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Repetitive DNA probes for the detection of Babesia equi
Molecular and Biochemical Parasitology, 34 (1989) 75-78 Elsevier
75
MBP 01118
R e p e t i t i v e D N A p r o b e s for the detection of Babesia equi Elsa S. Posnett and Riccardo E. Ambrosio Molecular Biology Section, Veterinary Research Institute, Onderstepoort, South Africa (Received 18 July 1988; accepted 9 November 1988)
This report describes DNA probes for the identification of Babesia equi. A genomic library of B. equi was constructed in pUC13. Several clones were identified that hybridized strongly to B. equi DNA. Clone pBE33 hybridized specifically to B. equi DNA and did not hybridize to horse DNA nor to DNA from Babesia caballi, Babesia bovis or Babesia bigernina. Two subclones of pBE33 (pSB20 and pEH21) containing B. equi repetitive sequences, could detect 0.49 ng and 0.97 ng B. equi DNA, respectively. Key words: Babesia equi; Equine babesiosis; DNA probe; Repetitive DNA
Introduction
Babesia equi and Babesia caballi are intraerythrocytic, tick-borne, protozoan parasites and are the causative agents of equine babesiosis [1]. Both species are endemic in equines throughout the tropics and subtropics [2]. In these areas the disease is transmitted by ticks of the genera Hyalomma, Derrnacentor and Rhipicephalus [3]. B. equi and B. caballi parasites can be demonstrated in stained blood smears 2-13 days post-infection. Due to the increasing international trade in horses, there is a high risk that the disease might spread to disease-free areas such as North America, Australia and Japan [3]. Importation is frequently restricted to animals that are seronegative by the complement fixation test [4]. Although this test rarely renders false-positive results, falsenegative results, which can be correctly diagnosed by the indirect immunofluorescence test, have been reported [2,4]. Although the indirect immunofluorescence test is more sensitive than Correspondence address: R.E. Ambrosio, Molecular Biology Department, Veterinary Research Institute, Onderstepoort 0110, South Africa. Abbreviations: SDS, sodium dodecyl sulphate; SSC, standard saline citrate; XGal, 5-bromo-4-chloro-3-indolyl-13-o-galactopyranoside; IPTG, isopropyl-13-D-thiogalactopyranoside.
the complement fixation test and produces no false-negative results, it cannot be standardized, and the reading of results is time-consuming [2]. Available serological tests cannot reliably distinguish between B. equi and B. caballi. A need therefore exists for a more rapid, accurate and sensitive test that would allow the detection of low levels of these parasites in infected horses. We describe here the isolation of two specific repetitive DNA probes for B. equi. Materials and Methods
Parasite isolation. B. equi and B. caballi parasites were isolated from the red blood cells of experimentally infected horses. In order to minimize contamination from equine leucocytes, the infected blood was centrifuged and the buffy coat removed. After lysing the red blood cells in distilled water, the suspension containing the parasites was passed through a Whatman CF-11 cellulose column [5]. DNA preparation. Parasites were lysed overnight in lysis buffer (0.32 M sucrose, 1% Triton X-100, 5 mM MgC12, 10 mM Tris-HCl, pH 7.4) containing 2% sodium dodecyl sulphate (SDS) and 100 txg m1-1 proteinase K. This was followed by multiple phenol, phenol-chloroform and chloroform
0166-6851/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
76 extractions. D N A was ethanol-precipitated and resuspended in TE (10 mM Tris-HC1, pH 7.4, 1 mM EDTA). The D N A was further purified by centrifugation in a caesium chloride/ethidium bromide density gradient [6]. Control DNA from uninfected horses was prepared by lysing white blood cells and extracting the D N A as described above. Restriction enzymes were used at 37°C in buffers, as recommended by the manufacturer. DNA was electrophoresed in 0.7% agarose gels and bands were visualized on a transilluminator after staining with ethidium bromide (1 p~g ml 1).
