Veterinary Parasitology, 36 (1990) 11-20 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
11
Characterization of a Repetitive D N A Probe for
Babesia bigemina GERALD M. BUENING 1., A. BARBET 2, P. MYLER 3, S. MAHAN 2, V. NENE 4 and T.C. McGUIRE 5
1Department of Veterinary Microbiology, University of Missouri Columbia, Columbia, MO (U.S.A.) 2Department of ln[ectious Diseases, University of Florida, Gainesville, FL (U.S.A.) :~Seattle Biomedical Research Institute, Seattle, WA (U.S.A.) 4ILRAD, P.O. Box 30709, Nairobi (Kenya) "~Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA (U.S.A.) (Accepted for publication 16 November 1989)
ABSTRACT Buening, G.M., Barbet, A., Myler, P., Mahan, S., Nene, V. and McGuire, T.C., 1990. Characterization of a repetitive DNA probe for Babesia bigemina. Vet. Parasitol., 36: 11-20. A plasmid (p 16) containing a Babesia bigemina DNA insert was selected and labeled with ;~2p. This probe was evaluated for specificity and sensitivity by dot blot hybridization. The probe was specific and hybridized with only Babesia bigemina DNA, and not DNA from Babesia bovis, bovine leukocyte, Trypanosoma brucei or A naplasma marginale. The DNA probe detected as little as 10 pg of Babesia bigemina DNA. The probe hybridized with Babesia bigemina isolates from Mexico, the Caribbean region and Kenya. Genomic Babesia bigemina DNA of a Kenyan isolate was digested with restriction endonucleases, and the fragments were separated by gel electrophoresis and Southern blotted. The filter was hybridized with labeled p16 and each endonuclease digestion produced at least 16 resolvable DNA fragments. The inserted Babesia bigemina DNA was approximately 6.3 kb in size. A partial restriction map was constructed. A simple whole blood dot blot procedure was utilized to evaluate the sensitivity of the DNA probe. This probe would detect as few as 150 Babesia bigemina infected erythrocytes contained in a 1-/~1sample. The DNA probe has the potential to be a very sensitive and specific diagnostic tool.
INTRODUCTION
Bovine babesiosis caused by the intraerythrocytic protozoans Babesia bovis and Babesia bigemina remains a major cattle disease in the world's tropical and *Author to whom correspondence should be addressed.
0304-4017/90/$03.50
© 1990 Elsevier Science Publishers B.V.
12
G.M. BUENING ET AL.
subtropical areas. It has been estimated that 500 million cattle are at risk. The U.S. National Research Council ranked this disease as a high priority for biotechnology research for international development (1982). Several investigators have reported the detection of the protozoans Plasmodium falciparum and B. bovis in blood by using DNA hybridization. Plasmids containing cloned repetitive DNA of P. falciparum were nick translated and used to detect as little as 25 pg ofP. falciparum DNA (Franzen et al., 1984 ). This would correspond to the DNA in 50 pl of blood having 0.001% infected erythrocytes. A 21-base long synthetic DNA from this repetitive family of genomic DNAs of P. [alciparum was synthesized and labeled isotopically (McLaughlin et al., 1985, 1987). This synthetic DNA probe detected 0.1-0.01 ng of P. falciparum DNA. McLaughlin et al. (1986) inserted B. bovis DNA into plasmids and obtained clones. Dot blot hybridization with DNA probes from these plasmids detected 100 pg of B. boris DNA. One hundred picograms of B. bovis DNA would correspond to the amount of DNA in 50/A of 0.01% parasitized erythrocytes (McLaughlin et al., 1986). The objectives of this study were to develop and evaluate the sensitivity and specificity of a DNA probe to detect B. bigemina in infected erythrocytes. MATERIALS AND METHODS
Construction of a Babesia bigemina DNA library Babesia bigemina DNA was obtained from the Seed isolate, a Mexican strain provided by Dr. K.L. Kuttler in 1979 (USDA, Hemoparasitic Diseases Unit, Pullman, WA 99164, U.S.A. ). Babesia bigemina was cultured in vitro in bovine erythrocytes according to the procedure of Vega et al. (1985). Infected erythrocytes were concentrated by density gradient centrifugation as described by Vega et al. (1986). Approximately 0.1 ml of concentrated infected erythrocytes were dispensed in cold 1 / 100 NaC1 and sodium citrate solution ( SSC ) (0.6 ml ) and gently mixed. The cells were solubilized in 2% sarcosyl in 0.2 M EDTA (pH 8.0) at 60 ° C. The mixture was treated with preboiled pancreatic RNase T1 ( 100 U. ml- 1) and proteinase K (2 mg m l - 1). The molarity of the solution was increased to 1 M with NaC1. The solution was extracted twice with phenol saturated with TE (10 mM Tris, 1 mM EDTA, pH 8.0). The DNA was precipitated by adding 2 volumes of cold ( - 20 ° C) 95% EtOH and the DNA isolated by spooling on a glass rod. The DNA concentration was determined by O.D. readings at 260, 280, 300 and 330 nm using an automated scanning spectrophotometer (Cary Model 118). The spooled DNA was resuspended in approximately 200/~l of TE and contained 103/~g of DNA per ml. A pilot endonuclease digestion was done using constant amounts of B. bigemina DNA (0.5 #g) with dilutions of Sau3A (0.005-0.5 U.) at 37°C for 1 h. The condition which achieved a maximum of B. bigemina DNA fragments in
REPETITIVE DNA PROBE FOR BABESIA BIGEMINA
13
the 3-6 kb size range was 0.1 U. of Sau3A ltg-1 DNA. A batch experiment using these conditions was done. The B. bigemina DNA fragments were ligated into BamHI cleaved and dephosphorylated plasmid vector pBR322 (New England Biolabs, Beverly, MA, U.S.A. ). The ligation mixture consisted of the three fold excess (based upon initial DNA concentration) of Sau3A digest to vector and T4 DNA ligase. Escherichia coli strain HB101 cells were grown, made competent, and transformed with 40 ng of plasmid vector, according to the procedure of Hanahan (1983). The transformed cells were plated on ampicillin agar plates. The colonies from representative plates were washed off with Luria-Bertani medium, mixed with 15% glycerol and stored a t - 70 ° C.
