혈청학적 진단을 위한 돼지 편충의 체항원, 배설/분비항원의 분리 및 비교

지차호,이철순,박승준,Jee, Cha-ho,Lee, Chul-soon,Park, Seung-jun 대한수의학회 1999 大韓獸醫學會誌 Vol.39 No.1

Swine whipworm(Trichuris suis) is cosmopolitan nematode which can cause serious pathology in immature stage(larva2~larva5) of infected pigs, such as anorexia, diarrhea, anemia, and death in heavy infections. In this larval stages, it is very difficult to diagnose the infection of whipworm and to differentiate from other common swine gastrointestinal disorders such as 21 day scours which are associated with TGE virus, rota virus, coccidium, and the stress of weaning. In this experiment, the isolated antigens of Trichuris spp. were carried out to examine the structure and specificity of antigens and to select the reasonable antigens which would be used in serological diagnosis by electrophoresis, Western blotting, ELISA. The results of this experiment were as follows; 1. The common fractions of each Trichuris suis antigen were identified 28,32,45, 80kDa by SDS-PAGE with silver stain and four major fractions could be detected in positive swine sera by Western blot analysis. 2. The OD(optical density) values of somatic and excretory-secretory antigens which were reacted against positive(negative) sera from pigs infected with Trichuris suis by ELISA reader were; 1) OD values($mean{\pm}SD$) of adult somatic antigen against positive(negative) sera were $0.30{\pm}0.12(0.09{\pm}0.006)$ and third-stage larva of somatic antigen were $0.28{\pm}0.038(0.10{\pm}0.005)$. And OD values of excretory-secretory antigens of adult and third-stage larva were $0.24{\pm}0.031(0.11{\pm}0.005)$ and $0.08{\pm}0.013(0.10{\pm}0.003)$, respectively. 2) OD values of adult somatic, larval somatic antigen and adult excretory-secretory antigen response to positive sera were significantly (p<0.01) associated with negative swine sera. And the Cut-off OD values(minimum positive value) were determined to be mean negative value plus 3 SD that would minimized the risk of false positives. 3. The OD values of somatic antigens of T suis and T vulpis against swine positive(negative) sera were $0.30{\pm}0.120(0.09{\pm}0.006)$ and $0.25{\pm}0.141(0.09{\pm}0.003)$. These data mean that the somatic antigens of T suis and T vulpis were able to diagnose T vulpis infection in dogs as well as T suis infection in pigs. These results suggest that somatic antigen of third-stage larva and excretory-secretory antigen of adult T suis could be used the diagnostic antigen by serological test(ELISA) in immature Trichuris spp. infection.

시험관내에서 돼지회충(Ascaris suum) 함자충란(L₂)의 인공배양

지차호,박승준,Jee, Cha-ho,Park, Seung-jun 대한수의학회 1998 大韓獸醫學會誌 Vol.38 No.1

The cultivation for development of Ascaris suum from the second-stage larvae($L_2$) embryonated egg and the third-stage of rat-derived larvae($L_3$) recovered from lung of rats were performed to use the screening test of anthelmintics in vitro. The preparations of larvae for cultivation were that the artificially-hatched $L_2$ incubated the embryonated eggs of Ascaris suum in 0.1% formalin solution at $25^{\circ}C$ for 28 days and the rat-derived larvae($L_3$) recovered from the lung of rat infected with the embryonated eggs of Ascaris suum on 7 days after infection(DAI). The cultivation for development of Ascaris suum from the embryonated eggs($L_2$) and the rat-derived larvae($L_3$) for 14 days in RPMI medium 1640(with 5% bovine calf serum) were as follows : 1. The sizes of the liberated larvae($L_2$) which were artificially hatched from embryonated eggs with glass beads(diameter 5mm) were $190{\sim}250{\mu}m$ on 1 days in culture(DIC). The second-stage larvae were molted into third-stage larvae(early $L_3$; $250{\sim}300{\mu}m$) and the features of these larvae were first observed such as cephalic cuticle, esophageal lumen and anus etc. on 5 DIC and the sizes of late third-stage larvae were $250{\sim}450{\mu}m$ on 10 DIC. The sizes of early fourth-stage larvae($L_4$) were $500{\sim}700{\mu}m$ and the features of these larvae were more pronounced in internal organs on 15 DIC. 2. The sizes of third-stage larvae($L_3$) recovered from the lung of rats were $1,340{\sim}1,370{\mu}m$ and the feartures of cephalic cuticle, esophageal lumen, intestine, rectum, anus were visualized by inverted microscope on 1 DIC. The fourth-stage larva($L_4$) completed by third ecdysis were recognizable and sizes of early fourth-stage larvae were developed as $1,400{\sim}2,200{\mu}m$ on 5 DIC. The sizes of middle fourth-stage of larva were $1,900{\sim}2,300{\mu}m$ and the thickened epithelial rectum was observed on 10 DIC. The rectum and anus of late fourth-stage larva($L_4$ $2,500{\sim}3,200{\mu}m$) had developed completely in RPMI medium 1640 on 15 DIC.