Construction and screening of B. equi p UC 13 library. B. equi DNA was digested with EcoRI and ligated to EcoRI-cut pUC13 [7]. After transformation into Escherichia coli JM105, recombinants were selected on ampicillin plates containing 5-bromo-4-chloro-3-indolyl-[3-I>galactopyranoside (XGal) and isopropyl-[3-D-thiogalactopyranoside (IPTG) [8]. Recombinant colonies were grown in LB-broth and plasmid DNA extracted using a rapid lysis protocol [9]. After EcoRI digestion, the D N A was electrophoresed, Southern-transferred to nylon [10] and sequentially hybridized to 32p-labelled horse and B. equi DNA. Recombinant clones containing B. equi D N A were identified by their lack of hybridization to horse DNA. Labelling of DNA and hybridization conditions. Horse, B. equi and plasmid D N A was labelled to high specific activity (1 × 10s dpm ~g-~) by nick translation [11] using [32p]dCTP (3000 Ci mmol 1, Amersham International). Membranes were hybridized with labelled probe at 65°C, in a hybridization solution containing 6 × SSC, 5 × Denhardt's [6] and 5% (w/v) SDS. Filters were washed at high stringency (0.1 × SSC, 0.1% (w/v) SDS at 65°C) and autoradiographed at -70°C on Cronex M R F 17 film with intensifying screens. Results and Discussion
Ligation of EcoRI-digested B. equi genomic D N A into similarly digested pUC13 resulted in 6-10 white, ampicillin-resistant colonies per ng of genomic D N A used in the ligation reaction. 200 white colonies were picked at random and their
D N A was extracted and electrophoresed in an agarose gel. Southern blots of D N A from these clones were screened with total genomic B. equi DNA as probe to select for B. equi-specific sequences. Strongly hybridizing bands were suggestive of repetitive sequences. To exclude the possibility that horse DNA sequences contaminating the B. equi DNA could give false positive results, a control hybridization was done using labelled horse DNA as probe. Three clones showing strong hybridization to B. equi DNA, and not to horse DNA, were isolated (pBE15, pBE33 and pBE37). When these clones were labelled and hybridized to EcoRI-digested B. equi genomic DNA, a different pattern of hybridization was obtained for each of the three clones. Bands of different sizes were detected by pBE33 and pBE37, whereas pBE15 hybridized to a single B. equi band. Hybridization of pBE33 and pBE37 to B. equi DNA remained strong even after washing at high stringency. However, when these three clones were used as probes to EcoRI digests of DNA from other Babesia spp., bovine and horse DNA, pBE37 cross-hybridized to Babesia boris, Babesia bigemina and bovine DNA. No cross-hybridization was detected with any of these clones to horse DNA. Clone pBE33 did not cross-hybridize to B. caballi DNA nor to DNA from other Babesia spp. and consistently hybridized strongly to B. equi DNA.
Characterization of pBE33. The size of the B. equi insert pBE33 was found to be 6.75 kb. The hybridization pattern obtained with pBE33 on EcoRI digests of B. equi genomic DNA is characteristic of tandemly repeated sequences (not shown). The large number of bands, more than 15, suggests a high level of distribution of this sequence through the parasite genome. The copy number of this repeat was determined by densitometric scanning [12] of dot blots containing dilutions of plasmid DNA (pBE33) and B. equi genomic DNA hybridized with 32p-labelled pBE33 (not shown). Assuming a genome size for B. equi of 1.2 × 10v bp [13] and 6.75 kb for the cloned fragment, we calculate a copy number of 8.7 × 102 per diploid genome. Fig. 1 shows the restriction map of the pBE33 insert. AluI, HinfI and Sau3A sites could not be
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Fig. 1. Restriction map of pBE33 insert. The map was constructed by digesting pBE33 insert D N A with the following enzymes: BarnHI, HindlII, HpalI, KpnI, PstI, SstI and XhoI. The following enzymes did not digest pBE33: BgilI, BstEII, ClaI, EcoRI, HaelII, Sail, SstlI, TaqI and XbaI.
