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Immunotherapy in calves experimentally infected with cryptosporidium parvm
위성환,주후돈,이정길,김종택,강영배,Wee, Sung-hwan,Joo, Hoo-don,Lee, Chung-gil,Kim, Jong-taek,Kang, Yung-bai The Korean Society of Veterinary Science 1998 大韓獸醫學會誌 Vol.38 No.2
7일령의 송아지 4마리에 실험적으로 크립토스포리디움을 감염시키고 면역혈청, 면역초유 그리고 단크론항체(C6)을 투여하여 이들의 면역요법제로서의 효과를 측정하였다. 크립토스포리디움 감염후 4일째부터 3일간 하루 2회(200~500ml)씩 3종의 면역요법제를 송아지 1마리씩 각각 경구로 투여하였으며, 나머지 한마리의 대조송아지에는 인산 완충액을 경구로 투여하였다. 크립토스포리디움에 감염된 송아지들은 설사를 나타냈는데 대조송아지의 경우 감염후 3일째부터 9일간 설사증상을 나타냈다. 면역혈청, 면역초유 그리고 C6로 치료한 송아지들은 치료후 각각 3일, 2일, 5일째부터 정상에 가까운 분변을 배출하기 시작하였다. 면역혈청과 면역초유로 치료한 송아지들의 경우, 분변으로 배출되는 오시스트의 수가 대조송아지에 비해 현저하게 줄어들었다. 이러한 결과들은 실험에 제공된 면역요법제중 면역초유나 면역혈청이 크립토스포리디움에 감염된 송아지의 설사증상과 오시스트의 배출을 억제하는 효과가 있음을 나타내는 것이다. To determine the efficacy of immunotherapeutic agents, four female Holstein calves 7-day-old were inoculated per os with $1{\times}10^7$ C parvum oocysts (VRI-CN91). Each calf received twice daily oral dosage of 200-500ml of the immune bovine serum, immune bovine colostrum, mAb C6, and phosphate-buffered saline, respectively. Treatment was initiated 4 days postinfection and laster 3 days. The clinical sign of the calf treated with phosphate-buffered saline lasted 9 days after the initial treatment. The calves treated with those immunotherapeutic agents, however, showed decreased severity of diarrhea at day 3, 2, 5 after the initial administration, respectively. The calves treated with immunotherapeutic agents showed reduced parasite loads compared to control calf. These results suggest that oral passive immunotherapy with immune bovine colostrum and immune bovine serum may be a useful treatment approach.
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세포배양에서 Cryptosporidium parvum의 발육
김보숙,주후돈,위성환,김태종,Kim, Bo-sook,Joo, Hoo-don,Wee, Sung-hwan,Kim, Tae-jong 대한수의학회 1995 大韓獸醫學會誌 Vol.35 No.2
The purpose of this study was to establish a method for in vitro culture of C parvum isolated in Korea by determination of suitable cell model to complete development of this parasite. The result obtained were summerized as follows: 1. To determine the most suitable cell line, six types of cell line were examined by microscopy. All cell lines were infected with C parvum and showed the highest infection score in HmLu cells. 2. The staining methods including DMSO-modified acid-fast(A-F) stain, hematoxylin-eosin(H & E) stain and immunofluorescence antibody(IFA) stain were applied to examine the infection of C parvum in cell culture. These staining methods were possible to examine the infection of C parvum in cell culture. The most sensitive one was IFA staining technique. 3. Developmental stages of C parvum in HmLu cell were observed. After the initial 8 hour incubation period, some trophozoites were observed. The meronts and gametes were appeared at 24-48 hour post inoculation(PI), and oocysts were observed firstly at 48-72 hour PI. 4. In H & E stain, the parasite appeared as basophilic within parasitophorous vacuole membrane(PVM) and lying in cytoplasm at near the nucleus of the host cells. It was able to distinguish the type I, type II meronts and gametes. 5. In DMSO-modified acid-fast stain, specific stained parasites were appeared firstly after 48 hour PI. The parasites were showed with different degrees of staining bright red color within PVM. 6. The endogenous stages of parasites in HmLu cell recovered at 48, 96, 120 and 144 hour after inoculation were reacted with rabbit immunized serum in immunofluorescence antibody and avidin-biotin complex peroxidase staining technique.
