ª ª (28) 48ƒ 2 Korean J Vet Res(28) 48(2) : 191~195 w1$3wxü/fptqpsbdbojovn Á 1ûw w 2 ( : 28 4 3) Detection of Neospora caninum in the blood of Korean native cattle and dairy cows using PCR Sang-Eun Lee 1,2, Jung-Youn Lee 1, * 1 Research Institute of Veterinary Medicine, Chungnam National University, Daejeon 35-764, Korea 2 Korea Centers for Disease Control and Prevention, Seoul 122-71, Korea (Accepted: April 3, 28) Abstract : This study was performed to detect Neospora caninum in blood of 61 Korean native cattle and 5 dairy cows in Chungnam province. All of them were healthy and did not show any clinical signs. DNA was isolated from blood samples and a 328 bp fragment was amplified by PCR using primer pair Np21 and Np6. The PCR positive rate was 14.8% in Korean native cattle and % in dairy cows. Cows with 15.6% were a little higher than bulls with 12.5% in gender. The detection rate of over 3-year-old Korean native cattle was 28.6% in age. The results demonstrate that N. caninum DNA can be detected in blood by PCR. PCR analysis in blood may be useful to annually screening test for N. caninum infection in clinically healthy cattle. Keywords : blood, Korean native cattle, Neospora caninum, PCR yš Neospora(N.) caninum w Apicomplexa Coccidia, Sarcocystidea w» j ¾,,,, š šš [8, 11-13, 15, 16, 2], s ùkü, w ƒ w ùkù [1]. N. caninum w» w xw z w, xÿw s y [1, 4, 1], ƒ ù x, PCR w N. caninum DNA w š [1, 14, 15, 21, 25]. N. caninum ƒ w wš, O'Handley [24] w x mw xü N. caninum wþš, Okeoma [26] xü N. caninum DNA ƒ š, w xü N. caninum DNA ƒ ƒwš šw. ù ü x PCR w N. caninum screening ƒ ƒ š, wz ü s w» w» w w w w PCR w x N. caninum w š w. *Corresponding author: Jung-Youn Lee Research Institute of Veterinary Medicine, Chungnam National University, Daejeon 35-764, Korea [Tel: +82-2-869-7582, Fax: +82-2-869-7583, E-mail: visionalive@hanmail.net] 191
192 Á Table 1. The detection rate of Neospora caninum using PCR in Korean native cattle and dairy cows in Chungnam province Sex Age Cows Bulls <3 >3 Korean native cattle Dairy cow NE * NP PR (%) NE * NP PR (%) 45 16 4 21 7 2 3 6 15.6 12.5 7.5 28.6 5 36 14 - - Total 61 9 14.8 5 * NE: number of examined, NP: number of positive, PR: positive rate v %/" 1$3 œ w w 61 y 5 w x w z, DNA extraction kit(bioneer Co., Korea) w DNA w. N. caninum w 3.125 1 2 tachyzoites DNA extraction kit(bioneer Co., Korea) w DNA w 28.5 ng/l(qubit Fluorometer; Invitrogen Co., USA). xü N. caninum DNA yw» w primer NP6 5'-CTCGCCAG TCAACCTACGTCTTCT-3' NP21+ 5'-CCCAGTGCGT CCAATCCTGTAAC-3' w [23, 25], PCR 94 o C 1 DNA wk z, 94 o C 1, 63 o C 1, 74 o C 3 4z w z 72 o C 1 g. s PCR 2% agarose gel»w z Ethidium bromide w UV transilluminator s w. œ w w 61 y 5 w PCR w xü N. caninum w. PCR w 15.6%(7/45), 12.5%(2/16) ùkü ƒ š, 3 w 7.5%(3/4), 3 28.6%(6/21) ùk ü. w s³ 14.8%(9/61). wr 5 ùkü(table 1). Fig. 1. Detection of Neospora caninum DNA in blood of Korean native cattle. The amplified 328 bp product was subjected to electrophoresis in 2% agarose gel. M: 1 bp maker; P: positive control; Lane 1: negative; Lanes 2-4: positive; Lane 5: negative; N: negative control. š x¾ s ùk ú w w xÿw, zxÿw x Á k w w ƒ yš [4, 1]. ü xÿw w N. caninum w k mw w üš [1-3, 5-7]. ù Dubey [1] w yw zxÿwù xÿw xw» w,», p ywšw ƒ, x PCR w N. caninum