mapped due to the large number of fragments generated by these enzymes. To locate the regions of specificity and of the repetitive elements within pBE33, restriction enzyme digests of the insert were probed with 32p-labelled B. equi genomic DNA. The repetitive elements were found on a 1.6-kb SstI/BamHI fragment and a 1.5-kb HindlII/EcoRI fragment (Fig. 1). These two pSB 20
fragments, termed pSB20 and pEH21, were subcloned into pUC13. Each of these subclones hybridized to a discrete set of EcoRI-digested B. equi bands sharing only a few bands of the same molecular weight (Fig. 2). pSB20 has restriction sites for XhoI, PstI, HpalI and KpnI, and only a single KpnI site occurs on pEH21. These results suggest that the repetitive sequences present in the subclones are not identical. The quantitative differences in the relative intensity of the bands could be due to variation in copy number of the repeat in the individual fragments. The hybridization pattern obtained with pSB20, pEH21 and pBE33 on D N A from two different isolates of B. equi suggests a difference in the distribution of the repetitive sequences in the B. equi genome (Fig. 2). This diversity in distribution implies that these probes could be used for differentiating between B. equi isolates. The use of repetitive DNA in the identification and comparison of different isolates has been reported for a number of parasites [14-16]. However, it has been reported that repetitive sequences may be unstable
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Fig. 2. Hybridization of 32p-labelled pSB20 and pEH21 to Southern blots of EcoRI-digested genomic DNA. Filters A and B were probed with pSB20 and pEH21 respectively• Lanes contain: (a) horse D N A ; (b) B. caballi DNA; and (c,d) two different isolates of B. equi DNA. Filters were washed at high stringency•
5h
5h
16h
116h
Fig. 3. Sensitivity: dilutions of (a) B. equi genomic DNA and (b) horse D N A were dot-blotted onto nylon and hybridized to labelled pEH21 (A and C) and pSB20 (B and D). Filters were washed at high stringency. Autoradiography was at -70°C with an intensifying screen. Filters A and B were exposed for 5 h, C and D for 16 h.
78 [12]. T h e r e f o r e , the p a t t e r n o b t a i n e d with these s e q u e n c e s a l o n e s h o u l d n o t be used to differentiate b e t w e e n isolates of the same species. No cross-hybridization of pSB20 and p E H 2 1 to D N A from o t h e r Babesia spp. or to horse D N A was obtained.
Sensitivity o f p E H 2 1 and pSB20. Since the sensitivity of a p r o b e is d e p e n d e n t o n the copy n u m ber of the s e q u e n c e to be d e t e c t e d , repetitive seq u e n c e s s h o u l d p r o v i d e the greatest sensitivity w h e n used as p r o b e s . T h e sensitivity of p E H 2 1 a n d pSB20 was d e t e r m i n e d by h y b r i d i z i n g the p r o b e s to dot blots c o n t a i n i n g d i l u t i o n s of B. equi D N A . A f t e r a 5-h e x p o s u r e , p E H 2 1 a n d pSB20 could detect 3.9 ng a n d 1.9 ng B. equi D N A , respectively (Fig. 3 A a n d B). A f t e r 16 h e x p o s u r e , 0.97 ng a n d 0.49 ng c o u l d be d e t e c t e d (Fig. 3C a n d D) a n d after 48 h, 0.24 ng a n d 0.12 ng could be detected (not shown). T h e sensitivity of pEH21 a n d pSB20 after 16 h e x p o s u r e c o r r e s p o n d s ap-
p r o x i m a t e l y to the a m o u n t of B. equi D N A in 50 txl of b l o o d with a 0.5% a n d 0.25% p a r a s i t a e m i a , respectively [17]. Clinical p a r a s i t a e m i a s in e q u i n e babesiosis due to B. equi vary b e t w e e n 5 % a n d 20% (T. de W a a l , p e r s o n a l c o m m u n i c a t i o n ) . T h e r e f o r e , the sensitivity a n d specificity of these B. equi clones m a k e s t h e m suitable for the dev e l o p m e n t of p r o b e s for specific a n d sensitive diagnostic tests. Possibly, pSB20 or p E H 2 1 could be used to detect the p r e s e n c e of parasites in cartier animals as well as tick vectors. This would aid e p i d e m i o l o g i c a l studies of B. equi a n d also lead to a b e t t e r u n d e r s t a n d i n g of this o r g a n i s m ' s dev e l o p m e n t a l cycle.