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Latex 응집반응을 이용한 동물의 톡소플라즈마병 진단용 kit 개발에 관한 연구
서명득,주후돈,데이빗 마-스,Suh, Myung-deuk,Joo, Hoo-don,Maass, Daivd 대한수의학회 1995 大韓獸醫學會誌 Vol.35 No.3
The present study was conducted to develop a toxoplasma latex agglutination test antigen(Test-MT) and evaluate the toxoplasma latex agglutination(LA) test using a newly-made "Test-MT kit" by comparing with the Toxo-MT kit(Eiken chemical co, Tokyo). Also, the specifity and sensitivity test were made by comparing with IFA test and IgG-ELISA. Tachyzoite suspensions of Toxoplasma gondii(RH strain) were ultracentrifuged for 30min at $60,000{\times}g(4^{\circ}C)$ and the supernatant was used as a water-lysate antigen. Polystyrene latex particles of $1.0{\mu}m$ in diameter(Polyscience co) were used for the preparation of sensitized latex-antigen supension(Test-MT). The frequency distribution of LA titers in Test-MT showed two peaks at <1:32 and 1:128. The borderline titer for positive test in Test-MT was determined to be 1:64. But the frequency distribution of LA tites in Toxo-MT showed two peaks at <1:16 and 1:64. The positive borderline was determined to be 1:32. Agreement of reactions between Test-MT and Toxo-MT kit by LA test was shown 92.5% in bovine sera and 97.0% in swine sera, respectively. From the results obtained here it was determined that the sensitized latex-antigen, Test-MT kit, for the microtiter agglutination test prepared as same as by the procedure described in the previous paper(Suh and Lee, 1993) was useful as a highly specific, sensitive and stable immunotiteration reagent for serodiagnosis of toxoplasma infection in animal sera.
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ELISA 법을 이용한 개 톡소플라즈마병의 조기진단에 관한 연구
서명득,주후돈,이병훈,Suh, Myung-deuk,Joo, Hoo-don,Lee, Byung-hoon 대한수의학회 1991 大韓獸醫學會誌 Vol.31 No.4
This study was conducted to detect the serum antibodies in the experimentally toxoplasma infected dogs and street dogs by use the of an enzyme-linked immunosorbent assay (ELISA). And this test was performed on the polystylene microplate by coating with the tachyzoites soluble antigen of T gondii (RH strain), incubated with sera diluted then, added with HPO-conjugated rabbit anti-dog IgG and o-phenylenediamine used as a substrate. Tachyzoites of T gondii harvested from mouse peritoneal cavity were purified by 30, 40 and 50% Percoll density gradient centrifugation and used as the source of antigen. The results obtained were summarized as follows; 1. The highest ratio of positive to negative (P/N ratio) was obtained at the level of $l{\mu}g/ml$ protein concentration of antigen with the 1/4000 dilution of the conjugate measured by checker-board titration. It was regarded as the optimum concentration of the antigen and conjugate. 2. Cut-off value in this IgG ELISA was 0.375 that was determined by mean absorbance (at 492nm) of IFA negative serum added with the dauble value of the standard deviation $(mean{\pm}2S.D.)$. 3. Serum ELISA IgG antibodies to T gondii in the exyerimentally infected dogs were detected firstly at the Week 3 after inoculation and the highest titer was recognized at the Week 4, 5 and 6 after inoculation. 4. Stability of the antigen absorbed in the microplates that were preserved at $4^{\circ}C$ and $-25^{\circ}C$ separately were prolonged up to 3 weeks and 10 weeks at $4^{\circ}C$ and $-25^{\circ}C$, respectively. However the reproducibility was not reliable after the preservation of 4 weeks and longer. 5. Positive rate of the specific antibodies in 312 test sera was 28.5% and there was no significant differences between the male (27.8%) and female (29.5%), respectively. 6. The IgG ELISA was proved to be a specific procedure for the detection of antibodies to canine toxoplasma infection and also evaluated as a screening test for the large scale of test samples in laboratory.
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Culicoides arakawae의 실험실내 colonization
최상호,주후돈,위성환,김기석,박근식,Choi, sang-ho,Joo, Hoo-don,Wee, Sung-hwan,Kim, Ki-seok,Park, Keun-sik 대한수의학회 1993 大韓獸醫學會誌 Vol.33 No.3
Culicoides arakawae is a kind of the main blood sucking insects of domestic fowls and serves as a vector of Leukocytozoom caulleryi, the causative protozoon of chicken leukocytozoonosis. In this study, the complete life history of C arakawae was cycled by laboratory colonization. Adult midges were collected from various poultry farm by light trap. The laboratory colonization was performed under the conditions of constant temperature of $25{\pm}1^{\circ}C$ and relative humidity of 80% or above. The hatched larvae were cultured in larval medium consisted of rice field mud and activated charcoal powder. The surface of medium was continuously flowed with biologically conditioned water. The fine powder meal composed of pellet feed for mice and equal mount of yeast was supplied for feeding larvae at every 72 hours. The life cycle completed at $25^{\circ}C$ in 35~35 days ; the period of preoviposition, egg. larval and pupal stage was 2~3, 3~4, 28~30 and 3 days, respectively. The measurements of the eggs, the lst instar larvae, the 4th instar larvae and pupae was $36.28{\mu}m{\pm}1.95$, $13.58{\mu}m{\pm}0.72$, $4000{\mu}m{\pm}1.47$ and $219.95{\mu}m{\pm}6.25$ in $mean{\pm}S.D.$, respectively. In order to confirm experimental colonization of C arakawae in laboratory, the colonized adult midges were allowed to suck blood from chicken infected with L caulleryi. The oocysts and sporozoites could be identified in midguts and salivary grands of engorged midges at 72 hours after blood sucking.
