• Korean Journal of Veterinary Research
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• v.37 no.4
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• pp.785-791
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• 1997

Theileria spp.의 생활환에 대해서는 여러종류의 책을 통해서 잘 소개되어 있다. 그중 Theileria spp.의 분열소체 즉, merozoite는 주로 숙주의 적혈구내에 존재하는데 진드기에 의해 흡혈되지 않으면 더이상 발육하지 못하고 생을 마감한다고 여겨 왔다. 그러나 적혈구내 merozoite가 임파구에 다시 들어가 schizont로 복귀하여 분열 증식된다는 가설은 아직까지 증명된 바 없다. 본 실험은 T sergenti merozoite의 schizont로의 복귀를 입증하고자 수행되었다. T sergenti에 감염되지 않은 3개월령의 송아지를 비장적출시킨 후 T sergenti merozoite에 감염된 순수적혈구를 인공감염시켰다. 인공감염후 경시적으로 혈액과 임파액을 채취하여 적혈구내 T sergenti 감염을 조사하고 백혈구 감별혈구를 계산하였으며 임파구내 schizont 출현을 관찰하여 다음과 같은 결과를 얻었다. 1. 혈구내원충 감염율(parasitemia : PE)은 인공감염후 28일째 최고치인 10.5%를 보였으며 그후 5%이내의 수준을 유지하다가 70일째 다시 8.5%의 상승점을 보였다. 2. 백혈구 감별혈구계산에서는 감염초기에는 호중구가 주종을 이루다가 감염후 19일을 기점으로 임파구(60~80%)가 급격히 증가하여 실험종료 때까지 유지되었다. 3. 인공감염후 19~23일, 59~63일 사이에 말초혈액내 임파구에서 분열 증식하고 있는 schizont를 관찰할 수 있었다. 4. 인공감염후 7일부터 림프액내 임파구의 크기가 커지면서 blast-formation이 진행되었으며 실험종료때까지 유지되었다. 이상의 결과로 보아 적혈구내 merozoite가 임파구에 다시 들어가 schizont로 복귀하여 분열 증식함을 입증하여 기존의 T sergenti 생활사는 수정되어야 된다고 사료된다.

• Korean Journal of Veterinary Research
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• v.36 no.2
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• pp.453-461
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• 1996

Eighteen holstein-calves(4~5 months old) in a divided groups including the matched control were immunized with $100{\mu}g/dose$ of 34kDa, 45kDa polypeptide and T sergenti merozoite vaccine(protein content $100{\mu}g/dose$) respectively, previously mixed with aluminium hydroxide to elicit antibodies. All groups of calves were boosted with same dose and intervals. The animals were challenged by tick infestations in the endemic pasture of theileriosis from March to September 1994. The animals were monitored for the erythrocyte count, parasitemia, hematocrit and the specific antibody reactions elicited by immunization. The immunological responses demonstrated that vaccination with 34kDa polypeptide and T sergenti merozoite derived vaccine inhibited to produce the 75kDa band immunological responds even in the vaccinated calves after being challenged by tick infestations in the pasture. However, the specific antibody reactions were detected at the 32kDa band in the nonimmunized calves and T sergenti merozoite derived vaccine by the western blot. The 34kDa polypeptide vaccine and T sergenti merozoite derived vaccine were evaluated to be able to protect inducing anemia and to decrease parasitemias level. These vaccines have the efficacy of inhibition to produce a certain antigen corresponding 75kDa band antigen of parasite in the calves as challenged with tick infestations.

• Korean Journal of Veterinary Research
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• v.30 no.4
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• pp.465-471
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• 1990

The fine structure of the inoculated Theileria sergenti on the splenectomized Korean native cattle was observed to delineate the morphological. characteristics with transmission and scanning electron microscope. The cattle was inoculated with 1.5ml cryopreserved stabilate ($5.63{\times}10^6/{\mu}l$, PE 3%). At peak parasitemia (40%), infected blood was collected, washed and then T sergenti was observed. Scanning electron micrograph of the erythrocytes infected with T sergenti appeared various irregular from involving specific swelling, and abnormal projections like acantocyte, echinocyte and knizocyte. Transmission electron microscopic studies of T sergenti showed that piroplasm possess intracytoplasmic food vacuole, rhoptries and tubule. Merozoite, $0.6{\sim}1.81{\mu}m$ to $0.4{\sim}1.21{\mu}m$ in length, surrounded by 10~15nm thickness of pellicula. which is surrounded by a single unit membrane. Various size of veil which was observed in stroma of erythrocytes infected with T sergenti, located at the proximate part of the merozoite. The merozoite multiplied by means of binary fission so that two and more oval-like merozoites in the stroma of infected erythrocyte could be observed.