Acknowledgements W e wish to t h a n k Dr. T. de W a a l for supplying the Babesia-infected b l o o d , a n d Miss M. Oosth u i z e n for technical assistance.
References 1 Purnell, R.E. (1981) Babesiosis in various hosts. In: Babesiosis (Ristic, M. and Krier, J.P., eds.), pp. 25-64, Academic Press, New York. 2 Tenter, A.M. and Friedhoff, K.T. (1986) Serodiagnosis of experimental and natural Babesia equi and Babesia caballi infections. Vet. Parasitol. 20, 49-61. 3 Friedhoff, K.T. (1982) Die Piroplasmen der Equiden-Bedeutung for den Internationalen Pferdeverkehr. Berl. Munch. Tierarztl. Wochenschr. 95,368-374. 4 Weiland, G. (1986) Species-specificserodiagnosis of equine piroplasma infections by means of complement fixation test (CFT), immunofluorescence (IIF), and enzyme-linked immunosorbent assay (ELISA). Vet. Parasitol. 20, 43-48. 5 Ambrosio, R.E., Potgieter, F.T. and Nel, N. (1986) A column purification procedure for the removal of leucocytes from parasite infected blood. Onderstepoort J. Vet. Res. 53, 119-120. 6 Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 7 Vieira, J. and Messing, J. (1982) The pUC plasmids, a M13 mp-7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19, 259-268. 8 Messing, J., Gronenborn, B., Muller-Hill, B. and Hofschneider, P.H. (1977) Filamentous coliphage M13 as a cloning vehicle: Insertion of a HindIII fragment of the lac regulatory region in the M13 replicative form in vitro. Proc. Natl. Acad. Sci. USA 75, 3642-3644. 9 Birnboim, C. and Doly, J. (1979) Rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1515-1523.
10 Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517. 11 Rigby, P.W.J., Dieckman, M., Rhode, S.C. and Berg, P. (1977) Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113,237-251. 12 Ellis, J. and Crampton, J. (1988) Characterization of a simple highly repetitive DNA sequence from the parasite Leishmania donovani. Mol. Biochem. Parasitol. 29, 9-18. 13 Allsopp, B.A. and Allsopp, M.T. (1988) Theileria parva: genomic studies reveal intra-specific sequence diversity. Mol. Biochem. Parasitol. 28, 77-84. 14 Kim Lee Sim, B., Piessens, W.F. and Wirth, D.F. (1986) A DNA probe cloned in E. coli for the identification of Brugia malayi. Mol. Biocfiem. Parasitol. 19, 117-123. 15 Oquendo, P., Goman, M., Mackay, M., Langsley, G., Walliker, D. and Scafie, J. (1986) Characterization of a repetitive DNA sequence from the malaria parasite, Plasmodiumfalciparum. Mol. Biochem. Parasitol. 18, 89-101. 16 Chambers, A.E., Almond, N.M., Knight, M., Simpson, A.J.G. and Parkhouse, R.M. (1986) Repetitive DNA as a tool for the identification and comparison of nematode variants: application to Trichinella isolates. Mol. Biochem. Parasitol. 21, 113-120. 17 Bone, N., Gibson, T., Gorman, M., Hyde, J.E., Langsley, G.W., Schaife, J.G., Walliker, D., Yanofsky, N.K. and Zolg, J.W. (1983) Investigation of the DNA of the human malaria parasite Plasmodium falciparum by in vitro cloning into phage lambda. In: Molecular Biology of Parasites (Guariola, J., Luzzatto, L. and Trager, W., eds.), pp. 125-134, Raven Press, New York.
