• Parasites, Hosts and Diseases
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• v.32 no.2
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• pp.111-116
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• 1994

Immunoblot analysis utilizing bovine sera from naturally or experimentally infected with Theileria sergenti were used to determine the immunodominant polypeptides of T sergenti (Korean isolated. The previously recognized major bands, 18 kDa,29 kDa, 34 kDa and 45 kDa, were excised after electrophoresis and transfer to PnF membrane. The individual bands were sequenced. The 34 kDa polypeptide which was the most antigenic and immunogenic peptide was observed in the Western blot. However, Chou-Fasman prediction sites (antigenic site) for antigen determinants of the 45 kDa, 34 kDa, 29 kDa and 18 kDa polypeptide were 6, 4, 2 and 0, respectively. However, the 45 kDa polypetide showed no reaction with anti- T sergenti hyperimmune serum.

• Korean Journal of Veterinary Research
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• v.51 no.2
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• pp.107-115
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• 2011

Theileria (T.) buffeli (formerly T. sergenti/T. orientalis) is the major hemo-protozoan distributed in the Far East Asian countries such as Korea, China and Japan. It is responsible for the clinical symptoms of anorexia, ateliosis, anemia, fever and icterus. It also causes abortion and sudden death under severe cases, resulting in economic losses for many livestock farms. The objective of this study was to analyze the genetic diversity of the major surface protein (Msp) gene in T. buffeli in Holstein in Korea, and we characterized the association of the diversification of the Msp gene and its relationship with the pathogenicity of Theileria. For this, complete blood counts and Theileria PCR sequence analysis were performed from 57 Holstein in Jeju Island. A total of 26 PCR positive Holstein (16 anemic and 10 non-anemic) were then randomly selected based on 18s rRNA sequence typing of the Theileria Msp gene. The DNA sequence of the T. buffeli Msp gene in Holstein showed 99.0%, 99.2%, 99.9%, 99.5%, 98.7%, 98.4% and 98.4% homology with T. sergenti, Theileria spp., T. sergenti, Theileria spp., Theileria spp., Theileria spp. and Theileria spp., respectively. The result showed a genetic variation of 57.7% (type I), 3.8% (type II), 15.4% (type III), 7.7% (type IV), 13.5% (type V) and 1.9% (type VI). Type I is the most frequent type in both anemic and non-anemic Holstein while type II was found in only non-anemic Holstein. This results of our study help confirm the diversity of Msp gene types and demonstrate that the gene type distribution of Msp genes varies among Theileria-infected Holstein in Jeju Island.

• Korean Journal of Veterinary Research
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• v.33 no.3
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• pp.479-486
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• 1993

This study was attempted co develope a method for detection of Theileria sergenti infection on the basis of hybridization of parasite DNA with a probe. For construction of a T sergenti genomic library, T sergenti DNA was digested completely with Bam-HI and the fragments were ligated into the Bam-HI site of pUC-19 before transformation of Escherichia colistrain JM83. To detect clones containing the parasite's DNA sequences, a genomic DNA library of T sergenti constructed in pUC-19 was screened by cracking and Southern hybridization. Seven colonies were chosen from 29 colonies which were screened by transformation of Escherichia coli strain JM83. Seven transformants were comfirmed from seven colonies by cracking. The sizes of transformants were about 5Kb, 5.7Kb, 4.3Kb, 7.75Kb, 7.85Kb, 5.8Kb, 3.8Kb, respectively. DNA inserts, T sergenti DNA, and bovine DNA were hybridized with radio-labelled T sergenti DNA. Two($pT_1$, $pT_1$) of the seven inserts and T sergenti DNA reacted strongly but another 5 inserts and bovine DNA showed weak reation. All of the DNA inserts were not reaction, but T sergenti DNA were very weakly and bovine DNA were strongly reacted to hybridization with radio-labelled bovine DNA. Therefore, we obtained total 7 T sergenti DNA fragments in this study.

• Parasites, Hosts and Diseases
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• v.35 no.2
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• pp.105-110
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• 1997

The gene encoding the 33 kDa piroplasm surface protein of Theileria sergenti isolated in Korea was cloned and the nucleotide sequence was determined by dideoxy chaill termination method. The cloned gene corresponds to 869 bps encoding an open reading frame of 283 amino acids. Comparison of the sequence between Korean and .Tapanese isolates showed 99.4% homology rate in the nucleotide sequence and 98.9% homology rate in the amino acid sequence.

• Korean Journal of Veterinary Research
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• v.36 no.1
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• pp.195-207
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• 1996

To make the genomic DNA probe of Theileria sergenti, the merozoites were purified from erythrocytes of Korean cattle, The previous studies on the probe of T sergenti had resulted in two probes as KTS1 and KTS3 DNA fragment. Nucleotide sequence of both ends of the KTS1 and KST3 were determined in order to design primers for polymerase chain reaction. A pair of an uper primer(5'-CCTCTTGAAGTCATCCATGT-3'; nucleotide position 48) and a lower primer(5'-CACTGAGCTG GAAAGAGCTA-3'; nucleotide position 156) in pKTS1 were synthesized. The anticipated PCR product was 128bp in length. To examine the sensitivity of the PCR, KTS1 DNA and purified T sergenti DNA were serially diluted by tenfolds with distilled water. The primers were sensitive enough to detect 4ag of the authentic template DNA and 4fg of the purified T sergenti DNA by PCR. Furthermore, when the blood was serially diluted by two-folds with 0.9% saline, the pair could detect up to 0.00029%(about 164 parasites in $10{\mu}l$ of blood) of T sergenti infection in bovine erythrocytes by PCR. In a comparison of microscopic and PCR detection of T sergenti in the same samples from Chonbuk area, 47 and 51 out of 70 sample(67.1%) were positive by the former and by the latter method, respectively.