Selection of colonies containing plasmids with Babesia bigemina DNA inserts Transformed cells were diluted and plated on ampicillin agar plates. Colonies were lifted on nitrocellulose filters, lysed, denatured, dried, and baked for 2 h in a vacuum oven at 80 ° C (Maniatis, 1982 ). A 32p labeled probe was made by nick translation of the isolated genomic B. bigemina DNA. A commercial nick translation kit was used (BRL, Gaithersburg, MD 20877, U.S.A. ) in conjunction with (~-32P) dCTP of 800 Ci mmol-1 specific activity (New England Nuclear, Wilmington, DE, U.S.A.). The filters were pre-hybridized for 4 h at 42 ° C with 5 × NaC1, NaH2PO4, H~O and EDTA solution ( SSPE ), 200 ~g m l - 1 denatured calf thymus DNA. The pre-hybridization solution was removed and then the filters were hybridized in an equal volume of 5 × SSPE, 0.25% sarcosyl and 100 #g m l - ' denatured calf thymus DNA containing the ~2p labeled probe for at least 16 h. The filters were initially washed twice in 1 x S S P E , 0.1% sarcosyl at 65 °C for 30 rain and then washed twice in 1 × SSPE, 0.0033% satcosyl at 65 ° C for 30 rain. The nitrocellulose filters were dried, identified with radioactive ink and placed in contact with X-ray film. Hybridizing colonies were identified by autoradiography. Sixteen colonies were selected based upon the intensity of the hybridization signal. Selected colonies were amplified in ampicillin broth overnight. Plasmids were isolated by the alkaline lysis minipreparation procedure (Maniatis, 1982). Ten microliters of each plasmid DNA was incubated with 0.5 #1 of HindIII 10 U. tt1-1 for 60 rain at 37°C. Conversion buffer and 0.5 zl of SalI 10 U. #1-1 was added to each tube and incubated for an additional 60 min at 37 ° C. The reaction was stopped and samples were run on a mid-size agarose gel at 200 V for 55 min. The separated plasmid DNA was transferred to nitrocellulose by the Southern blot technique and hybridized to the ~2p labeled B. bigemina genomic probe.
Evaluation of plasmid DNA probes for specificity and sensitivity to detect isolated DNA Four plasmids containing B. bigemina DNA inserts were extracted from amplified cultures and nick translated and labeled with ~2p dCTP. Various con-
]4
G.M. BUENING ET AI,.
centrations (100 ttg-10 pg) of denatured B. bigemina, B. bovis, bovine leukocyte and Trypanosoma brucei DNA were spotted on nitrocellulose filters. A separate filter was hybridized with each of four individual DNA probes. The probes were heat denatured and placed in plastic bags with carrier DNA. Filters were hybridized for at least 16 h at 65 ° C. Filters were washed and prepared for autoradiography as described previously. The labeled plasmid probe (p16) was hybridized with filters containing various concentrations of DNA extracted from 10~ to 103 infected erythrocytes from different isolates ofB. bigemina. The Babesia isolates and dates obtained in our laboratory are indicated: Texas 651 isolate provided by Dr. G. Wagner in July 1984 (Center for Tropical Animal Health, College of Veterinary Medicine, Texas A & M University, College Station, TX 77843, U.S.A. ); Texcoco isolate from a clinical case at the municipality of Texcoco, Mex., Mexico, was donated by Dr. G.J. Canto in September 1984 (International Hemoprotozoan Project, Instituto Nacional de Investigaciones Pecuarias, Palo Alto, D.F. 05110, Mexico); Puerto Rico and St. Croix isolates provided by Dr. J. Brown in June 1985 (USDA-APHIS, National Veterinary Services Laboratory, Ames, IA 50010, U.S.A. ); Costa Rican isolate obtained from the infected blood of a clinical case at a farm in Costa Rica kindly provided by Dr. C.A. Carson in March 1987 (Department of Veterinary Microbiology, UM/C, Columbia, MO 65211, U.S.A.). As specificity controls, B. bovis-KBb Mexican isolate DNA, Anaplasma marginale DNA and bovine white cell DNA were included on each illter. The isolated DNA was applied to nitrocellulose filters with a slot blot apparatus (Hybri-Slot, BRL ). The filters were processed as previously described.
Evaluation of candidate plasmid probe by Southern blotting DNA was prepared from a Kenyan isolate of B. bigemina DNA. DNA preparations were digested with BamHI/BstEII, HindIII and EcoRI. In addition, as specificity controls, bovine DNA, A. marginale DNA (Kenyan isolate), Theileria parva DNA, Theileria mutans DNA and P. falciparum DNA were digested with EcoRI. The digests were separated on a 1.0% agarose gel and the DNA was transferred to a nylon membrane. The membrane was exposed to UV light for 2 min and then pre-hybridized (5 ×Denhart's solution with 100 zg m1-1 salmon sperm DNA) for 4 h at 68°C. The p16 plasmid was labeled with 32p by nick translation. The hybridization solution (Denhardt's solution with 9% dextran sulfate) contained 100 ng of the probe solution with a specific activity of 10Gcpm ml- '. The filters were incubated overnight at 68 ° C, washed at high stringency, dried and exposed for autoradiography.
Detection of infected erythrocytes In vitro cultures of B. bigemina and B. bovis were used as the source of infected erythrocytes. The percent of infected erythrocytes in B. bigemina cultures was 0.7% and in B. boris cultures was 8.0%. The whole cultures (approx-
REPETITIVE DNA PROBE FOR BABESIA BIGEMINA
15
imately 106 bovine erythrocytes p l - 1 ) were diluted in phosphate buffer saline to the desired concentration of infected erythrocytes. The plasmid (p16) was labeled with 32p and hybridized with nitrocellulose or nylon filters spotted with various concentrations of B. bigemina (1.2 X 104-1.8X 101) and B. bovis (6.5 X 104-1 X 102 ) infected erythrocytes. Controls consisted of isolated DNA from B. bigemina, B. bovis and A. marginale. The filters were placed in a commercial filter holder (Hybrid-Ease, Hoefner Sci. Inst., San Francisco, CA 94107, U.S.A. ). The pre-hybridization conditions consisted of a solution containing 50% deionized formamide, 10% dextran sulfate, 1% SDS and 1 M NaC1, and 20 pg m l - 1 of boiled, sheared salmon sperm DNA. The apparatus was placed in a 42 °C water-bath for 5 h. The labeled probe (specific activity > 10s cpm pg- 1) was denatured by boiling in a solution containing carrier DNA. The probe was immediately placed on ice for 10 min and then added to the apparatus. The filters were hybridized at 42 ° C for 18-24 h. The hybridization mix was removed and the filters washed with 2 X SSC at room temperature (20-40 rinse cycles). The filters were then washed with 2×SSC, 1% SDS at 65°C. The apparatus was placed in a 65°C water-bath for 30 min and then washed (20-40 rinse cycles). This procedure was repeated twice. Next the apparatus was placed at room temperature and the filters washed twice (20-40 cycles) with 0.1 X SSC at room temperature. The apparatus was left undisturbed for 30 min between the washings. The filters were then dried, placed on cardboard or covered with plastic wrap and processed for autoradiography as previously described.
