Veterinary Parasitology, 42 (1992) 189-198 Elsevier Science Publishers B.V., Amsterdam

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A specific DNA probe which identifies Babesia bovis in whole blood Wasana Petchpooa, Peerapan Tan-ariyab, Vichai Boonsaengc, Chariya R. Brockelman b, P r a p o n Wilairat c and Sakol P a n y i m c aDepartment of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok 10110, Thailand bDepartment of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand CDepartmentof Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand (Accepted 15 November 1991 )

ABSTRACT Petchpoo, W., Tan-ariya, P., Boonsaeng, V., Brockelman, C.R., Wilairat, P. and Panyim, S., 1992. A specific DNA probe which identifies Babesia bovis in whole blood. Vet. Parasitol., 42:189-198. A genomic library of Babesia bovis DNA from the Mexican strain M was constructed in plasmid pUN 121 and cloned in Escherichia coli. Several recombinants which hybridized strongly to radioactively labeled B. boris genomic DNA in an in situ screening were selected and further analyzed for those which specifically hybridized to B. bovis DNA. It was found that pMU-B 1 had the highest sensitivity, detecting 25 pg of purified B. bovis DNA, and 300 parasites in 10/d of whole infected blood, or 0.00025% parasitemia, pMU-BI contained a 6.0 kb B. bovis DNA insert which did not crosshybridize to Babesia bigernina, Trypanosorna evansi, Plasmodiumfalciparum, Anaplasma marginale, Boophilus microplus and cow DNA. In the Southern blot analysis of genomic DNA, pMU-B 1 could differentiate between two B. bovis geographic isolates, Mexican strain M and Thai isolate TS4. Thus, the pMU-B 1 probe will be useful in the diagnosis of Babesia infection in cattle and ticks, and in the differentiation of B. bovis strains.

INTRODUCTION

Recent developments in biotechnology, particularly genetic engineering and techniques of DNA hybridization, have provided new tools for differential diagnosis of human hemoprotozoan diseases. For example, nucleic acid probes have been developed for the detection of malaria (Barker et al., 1986; Boonsaeng et al., 1989) and leishmaniasis (Kennedy, 1984). This novel methodology for identification of parasites will also have a significant impact in the Correspondence to: Dr. C.R. Brockelman, Department of Microbiology, Faculty of Science, Mahidol University, R a m a VI Road, Bangkok 10400, Thailand. Fax Number: 66 2 247 7051.

© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00

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diagnosis of cattle diseases as herds are generally comprised of a large number of animals which cannot be pre-screened for overt signs of infection. Thus far, recombinant DNA technology has been applied to the development of diagnostic probes for bovine trypanosomiasis (Viseshakul and Panyim, 1990 ), anaplasmosis (Goff et al., 1988), and babesiosis (McLaughlin et al., 1986; Posnett and Ambrosio, 1989; Buening et al., 1990; Jasmer et al., 1990). Bovine babesiosis is caused by protozoan parasites of the genus Babesia, namely Babesia bovis and Babesia bigemina. Babesia bovis is more virulent than B. bigemina and the former species is also less sensitive to some babesiacidal compounds (Brockelman and Tan-ariya, 1991 ) making it difficult to radically cure the infected animals. The two parasite species cannot be readily distinguished by conventional microscopic methods, and failure to identify parasite species accurately poses a constraint in chemotherapeutic treatment. In view of the fact that babesiosis is one of the major hindrances to the development of the cattle industry in Thailand, as well as other developing countries in Asia (Brockelman, 1989), Africa and South America (Thompson, 1979; Bram, 1983) there is a need for a specific, sensitive and simple method for detection of the parasite. We describe the construction of a DNA probe which could specifically identify B. bovis infection. The probe was sensitive enough to detect low parasitemia in whole infected blood without requiring laborious procedures for DNA extraction. MATERIALS AND METHODS

Parasites and DNA isolation Babesia bovis parasites used in this study included a Mexican strain M (Rodriguez et al., 1986) and two Thai isolates, TS2 and TS4 (Brockelman and Tan-ariya, 1991 ). Continuous cultivation procedures to maintain parasite stocks have been described elsewhere (Brockelman and Tan-ariya, 1991 ). Cultures harboring 8-10% parasitemia were pooled and washed twice in VyM buffer (Vega et al., 1985) by repeated centrifugation at 4°C. The packed erythrocytes were lyzed with 1% acetic acid containing 1% Triton X- 100. The released parasites were centrifuged at 400 X g and the pellets were digested with 500/tg m l - ~proteinase K solution in 10 mM Tris-HC1, pH 8.0, containing 2% sodium dodecyl sulfate (SDS), at 37 ° C for 15-18 h. The solution was subjected to three extractions each of phenol, chloroform and ether. The aqueous phase was treated with RNase, followed by phenol-chloroform extractions as described above. DNA was precipitated by ethanol, air dried then redissolved in 10 mM Tris-HC1, pH 7.4, and 1 mM ethylenediaminetetraacetic acid (EDTA) (TE buffer) and stored at 4°C until use.

