| Home | E-Submission | Sitemap | Contact us |  
Korean J Parasitol > Volume 35(2):1997 > Article

Original Article
Korean J Parasitol. 1997 Jun;35(2):111-117. English.
Published online Jun 20, 1997.  http://dx.doi.org/10.3347/kjp.1997.35.2.111
Copyright © 1997 by The Korean Society for Parasitology
Rapid detection of Theileria sergenti by polymerase chain reaction
E J Choi,1S W Kang,*1C H Kweon,1W S Jeong,1Y D Yoon,1 and H J Song2
1National Veterinary Research Institute, RDA, Anyang 430-016, Korea.
Received February 06, 1997; Accepted May 09, 1997.


Four separate pairs of oligonucleotide primers within the coding region in a T. sergenti 33-kDa surface protein gene were selected to detect T. sergenti by PCR. The specificity of PCR-amplified DNA was examined by digestion with restriction enzyme and Southern blot hybridization using T. sergenti p33 DNA probe. PCR appears to be specific for T. sergenti, without detectable signals from uninfected erythrocytes, uninfected bovine leukocytes and other hemoparasites, including A. marginale and B. ovata. Although 46 of 71 specimens (64.8%) from grazing cattle were microscopically positive, PCR in this study showed that 64 specimens (88.7%) were positive. Therefore, PCR proves a useful diagnostic tool for detecting T. sergenti-infected cattle. In addition, it is also revealed that PCR was significantly more sensitive than traditional microscopic examination using Giemsa's stain.


Fig. 1
Disign of primer sets for diagnosis on T. sergenti by PCR.

*Restriction endonuclease site with p33 gene is indicated

Fig. 2
Specificity of PCR product with C4 primer set by restriction enzyme (Msp I) digestion. M; 1Kb DNA ladder, lane 1; standard Korean isolate, lane 2; two fragments cutting by Msp I

Fig. 3
Specificity of PCR products by southern blot using digoxigenin-labeled standard T. sergenti p33 DNA (Korean isolate). a: PCR products using TS2 and TS4 primer, b: PCR products using TS1 and TS4, c: PCR products using TS1 and TS3 primer, d: PCR products using TS2 and TS3 primer. Lane 1, 4, 7, 10; standard Korean isolate, lane 2, 8, 12; No. 37, lane 3; No. 34, lane 5, 11; No. 42, lane 6, 13; No. 15, lane 9; No. 1, a; 365bps, b; 499bps, c; 381bps, d; 247bps.

Fig. 4
Specificity of PCR using primers with specimens from T. sergenii-uninfected leukocytes, A. marginale, and B. ovata. A:B. ovata,B:A. marginale,C:T. sergenti-uninfected erythrocyte, D:T. sergenti-uninfected leukocytes, E:T. sergenti-infected erythrocytes. F:T. sergenti-infected leukocytes. Lane 1: PCR products using Ka1 and Ka2 primer, lane 2: PCR products using TS1 and TS4 primer, lane 3: PCR products using TS1 and TS3 primer, lane 4: PCR products using TS2 and TS4 primer, lane 5: PCR products using TS2 and TS3 primer.

Fig. 5
Detection of T. sergenti-negative infection by PCR. M; 1Kb DNA ladder, lane 1-25; No. 1, 2, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 16, 19, 24, 26, 28, 33, 35, 37, 41, 42, 43, 44.

Fig. 6
Detection of T. sergenti by PCR using different sets of primers. M: 1 Kb DNA ladder, A: PCR products using TS2 and TS3 primer, B: PCR products using TS1 and TS3 primer, C: PCR products using TS1 and TS4 primer, D: PCR products using TS2 and TS4 primer. Lane 1, 6, 12, 17, No. 1, lane 2, 7, 13; No. 15, lane 3, 9, 14, 19; No. 37, lane 4, 10, 15; No. 42, lane8, 18; No. 34, lane 5, 11, 16, 20; standard Korean isolate.