w PCR w xü Neospora caninum 193 DNA wü yw w š šw [21, 27]. N. caninum PCR w» w w primerƒ [25, 26, 3], NP6 NP21+ primer N. caninum Nc5 r s j p primer š š [17, 19, 3]. NP6 NP21+ primer w 328 bp p s xü N. caninum wš yw. wr w, w xü N. caninum 14.8%(9/61) x [6] šw û w w 47.8%(11/23) ûš, y [7] šw ü w w 17.9%(145/868) w, ½ [18] šw û w w 8.9%(11/123). w w x N. caninum w N. caninum s, w y,. y [7] (19.6%)ƒ (17.3%) š šw, (15.6%)ƒ (12.5%) ù, y [7] ùƒ s³ 2ƒ š w. 3 28.6%(6/21) N. caninum q. ù 3 w ƒ 3 2 ƒ w. wr N. caninum w š šù [6, 22, 28], ƒ, qƒ ù N. caninum» y y. N. caninum w w y w sw w q. w, ü N. caninum y w ƒ wš, O'Handley [24] Serrano-Martinez [29] N. caninum oocystes x z,» xl N. caninum š šw, Okeoma [25] Ferre [14] x xü N. caninum wš šw. ù x w» w, Okeoma [25] xü N. caninum,, š šw, N. caninum 9 š w, N. caninum. w sww ƒ w. w ¾ zw ù e ƒ» wù PCR ùkü z kjš, qj w w w ƒ w w, l ù y w w [4, 1]. w q w» w wz w w w w ƒ w. œ w y w 61 w 5 x N. caninum w» w NP21+ NP6 primer w PCR w. w x 14.8% N. caninum ùk ü, ùkü. (15.6%)ƒ (12.5%) ù kü, 3 w (28.6%) ùkü., xü N. caninum DNAƒ N. caninum š q, wz N. caninum» jw x w PCR w. šx 1., ½z, y, ûy,,, ½. Neospora w s xw. wwz 23, 43, 677-681. 2. ½, y, ½z, x«, y,, y,, y. Neospora w. wwz 1997, 37, 67-612. 3. ½z, y, x, y,. ½. Neospora caninum w. w wz 1998, 38, 853-858.
194 Á 4., ½x,.»w. 3q. pp. 567-574, ù, 27. 5. x«, ½z, y, y, y,,,,, ½. xÿw w ü Neospora caninum w x w. wwz 1998, 38, 859-866. 6. x, ½, ½, xz, ³,,. Neospora caninum w wƒ, w ƒwz 21, 24, 9-14. 7. y. w Neospora caninum w w. wwz 23, 43, 283-288. 8. Barr BC, Anderson ML, Woods LW, Dubey JP, Conrad PA. Neospora-like protozoal infections associated with abortion in goats. J Vet Diagn Invest 1992. 4, 365-367. 9. Cho JH, Chung WS, Song KJ, Na BK, Kang SW, Song CY, Kim TS. Protective efficacy of vaccination with Neospora caninum multiple recombinant antigens against experimental Neospora caninum infection. Korean J Parasitol 25, 43, 19-25. 1. Dubey JP. Review of Neospora caninum and neosporosis in animals. Korean J Parasitol 23, 41, 1-16. 11. Dubey JP, Hartley WJ, Lindsay DS, Topper MJ. Fatal congenital Neospora caninum infection in a lamb. J Parasitol 199, 76, 127-13. 12. Dubey JP, Lindsay DS, Adams DS, Gay JM, Baszler TV, Blagburn BL, Thulliez P. Serologic responses of cattle and other animals infected with Neospora caninum. Am J Vet Res 1996, 57, 329-336. 13. Dubey JP, Rigoulet J, Lagourette P, George C, Longeart L, LeNet JL. Fatal transplacental neosporosis in a deer (Cervus eldi siamensis). J Parasitol 1996, 82, 338-339. 14. Ferre I, Aduriz G, del-pozo I, Regidor-Cerrillo J, Atxaerandio R, Collantes-Fernández E, Hurtado A, Ugarte-Garagalza C, Ortega-Mora LM. Detection of Neospora caninum in the semen and blood of naturally infected bulls. Theriogenology 25, 63, 154-1518. 