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Construction ofB. bovis library Endonuclease digestion of B. boris DNA (Mexican strain M) was carried out in a reaction mixture which contained 5/zg of DNA, 600 units of EcoR I, and 6% glycerol in a low salt buffer. After 3 h of incubation at 37°C, the solution was extracted with phenol and DNA precipitated. The DNA pellet was washed with 70% ethanol, dissolved in TE buffer and analyzed by agarose gel electrophoresis. The B. bovis DNA fragments were ligated with EcoR Idigested plasmid pUN 121, at a B. bovis DNA fragments to vector ratio of 1 : 1, and employing 1 unit of T 4 DNA ligase. Escherichia coli JM 107 was transformed using 100-300 ng of recombinant plasmid and selected on tetracycline LB-agar plates at 37 °C overnight.

Selection of colonies containing plasmids with B. bovis insert Colonies grown on tetracycline plates were individually transferred onto nitrocellulose (Schleicher and Schuell, Gmbh, Dassel, Germany) or nylon membranes (Gene screen plus, NEN, Boston, MA) in duplicate and one set of filters was hybridized with B. bovis DNA labeled with 3ap-dATP by nick translation to a specific activity of 3 X 107 cpm/tg- 1 DNA (Rigby et al., 1977 ). Prehybridization and hybridization procedures were both carried out at 42 ° C in a buffer containing 50% formamide, 5 × SSC ( 1 × SSC: 150 mM NaC1, 15 mM trisodium citrate, pH 7.5 ), 5 × Denhardt's solution (50 X Denhardt's solution: 1% Ficoll 400, 1% PVP, 1% BSA fraction V), 5 mM EDTA, 20 mM Tris-HC1, pH 8.0, 2% SDS (for nylon ) and 100/tg ml-1 heat-denatured sonicated salmon sperm DNA. Hybridized filters were washed twice in 0.1 × SSC containing 0.1% SDS at 55 °C for 30 min, air dried and autoradiographed at - 7 0 ° C on Kodak XOMAT film.

Detection ofB. bovis DNA by pMU-B1 probe pMU-B1 was selected for use as a probe in detection ofB. bovis by dot blot hybridization technique using either purified B. bovis DNA or B. bovis infected bovine blood. The concentration of purified parasite DNA was estimated by measuring absorbance at 260 nm. DNA dilution was made by using TE buffer containing 50 ng m l - ~ of sonicated salmon sperm DNA. The diluted DNA samples were spotted in duplicate onto nylon membranes, then hybridized with pMU-B 1 probe which had been labeled by the multiprime labeling method (Feinberg and Vogelstein, 1983) to a specific activity of 4 X 108 cpm/tg- 1 DNA. For detection of B. bovis in infected blood from cultures, isolates TS2 and TS4 collected from Tong Song, South Thailand, and a Mexican strain M, were used. The number of parasites in infected blood was estimated by microscopy, and then the sample was serially diluted using whole

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normal bovine blood. After dilution, the number of parasites in each diluted sample was reconfirmed by microscopy. Four 10-/zl samples were taken and counted in a Coulter counter which yielded a mean of 6 X 107 red blood cells per 10/zl. DNA from other blood protozoan parasites (Babesia bigemina, Trypanosoma evansi, Anaplasma marginale, Plasmodium falciparum ), and host DNA (tick and cow) were spotted in duplicate onto nylon membranes and were hybridized with radioactively labeled pMU-B 1 probe.

Evaluation of pMU-B1 probe by Southern blot hybridization Genomic DNA of B. bovis, Mexican strain M and Thai isolate TS4, were digested to completion with Rsa I, Hinf I, Nde I and Hha I, respectively, at 37°C for 3 h. Digested DNA was electrophoretically separated in a 0.6% agarose gel at 40 V for 15 h, transferred onto nylon membranes using a vacuum blotting apparatus and hybridized with pMU-B 1 probe (specific activity of 3 X 108 cpm ltg- 1 ) overnight at 42 ° C. The filter was washed, air dried and autoradiographed.

pMU-B1 restriction map pMU-B 1 plasmid was digested with various restriction enzymes and analyzed by agarose gel electrophoresis. The following endonucleases were used in single or double digestion experiments: Acc I, Ava I, Ava II, BamH I, Cla I, Cfo I, EcoR I, Hind III, Kpn I, Mbo I, Nae I, Nde I, Sau 961, Spe I, Sst I, Xba I and Xho I. RESULTS

Construction ofB. bovis DNA library Recombinant plasmids were constructed by ligation of EcoR I-digested B. bovis DNA (Mexican strain M ) into EcoR I site of p U N 121 and transformed into E. coli JM 107. By counting the colonies grown on 17 tetracycline agar plates, it was calculated that the transformation efficiency was 1 X 104 colonies per microgram of B. bovis DNA. The library consisted of approximately 3.7 × 10 3 transformants.