연충감염우에 있어서 충란검사(蟲卵檢査)에 의한 구충효능(驅蟲效能) 평가방법(評價方法)에 관한 연구(硏究)

지차호,장두환,윤희정,Jee, Cha-ho,Jang, Du-hwan,Youn, Hi-jeong 대한수의학회 1984 大韓獸醫學會誌 Vol.24 No.1

In the evaluation of anthelmintic efficacy by fecal egg counts for cattle naturally infected with helminths, the reasonable technique of fecal egg counts and the reliable guidelines were determined as follows; 1. Modified technique of Harashigeru and Kim's sedimental tube was the most reasonable in fluke egg counts. 2. Universal egg counting technique was preciser than McMaster egg counting technique and was lower in coefficient variation. 3. Fecal egg counts of pretreatment should be carried out twice and mean of the epg should be calculated. 4. Fecal egg counts of posttreatment should be carried out 3 times and established at suitable intervals in consideration of anthelmintic mechanism and withdrawal days of anthelmintics. 5. If nematodal eggs are not found by UECT in posttreatment, direct flotation method should be carried out. And if positive, this epg was calculated at 20 (factor of UECT; $40{\times}1/2$).

시험관내에서 인공배양한 제 3기 자충 및 성충을 이용한 구충효능 선발시험

지차호,박승준,Jee, Cha-ho,Park, Seung-jun 대한수의학회 1998 大韓獸醫學會誌 Vol.38 No.3

The in vitro screening tests against the in vitro cultivated $L_3$ of Ascaris suum (in vitro $L_3$), which were cultivated from the embryonated egg to third-stage larva on 7 days in culture(DIC) and the in vivo rat's lung-derived $L_3$ of Ascaris suum (in vivo $L_3$), which were recovered from the lungs of rat on 7 days after infection, carried out in order to compare the anthelmintic efficacy of in vitro $L_3$ and that of in vivo $L_3$ in RPMI medium 1640 with 5% bovine calf serum. And also a screening test of efficacy against adult worms of Trichuris suis performed. The efficacies of screening tests were as follows : 1. The screening efficacies of abamectin and ivermectin against the in vitro $L_3$ were all 100% at the 10ppm concentration in RPMI medium 1640 on 5 DIC. 2. The screening efficacies of abamectin and ivermectin against the in vivo $L_3$ were all 100% at the 20ppm on 5 DIC or at 40ppm on 3 DIC. 3. The screening efficacies of abamectin and ivermectin against the adult worms of Trichuris suis were all 100% at 20ppm on 4 DIC. And therefore, the in vitro cultivated $L_3$ of Ascaris suum were used in the screening test as well as the in vivo rat's lung-derived $L_3$ of Ascaris suum. And also the adult worms such as Trichuris suis and filaroids which is small size and difficult to cultivate to vitro, were used in the screening test in vitro.

ELISA를 이용한 돼지 옴 (Sarcoptes scabiei var. suis) 감염증의 진단

지차호,이삼선,장래훈,Jee, Cha-Ho,Lee, Sam-sun,Chang, Lai-hun 대한수의학회 2001 大韓獸醫學會誌 Vol.41 No.4

The diagnosis of swine sarcoptic mange (Sarcoptes scabiei var. suis) was investigated by ELISA in order to replace current diagnostic methods such as skin scraping, scratching index, or lesion score of dermatitis. The current methods need many efforts and much times and cost much. They can not handle many samples simultaneously. Therefore, in this research we developed ELISA that can handle many samples at a time. The antigens of swine sarcoptic mite were isolated and examined by 12.5% SDS-PAGE and silver staining. The antigenicity of antigen was confirmed by Western blotting using the swine from the artificailly-infested swine with swine sarcoptic mite. The optical density (OD) values of the artificailly-infested positive sera and the naturally-infested positive sera of sows were measured and read in order to confirm the stability of antigens, the reproducibility and validity (sensitivity and specificity) of the manufactured ELISA of swine sarcoptic antigens. In above results, the developed ELISA would be possible to use the diagnostic tool of sarcoptic mange if OD values of piglets, fattening pigs and sows are interpreted reasonably and classified as mange-free and infested.

