia ovis; Sheep and Goats; PCR; Microscopy; Specific primers
1. Introduction Ovine babesiosis is the most important hemoparasitic tick-borne disease of small ruminants caused by Babesia ovis, Babesia motasi and Babesia crassa. These parasites are widespread in tropical and subtropical areas of the world (Uilenberg, 2001). * Corresponding author. Tel.: +90 424 237 0000; fax: +90 424 238 8173. E-mail address: [email protected] (M. Aktas¸).
B. ovis is highly pathogenic especially in sheep and causes severe infections which is charecterised by fever, anemia, icterus and hemoglobinuria. Mortality rates in susceptible hosts range from 30 to 50% in field infections. The pathogenecity of B. motasi is not high and appears to be moderately virulent. In contrast, B. crassa is considered as being non-pathogenic to small ruminants (Friedhoff, 1997; Hashemi-Fesharki, 1997). Microscopic examination of Giemsa stained blood smears remains the most appropriate for the diagnosis of acute babesiosis, but the low sensitivity of the
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method does not permit its use in epidemiological investigations (Almeria et al., 2001). Serological methods are frequently employed in determining subclinical infections in epidemiological studies. However, these methods lack specificity due to cross reactivity with other Babesia species (Passos et al., 1998). Furthermore, false positive and negative results are commonly observed in these tests. Therefore, development of a highly specific and sensitive method for the diagnosis of Babesia infections is required. Recently, molecular techniques have become the preferred methods for diagnosis of babesiosis and theileriosis, because these techniques are more sensitive and specific than other conventional methods (Nagore et al., 2004; Schnittger et al., 2004; Alhassan et al., 2005; Altay et al., 2005). In this study, the use of PCR for the specific detection of B. ovis in sheep and goats infected erythrocytes using oligonucleotides designed from the sequence of the small subunit ribosomal RNA (ssu rRNA) gene of B. ovis isolated from sheep in eastern Turkey is described.
2. Materials and methods Blood samples were obtained from two sheep with clinical babesiosis in Elazig province, Turkey. DNAs extracted from the samples were revealed to be Babesia ovis by ssu rRNA gene sequence analysis. The nucleotide sequences of ssu rRNA gene of B. ovis have been submitted to the GenBank data base under accession no. AY998123 and AY998124. In this study, DNAs from these samples were used as positive controls for B. ovis specific PCR. Plasmid DNAs of Babesia motasi, Theileria sp. OT1 and Theileria sp. OT3 were kindly provided by Dr. Ana Hurtado (Department of Animal Health, Instituto Vasco de Investigacion y Desarrollo Agrario (NEIKER) Berreaga 1, 48160 Derio, Bizkaia, Spain). Genomic DNA of T. lestoquardi was kindly provided by Prof. Dr. J.S. Ahmed (Department of Immunology and Cell biology, Research Center, Borstel, Germany) and Babesia canis and Babesia microti were kindly provided by Dr. Bernard Carcy (Faculte´ de Pharmacie, Laboratoire de Biologie Cellulaire et Mole´culaire, Universite Montpellier, France). T. annulata and T. ovis were isolated from a cow and sheep at our laboratory (Department of
Parasitology, Faculty of Veterinary Medicine, University of Firat, Elazig, Turkey). Venous blood sample, taken from a healthy sheep without contact with ticks, was used as the negative control. Extraction of DNA was performed according to the method described by Clausen et al. (1999) with minor modifications. Briefly, 125 ml of blood was added to 250 ml of lysis mixture (0.32 M sucrose, 0.01 M Tris, 0.005 M MgCl2, 1% Triton X-100, pH 7.5) and 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 then incubated at 56 8C for 1 h. Lastly, the sample was boiled at 95 8C for 10 min. Two oligonucleotide primer targets for the 549-bp fragment were designed based on the ssu rRNA gene sequence of B. ovis isolated from sheep in eastern Turkey (GenBank accession numbers AY998123 and AY998124). Forward strand primer Bbo-F 50 TGGGCAGGACCTTGGTTCTTCT-30 , and reverse strand primer Bbo-R 50 -CCGCGTAGCGCCGGCTAAATA-30 were used in the PCR amplification. The PCR was performed in a touchdown thermocycler in a total reaction volume of 50 ml containing 5 ml of 10X PCR buffer [750 mM Tris–HCl (pH 8.8), 200 