Detection of Neospora caninum in the blood of Korean native cattle and dairy cows using PCR

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1 ª ª (28) 48ƒ 2 Korean J Vet Res(28) 48(2) : 191~195 w1$3wxü/fptqpsbdbojovn Á 1ûw w 2 ( : ) 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 , Korea 2 Korea Centers for Disease Control and Prevention, Seoul , 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 , Korea [Tel: , Fax: , visionalive@hanmail.net] 191

2 192 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 (%) Total * 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 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), %(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

3 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 %(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, ½, y, ½z, x«, y,, y,, y. Neospora w. wwz 1997, 37, ½z, y, x, y,. ½. Neospora caninum w. w wz 1998, 38,

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