Selection of recombinant plasmids To detect plasmids containing B. bovis repetitive DNA sequences, colonies were first screened by a colony hybridization assay using radiolabeled genomic B. bovis DNA probe. Thirty-three colonies which gave a strong hybridization signal were selected. They were individually grown in tetracycline broth

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and plasmids were isolated. Ten candidate plasmids were then selected based upon fragment size and intensity of Southern blot hybridization signal. These plasmids were then labeled with 32p-dATP by nick translation and hybridized with B. boris, B. bigemina and cow DNA. Plasmid pMU-B1 gave the strongest and most specific hybridization signal and thus was selected for B. bovis detection.

Detection of isolated DNA using pMU-B1 probe One-microliter aliquots containing various concentrations ofB. bovis DNA (0.001-5 ng) were spotted onto nylon membranes and hybridized with pMUB 1 probe. Figure 1 shows that the probe could detect 25 pg of purified B. bovis DNA. There was no cross-hybridization signal with DNA from Babesia bigemina ( 100 ng), Trypanosoma evansi (250 ng), Anaplasma marginale (2 X 106 parasites), Plasmodiumfalciparum (300 ng), cow (450 ng), and Boophilus microplus ( 1/ 10 of tick). Self-hybridization of pMU-B 1 gave a signal at 1.5

ng

M

C

5

Bbi

1

Te

.5

Am

.1

Pf

.05

Cw

.025

Bm

.010

1.50 pg

.001

0.75 pg

Fig. 1. Sensitivity and specificity of pMU-B 1 probe for detection of purified B. boris DNA. Purified DNA was spotted onto nylon membranes and hybridized with s2p-dATP labeled pMUB 1 probe (specific activity was 4 X 108 c p m / t g - l ). Filter was stringently washed at 55 ° C and exposed to X-ray film (Kodak XOMAT) overnight at - 7 0 ° C . M, Babesia bovis Mexican strain; C, Babesia bigemina (B bi) 100 ng, Trypanosoma evansi (Te) 250 ng, Anaplasma marginale (Am) 2 X 106 parasites, Plasmodiumfalciparum (Pf) 300 ng, cow (Cw) 450 ng and Boophilus microplus (Bm) 1/10 of tick; S, pMU-B1.

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pg which was equivalent to 100 pg of B. bovis DNA. Based on a genome size of 1.2 × 107 for B. boris (Posnett and Ambrosio, 1989) and 104 for pMU-B l, we estimated a copy number of 18 per haploid genome.

Detection of infected erythrocytes The pMU-B 1 probe was labeled with 32p-dATP to a specific activity of 2 × 108 cpm/zg- 1 and hybridized with B. bovis-infected erythrocytes obtained from continuous culture and directly spotted onto nylon membranes. Figure 2 shows that the pMU-B1 probe detected the three isolates of B. bovis (M, TS2, and TS4) with equal sensitivity. No hybridization signal was obtained with uninfected bovine blood (Row C). The probe could detect as little as 300 parasites in 10/zl of infected blood which is equivalent to approximately 150 infected erythrocytes because the majority of the infected cells harbored paired parasites or a parasitemia of 0.00025%.

Evaluation of pMU-B1 probe by Southern blotting To compare the Southern hybridization profile of pMU-B 1 between Mexican strain M and Thai isolate TS4, five restriction enzymes (EcoR I, Hha I, Hinf I, Nde I and Rsa I ) were used. EcoR I-digested DNA showed no differ-

M

TS2

TS4

ParasitesllO pl 3.0 XIO

5

7.5 XlO 4

4 1.9 Xl 0 4.7X1~

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3 2

C Fig. 2. Sensitivity of pMU-B 1 probe for detection of B. bovis-infected blood. Erythrocytes infected with Babesia bovis of Mexican strain M, and two Thai isolates TS2 and TS4, were serially diluted in normal whole bovine blood. The number of parasites per 10/d is indicated. Row C contained normal bovine blood ( 10 #l ). Filter was hybridized with 32p-dATP labeled pMU-B 1 probe (specific activity was 2 × 108 cpm #g- t ), washed and autoradiographed as described in the legend for Fig. 1.