p16 restriction map Isolated p16 plasmid DNA was digested with a selected group of restriction endonucleases. The following endonucleases were used in single or double digestion experiments: EcoRV, BamHI, BanII, SphI, PvuI, PvuII, PstI, BsmI, NdeI, XmaIII, AatII, DraI, HincII, Tth111I, EcoRI, HindIII, AvaI, SalI, MluI, SmaI, SstI, XbaI, ApaI, BglII, HpaI, KpnI, BstEII, XhoI. Isolation of the Babesia bigemina DNA insert from p16 Purified p16 (5 pg) was double digested with BamHI and BstEII for 3 h at 37°C. The reaction was stopped and samples run on a mid-size 0.8% agarose gel at 25 V overnight. Gel plugs containing the large fragment representing the B. bigemina insert were removed. The gel plugs were processed by the procedure of Benson (1984) to isolate the DNA. RESULTS
Construction of Babesia bigemina DNA library By counting the colonies on 10 ampicillin agar plates, it was calculated that transformation rate of E. coli strain HB101 was 3.6X 10Gcolonies pg-1 of B.
16
G.M. BUENING ET AL.
bigemina DNA. The primary library consisted of approximately 1.44× 10:' transformants. Selection of repetitive plasmids The specific activity of the 32p labeled probe made by nick translation of the isolated genomic B. bigemina DNA was 2.44 × l0 s cpm ~g 1 DNA. Sixteen colonies which gave strong signals by hybridization were selected; all 16 grew in ampicillin broth and plasmids were isolated. Four candidate plasmids were selected from the 16 based upon fragment size of agarose gel electrophoresis of HindIII and SalI digest and intensity of hybridization signal with the Southern blot. Plasmids pl, p5, p9 and p16 were amplified and extracted to test as candidate B. bigemina DNA probes.
Detection of isolated D NA The specific activity of the plasmid DNA probes (pl, p5, p9 and p16) was > l0 s cpm #g- 1. One-microliter aliquots containing various concentrations of DNA (100 zg-10 pg) were spotted on nitrocellulose filters. The DNAs tested were isolated from: B. bigernina, B. boris, Trypanosoma brucei and bovine white cell. The four plasmid probes hybridized only with B. bigemina DNA and would detect at least 10 pg of B. bigemina DNA (data not shown). The p16 plasmid was selected based upon the intensity of the hybridization signal and growth of the parent colony. In the experiment where DNA was extracted from B. bigemina infected erythrocyte suspensions containing known parasitemias, the hybridization procedure detected as few as 103 infected erythrocytes. The p16 probe appeared to react with the four isolates tested with equal sensitivity (data not shown). There was minimum background with DNA from 106 B. bovis infected erythrocytes. There was no hybridization with purified DNAs from A. marginale, bovine white cells or B. boris.
Evaluation of candidate plasmid probe by Southern blotting The probe hybridized only with restriction fragments derived from B. bigemina DNA (Fig. 1). It can be observed in Lane 2, that at least 18 reiterated sequences were formed by BamHI/BstEII digestion. When HindIII or EcoRI endonucleases were used for digestion of genomic B. bigemina at least 16 fragments were observed. The presence of numerous bands indicates that repetitive sequences are present in the B. bigemina genome and the p16 clone.
REPETITIVEDNAPROBEFORBABESIA BIGEMINA
17
].
Fig. 1. Southern blot analysis of genomic DNAs digested with endonucleases and hybridized against the p16 32p labeled probe. Lane 1 = molecular weight markers 2-phage DNA. Lane 2 =B. bigemina (Kenya) digested with BamHI/BstEII. Lane 3 =B. bigemina (Kenya) digested with HindIII. Lane 4 = B. bigemina (Kenya) digested with EcoRI. Lane 5-- Bovine DNA digested with EcoRI. Lane 6=A. marginale digested with EcoRI. Lane 7-- T. parva digested with EcoRI. Lane 8-- T. mutans digested with EcoRI. Lane 9 =P. [alciparum digested with EcoRI. Lane 10 = molecular weight markers 2-phage DNA. Note that hybridization signals are observed only with bands of B. bigemina DNA.
Detection of infected erythrocytes Plasmid p16 DNA probes were constructed by labeling with 32p by the nick translation or random primer techniques. Both labeling techniques resulted in probes of equivalent sensitivity. The constructed probes hybridized only with B. bigemina infected erythrocytes and not with B. boris infected erythrocytes (Fig. 2). It should be stressed that each spotted area of B. bigemina and B. boris contained a ratio of at least nine non-infected to one infected bovine erythrocyte. No hybridization was observed in the B. boris infected row which contained an equivalent number of bovine erythrocytes as the B. bigemina infected row. The hybridization signal decreased in intensity, possibly not in
18
G.M. BUENINGET AI..
Sensitivity of DNA probe Babesia bigemina 120(O
1200
600
300
150
75
37
18
65000
6500
3250
1625
812
406
203
102
B. bigemina iPd3C's
B. bovis iRBC's Fig. 2. Dot blot hybridization assay of plasmid p 16 DNA probe labeled with 32p. The concentration of infected erythrocytes is indicated by the number above the columns. It should be emphasized that each dot contained a ratio of greater than nine normal erythrocytes to one infected erythrocyte. The first row contains various dilutions of B. bigemina infected erythrocytes and the second row B. bovis infected erythrocytes. The autoradiograph shows specificity for B. bigemina and the signal intensity varies with the concentration of infected erythrocytes.