• Microbiology and Biotechnology Letters
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• v.17 no.5
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• pp.499-503
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• 1989

Relatively little is known about the antigenic relatedness of the different developmental stages of Cryptosporidium. A monoclonal antibody (mAb), an IgG3, was produced against the Cryp-tosporidium merozoite stage by immunizing mice with merozoite preparation. This monoclonal was reacted with sporozoite antigens in Western blotting resulting in recognition of an epitope on a 3.5-kDa antigen. An immunoelectron microscopic technique was used to investigate the antigenic relatedness of Cryptosporidium Sporozoites and merozoites. Mouse intestine was fixed with 1 ％ glutaraldehyde and embedded in LR White. Thin sections were then sequentially treated with murine IgG3 mAb and anti-mouse IgG conjugated to 15-nm diameter colloidal gold. This mAb showed similar (sur-face/cytoplasmic) immunoelectron microsropic colloidal gold labeling patterns with sporozoites and merozoites, indicalting epitope sharing between these two stages. This information might be useful for identifying possible epitopes to which a vaccine could be developed.

• Parasites, Hosts and Diseases
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• v.30 no.3
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• pp.201-208
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• 1992

A Theileria sergenti soluble merozoite preparation containing the 29, 34, 35 and 105 KD as the immunodominant polypeptides, was evaluated for efficacy, safety and protectivity in Holstein calves against virulent field tick challenge. The soluble antigens (100 mg/dose) were fortified with either complete or incomplete Freund's adjuvant. Twenty naive calves, aged one month, were subcutaneously inoculated with the preparation and a booster dose was administered 4 weeks later. Twenty additional calves served as controls. Five weeks after the booster dose, vaccinates and uninoculated controls were moved to a pasture, a heavily tick infested area in Cheju-do, Korea. The vaccinates showed negligible change in hematocrit and total RBC count whereas control animals showed significant (p<0.05) hematological changes and associated anemia. Only 30% of vaccinates required chemotherapy after the experiment was terminated. All control animals required chemotherapy and 25% received blood transfusion. The highest percent parasitized erythrocytes in vaccinated cattle was 0.4% as compared with 3.6% among controls during the month of July. A significant difference (p<0.05) was observed in the rate of body weight increase. Significant difFerences were also noted in serum albumin, aspartate aminotransferase, total protein and bilirubin. Significantly more vaccinated cattle maintained normal ranges of hematological and biochemical values as compared with the control group. It is suggested that soluble merozoite T. sergenti antigens may be potential vaccine candidates for developing a genetic vaccine in Korea.

• Parasites, Hosts and Diseases
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• v.45 no.1 s.141
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• pp.59-63
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• 2007

Merozoite surface protein-1(MSP-1) and merozoite surface protein-2(MSP-2) were used to develop vaccines and to investigate the genetic diversity in Plasmodium falciparum malaria in Iran. Nested polymerase chain reaction amplification was used to determine polymorph isms of block 2 of the MSP-1 and the central domain of MSP-2 genes. A total of 67 microscopically positive P. falciparum infected individuals from a major endemic region, southeast Iran, were included in this trial. Nine alleles of MSP-1 and 11 alleles of MSP-2 were identified. The results showed that amplified product from these surface antigen genes varied in size and there was specific pattern for each isolate. Besides, regarding this pattern, 23 multiple infections with at least 2 alleles were observed. While the endemic regions of malaria in Iran is classified in low to moderate group, but extensive polymorphism was observed for each marker and the MSP-2 central repeat was the most diverse that could be considered in designing malaria vaccine.

• Parasites, Hosts and Diseases
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• v.52 no.1
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• pp.105-109
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• 2014

Plasmodium falciparum malaria is a major public health problem in Thailand due to the emergence of multidrug resistance. The understanding of genetic diversity of malaria parasites is essential for developing effective drugs and vaccines. The genetic diversity of the merozoite surface protein-1 (PfMSP-1) and merozoite surface protein-2 (PfMSP-2) genes was investigated in a total of 145 P. falciparum isolates collected from Mae Sot District, Tak Province, Thailand during 3 different periods (1997-1999, 2005-2007, and 2009-2010). Analysis of genetic polymorphisms was performed to track the evolution of genetic change of P. falciparum using PCR. Both individual genes and their combination patterns showed marked genetic diversity during the 3 study periods. The results strongly support that P. falciparum isolates in Thailand are markedly diverse and patterns changed with time. These 2 polymorphic genes could be used as molecular markers to detect multiple clone infections and differentiate recrudescence from reinfection in P. falciparum isolates in Thailand.