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ELISA를 이용한 돼지 톡소플라스마병의 조기 진단에 관한 연구
서명득,장동화,주후돈,Suh, Myung-deuk,Jang, Dong-hwa,Joo, Hoo-don 대한수의학회 1989 大韓獸醫學會誌 Vol.29 No.4
This study was conducted to evaluate the possibility of application of a microenzyme-linked immunosorbent assay(micro-ELISA) for the serodiagnosis of specific toxoplasma antibodies in swine sera and this test was performed as a microplate system by coating the polystyrene plates with toxoplasma soluble antigen, incubated serially diluted sera, then added horse radish peroxidase labelled goat anti-swine IgG(r) conjugate followed by o-phenylenediamine as substrate. The color development by enzyme-substrate reaction was determined by the photometric reading [ELISA reader at 490nm (OD)] and visual reading. The soluble antigen was prepared from the tachyzoites in mouse peritoneal cavity. A total of 1,200 swine sera from pig slaughter-house and a total of 116 swine sera from pig breeding station (S-C farm) were tested for the detection of antibodies to Toxoplasma gondii. The results obtained were summarized as follows: 1. The optimal reactions of indirect ELISA for the test sera were determined by the dilution of antigen 1:256 and 1:3,200 of horse radish peroxidase conjugate [anti-swine IgG(r)]. 2. The specific togoplasma antibody(IgG) in pigs infected with Tp artificially were detected as the serum titers of 1:64 or 1:128 at one week postinfection. 3. Of a total of 1,200 swine sera from pig slaughter-house 505 samples of sera were detected as positive (42.1%) and of a total of 116 swine sera from S-C pig breeding station 68 samples of sera as positive (58.6%). 4. The specific antibody(IgG) detection rates against a total of 1,200 test sera from pig slaughter-house were not significant between male (43.1%) and female (40.7%). 5. The indirect ELISA was proved to be a sensitive and specific procedure for the serodiagnosis of swine toxoplasmosis and also evaluated as an effective screening test for the large scale of test samples in laboratory.
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위성환,이정길,강영배,주후돈,주이석,박용호,최상호,Wee, Sung-hwan,Lee, Chung-gil,Kang, Yung-bai,Joo, Hoo-don,Joo, Yi-seok,Park, Yong-ho,Choi, Sang-ho 대한수의학회 1995 大韓獸醫學會誌 Vol.35 No.1
Four Holstein calves 7-day-old were infected with C parvum oocysts for parasitological and pathological investigations of bovine cryptosporidiosis. Of those calf 1 was orally administered with $7{\times}10^6$ oocysts of C parvum isolated from a Korean mouse (VRI-CN91), and calf 2 with same number of C parvum oocysts provided by Washington State University(WSU). The rest (calf 3 and 4) were orally administered with $1{\times}10^8$ oocysts of VRI-CN91 strain. Calf 1 commenced to discharge oocysts in feces at days 6 post inoculation(PI), and it reached a peak $1.4{\times}10^7$ oocysts per gram of feces(OPG) on day 8 PI. Calf 2 commenced to discharge oocysts in feces at day 4 PI, and it reached a peak $3.75{\times}10^6$ OPG on day 7 PI. Calf 3 and 4 commenced to discharge oocysts in feces at day 3 and day 4 PI, and it reached a peak on day 7 PI (calf 3, $7.8{\times}10^6$ OPG; calf 4, $1.7{\times}10^6$ OPG). Clinically, the calves began to show mucoid-watery diarrhea at day 3 to 5 PI, and the sign lasted 5 to 7 days. Calf 2 died on day 9 PI with a severe dehydration. On necropsy the intestine was found to be congested and hemorrhagic. Protozoan oocysts were observed mainly in the ileum and occasionally in jejunum. The results in the present study indicate that the Korean isolate was pathogenic in calves.
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