Table 1
Nucleotide sequences of PCR primers for the amplification of T. sergenti

Table 2
Comparison of the PCR and microscorpic examination for diagnosis of T. sergenti infection

1. Baek BG, et al. Korean J Vet Res 1990;30:465–471.
2. Baek BG, et al. Korean J Vet Res 1993;33:665–671.
3. Chae JS, et al. Korean J Vet Res 1996;36(1):187–193.
4. Chae JS, et al. Korean J Vet Res 1996;36(1):195–207.
5. Chon Y. Res Rep off RDA 1970;13:81–87.
6. Chon Y. Res Rep off RDA 1978;20:1–3.
7. Hirano A, Kirisawa R, Matsuba T, Komatsu R, Tanaka M, Takahashi K, Kawakami Y, Onuma M. Evaluation of high sensitive DNA probe for the detection of Theileria sergenti infection in cattle. J Vet Med Sci 1991;53(5):933–935.
8. Kajiwara N, Kirisawa R, Onuma M, Kawakami Y. Specific DNA probe for the detection of Theileria sergenti infection in cattle. Nippon Juigaku Zasshi 1990;52(6):1199–1204.
9. Kang SW, Choi EJ, Kweon CH. [Cloning and sequencing of p33 in a Korean isolate of Theileria sergenti]. Korean J Parasitol 1997;35(2):105–110.
10. Kawazu S, Sugimoto C, Kamio T, Fujisaki K. Analysis of the genes encoding immunodominant piroplasm surface proteins of Theileria sergenti and Theileria buffeli by nucleotide sequencing and polymerase chain reaction. Mol Biochem Parasitol 1992;56(1):169–175.
11. Kawazu S, Kamio T, Sekizaki T, Fujisaki K. Theileria sergenti and T. buffeli: polymerase chain reaction-based marker system for differentiating the parasite species from infected cattle blood and infected tick salivary gland. Exp Parasitol 1995;81(4):430–435.
12. Kim YC, et al. Korean J Ani Sci 1984;26:137–144.
13. Kobayashi N, Onuma M, Kirisawa R, Ohgitani T, Takahashi K, Sasaki N, Kawakami Y. Monoclonal antibodies against intraerythrocytic merozoites (piroplasms) of Theileria sergenti. Nippon Juigaku Zasshi 1987;49(4):697–702.
14. Kubota S, Sugimoto C, Onuma M. A genetic analysis of mixed population in Theileria sergenti stocks and isolates using allele-specific polymerase chain reaction. J Vet Med Sci 1995;57(2):279–282.
15. Lee JM, et al. Korean J Vet Res 1987;27:321–330.
16. Matsuba T, Kawakami Y, Iwai H, Onuma M. Genomic analysis of Theileria sergenti stocks in Japan with DNA probes. Vet Parasitol 1992;41(1-2):35–43.
17. Matsuba T, Kubota H, Tanaka M, Hattori M, Murata M, Sugimoto C, Onuma M. Analysis of mixed parasite populations of Theileria sergenti using cDNA probes encoding a major piroplasm surface protein. Parasitology 1993;107(Pt 4):369–377.
18. Ohgitani T, Okabe T, Sasaki N. Antigenic properties of Theileria sergenti in ELISA serodiagnosis. Nippon Juigaku Zasshi 1987;49(3):531–534.
19. Persing DH, Mathiesen D, Marshall WF, Telford SR, Spielman A, Thomford JW, Conrad PA. Detection of Babesia microti by polymerase chain reaction. J Clin Microbiol 1992;30(8):2097–2103.
20. Tanaka M, et al. J Protozool Res 1992;2:34–39.
21. Tanaka M, Onoe S, Matsuba T, Katayama S, Yamanaka M, Yonemichi H, Hiramatsu K, Baek BK, Sugimoto C, Onuma M. Detection of Theileria sergenti infection in cattle by polymerase chain reaction amplification of parasite-specific DNA. J Clin Microbiol 1993;31(10):2565–2569.
22. Yoshihara K, Nakamura Y, Fujisaki K, Onodera T. Bovine monoclonal antibody specific to Theileria sergenti. Nippon Juigaku Zasshi 1990;52(6):1333–1335.
Editorial Office
Department of Molecular Parasitology, Samsung Medical Center, School of Medicine, Sungkyunkwan University,
2066 Seobu-ro, Jangan-gu, Suwon 16419, Gyeonggi-do, Korea.
Tel: +82-31-299-6251   FAX: +82-1-299-6269   E-mail: kjp.editor@gmail.com
About |  Browse Articles |  Current Issue |  For Authors and Reviewers
Copyright © 2022 by The Korean Society for Parasitology and Tropical Medicine.     Developed in M2PI