제1위 섬모충(rumen ciliates)을 이용한 동물성 단백질(치어용 사료) 개발

지차호,현공율,Jee, Cha-ho,Hyun, Gong-yool 대한수의학회 1995 大韓獸醫學會誌 Vol.35 No.2

This study was carried out to develop the animal protein(feed for fry) that was isolated, purified and lyophilized the rumen ciliates from the healthy rumen contents which have $10^5-10^6/g$ ciliates and were discarded in abattoirs. The rumen ciliates are non-pathogenic, anaerobic and the weight of this protozoa is 2% of rumen content. The rumen protozoan and bacterial proteins both have a biological value for rats of 80-81, which is higher than the 72 of brewer's yeasts. Furthermore, the true digestibility and net protein utility of the protozoan protein are 91 and 73, much higher than those of bacterial(74 and 60) or yeast(84 and 60) proteins. The amino acids of rumen protozoa is nutritionally superior than the others. The size of rumen ciliates is $30-200{\times}20-110{\mu}m$ and so we had isolated and purified the rumen ciliates from the rumen contents by the physical methods. The purified rumen protozoa was lyophilized with freezing dryer. The results of this experiment were as follows : 1. Population dynamics of protozoan ciliates in slaughtered rumens; % of samples which small ciliates were predominated was 82.5%(52/63) and that of large ciliates was 17.5%(11/63). 1) predominant species of small ciliates were Entodinium ovinum and E nanellum. 2) predominant species of large ciliates were Epidinium ecaudatum and Diploplastron affine. 2. The lyophilized rumen ciliates which were isolated and purified from 1 kg of rumen content at the pH 6.2-6.8 was about 7.0 gram. 3. The nutrient analysis of lyophilized rqmen ciliates(LRC) was as follows: 1) Proximate analysis of the LRC and the composition of fry feed; moisture 8.05%(below 10.0), protein 35.37%(45), fat 5.39%(4.5), fiber 1.23%(below 2.5), ash 2.25%(below 15.0), Ca 0.26%(below 2.0), P 0.14%(below 1.1), energy 4,608.11(fish meal 5000 cal/g) 2) Amino acids (% in crude protein) of the LRC and the rotifer(Brachionus plicatilis); Arg 5.19%(4.50), His 2.50%(1.55), Ile 5.29%(3.45), Leu 8.11%(5.85), Lys 10.34%(6.15), Met 2.25% (0.85), Phe 5.66%(3.80), Thr 5.14% (3.45), Val 4.18%(3.90), Ala 4.13%(3.35), Asp 13.26%(8.25), Glu 16.62%(9.20), Gly 4.23%(3.10), Pro 3.25%(5.05), Ser 4.85%(3.85), Tyr 5.04%(3.05) 3) Fatty acids(% in fat) of the LRC and the rotifer(biological feed ; Brachionus plicatilis); myristic acid(C14:0) 3.27%(0.3), myristoleic acid(C14:1) 0.83%(-), palmitic acid(C16:0) 39.11% (23.5), palmitoleic acid(C16:1) 2.81%(2.0), stearic acid(C18:0) 9.36%(5.6), oleic acid(C18:1) 25.54%(3.5), linoleic acid(C18:2) 15.05%(32.9), linolenic acid(C18:3) 1.74%(9.8). Judging from the above investigated results, the analytical data of proximate analysis, amino acids, fatty acids of the purified and lyophilized rumen protozoa are reasonable for the feed of freshwater fishes(fry and fingerling). But it was disappointed of our expectation that the crude protein of lyophilized rumen ciliates contains low percentage, it was thought that because of the small ciliates(starch digester) in beef cattle rumens which were administered the concentrated feed, is much difficult to isolate and purify than the large ciliates(fiber digester).

