1000070109510

3989

3990

3991

3992

3993

3994

70109510

9000014483774

90231373

Matsuba, Takashi

Okabe, Tatsuji

Onoe, Sadao

Onuma, Misao

read0166752

read0166758

Sasaki, Norimasa

Sugimoto, Chihiro

Tanaka, Masayuki

Yonemichi, Hiromi

English

杉本, 千尋

OAK Obihiro University Archives of Knowledge

34 J. Protozool. Res., 2, 34-39 (1992) Copyright © 1992, Research Center for Protozoan Molecular Immunology Biotin-labeled Genomic DNA Probe for the Detection of Theileria sergenti and its Nucleotide Sequence  MASAYUKI TANAKA1,2 ,TAKASHI MATSUBA3, SADAO ONOE4,HIROMI YONEMICHI4, TATSUJI OKABE2, NORIMASA SASAKI2,CHIHIRO SUGIMOTO1, and MISAO ONUMA11Department of Epizootiology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido 060, 2Division of Veterinary Microbiology, Kyoto Biken Laboratrories, Uji, Kyoto 611, 3Department of Veterinary Microbiology, School of Veterinary Medicine, Rakunou Gakuen University, Ebetsu, Hokkaido 069, 4Hokkaido Prefectuaral Shintoku Animal Husbandry Experimental Station, Shintoku, Hokkaido 081, JAPAN Recieved 17 May 1991 / Accepted 28 October 1991  Key words: DNA probe diagnosis, nucleotide sequence, Theileria sergenti Bovine theileriosis is economically one of the most important tick-borne protozoan parasite disease of grazing cattle in Japan. It is caused by Theileria sergenti, and brings about mild hyperthermia and anemia in cattle. The diagnosis of T. sergenti has routinely relied upon laborious microscopical examination of Giemsa-stained blood smears. Recent advances in molecular biology have made it possible to develop diagnostic assays that use nucleic acid probes to detect pathogens directly, specifically and sensitively in clinical specimens. The utility of nucleic acid probes for routine diagnosis of pathogens has been limited by the use of radiolabeled probes for their detection. However, this limitation has been overcome by the development of non-radioactive detection systems (Langer et al. 1981, Leary et al. 1983). We previously reported the molecular cloning of genomic DNA of T. sergenti (Kajiwara et al. 1990). One of the cloned genomic DNA fragments (pTs-11-D1) was used to detect T. sergenti in a dot blot hybridization assay (Hirano et al. 1991). This pTs-11-D1 probe did not react with DNA of Babesia ovata or Anaplasma centrale by dot blot and Southern hybridization as reported by Hirano et al.(1991). However, the same probe hybridized to DNA of Theileria buffeli which may be taxonomically related to T. sergenti (Matsuba et al. 1991). In the present study, we evaluated a dot blot hybridization assay using a genomic DNA pTs-11-D1 probe for the detection of T. sergenti as a practical diagnostic method. The nucleotide sequence of this probe is also described. T. SERGENTI GENOMIC DNA PROBE We compared the dot blot hybridization using a pTs-11-D1 probe for T. sergenti with routine microscopic diagnosis. A splenectomized calf was infected with T. sergenti (Chitose stock) by nymphal ticks of Haemophysalis longicornis which had been infected with T. sergenti. The calf was kept under daily clinical and hematological observations and treated with blood transfusion or anti-parasite drug if it showed severe anemia. The piroplasm DNA was prepared from parasitized erythrocytes as described previously (Kajiwara et al. 1990) with slight modifications; infected erythrocytes were lysed by Aeromonas hydrophila hemolysin (Sugimoto et al. 1990) instead of 0.2% NaCl, and purified parasites were digested with proteinase K (0.3 mg/ml) instead of pronase E. The DNA was spotted on a sheet of cellulosenitron (E) filter (Schleicher & Schuell, F. R. G.), denatured in 0.5N NaOH, and fixed onto the membrane by cross-linking with short wavelength ultraviolet light. Genomic DNA pTs-11-D1 probe was labeled with biotin 16-dUTP (Boehringer, USA) by random priming (Feiberg and Vogelstein 1983). Hybridization was performed as described previously (Kajiwara et al. 1990). The hybridization solution containing 4x SSPE (0.6M NaCl, 0.04M NaH2PO4 and 4mM EDTA) , 10x Denhardt's solution (0.2% Ficoll, 0.2% polyvinylpyrrolidone and 0.2% bovine serum albumin), 150 µg/ml salmon sperm DNA, and 50% formamide was used to prehybridize filters at 42 OC for 2 h. After prehybridization, the buffer was removed and fresh hybridization solution was added with the biotinylated probes. After incubation at 42 OC for 18 h, the filters were washed three times at room temperature for 5 min and twice at 55 OC for 15 min each in 0.2x SSC (0.03M NaCl and 3mM sodium citrate) containing 0.1% SDS. The biotinylated probes were visualized using the BluGENE nonradioactive nucleic acid detection system (BRL, USA). T. sergenti piroplasms were detected at day 11 after inoculation by the microscopical examination of Giemsa-stained blood smear. T. sergenti DNA was first detected at day 7 after inoculation by using pTs-11-D1 probe (Table 1.).  Table 1. Detection of Theileria sergenti from calf erythrocytes  T. SERGENTI GENOMIC DNA PROBE Since clone pTs-11-D1 was derived from DMA of T. sergenti (Chitose stock) which was isolated in Hokkaido, we examined the reactivity of the DNA probe with DNA of T. sergenti isolates from 11 geographically different districts in Japan. Purified piroplasm DNA was applied onto the membrane with HYBRI-SLOTTM MANIFOLD (BRL, USA) and probed with pTs-11-D1. The probe hybridized with DNA from all the isolates at a similar intensity. This result suggested that the sequence detected by the DNA probe is conserved among the T. sergenti isolates in Japan (Fig. 1.). The probe did not hybridized with DNA prepared from bovine erythrocyte infected with Babesia ovata or Anaplasma centrale under the same hybridization condition (data not shown).  Fig. 1. Reactivity of pTs-11-D1 for DNA of T. sergenti isolates from geographically different districts. Each piroplasm DNA was extracted from purified parasites and 0.1-0.2 µg applied onto the membrane. The filters were hybridized with biotin 16-dUTP-labeled pTs-11-D1. Slots 1 to 12 correspond to T. sergenti isolates collected from 1; Hokkaido (Chitose), 2; Hokkaido (Hiyama), 3; Hokkaido (Oshima), 4; Hokkaido (Shintoku), 5; Hokkaido (Abashiri), 6; Aomori, 7; Fukushima, 8; Tochigi, 9; Shizuoka, 10; Okayama, 11; Fukuoka, 12; Kagoshima.  As the pTs-11-D1 fragment hybridized with more than 14 BamH I restriction fragments of T. sergenti genomic DNA, it detects a repetitive sequence of the genome (Hirano et al. 1991). Furthermore, it hybridized with a 6 kb fragment of piroplasm mRNA by Northern blotting (unpublished observation). To elucidate whether there are internal repeats within the probe sequence as known in diagnostic DNA probes of other parasitic organisms (Barker 1990, Klion et al. 1991), and whether this DNA fragment of 2 kbp in length contains protein coding region, we sequenced the pTs-11-D1 fragment. Clone pTs-11-D1 was subcloned into the Bluescript KS+ vector and deletion clones were generated for dideoxy sequencing (Henikoff 1984). Sequencing by the dideoxy method was performed using [α-32P]dCTP and 7-deaza-sequencing kits (TOYOBO Co., Ltd., Japan). Sequence analysis verified the cloned fragment to be 1,998 base pairs in length (Fig. 2.). The A/T content was 63%, and the G/C content was 37%. The pTs-11-D1 did not contain portions of a long open reading frame in both strands (Fig. 3.). No internal repeats or inverted repeats were recognized. Significant homology was not detected with sequences stored in the GenBank data bases using the DNASIS program (Hitachi Software Engineering Co., Ltd., Japan). Recently, a genomic DNA encoding a 41 kDa protein of Plasmodium falciparum was cloned and sequenced (Knapp et al. 1991). T. SERGENTI GENOMIC DNA PROBE Fig.2. Nucleotide sequence of clone pTs-11-D1. clone pTs-11-D1 was subcloned into Bluescript KS+ vector and deletion clones were generated for dideoxy sequencing.  