Plasmid p 1 6
Sall EcoRI Aval I ~ I I. ]1 B.~HL Hind.III -Nincll I
BstEll 0 ,.
10.7 K b
I i Th__~t Ill
I
pBr~Z2 l 0 ,
Babesia I)~gemina Insert
6.3 K b Fig. 3. A partial restriction map of the plasmid (p16) It should be noted that the majority of the insert can be removed by a double digest of BstEII and BamHI. In addition there were no sites detected between the ThtIII and AvaI site.
proportion, with the dilution of B. bigemina infected erythrocytes. However, repeatable experiments demonstrated a sensitivity to detect as few as 150 infected erythrocytes (Fig. 2).
Characterization o[ a selected plasmid by constructing a restriction map The inserted B. bigemina D N A was determined to be approximately 6.3 kb in size. A restriction map was constructed and the location of restriction sites
REPETITIVE DNA PROBE FOR BABESIA BIGEMINA
19
for the following endonucleases were determined: BstEII, Tth111I, AvaI, HindIII, SalI, HincII, BamHI. There was approximately a 4.8 kb segment that did not cut with any of the nucleases tested. It has been possible to cut the insert out of the plasmid using BamHI and BstEII. These two restriction sites were located close to the ends of the insert (Fig. 3). This fragment has been labeled with 32p and achieved similar sensitivity compared with the plasmid p16 probe (data not shown). DISCUSSION
Genomic B. bigemina DNA was used to construct a genomic library in the plasmid vector pBR322. Transformed E. coli HB101 containing inserts of B. bigemina DNA were selected by hybridization with genomic DNA. The candidate probe hybridized only with B. bigemina DNA and not with DNA or infected erythrocytes from B. bovis, A. marginale, P. falciparum, Trypanosoma brucei, Theileria parva or Theileria mutans. These observations document the specificity of the probe. In addition, the probe hybridized with at least 16 repetitive DNA families of genomic B. bigemina. The Southern blotting analysis (Fig. 1 ) probably underestimated the number of repetitive families. At the top of the gel, large poorly resolved fragments were observed. DNA probes that would be candidates to be used as diagnostic aids should hybridize with isolates from different areas of the world. The present candidate probe hybridized with isolates from Texas, Mexico, Puerto Rico, St. Croix, Costa Rica and Kenya. This would indicate that this probe could be used in world-wide epidemiological studies. The candidate DNA probe appeared to be highly sensitive in that it would detect 10 pg ofB. bigemina DNA and as few as 150 infected erythrocytes. This probe could be used in epidemiological studies to detect carrier animals. In a recent study Eriks et al. (1989) characterized an A. marginale nucleic acid probe to detect carrier animals, which was specific and could detect 10 pg of genomic DNA and 500-1000 infected erythrocytes in 0.5 ml of blood. Previous reports by McLaughlin et al. (1986) concerning DNA probes for detecting B. bovis DNA indicated that the probes detected 100 pg of B. boris DNA which corresponded to a parasitemia of 0.01%. In epidemiological studies it would be desirable to have a nucleic acid probe which would detect carrier animals that would have a very low level of parasitemia. However, with the development of the polymerase chain reaction (PCR) the presence of specific DNA in a sample can be amplified up to 200 000-fold (Kogan et al., 1987). It may be possible to adapt PCR technology for use with the p16 probe. Therefore one of the major requirements for a diagnostic probe would be its specificity for the target organism and ability to react with many different isolates. The candidate probe described in this report fulfills these requirements.
20
G.M.BUENINGET AL.
ACKNOWLEDGMENTS
We thank Karen McLaughlin for excellent technical assistance and Ellen Swanson for typing the manuscript. The Kenyan isolates of Theileria, Anaplasma and Babesia were provided by S.P. Morzaria and Plasmodium by B. Khan. This investigation was supported in part by Burroughs-Wellcome Fund; U.S. Agency for International Development Grant DPE-5542-G-SS-4026-00, and USAID Prime Agreement No. DAN 4178-A-00-7056-00.
REFERENCES Benson, S.A., 1984. A rapid procedure for isolation of DNA fragments from agarose gels. Biotechniques, 2 (March/April). Eriks, I.S., Palmer, G.H., McGuire, T.C., Allred, D.R. and Barbet, A.F., 1989. Detection and quantitation of A naplasma marginale in carrier cattle by using a nucleic acid probe. J. Clin. Microbiol., 27: 279-284. Franzen, L., Westin, G., Shabo, R., Aslund, L., Perlmann, H., Persson, T., Wigzell, H. and Pettersson, U., 1984. Analysis of clinical specimens by hybridization with probe containing repetitive DNA from Plasmodium [alciparum. Lancet, 8376 (i): 525-528. Hanahan, D., 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166: 557. Kogan, S.C., Doherty, M. and Gitschier, J., 1987. An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences: application of hemophilia A. N. Engl. J. Med., 317: 985-990. Maniatis, T., Fritsche, E.F. and Sambrook, J., 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory. McLaughlin, G.L., Edlind, T.D., Campbell, G.H., Eller, R.F. and Ihler, G.M., 1985. Detection of Plasmodium falciparum using a synthetic DNA probe. Am. J. Trop. Med. Hyg., 34 (5): 837 840. McLaughlin, G.L., Edlind, T.D. and Ihler, G.M., 1986. Detection of Babesia boris using DNA hybridization. J. Protozool., 33 (1): 125-128. McLaughlin, G.L., Collins, W.E. and Campbell, G.H., 1987. Comparison of genomic plasmid, synthetic and combined DNA probes for detecting Plasmodium [alciparum DNA. J. Clin. Microbiol., 25:791 795. National Research Council, 1982. Priorities in Biotechnology Research for International DeveL opment Proceedings of a Workshop. Directed by the Board on Science and Technology h)r International Development, Office of International Affairs, National Academy Press, Washington, DC. Vega, C.A., Buening, G.M., Green, T.J. and Carson, C.A., 1985. In vitro cultivation of Babesia bigemina. Am. J. Vet. Res., 46: 416-420. Vega, C.A., Buening, G.M., Rodriguez, S.D. and Carson, C.A., 1986. Concentration and enzyme content of in vitro-cultured Babesia bigemina-infected erythrocytes. J. Protozool. 33:514 518.