mM (NH4)2SO4, 0.1% Tween 20], 2 mM MgCl2, 250 mM of each of the four deoxynucleotide triphosphates, 1.25 U Taq DNA polymerase (Fermentas) and 50 pmol of each primer and 5 ml of template DNA. The reaction mixture was overlaid with 100 ml mineral oil and amplification was carried out in a no hot-lid minicycler (MJ Research, US). The reaction was repeated for 35 cycles under the following conditions: 1 min at 94 8C, 1 min at 62 8C and 1 min at 72 8C. PCR products were visualized by UV transillumination in a 1.5% agarose gel following electrophoresis and staining with ethidium bromide. DNAs from Babesia motasi, T. ovis, Theileria sp. OT1, Theileria sp. OT3, T. lestoquardi, B. canis, B. microti, T. annulata, and blood from a normal sheep with no exposure to ticks were tested in the PCR along with B. ovis DNA using the procedure described above. Primers RLB-F and -R (50 -GAGGTAGTGACAAGAAATAACAATA-30 and 50 -TCTTCGATCCC-
M. Aktas¸ et al. / Veterinary Parasitology 133 (2005) 277–281
CTAACTTTC-30 , described as primers for specific amplification (460–520-bp) of Babesia and Theileria species 18S ssu rRNA genes (Gubbels et al., 1999), were used in the PCR assay as a positive quality control for the Babesia and Theileria species. To determine the detection limit of the PCR assay, B. ovis-infected erythrocytes with 1% parasitemia calculated according to Oura et al. (2004) were subjected to 10-fold serial dilutions (from 101 to 109) using an uninfected sheep erythrocytes, and DNA was extracted from each diluted sample and processed for PCR as described above. A field study was conducted in Elazig province of eastern Turkey, to assess the suitability of the PCR for use with field samples. Blood samples were collected in tubes with EDTA from 98 small ruminants (68 sheep, 30 goats). These samples were used to prepare blood smears for microscopic examination and to extract DNA for PCR analysis. The blood smears were prepared immediately after taking the blood samples. The blood smears were fixed with methanol for 5 min, stained with Giemsa at a dilution of 5% in buffer solution for 30 min, and then examined at 1000 magnification for the presence of Babesia piroplasms by microscopy. The blood smears were recorded as negative for Babesia spp., if no piroplasms were observed in 200 oil-immersion fields.
3. Results The specificity of the primer pair was tested with DNA from B. ovis, B. motasi, B. canis, B. microti, Theileria sp. OT1, Theileria sp. OT3, T. lestoquardi, T. ovis and T. annulata. The expected 549-bp fragment was generated from B. ovis DNA, but was not generated from the other Babesia and Theileria DNAs (Fig. 1B). The Babesia and Theileria-specific primers RLB-F/-R were used as positive control and amplified a 460–520-bp fragment from the Babesia and Theileria species examined (Fig. 1A). The results of the detection limit of the assay are shown in Fig. 2. The blood dilutions between 1 and 105% are clearly positive with a diminishing signal as the target DNA concentration decreases. The lowest concentration of piroplasm detected was 0.00001% parasitemia in PCR. The PCR results for B. ovis showed that one infected cell out of 107 sheep erythrocytes or that
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Fig. 1. Specificity of the PCR method. Ethidium bromide stained agarose gel electrophoresis of amplication products from Babesia and Theileria species using Babesia and Theileria-specific primers or B. ovis-specific primers. (A) PCR with Babesia- and Theileriaspecific primer pair RLB-F/-R. (B) PCR with B. ovis-specific primer pair Bbo-F and -R. Lane M, 100-bp ladder DNA marker; lane 1, uninfected sheep blood (negative control); lane 2, B. ovis; lane 3, B. motasi; lane 4, T. ovis; lane 5, Theileria sp. OT1; lane 6, Theileria sp. OT3; lane 7, T. lestoquardi; lane 8, B. canis; lane 9, B. microti; lane 10, T. annulata.
parasites in blood with an equivalent parasitemia of 0.00001% could be detected. Thin blood smear examination of 98 small ruminants (68 sheep, 30 goats) from eastern Turkey showed that four animals (three sheep, one goat) were positive for Babesia spp. piroplasms. Using PCR, 21 of 98 (21.42%) animals (19 sheep, two goats) were positive. All of the positive samples by thin blood smears were also determined to be positive by PCR, whereas no piroplasms were seen by light microscopy in 17 of the 21 PCR positive animals.