75
MBP 01118
R e p e t i t i v e D N A p r o b e s for the detection of Babesia equi Elsa S. Posnett and Riccardo E. Ambrosio Molecular Biology Section, Veterinary Research Institute, Onderstepoort, South Africa (Received 18 July 1988; accepted 9 November 1988)
This report describes DNA probes for the identification of Babesia equi. A genomic library of B. equi was constructed in pUC13. Several clones were identified that hybridized strongly to B. equi DNA. Clone pBE33 hybridized specifically to B. equi DNA and did not hybridize to horse DNA nor to DNA from Babesia caballi, Babesia bovis or Babesia bigernina. Two subclones of pBE33 (pSB20 and pEH21) containing B. equi repetitive sequences, could detect 0.49 ng and 0.97 ng B. equi DNA, respectively. Key words: Babesia equi; Equine babesiosis; DNA probe; Repetitive DNA
Introduction
Babesia equi and Babesia caballi are intraerythrocytic, tick-borne, protozoan parasites and are the causative agents of equine babesiosis [1]. Both species are endemic in equines throughout the tropics and subtropics [2]. In these areas the disease is transmitted by ticks of the genera Hyalomma, Derrnacentor and Rhipicephalus [3]. B. equi and B. caballi parasites can be demonstrated in stained blood smears 2-13 days post-infection. Due to the increasing international trade in horses, there is a high risk that the disease might spread to disease-free areas such as North America, Australia and Japan [3]. Importation is frequently restricted to animals that are seronegative by the complement fixation test [4]. Although this test rarely renders false-positive results, falsenegative results, which can be correctly diagnosed by the indirect immunofluorescence test, have been reported [2,4]. Although the indirect immunofluorescence test is more sensitive than Correspondence address: R.E. Ambrosio, Molecular Biology Department, Veterinary Research Institute, Onderstepoort 0110, South Africa. Abbreviations: SDS, sodium dodecyl sulphate; SSC, standard saline citrate; XGal, 5-bromo-4-chloro-3-indolyl-13-o-galactopyranoside; IPTG, isopropyl-13-D-thiogalactopyranoside.
the complement fixation test and produces no false-negative results, it cannot be standardized, and the reading of results is time-consuming [2]. Available serological tests cannot reliably distinguish between B. equi and B. caballi. A need therefore exists for a more rapid, accurate and sensitive test that would allow the detection of low levels of these parasites in infected horses. We describe here the isolation of two specific repetitive DNA probes for B. equi. Materials and Methods
Parasite isolation. B. equi and B. caballi parasites were isolated from the red blood cells of experimentally infected horses. In order to minimize contamination from equine leucocytes, the infected blood was centrifuged and the buffy coat removed. After lysing the red blood cells in distilled water, the suspension containing the parasites was passed through a Whatman CF-11 cellulose column [5]. DNA preparation. Parasites were lysed overnight in lysis buffer (0.32 M sucrose, 1% Triton X-100, 5 mM MgC12, 10 mM Tris-HCl, pH 7.4) containing 2% sodium dodecyl sulphate (SDS) and 100 txg m1-1 proteinase K. This was followed by multiple phenol, phenol-chloroform and chloroform
0166-6851/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
76 extractions. D N A was ethanol-precipitated and resuspended in TE (10 mM Tris-HC1, pH 7.4, 1 mM EDTA). The D N A was further purified by centrifugation in a caesium chloride/ethidium bromide density gradient [6]. Control DNA from uninfected horses was prepared by lysing white blood cells and extracting the D N A as described above. Restriction enzymes were used at 37°C in buffers, as recommended by the manufacturer. DNA was electrophoresed in 0.7% agarose gels and bands were visualized on a transilluminator after staining with ethidium bromide (1 p~g ml 1).