11
Characterization of a Repetitive D N A Probe for
Babesia bigemina GERALD M. BUENING 1., A. BARBET 2, P. MYLER 3, S. MAHAN 2, V. NENE 4 and T.C. McGUIRE 5
1Department of Veterinary Microbiology, University of Missouri Columbia, Columbia, MO (U.S.A.) 2Department of ln[ectious Diseases, University of Florida, Gainesville, FL (U.S.A.) :~Seattle Biomedical Research Institute, Seattle, WA (U.S.A.) 4ILRAD, P.O. Box 30709, Nairobi (Kenya) "~Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA (U.S.A.) (Accepted for publication 16 November 1989)
ABSTRACT Buening, G.M., Barbet, A., Myler, P., Mahan, S., Nene, V. and McGuire, T.C., 1990. Characterization of a repetitive DNA probe for Babesia bigemina. Vet. Parasitol., 36: 11-20. A plasmid (p 16) containing a Babesia bigemina DNA insert was selected and labeled with ;~2p. This probe was evaluated for specificity and sensitivity by dot blot hybridization. The probe was specific and hybridized with only Babesia bigemina DNA, and not DNA from Babesia bovis, bovine leukocyte, Trypanosoma brucei or A naplasma marginale. The DNA probe detected as little as 10 pg of Babesia bigemina DNA. The probe hybridized with Babesia bigemina isolates from Mexico, the Caribbean region and Kenya. Genomic Babesia bigemina DNA of a Kenyan isolate was digested with restriction endonucleases, and the fragments were separated by gel electrophoresis and Southern blotted. The filter was hybridized with labeled p16 and each endonuclease digestion produced at least 16 resolvable DNA fragments. The inserted Babesia bigemina DNA was approximately 6.3 kb in size. A partial restriction map was constructed. A simple whole blood dot blot procedure was utilized to evaluate the sensitivity of the DNA probe. This probe would detect as few as 150 Babesia bigemina infected erythrocytes contained in a 1-/~1sample. The DNA probe has the potential to be a very sensitive and specific diagnostic tool.
INTRODUCTION
Bovine babesiosis caused by the intraerythrocytic protozoans Babesia bovis and Babesia bigemina remains a major cattle disease in the world's tropical and *Author to whom correspondence should be addressed.
0304-4017/90/$03.50
© 1990 Elsevier Science Publishers B.V.
12
G.M. BUENING ET AL.
subtropical areas. It has been estimated that 500 million cattle are at risk. The U.S. National Research Council ranked this disease as a high priority for biotechnology research for international development (1982). Several investigators have reported the detection of the protozoans Plasmodium falciparum and B. bovis in blood by using DNA hybridization. Plasmids containing cloned repetitive DNA of P. falciparum were nick translated and used to detect as little as 25 pg ofP. falciparum DNA (Franzen et al., 1984 ). This would correspond to the DNA in 50 pl of blood having 0.001% infected erythrocytes. A 21-base long synthetic DNA from this repetitive family of genomic DNAs of P. [alciparum was synthesized and labeled isotopically (McLaughlin et al., 1985, 1987). This synthetic DNA probe detected 0.1-0.01 ng of P. falciparum DNA. McLaughlin et al. (1986) inserted B. bovis DNA into plasmids and obtained clones. Dot blot hybridization with DNA probes from these plasmids detected 100 pg of B. boris DNA. One hundred picograms of B. bovis DNA would correspond to the amount of DNA in 50/A of 0.01% parasitized erythrocytes (McLaughlin et al., 1986). The objectives of this study were to develop and evaluate the sensitivity and specificity of a DNA probe to detect B. bigemina in infected erythrocytes. MATERIALS AND METHODS
Construction of a Babesia bigemina DNA library Babesia bigemina DNA was obtained from the Seed isolate, a Mexican strain provided by Dr. K.L. Kuttler in 1979 (USDA, Hemoparasitic Diseases Unit, Pullman, WA 99164, U.S.A. ). Babesia bigemina was cultured in vitro in bovine erythrocytes according to the procedure of Vega et al. (1985). Infected erythrocytes were concentrated by density gradient centrifugation as described by Vega et al. (1986). Approximately 0.1 ml of concentrated infected erythrocytes were dispensed in cold 1 / 100 NaC1 and sodium citrate solution ( SSC ) (0.6 ml ) and gently mixed. The cells were solubilized in 2% sarcosyl in 0.2 M EDTA (pH 8.0) at 60 ° C. The mixture was treated with preboiled pancreatic RNase T1 ( 100 U. ml- 1) and proteinase K (2 mg m l - 1). The molarity of the solution was increased to 1 M with NaC1. The solution was extracted twice with phenol saturated with TE (10 mM Tris, 1 mM EDTA, pH 8.0). The DNA was precipitated by adding 2 volumes of cold ( - 20 ° C) 95% EtOH and the DNA isolated by spooling on a glass rod. The DNA concentration was determined by O.D. readings at 260, 280, 300 and 330 nm using an automated scanning spectrophotometer (Cary Model 118). The spooled DNA was resuspended in approximately 200/~l of TE and contained 103/~g of DNA per ml. A pilot endonuclease digestion was done using constant amounts of B. bigemina DNA (0.5 #g) with dilutions of Sau3A (0.005-0.5 U.) at 37°C for 1 h. The condition which achieved a maximum of B. bigemina DNA fragments in
REPETITIVE DNA PROBE FOR BABESIA BIGEMINA
13
the 3-6 kb size range was 0.1 U. of Sau3A ltg-1 DNA. A batch experiment using these conditions was done. The B. bigemina DNA fragments were ligated into BamHI cleaved and dephosphorylated plasmid vector pBR322 (New England Biolabs, Beverly, MA, U.S.A. ). The ligation mixture consisted of the three fold excess (based upon initial DNA concentration) of Sau3A digest to vector and T4 DNA ligase. Escherichia coli strain HB101 cells were grown, made competent, and transformed with 40 ng of plasmid vector, according to the procedure of Hanahan (1983). The transformed cells were plated on ampicillin agar plates. The colonies from representative plates were washed off with Luria-Bertani medium, mixed with 15% glycerol and stored a t - 70 ° C.