Fig. 3. The occurrence of initiation (ATG) and termination (TAG, TGA, TAA) codons of both strands. Nucleotide numbers are shown at the bottom. Vertical lines initiation (above the horizontal lines) and termination (under the horizontal lines) codons in three frames in both strands. T. SERGENTI GENOMIC DNA PROBE The gene consists of 9 exons with short open reading frames coding for 23 to 109 amino acids, respectively. If the probe DNA of T. sergenti is a part of a gene carrying several intervening sequences, disagreement between our sequence data indicating that the sequence does not contain long open reading frames, and the result of Northern blot analysis could be explained. To elucidate this, cDNA library of T. sergenti should be screened with this DNA probe and DNA sequence of the transcript should be compared with that of genomic DNA presented in this paper. In conclusion, the use of the pTs-11-D1 DNA probe is practical for early diagnosis of T. sergenti infection with high sensitivity and specificity. As the probe hybridized with DNA of T. sergenti isolates collected from various regions in Japan, it is also useful for epidemiological studies of T. sergenti infection in Japan. Furthermore, the use of DNA probes will elucidate taxonomical relationships among benign Theileria species, T. sergenti/buffeli/ orientalis, which are distributed in Asian and European countries and Australia (Stewart et al. 1988, Uilenberg et al. 1985), at the molecular level.  ACKNOWLEDGMENTS :We are grateful to Dr. S. Mori, Bayer Japan Ltd., for arranging the collection of T. sergenti-infected blood from different districts in Japan.  REFERENCES :Barker, R. H. 1990. DNA probe diagnosis of parasitic infections. Exp. Parasitol. 70: 494-499. Feinberg, A. P. & Vogelstein, B. 1983. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13 Henikoff, S. 1984. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28: 351-359. Hirano, A., Kirisawa, R., Matsuba, T., Komatsu, R., Tanaka, M., Takahashi, K., Kawakami, Y. & Onuma, M. 1991. Evaluation of high sensitive DNA probe for the detection of Theileria sergenti infection in cattle. J. Vet. Med. Sci. 53: 933-935. Kajiwara, N., Kirisawa, R., Onuma, M. & Kawakami, Y. 1990. Specific DNA probe for the detection of Theileria sergenti infection in cattle. Jpn. J. Vet. Sci. 52: 1199-1204. Klion, A., D., Raghaven, N., Brindley, P., J. & Nutman, B. 1991. Cloning and characterization of a species-specific repetitive DNA sequence from Loa loa. Mol. Biochem. Parasitol. 45: 297-306. T. SERGENTI GENOMIC DNA PROBE Knapp, B., Nau, U., Hundt, E. & Küpper H. A. 1991. Demonstration of alternative splicing of a pre-mRNA expressed in the blood stage form of Plasmodium falciparum, J. Biol. Chem. 266:7148-7154. Langer, P. R., Waldrop, A. A. & Ward, D. C. 1981.  Enzymatic synthesis of biotin-labeled polynucleotides: Novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. USA. 78: 6633-6637. Leary, J. J., Brigait, D. J. & Ward, D. C. 1983. Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots. Proc. Natl. Acad. Sci. USA. 80: 4045-4049. Matsuba, T., Kawakami, Y., Iwai, H. & Onuma, M. 1991. Genomic analysis of Theileria sergenti stocks in Japan with DNA probes. Vet. Parasitol. (in press) Stewart, N. P., Devos, A. J., McGregor, W. & Shiels, A. 1988. Observations on the development of tick-transmitted Theileria buffeli (syn T. orientalis ? ) in cattle. Res. Vet. Sci. 44: 338-342. Sugimoto, C., Sato, M., Kawazu, S., Kamio, T. & Fujisaki, K. 1991. Purification of merozoites of Theileria sergenti from infected bovine erythrocytes. Parasitol. Res. 77: 129-131. Uilenberg, G., Perie, N. M., Spanjer, A. A. M. & Franssen, F. F. J. 1985. Theileria orientalis, acosmopolitan blood parasite of cattle: demonstration of the schizont stage. Res. Vet. Sci. 38: 352-357.  Address corespondence and reprint requests to Dr. Misao Onuma, Department of Epizootiology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo/ Hokkaido 060, Japan.  

Biotin-labeled Genomic DNA Probe for the Detection of Theileria sergenti and its Nucleotide Sequence

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Biotin-labeled Genomic DNA Probe for the Detection of Theileria sergenti and its Nucleotide Sequence

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