개 심장사상충(Dirofilaria immitis) 진단을 위한 항원성 조사 및 단크론항체 생산

이철순,지차호,Lee, Cheol-soon,Jee, Cha-ho 대한수의학회 2000 大韓獸醫學會誌 Vol.40 No.1

In order to diagnose canine heartworm infection by antigen capture ELISA, the crude somatic(S), partial somatic(below 45kDa) and excretory/secretory(E/S) antigen of adult heartworm were identified and the antigenicity was examined by silver stain, immunoblot and ELISA. Then, production of monoclonal antibody to specific antigen carried out in this experiment. The bands to S antigen and E/S antigen were recognized between 10 and 200kDa and common bands were recognized strongly 14, 18, 28, 43kDa by silver stain. By western blot analysis, fractions to S antigen were recognized 14, 16, 18, 20, 24, 28, 32, 43, 50, 55kDa, etc. and only a 14kDa to E/S antigen in positive sera which were positive in modified Knott's test and necropsy. In ELISA, the positive sera reacted to antigens(SA, $SA_{45}$, E/S) were significantly different from negative sera by Student's t-test(p<0.05). Four hybridoma cell lines(14, 16, 17, 32kDa) than produce specific monoclonal antibodies for these antigens were obtained by immunizing BALB/c mice with a partially purified somatic antigen (below 45kDa) preparation, by fusing spleen cells with SP2/O cell myeloma cells, and by screening cell culture supernatants for antibody. In these results, it was confirmed that partial somatic antigen(below 45kDa) or E/S antigen can be used for serologic diagnosis of heartworm infection and monoclonal antibody reacting with specific antigen(14kDa) can be used for antigen capture ELISA in prepatent period of canine heartworm infection.

소(우)의 흡충류 및 소화관내선충류에 대한 Levamisol HCl 및 Oxyclozanide 합제의 구충효능시험

장두환,지차호,윤희정,Jang Du-Hwan,Jee Cha-Ho,Youn Hi-Joeng 대한수의사회 1984 대한수의사회지 Vol.20 No.5

A total of 290 cattle in several districts was selected for treatment against the liver flukes, paramphistomes, and the gastrointestinal roundworms from March to september, 1983. 1. Egg reduction rates against the liver flukes, paramphistomes, and gastroi

시험관내에서 이버멕틴, 도라멕틴, 에타놀에 대한 아나사키스 유충의 운동성 억제효과

전재형,지차호,Jeon, Jae-Hyung,Jee, Cha-Ho 대한수의학회 2007 大韓獸醫學會誌 Vol.47 No.2

This experiment has been investigated in order to examine larval migration inhibition activity of ivermectin, doramectin and ethanol against Anisakis simplex (A. simplex) in vitro. A. simplex larvae were obtained from the mackerel acquired from the fish market of Cheongju. They were divided into many groups and placed in culture dishes (40 larvae each) containing RPMI-1640, in the absence or presence of different concentrations of ivermectin, doramectin and ethanol. Ivermectin had a complete inhibition of larval migration at 72 h in all groups ($10-300{\mu}g/ml$). Ethanol reduced the migration of the larvae, its maximum activity being an high doses (7.5%, 10% ethanol) when it was 100% efficacy at 4 h. Doramectin had no efficacy in vitro. Being needed that further studies with ivermectin and doramectin, it is recommended that in vivo test with laboratory animals should be carried.

시험관내에서 아니사키스 유충의 운동성에 대한 고련피, 관중, 사군자의 억제효과

권희녕,지차호,Kwon, Hee-Nyung,Jee, Cha-Ho 대한수의학회 2008 大韓獸醫學會誌 Vol.48 No.4

A high incidence of Anisakiasis has been reported in many countries where people eat frequently raw or undercooked seafood. Anisakis spp. larvae were obtained from the mackerel acquired from a fish market of Cheongju city. They were divided into several groups and placed in culture dishes containing RPMI-1640 (culture media), in the presence or absence of different concentrations of herbal extracts (Meliae ezadarach, Dryopteris crassirhizoma, Quisqualis indica var villosa). The objective of the present study was to investigate the activity of larval migration inhibition in vitro. Meliae ezadarach at the concentrations of 7.5, 15, and 30 mg/ml effectively inhibited the larvae migration in time-dependent manner during experimental period of 0-24 h. Treatment of Meliae ezadarach at the three concentrations completely inhibited the larvae migration in vitro. Dryopteris crassirhizoma at the concentrations of 5, 10, and 20 mg/ml also effectively inhibited the larvae migration in a time-dependent manner. The treatment of Dryopteris crassirhizoma for 12 h completely inhibited the larvae migration. The inhibitory effect of Dryopteris crassirhizoma was stronger than that of Meliae ezadarach. Although Quisqualis indica var villosa also showed the inhibitory effect on larvae migration, its inhibitory efficacy was the weakest among tested herbal extracts. These results indicated that some herbal extracts may be useful in controlling human anisakiasis.