Selection of colonies containing plasmids with Babesia bigemina DNA inserts Transformed cells were diluted and plated on ampicillin agar plates. Colonies were lifted on nitrocellulose filters, lysed, denatured, dried, and baked for 2 h in a vacuum oven at 80 ° C (Maniatis, 1982 ). A 32p labeled probe was made by nick translation of the isolated genomic B. bigemina DNA. A commercial nick translation kit was used (BRL, Gaithersburg, MD 20877, U.S.A. ) in conjunction with (~-32P) dCTP of 800 Ci mmol-1 specific activity (New England Nuclear, Wilmington, DE, U.S.A.). The filters were pre-hybridized for 4 h at 42 ° C with 5 × NaC1, NaH2PO4, H~O and EDTA solution ( SSPE ), 200 ~g m l - 1 denatured calf thymus DNA. The pre-hybridization solution was removed and then the filters were hybridized in an equal volume of 5 × SSPE, 0.25% sarcosyl and 100 #g m l - ' denatured calf thymus DNA containing the ~2p labeled probe for at least 16 h. The filters were initially washed twice in 1 x S S P E , 0.1% sarcosyl at 65 °C for 30 rain and then washed twice in 1 × SSPE, 0.0033% satcosyl at 65 ° C for 30 rain. The nitrocellulose filters were dried, identified with radioactive ink and placed in contact with X-ray film. Hybridizing colonies were identified by autoradiography. Sixteen colonies were selected based upon the intensity of the hybridization signal. Selected colonies were amplified in ampicillin broth overnight. Plasmids were isolated by the alkaline lysis minipreparation procedure (Maniatis, 1982). Ten microliters of each plasmid DNA was incubated with 0.5 #1 of HindIII 10 U. tt1-1 for 60 rain at 37°C. Conversion buffer and 0.5 zl of SalI 10 U. #1-1 was added to each tube and incubated for an additional 60 min at 37 ° C. The reaction was stopped and samples were run on a mid-size agarose gel at 200 V for 55 min. The separated plasmid DNA was transferred to nitrocellulose by the Southern blot technique and hybridized to the ~2p labeled B. bigemina genomic probe.
Evaluation of plasmid DNA probes for specificity and sensitivity to detect isolated DNA Four plasmids containing B. bigemina DNA inserts were extracted from amplified cultures and nick translated and labeled with ~2p dCTP. Various con-
]4
G.M. BUENING ET AI,.
centrations (100 ttg-10 pg) of denatured B. bigemina, B. bovis, bovine leukocyte and Trypanosoma brucei DNA were spotted on nitrocellulose filters. A separate filter was hybridized with each of four individual DNA probes. The probes were heat denatured and placed in plastic bags with carrier DNA. Filters were hybridized for at least 16 h at 65 ° C. Filters were washed and prepared for autoradiography as described previously. The labeled plasmid probe (p16) was hybridized with filters containing various concentrations of DNA extracted from 10~ to 103 infected erythrocytes from different isolates ofB. bigemina. The Babesia isolates and dates obtained in our laboratory are indicated: Texas 651 isolate provided by Dr. G. Wagner in July 1984 (Center for Tropical Animal Health, College of Veterinary Medicine, Texas A & M University, College Station, TX 77843, U.S.A. ); Texcoco isolate from a clinical case at the municipality of Texcoco, Mex., Mexico, was donated by Dr. G.J. Canto in September 1984 (International Hemoprotozoan Project, Instituto Nacional de Investigaciones Pecuarias, Palo Alto, D.F. 05110, Mexico); Puerto Rico and St. Croix isolates provided by Dr. J. Brown in June 1985 (USDA-APHIS, National Veterinary Services Laboratory, Ames, IA 50010, U.S.A. ); Costa Rican isolate obtained from the infected blood of a clinical case at a farm in Costa Rica kindly provided by Dr. C.A. Carson in March 1987 (Department of Veterinary Microbiology, UM/C, Columbia, MO 65211, U.S.A.). As specificity controls, B. bovis-KBb Mexican isolate DNA, Anaplasma marginale DNA and bovine white cell DNA were included on each illter. The isolated DNA was applied to nitrocellulose filters with a slot blot apparatus (Hybri-Slot, BRL ). The filters were processed as previously described.
Evaluation of candidate plasmid probe by Southern blotting DNA was prepared from a Kenyan isolate of B. bigemina DNA. DNA preparations were digested with BamHI/BstEII, HindIII and EcoRI. In addition, as specificity controls, bovine DNA, A. marginale DNA (Kenyan isolate), Theileria parva DNA, Theileria mutans DNA and P. falciparum DNA were digested with EcoRI. The digests were separated on a 1.0% agarose gel and the DNA was transferred to a nylon membrane. The membrane was exposed to UV light for 2 min and then pre-hybridized (5 ×Denhart's solution with 100 zg m1-1 salmon sperm DNA) for 4 h at 68°C. The p16 plasmid was labeled with 32p by nick translation. The hybridization solution (Denhardt's solution with 9% dextran sulfate) contained 100 ng of the probe solution with a specific activity of 10Gcpm ml- '. The filters were incubated overnight at 68 ° C, washed at high stringency, dried and exposed for autoradiography.
Detection of infected erythrocytes In vitro cultures of B. bigemina and B. bovis were used as the source of infected erythrocytes. The percent of infected erythrocytes in B. bigemina cultures was 0.7% and in B. boris cultures was 8.0%. The whole cultures (approx-
REPETITIVE DNA PROBE FOR BABESIA BIGEMINA
15
imately 106 bovine erythrocytes p l - 1 ) were diluted in phosphate buffer saline to the desired concentration of infected erythrocytes. The plasmid (p16) was labeled with 32p and hybridized with nitrocellulose or nylon filters spotted with various concentrations of B. bigemina (1.2 X 104-1.8X 101) and B. bovis (6.5 X 104-1 X 102 ) infected erythrocytes. Controls consisted of isolated DNA from B. bigemina, B. bovis and A. marginale. The filters were placed in a commercial filter holder (Hybrid-Ease, Hoefner Sci. Inst., San Francisco, CA 94107, U.S.A. ). The pre-hybridization conditions consisted of a solution containing 50% deionized formamide, 10% dextran sulfate, 1% SDS and 1 M NaC1, and 20 pg m l - 1 of boiled, sheared salmon sperm DNA. The apparatus was placed in a 42 °C water-bath for 5 h. The labeled probe (specific activity > 10s cpm pg- 1) was denatured by boiling in a solution containing carrier DNA. The probe was immediately placed on ice for 10 min and then added to the apparatus. The filters were hybridized at 42 ° C for 18-24 h. The hybridization mix was removed and the filters washed with 2 X SSC at room temperature (20-40 rinse cycles). The filters were then washed with 2×SSC, 1% SDS at 65°C. The apparatus was placed in a 65°C water-bath for 30 min and then washed (20-40 rinse cycles). This procedure was repeated twice. Next the apparatus was placed at room temperature and the filters washed twice (20-40 cycles) with 0.1 X SSC at room temperature. The apparatus was left undisturbed for 30 min between the washings. The filters were then dried, placed on cardboard or covered with plastic wrap and processed for autoradiography as previously described.