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Fig. 2. Sensitivity of the PCR method. Ethidium bromide stained agarose gel electrophoresis of amplication products from 10-fold serial diluted samples (from 101 to 109%). Lane M, 100-bp ladder DNA marker; lane 1, undiluted blood with 1% parasitemia; lane 2, dilution of 101 with 0.1% parasitemia; lane 3, dilution of 102 with 0.01% parasitemia; lane 4, dilution of 103 with 0.001% parasitemia; lane 5, dilution of 104 with 0.0001% parasitemia; lane 6, dilution of 105 with 0.00001% parasitemia; lane 7, dilution of 106 with 0.000001% parasitemia; lane 8, dilution of 107 with 0.0000001% parasitemia; lane 9, dilution of 108 with 0.00000001% parasitemia; lane 10, dilution of 109 with 0.000000001% parasitemia; lane 11, positive control; lane 12, uninfected sheep blood (negative control).
4. Discussion The diagnosis of piroplasm infections in vertebrate hosts has been mainly carried out by microscopic examination of blood smear. However, the method requires expertise because these parasites have similar morphological features and therefore may confuse when mixed infections occur. Serological tests have also been used, but there are some difficulties with specificity and sensitivity (Passos et al., 1998). An exact differentiation between these parasites is crucial to understand their epidemiology. The detection of Babesia infection in carrier animals by DNA amplification was a powerful tool for epidemiological investigation, since these animals represent an important source of alimentary infection of Ixodid ticks. It is more sensitive and specific than detection of parasite by conventional methods (d’Oliveira et al., 1995; Aktas et al., 2002). Amplification of parasite DNA using specific PCR has been applied to various Babesia and Theileria species (Kirvar et al., 1998, 2000; Schnittger et al., 2000; Fukumoto et al., 2001; Oliveira-Sequira et al., 2005; Alhassan et al., 2005; Altay et al., 2005), but PCR specific for B. ovis
has not been available until now. In this study, a highly specific and sensitive PCR assay was developed for the detection of B. ovis in sheep and goats using a pair of oligonucleotide primers which target the 549-bp fragment in the ssu rRNA gene of the parasite. The specificity of the primers used in this study for B. ovis was determined by successful amplification of a 549-bp product from positive control and collected field samples. In contrast, no PCR products were seen with B. motasi, B. canis, B. microti, Theileria sp. OT1, Theileria sp. OT3, T. lestoquardi, T. ovis and T. annulata DNAs. The successful amplification of only B. ovis products indicates that the designed primers are specific for B. ovis DNA. The Babesia and Theileriaspecific primers RLB-F/-R, described by Gubbels et al. (1999), generated the expected a 460–520-bp fragment of Babesia and Theileria species examined, including B. ovis, confirming the integrity of the DNAs used to validate the specificity of the assay. We found that the detection limit of the test was 0.00001% parasitemia. This is equal to a parasitemia at the level of 105% and is over 100 times greater than can be attained by microscopic examination of blood smears. These results demonsrate that PCR using B. ovis-specific primers is capable of detecting B. ovis parasites present at extremely low parasitemias in sheep and goats and can be applied to samples collected under field conditions. Similar sensitivities in Theileria and Babesia PCR assays have been reported by d’Oliveira et al. (1995) for T. annulata, Kirvar et al. (1998, 2000) for T. lestoquardi and T. annulata, Fukumoto et al. (2001) for B. gibsoni, Oliveira-Sequira et al. (2005) for B. bigemina and B. bovis, Alhassan et al. (2005) for Babesia caballi and Babesia equi, Altay et al. (2005) for T. ovis. The results obtained from field samples indicated that the frequency of B. ovis detected by DNA amplification method was significantly higher than detection by light microscopy, because the latter method does not detect positive animals in the early phase of infection and the long-term carrier status, when the parasitemia is very low. The result agrees with previous report about the B. bovis (Calder et al., 1996). In conclusion, the primer pair described here could be useful tools for clarifiying the epidemiology of ovine babesiosis caused by B. ovis and for exact discrimination between B ovis and B. motasi infections in sheep and goats.
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Acknowledgements The authors would like to thank all veterinarians and technicians for their kind help during sample collection for this study. For the gifts of genomic DNA from Jabbar Ahmed, Ana Hurtado and Bernard Carcy are gratefully acknowledged. This study was supported by a grant (104 O 393) from The Scientific and ´ TAK). ¨ BY Technical Research Council of Turkey (TU
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