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M TS4 Kb

...............

iii iliiil iii!

3.5,

1.5,

11.5, Fig. 3. Southern blot analysis of the pMU-B 1 sequence in genomic DNA of Babesia bovis of Mexican strain M and Thai isolate TS4. The purified genomic DNA ( 1.5/zg) was digested with Rsa I and hybridized with pMU-B1 probe (specific activity was 3 × l0 8 cpm/zg- ] ) in a Southern blot analysis. Numbers on the left refer to the size of DNA fragments based on Lambda Hind III size standards. ence in h y b r i d i z a t i o n patterns b e t w e e n these isolates whereas Rsa 1-digested D N A d e m o n s t r a t e d a hybridizable b a n d at 3.5 kb in the M e x i c a n strain, b u t this b a n d was absent in the T h a i isolate (Fig. 3 ). It was also noticeable t h a t the d o u b l e t b a n d at 1.5 kb was m o r e intense in the TS4 isolate. Thus, p M U B 1 could distinguish between isolates f r o m two different geographic locations. T h e h y b r i d i z a t i o n patterns resulting f r o m Hha I, Hinf I a n d Nde I digestions showed smear patterns.

Characterization of pMU-B1 by restriction endonuclease digestion T h e inserted B. bovis D N A was d e t e r m i n e d to be 6.0 kb in length. T h e locations o f restriction sites were a n a l y z e d a n d a restriction m a p was constructed (Fig. 4).

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EcoR I

T

Acc I

o"

.L Kpn I Nde I Acc I Nde I Soe I Nde I Nde I Acc I Avo I L

EcoR I

Fig. 4. Restriction map of pMU-B1 insert. The following enzymes did not digest pMU-BI: B a m H I, Cla I, Hind III, M b o I, Pst I, Pvu II, Sst I, Xba I, and Xho I.

DISCUSSION

Of recent attempts to develop diagnostic probes for Babesia boris, there are reports by McLaughlin et al. ( 1986 ) and Jasmer et al. ( 1990 ). The first group reported a DNA probe which detected 100 pg of B. boris DNA but showed cross-hybridization with 10 ng orB. bigemina. Jasmer et al. ( 1990 ) described two B. bovis DNA sequences which could be used as DNA probes, namely Bo25 and Bo6. Both sequences detected 100 pg of B. bovis DNA, but Bo6 cross-hybridized to 1.0 ng ofB. bigemina. Probe pMU-B 1 reported in this study detected 25 pg of purified B. boris DNA from homologous Mexican strain, pMU-B 1 did not cross-hybridize with B. bigemina DNA at any concentration tested, ranging from 1 to 100 ng. Based on the restriction map, pMU-B 1 was not related to other previously reported B. boris DNA sequences. Southern hybridization patterns using pMU-B1 could differentiate between isolates from Mexico and Thailand (Fig. 3 ), but that of the previously described B. boris probe (McLaughlin et al., 1986 ) was not reported. The use of repetitive DNA probes in the identification and comparison of different isolates has been reported for a number of other parasites

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( C h a m b e r s et al., 1986; K i m Lee Sim et al., 1986; F u c h a r o e n et al., 1988; J a s m e r et al., 1 9 9 0 ) . M o r e o v e r , the p M U - B 1 p r o b e has p o t e n t i a l for use in e n d e m i c areas b e c a u s e it c o u l d d e t e c t B. boris in whole i n f e c t e d blood. T h i s result is i m p o r t a n t for field a p p l i c a t i o n s w h e r e the D N A e x t r a c t i o n step can be o m i t t e d . H e n c e , the p r o b e can be u s e d directly o n b l o o d samples collected f r o m h e r d s s u s p e c t e d o f h a r b o r i n g B. bovis infection. ACKNOWLEDGMENTS T h e w o r k was s u p p o r t e d b y the Science T e c h n o l o g y D e v e l o p m e n t B o a r d o f T h a i l a n d , grant no. C P T 87A-1-05-006. We t h a n k Dr. V i c h i t r S u k h a p e s a n a a n d Dr. N o p p o r n S a r a t a p h a n , the N a t i o n a l A n i m a l H e a l t h a n d P r o d u c t i o n Institute, Dr. U s a C h e t a n o n d , S o u t h e r n D i a g n o s t i c Center, for supplies o f b o v i n e b l o o d a n d sera, a n d Dr. A n c h a l e e T a s s a n a k a j o n for helpful c o m m e n t s .

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