Construction and screening of B. equi p UC 13 library. B. equi DNA was digested with EcoRI and ligated to EcoRI-cut pUC13 [7]. After transformation into Escherichia coli JM105, recombinants were selected on ampicillin plates containing 5-bromo-4-chloro-3-indolyl-[3-I>galactopyranoside (XGal) and isopropyl-[3-D-thiogalactopyranoside (IPTG) [8]. Recombinant colonies were grown in LB-broth and plasmid DNA extracted using a rapid lysis protocol [9]. After EcoRI digestion, the D N A was electrophoresed, Southern-transferred to nylon [10] and sequentially hybridized to 32p-labelled horse and B. equi DNA. Recombinant clones containing B. equi D N A were identified by their lack of hybridization to horse DNA. Labelling of DNA and hybridization conditions. Horse, B. equi and plasmid D N A was labelled to high specific activity (1 × 10s dpm ~g-~) by nick translation [11] using [32p]dCTP (3000 Ci mmol 1, Amersham International). Membranes were hybridized with labelled probe at 65°C, in a hybridization solution containing 6 × SSC, 5 × Denhardt's [6] and 5% (w/v) SDS. Filters were washed at high stringency (0.1 × SSC, 0.1% (w/v) SDS at 65°C) and autoradiographed at -70°C on Cronex M R F 17 film with intensifying screens. Results and Discussion
Ligation of EcoRI-digested B. equi genomic D N A into similarly digested pUC13 resulted in 6-10 white, ampicillin-resistant colonies per ng of genomic D N A used in the ligation reaction. 200 white colonies were picked at random and their
D N A was extracted and electrophoresed in an agarose gel. Southern blots of D N A from these clones were screened with total genomic B. equi DNA as probe to select for B. equi-specific sequences. Strongly hybridizing bands were suggestive of repetitive sequences. To exclude the possibility that horse DNA sequences contaminating the B. equi DNA could give false positive results, a control hybridization was done using labelled horse DNA as probe. Three clones showing strong hybridization to B. equi DNA, and not to horse DNA, were isolated (pBE15, pBE33 and pBE37). When these clones were labelled and hybridized to EcoRI-digested B. equi genomic DNA, a different pattern of hybridization was obtained for each of the three clones. Bands of different sizes were detected by pBE33 and pBE37, whereas pBE15 hybridized to a single B. equi band. Hybridization of pBE33 and pBE37 to B. equi DNA remained strong even after washing at high stringency. However, when these three clones were used as probes to EcoRI digests of DNA from other Babesia spp., bovine and horse DNA, pBE37 cross-hybridized to Babesia boris, Babesia bigemina and bovine DNA. No cross-hybridization was detected with any of these clones to horse DNA. Clone pBE33 did not cross-hybridize to B. caballi DNA nor to DNA from other Babesia spp. and consistently hybridized strongly to B. equi DNA.
Characterization of pBE33. The size of the B. equi insert pBE33 was found to be 6.75 kb. The hybridization pattern obtained with pBE33 on EcoRI digests of B. equi genomic DNA is characteristic of tandemly repeated sequences (not shown). The large number of bands, more than 15, suggests a high level of distribution of this sequence through the parasite genome. The copy number of this repeat was determined by densitometric scanning [12] of dot blots containing dilutions of plasmid DNA (pBE33) and B. equi genomic DNA hybridized with 32p-labelled pBE33 (not shown). Assuming a genome size for B. equi of 1.2 × 10v bp [13] and 6.75 kb for the cloned fragment, we calculate a copy number of 8.7 × 102 per diploid genome. Fig. 1 shows the restriction map of the pBE33 insert. AluI, HinfI and Sau3A sites could not be
77
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6
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=-
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-'
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Fig. 1. Restriction map of pBE33 insert. The map was constructed by digesting pBE33 insert D N A with the following enzymes: BarnHI, HindlII, HpalI, KpnI, PstI, SstI and XhoI. The following enzymes did not digest pBE33: BgilI, BstEII, ClaI, EcoRI, HaelII, Sail, SstlI, TaqI and XbaI.