• Korean Journal of Veterinary Research
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• v.33 no.4
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• pp.665-671
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• 1993

The rate of 67 neonatal calves's infection of Theileria sergenti was investigated in random samples on the farms located in Kyeongki, Chonbuk and Jeju districts of Korea. The criteria used in verifying infection with T sergenti included the detection of parasites by giemsa's stain and acridine orange stain in the blood smear slides. Further evidence of current or previous exposure to T sergenti was based on the demonstration of T sergenti-specific antibody and antigen by the western immunoblot and the indirect immunofluorescence antibody test in the peripheral blood of the calves. The prevalence rates were 35%, 50% and 100% in Kyeongki, Chonbuk and Jeju provinces respectively and the overall prevalence in all the farms was 43.2% by means of acridine orange stain. The parasites that were observed in the peripheral blood of calves was shown surely by the western immunoblot to the characteristic 34KD antigen among the proteins of T sergenti (Korean Isolate). And the antibody of the neonatal calves reacted at the very highest titer(1 : >2,520). These data highlight the significance of T sergenti in the neonatal calf disease in Korea.

• 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.

• Parasites, Hosts and Diseases
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• v.35 no.2
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• pp.111-118
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• 1997

Four separate pairs of oligonucleotide primers within the coding region in a T sergenti 33-kDa surface protein gene were selected to detect T. sergenti by PCR. The specificity of PCR-amplified DNA was examined by digestion with restriction enzyme 3nd Southern blot hybridization using T. sergenti p33 DNA probe. PCR appears to be specific for T. sergenti, without detectable signals from uninfected erythrocytes, uninfected bovine leukocytes and other hemoparasites, including A. morginnle and 3. ouata. Although 46 of 71 specimens (64.8%) from grazing cattle were microscopically positive. PCR in this study showed that 64 specimens (88.7%) were positive. Therefore, PCR proves a useful diagnostic tool for detecting T sergenti-infected cattle. In addition, it is also revealed that PCR was significantly more sensitive than traditional microscopic examination using Giemsa's stain.

• Korean Journal of Veterinary Research
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• v.34 no.2
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• pp.387-394
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• 1994

To make the genomic DNA probe of Theileria sergenti, the merozoites were purified from bovine erythrocytes. The infected erythrocytes were lysed by Aeromonas hydrophila(Ah-1) hemolysin, and the parasites were isolated by ultracentrifugation on a Percoll discontinuous density gradient. For construction of a T sergenti genomic DNA library, T sergenti DNA was digested with Pstl and the fragments were ligated into the PstI site of pUC19 before transformation of Escherichia coli JM83. Out of thousands of transformants obtained by transformation of E coli JM83 with the genomic library, three plasmids were chosen. The sizes of the inserted DNAs were 2.9kb(2.4kb and 0.5kb) in pKTS1, 4.3kb in pKTS2 and 1.5kb in pKTS3, respectively. The DNA fragments used as probe KTS1(2.4kb), KTS2(4.3kb) and KTS3(1.5kb) were labeled digoxigenin-11-dUTP for the Southern hybridization. In Southern hybridization, all of the probes(KTS1, KTS2 and KTS3) reacted specifically to T sergenti DNA, but not to bovine leucocyte DNA. In order to find out the sensitivities of the digoxigenin-11-dUTP-labeled KTS1 and KTS3 as the probes, purified merozoite DNA and bovine DNA (control) were checked by dot blot hybridization with the probes. Both of the probes, KTS1 and KTS3, detected as minimum amount of 975pg of the T sergenti DNA, but not bovine DNA even to 500ng.

• Parasites, Hosts and Diseases
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• v.37 no.4
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• pp.277-283
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• 1999

To investigate the development of a subunit vaccine against theileriosis in cattle, the DNA fragments encoding piroplasm surface protein (p33) of Theileria sergenti of a Korean isolate were expressed in baculoviruses. The expressed p33 was characterized by indirect fluorescent antibody (IFA) and western blotting analysis. The expression of p33 was mainly detected on the surface of infected Sf21 cells by IFA. The immunoblotting analysis revealed the presence of a same molecular weight protein band of p33. The antigenicity of expressed polypeptide was further examined through the inoculation of a guinea pig. The sera of guinea pigs immunized with p33 expressed cell Iysate showed similar fluorescent antibody patterns and reacted with the same molecular weight protein of T. sergenti in immunoblotting analysis, thus indicating that this protein can be a promising candidate for a subunit vaccine in the future.