• Korean Journal of Veterinary Research
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• v.30 no.2
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• pp.223-229
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• 1990

A splenectomized 5-month-old calf was inoculated with cryopreserved Theileria sergenti infected blood originated from naturally infected Korean native cattle in Chonbuk district. At peak parasitemia (40.1%), blood was collected, washed, lysed and then the T sergenti merozoite was isolated by differential centrifugation. Antigenic profile of isolated T sergenti organism was analized by SDS-PAGE and western blotting techniques. Coomassie blue stained SDS-PAGE gel revealed at least twelve protein bands of approximately 14Kd, 28Kd, 30Kd, 34Kd, 36Kd, 38Kd, 41Kd, 56Kd, 66Kd, 72Kd, 97Kd and 116Kd in the merozoite homogenate. In western blot, although T sergenti antigen recognized by specific anti-T sergenti antibodies demonstrated 28Kd, 30Kd, 38Kd, 56Kd, 58Kd, 66Kd, 97Kd and 116Kd proteins. False positive reactions were also observed in normal bovine serum with T sergenti and normal erythrocytic antigens. Therefore, predominant proteins of T sergenti merozoite antigen were found to be 28Kd, 30Kd, and 41Kd proteins of molecular weights. On going studies we will analyze the relative importance of those antigens for immunity of T sergenti in Korean native cattle.

• Parasites, Hosts and Diseases
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• v.54 no.4
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• pp.385-391
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• 2016

The discovery and understanding of antigenic proteins are essential for development of a vaccine against malaria. In Plasmodium falciparum, Pf92 have been characterized as a merozoite surface protein, and this protein is expressed at the late schizont stage, but no study of Pv92, the orthologue of Pf92 in P. vivax, has been reported. Thus, the protein structure of Pv92 was analyzed, and the gene sequence was aligned with that of other Plasmodium spp. using bioinformatics tools. The recombinant Pv92 protein was expressed and purified using bacterial expression system and used for immunization of mice to gain the polyclonal antibody and for evaluation of antigenicity by protein array. Also, the antibody against Pv92 was used for subcellular analysis by immunofluorescence assay. The Pv92 protein has a signal peptide and a sexual stage s48/45 domain, and the cysteine residues at the N-terminal of Pv92 were completely conserved. The N-terminal of Pv92 was successfully expressed as soluble form using a bacterial expression system. The antibody raised against Pv92 recognized the parasites and completely merged with PvMSP1-19, indicating that Pv92 was localized on the merozoite surface. Evaluation of the human humoral immune response to Pv92 indicated moderate antigenicity, with 65% sensitivity and 95% specificity by protein array. Taken together, the merozoite surface localization and antigenicity of Pv92 implicate that it might be involved in attachment and invasion of a merozoite to a new host cell or immune evasion during invasion process.

• Korean Journal of Veterinary Research
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• v.35 no.1
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• pp.87-94
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• 1995

Western immunoblot analysis of antigen of T sergenti merozoite revealed that the immunodominant proteins of this organism were characterized as the 18KD, 29KD, 34KD, 45KD and 105KD in Korea. The 34KD and 45KD among those immunodominant proteins of the parasite were isolated and their amino acid sequences from the $NH_2$-terminus were determined and synthesized. They respective polypeptides were cationized to enhance their antigenicity, fortified with Freund's adjuvant and tested for immunogenicity in rabbits and cattle. The results obtained were as follows; 1. Theileria sergenti merozoite antigen was shown in 120KD, 100KD, 66KD, 45KD, 34KD and 30KD in western immunoblot using serum of rabbits immunized with 34KD synthetic polypeptide and 70KD, 58KD, 55KD and 45KD using bovine serum. In western immunoblot, 45KD, 34KD and 30KD were recognized by immunized rabbits, and 50KD and 45KD by cattle sera immunized with 45KD synthetic polypeptide, respectively. 2. The ELISA utilizing the synthetic polypeptides demonstrated significant antibody response to the respective peptides. After the 2nd booster injection, an OD of 0.760(preimmunization 0.132) in rabbits and an OD of 0.645(preimmunization 0.488) to 34KD synthetic polypeptide in cattle were observed. In animals immunized with 45KD synthetic polypeptide, after the 2nd booster injection, an OD of 0.640(preimmunization 0.144) in rabbit, and an OD of 0.776 (preimmunization 0.477) in cattle were measured. 3. After the 2nd booster the reciprocal IFA titer was 1:64 in rabbits and 1:512 in cattle immunized with the 34KD synthetic polypeptide. The IFA titre was observed as 1:512 in rabbit and 1:1,024 in cattle in immunized with the 45KD synthetic polypeptide.