mapped due to the large number of fragments generated by these enzymes. To locate the regions of specificity and of the repetitive elements within pBE33, restriction enzyme digests of the insert were probed with 32p-labelled B. equi genomic DNA. The repetitive elements were found on a 1.6-kb SstI/BamHI fragment and a 1.5-kb HindlII/EcoRI fragment (Fig. 1). These two pSB 20
fragments, termed pSB20 and pEH21, were subcloned into pUC13. Each of these subclones hybridized to a discrete set of EcoRI-digested B. equi bands sharing only a few bands of the same molecular weight (Fig. 2). pSB20 has restriction sites for XhoI, PstI, HpalI and KpnI, and only a single KpnI site occurs on pEH21. These results suggest that the repetitive sequences present in the subclones are not identical. The quantitative differences in the relative intensity of the bands could be due to variation in copy number of the repeat in the individual fragments. The hybridization pattern obtained with pSB20, pEH21 and pBE33 on D N A from two different isolates of B. equi suggests a difference in the distribution of the repetitive sequences in the B. equi genome (Fig. 2). This diversity in distribution implies that these probes could be used for differentiating between B. equi isolates. The use of repetitive DNA in the identification and comparison of different isolates has been reported for a number of parasites [14-16]. However, it has been reported that repetitive sequences may be unstable
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Fig. 2. Hybridization of 32p-labelled pSB20 and pEH21 to Southern blots of EcoRI-digested genomic DNA. Filters A and B were probed with pSB20 and pEH21 respectively• Lanes contain: (a) horse D N A ; (b) B. caballi DNA; and (c,d) two different isolates of B. equi DNA. Filters were washed at high stringency•
5h
5h
16h
116h
Fig. 3. Sensitivity: dilutions of (a) B. equi genomic DNA and (b) horse D N A were dot-blotted onto nylon and hybridized to labelled pEH21 (A and C) and pSB20 (B and D). Filters were washed at high stringency. Autoradiography was at -70°C with an intensifying screen. Filters A and B were exposed for 5 h, C and D for 16 h.
78 [12]. T h e r e f o r e , the p a t t e r n o b t a i n e d with these s e q u e n c e s a l o n e s h o u l d n o t be used to differentiate b e t w e e n isolates of the same species. No cross-hybridization of pSB20 and p E H 2 1 to D N A from o t h e r Babesia spp. or to horse D N A was obtained.
Sensitivity o f p E H 2 1 and pSB20. Since the sensitivity of a p r o b e is d e p e n d e n t o n the copy n u m ber of the s e q u e n c e to be d e t e c t e d , repetitive seq u e n c e s s h o u l d p r o v i d e the greatest sensitivity w h e n used as p r o b e s . T h e sensitivity of p E H 2 1 a n d pSB20 was d e t e r m i n e d by h y b r i d i z i n g the p r o b e s to dot blots c o n t a i n i n g d i l u t i o n s of B. equi D N A . A f t e r a 5-h e x p o s u r e , p E H 2 1 a n d pSB20 could detect 3.9 ng a n d 1.9 ng B. equi D N A , respectively (Fig. 3 A a n d B). A f t e r 16 h e x p o s u r e , 0.97 ng a n d 0.49 ng c o u l d be d e t e c t e d (Fig. 3C a n d D) a n d after 48 h, 0.24 ng a n d 0.12 ng could be detected (not shown). T h e sensitivity of pEH21 a n d pSB20 after 16 h e x p o s u r e c o r r e s p o n d s ap-
p r o x i m a t e l y to the a m o u n t of B. equi D N A in 50 txl of b l o o d with a 0.5% a n d 0.25% p a r a s i t a e m i a , respectively [17]. Clinical p a r a s i t a e m i a s in e q u i n e babesiosis due to B. equi vary b e t w e e n 5 % a n d 20% (T. de W a a l , p e r s o n a l c o m m u n i c a t i o n ) . T h e r e f o r e , the sensitivity a n d specificity of these B. equi clones m a k e s t h e m suitable for the dev e l o p m e n t of p r o b e s for specific a n d sensitive diagnostic tests. Possibly, pSB20 or p E H 2 1 could be used to detect the p r e s e n c e of parasites in cartier animals as well as tick vectors. This would aid e p i d e m i o l o g i c a l studies of B. equi a n d also lead to a b e t t e r u n d e r s t a n d i n g of this o r g a n i s m ' s dev e l o p m e n t a l cycle.
Acknowledgements W e wish to t h a n k Dr. T. de W a a l for supplying the Babesia-infected b l o o d , a n d Miss M. Oosth u i z e n for technical assistance.
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