p16 restriction map Isolated p16 plasmid DNA was digested with a selected group of restriction endonucleases. The following endonucleases were used in single or double digestion experiments: EcoRV, BamHI, BanII, SphI, PvuI, PvuII, PstI, BsmI, NdeI, XmaIII, AatII, DraI, HincII, Tth111I, EcoRI, HindIII, AvaI, SalI, MluI, SmaI, SstI, XbaI, ApaI, BglII, HpaI, KpnI, BstEII, XhoI. Isolation of the Babesia bigemina DNA insert from p16 Purified p16 (5 pg) was double digested with BamHI and BstEII for 3 h at 37°C. The reaction was stopped and samples run on a mid-size 0.8% agarose gel at 25 V overnight. Gel plugs containing the large fragment representing the B. bigemina insert were removed. The gel plugs were processed by the procedure of Benson (1984) to isolate the DNA. RESULTS
Construction of Babesia bigemina DNA library By counting the colonies on 10 ampicillin agar plates, it was calculated that transformation rate of E. coli strain HB101 was 3.6X 10Gcolonies pg-1 of B.
16
G.M. BUENING ET AL.
bigemina DNA. The primary library consisted of approximately 1.44× 10:' transformants. Selection of repetitive plasmids The specific activity of the 32p labeled probe made by nick translation of the isolated genomic B. bigemina DNA was 2.44 × l0 s cpm ~g 1 DNA. Sixteen colonies which gave strong signals by hybridization were selected; all 16 grew in ampicillin broth and plasmids were isolated. Four candidate plasmids were selected from the 16 based upon fragment size of agarose gel electrophoresis of HindIII and SalI digest and intensity of hybridization signal with the Southern blot. Plasmids pl, p5, p9 and p16 were amplified and extracted to test as candidate B. bigemina DNA probes.
Detection of isolated D NA The specific activity of the plasmid DNA probes (pl, p5, p9 and p16) was > l0 s cpm #g- 1. One-microliter aliquots containing various concentrations of DNA (100 zg-10 pg) were spotted on nitrocellulose filters. The DNAs tested were isolated from: B. bigernina, B. boris, Trypanosoma brucei and bovine white cell. The four plasmid probes hybridized only with B. bigemina DNA and would detect at least 10 pg of B. bigemina DNA (data not shown). The p16 plasmid was selected based upon the intensity of the hybridization signal and growth of the parent colony. In the experiment where DNA was extracted from B. bigemina infected erythrocyte suspensions containing known parasitemias, the hybridization procedure detected as few as 103 infected erythrocytes. The p16 probe appeared to react with the four isolates tested with equal sensitivity (data not shown). There was minimum background with DNA from 106 B. bovis infected erythrocytes. There was no hybridization with purified DNAs from A. marginale, bovine white cells or B. boris.
Evaluation of candidate plasmid probe by Southern blotting The probe hybridized only with restriction fragments derived from B. bigemina DNA (Fig. 1). It can be observed in Lane 2, that at least 18 reiterated sequences were formed by BamHI/BstEII digestion. When HindIII or EcoRI endonucleases were used for digestion of genomic B. bigemina at least 16 fragments were observed. The presence of numerous bands indicates that repetitive sequences are present in the B. bigemina genome and the p16 clone.
REPETITIVEDNAPROBEFORBABESIA BIGEMINA
17
].
Fig. 1. Southern blot analysis of genomic DNAs digested with endonucleases and hybridized against the p16 32p labeled probe. Lane 1 = molecular weight markers 2-phage DNA. Lane 2 =B. bigemina (Kenya) digested with BamHI/BstEII. Lane 3 =B. bigemina (Kenya) digested with HindIII. Lane 4 = B. bigemina (Kenya) digested with EcoRI. Lane 5-- Bovine DNA digested with EcoRI. Lane 6=A. marginale digested with EcoRI. Lane 7-- T. parva digested with EcoRI. Lane 8-- T. mutans digested with EcoRI. Lane 9 =P. [alciparum digested with EcoRI. Lane 10 = molecular weight markers 2-phage DNA. Note that hybridization signals are observed only with bands of B. bigemina DNA.
Detection of infected erythrocytes Plasmid p16 DNA probes were constructed by labeling with 32p by the nick translation or random primer techniques. Both labeling techniques resulted in probes of equivalent sensitivity. The constructed probes hybridized only with B. bigemina infected erythrocytes and not with B. boris infected erythrocytes (Fig. 2). It should be stressed that each spotted area of B. bigemina and B. boris contained a ratio of at least nine non-infected to one infected bovine erythrocyte. No hybridization was observed in the B. boris infected row which contained an equivalent number of bovine erythrocytes as the B. bigemina infected row. The hybridization signal decreased in intensity, possibly not in
18
G.M. BUENINGET AI..
Sensitivity of DNA probe Babesia bigemina 120(O
1200
600
300
150
75
37
18
65000
6500
3250
1625
812
406
203
102
B. bigemina iPd3C's
B. bovis iRBC's Fig. 2. Dot blot hybridization assay of plasmid p 16 DNA probe labeled with 32p. The concentration of infected erythrocytes is indicated by the number above the columns. It should be emphasized that each dot contained a ratio of greater than nine normal erythrocytes to one infected erythrocyte. The first row contains various dilutions of B. bigemina infected erythrocytes and the second row B. bovis infected erythrocytes. The autoradiograph shows specificity for B. bigemina and the signal intensity varies with the concentration of infected erythrocytes.