15. Ferroglio E, Pasino M, Romano A, Grande D, Pregel P, Trisciuoglio A. Evidence of Neospora caninum DNA in wild rodents. Vet Parasitol 27, 148, 346-349. 16. Jenkins MC, Parker C, Hill D, Pinckney RD, Dyer R, Dubey JP. Neospora caninum detected in feral rodents. Vet Parasitol 27, 143, 161-165. 17. Kaufmann H, Yamage M, Roditi I, Dobbelaere D, Dubey JP, Holmdahl JMO, Trees A, Gottstein B. Discrimination of Neospora caninum from Toxoplasma gondii and other apicomplexan parasites by hybridization and PCR. Mol Cell Probes 1996, 1, 289-297. 18. Kim JH, Lee JK, Hwang EK, Kim DY. Prevalence of antibodies to Neospora caninum in Korean native beef cattle. J Vet Med Sci 22, 64, 941-943. 19. Liddell S, Jenkins MC, Dubey JP. A competitive PCR assay for quantitative detection of Neospora caninum. Int J Parasitol 1999, 29, 1583-1587. 2. Marsh AE, Barr BC, Madigan J, Lakritz J, Nordhausen R, Conrad PA. Neosporosis as a cause of equine protozoal myeloencephalitis. J Am Vet Med Assoc 1996, 29, 197-1913. 21. McInnes LM, Ryan UM, O'Handley R, Sager H, Forshaw D, Palmer DG. Diagnostic significance of Neospora caninum DNA detected by PCR in cattle serum. Vet Parasitol 26, 142, 27-213. 22. Moore DP, Campero CM, Odeón AC, Posso MA, Cano D, Leunda MR, Basso W, Venturini MC, Späth E. Seroepidemiology of beef and dairy herds and fetal study of Neospora caninum in Argentina. Vet Parasitol 22, 17, 33-316. 23. Müller N, Zimmermann V, Hentrich B, Gottstein B. Diagnosis of Neospora caninum and Toxoplasma gondii infection by PCR and DNA hybridization immunoassay. J Clin Microbiol 1996, 34, 285-2852. 24. O'Handley R, Liddell S, Parker C, Jenkins MC, Dubey JP. Experimental infection of sheep with Neospora caninum oocysts. J Parasitol 22, 88, 112-1123. 25. Okeoma CM, Williamson NB, Pomroy WE, Stowell KM, Gillespie L. The use of PCR to detect Neospora caninum DNA in the blood of naturally infected cows. Vet Parasitol 24, 122, 37-315. 26. Okeoma CM, Stowell KM, Williamson NB, Pomroy WE. Neospora caninum: Quantification of DNA in the blood of naturally infected aborted and pregnant cows using real-time PCR. Exp Parasitol 25, 11, 48-55. 27. Ortega-Mora LM, Ferre I, del-pozo I, Caetano-da- Silva A, Collantes-Fernández E, Regidor-Cerrillo J, Ugarte-Garagalza C, Aduriz G. Detection of Neospora caninum in semen of bulls. Vet Parasitol 23, 117, 31-38. 28. Osawa T, Wastling J, Acosta L, Ortellado C, Ibarra J, Innes E. Seroprevalence of Neospora caninum infection in dairy and beef cattle in Paraguay. Vet Parasitol 22, 11, 17-23.
w PCR w xü Neospora caninum 195 29. Serrano-Martínez E, Ferre I, Martínez A, Osoro K, Mateos-Sanz A, Del-Pozo I, Aduriz G, Tamargo C, Hidalgo CO, Ortega-Mora LM. Experimental neosporosis in bulls: parasite detection in semen and blood and specific antibody and interferon-gamma responses. Theriogenology 27, 67, 1175-1184. 3. Yamage M, Flechtner O, Gottstein B. Neospora caninum: specific oligonucleotide primers for the detection of brain "cyst" DNA of experimentally infected nude mice by the polymerase chain reaction (PCR). J Parasitol 1996, 82, 272-279.