Plasmid p 1 6
Sall EcoRI Aval I ~ I I. ]1 B.~HL Hind.III -Nincll I
BstEll 0 ,.
10.7 K b
I i Th__~t Ill
I
pBr~Z2 l 0 ,
Babesia I)~gemina Insert
6.3 K b Fig. 3. A partial restriction map of the plasmid (p16) It should be noted that the majority of the insert can be removed by a double digest of BstEII and BamHI. In addition there were no sites detected between the ThtIII and AvaI site.
proportion, with the dilution of B. bigemina infected erythrocytes. However, repeatable experiments demonstrated a sensitivity to detect as few as 150 infected erythrocytes (Fig. 2).
Characterization o[ a selected plasmid by constructing a restriction map The inserted B. bigemina D N A was determined to be approximately 6.3 kb in size. A restriction map was constructed and the location of restriction sites
REPETITIVE DNA PROBE FOR BABESIA BIGEMINA
19
for the following endonucleases were determined: BstEII, Tth111I, AvaI, HindIII, SalI, HincII, BamHI. There was approximately a 4.8 kb segment that did not cut with any of the nucleases tested. It has been possible to cut the insert out of the plasmid using BamHI and BstEII. These two restriction sites were located close to the ends of the insert (Fig. 3). This fragment has been labeled with 32p and achieved similar sensitivity compared with the plasmid p16 probe (data not shown). DISCUSSION
Genomic B. bigemina DNA was used to construct a genomic library in the plasmid vector pBR322. Transformed E. coli HB101 containing inserts of B. bigemina DNA were selected by hybridization with genomic DNA. The candidate probe hybridized only with B. bigemina DNA and not with DNA or infected erythrocytes from B. bovis, A. marginale, P. falciparum, Trypanosoma brucei, Theileria parva or Theileria mutans. These observations document the specificity of the probe. In addition, the probe hybridized with at least 16 repetitive DNA families of genomic B. bigemina. The Southern blotting analysis (Fig. 1 ) probably underestimated the number of repetitive families. At the top of the gel, large poorly resolved fragments were observed. DNA probes that would be candidates to be used as diagnostic aids should hybridize with isolates from different areas of the world. The present candidate probe hybridized with isolates from Texas, Mexico, Puerto Rico, St. Croix, Costa Rica and Kenya. This would indicate that this probe could be used in world-wide epidemiological studies. The candidate DNA probe appeared to be highly sensitive in that it would detect 10 pg ofB. bigemina DNA and as few as 150 infected erythrocytes. This probe could be used in epidemiological studies to detect carrier animals. In a recent study Eriks et al. (1989) characterized an A. marginale nucleic acid probe to detect carrier animals, which was specific and could detect 10 pg of genomic DNA and 500-1000 infected erythrocytes in 0.5 ml of blood. Previous reports by McLaughlin et al. (1986) concerning DNA probes for detecting B. bovis DNA indicated that the probes detected 100 pg of B. boris DNA which corresponded to a parasitemia of 0.01%. In epidemiological studies it would be desirable to have a nucleic acid probe which would detect carrier animals that would have a very low level of parasitemia. However, with the development of the polymerase chain reaction (PCR) the presence of specific DNA in a sample can be amplified up to 200 000-fold (Kogan et al., 1987). It may be possible to adapt PCR technology for use with the p16 probe. Therefore one of the major requirements for a diagnostic probe would be its specificity for the target organism and ability to react with many different isolates. The candidate probe described in this report fulfills these requirements.
20
G.M.BUENINGET AL.
ACKNOWLEDGMENTS
We thank Karen McLaughlin for excellent technical assistance and Ellen Swanson for typing the manuscript. The Kenyan isolates of Theileria, Anaplasma and Babesia were provided by S.P. Morzaria and Plasmodium by B. Khan. This investigation was supported in part by Burroughs-Wellcome Fund; U.S. Agency for International Development Grant DPE-5542-G-SS-4026-00, and USAID Prime Agreement No. DAN 4178-A-00-7056-00.
REFERENCES Benson, S.A., 1984. A rapid procedure for isolation of DNA fragments from agarose gels. Biotechniques, 2 (March/April). Eriks, I.S., Palmer, G.H., McGuire, T.C., Allred, D.R. and Barbet, A.F., 1989. Detection and quantitation of A naplasma marginale in carrier cattle by using a nucleic acid probe. J. Clin. Microbiol., 27: 279-284. Franzen, L., Westin, G., Shabo, R., Aslund, L., Perlmann, H., Persson, T., Wigzell, H. and Pettersson, U., 1984. Analysis of clinical specimens by hybridization with probe containing repetitive DNA from Plasmodium [alciparum. Lancet, 8376 (i): 525-528. Hanahan, D., 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166: 557. Kogan, S.C., Doherty, M. and Gitschier, J., 1987. An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences: application of hemophilia A. N. Engl. J. Med., 317: 985-990. Maniatis, T., Fritsche, E.F. and Sambrook, J., 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory. McLaughlin, G.L., Edlind, T.D., Campbell, G.H., Eller, R.F. and Ihler, G.M., 1985. Detection of Plasmodium falciparum using a synthetic DNA probe. Am. J. Trop. Med. Hyg., 34 (5): 837 840. McLaughlin, G.L., Edlind, T.D. and Ihler, G.M., 1986. Detection of Babesia boris using DNA hybridization. J. Protozool., 33 (1): 125-128. McLaughlin, G.L., Collins, W.E. and Campbell, G.H., 1987. Comparison of genomic plasmid, synthetic and combined DNA probes for detecting Plasmodium [alciparum DNA. J. Clin. Microbiol., 25:791 795. National Research Council, 1982. Priorities in Biotechnology Research for International DeveL opment Proceedings of a Workshop. Directed by the Board on Science and Technology h)r International Development, Office of International Affairs, National Academy Press, Washington, DC. Vega, C.A., Buening, G.M., Green, T.J. and Carson, C.A., 1985. In vitro cultivation of Babesia bigemina. Am. J. Vet. Res., 46: 416-420. Vega, C.A., Buening, G.M., Rodriguez, S.D. and Carson, C.A., 1986. Concentration and enzyme content of in vitro-cultured Babesia bigemina-infected erythrocytes. J. Protozool. 33:514 518.
