• 대한수의학회지
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• 제34권3호
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• pp.529-536
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• 1994

To establish more specific and simple diagnostic methods for detection of the antibodies and antigens of Aujeszky's disease virus(ADV), we designed indirect dot-immunoassay(IDI) and double sandwich dotimmunoassay(DSDI) using the solid phases of nitrocellolose paper and polystyrene plate. The diagnostic efficacy of these methods was investigated. As the sensitivity of IDI was tested by various virus concentration, the specimens with the virus titer above $10^{4.0}TCID_{50}/0.2ml$ showed positive reaction, but that below $10^{1.0}TCID_{50}/ml$ revealed negative. Tonsil emulsion at the virus titer of $10^{4.5}TCID_{50}/0.2ml$ showed the highest sensitivity as diluted by 1/100. In detection of ADV antigens from the various tissues of the rats and pigs infected with ADV, IDI using monoclonal antibody showed the higher specificity as compared with IDI using polyclonal antibody and virus isolation method. The efficacy of the DSDI for detection of ADV antibody was compared with other tests. The sensitivity of DSDI was higher than virus neutralization(VN) and agar gel immunodiffusion test(AGID). Meanwhile, specificity of DSDI was lower than AGID, but similar to IDEA. In comparison with VN test, DSDI showed 96.9% agreement to VN test that is the highest of three tests. In general, application of polyclonal antibody in both tests caused the higher sensitivity but the lower specificty.

• 대한수의학회지
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• 제45권4호
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• pp.569-574
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• 2005

Enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to reticuloendotheliosis virus (REV) at single serum dilution was standardized. REV HI, one of the Korean field isolates, was inoculated into chicken embryo fibroblast (CEF) cells and was harvested from the culture fluids and cells after 10 to 12 days. Viruses were purified by centrifugation at the $107,000{\times}g$ for 12 hours on 20, 30, 45% (W/V) sucrose gradient. Virus specific fraction was collected and used as ELISA antigen. To standardize ELISA, the optimal concentration of coating antigen ($1{\mu}g/well$) and conjugate (1/1000) was determined by corrected OD (OD value of positive serum-OD value of negative serum) and P/N ratio (OD value of positive serum/OD value of negative serum). To calculate ELISA titer by measuring absorbance at 1/400 single serum dilution, serum titrations were carried out for various sample sera together with standard positive and negative sera. The observed titers of serum samples were plotted against sample/positive (s/p) ratios at 1/400 serum dilution. From the above data, the ELISA titers could be calculated by the equation of $log_{10}$ ELISA titer = 2.2763 ($log_{10}$ s/p) + 3.482 (r = 0.93). For evaluating the sensitivity, the standardized method were compared with conventional agar gel immunodiffusion (AGID) test method using serum samples collected from REV infected field chicken flocks. Fifty seven of 60 samples (95%) were positive for REV by ELISA, whereas only 11 (18.3%) samples were positive by AGID test. This results suggested that the ELISA tests developed in this study could be used for detection of antibodies to REV with high sensitivity.

• 대한미생물학회지
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• 제18권1호
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• pp.23-29
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• 1983

An epidemiological survey was carried out to identify the serotypes of Streplococcus mutans isolated from carious lesions of 65 caries-active subjects(CAS) and sound tooth surfaces of 40 caries-free subjects(CFS). The autoclaved antigen extract was performed on each isolate, and then, serotypes of the isolates were determined in agar-gel immunodiffusion test. The results were as follows: 1. S. mutans was found in 78% of the samples of CAS, and of CFS. The difference of isolation frequency between CAS and CFS was not observed. 2. Only one serotype per single subject was detected in 61% of total samples, in remaining 39% of samples two or more serotypes were detected. 3. In 41.2% of CAS samples plural serotypes of S. mutans were found, whereas 35.5% of CFS samples showed plural serotypes distribution. 4. The most frequently identified serotype in each subject was serotype c; 69.5% of subjects harbored serotype c S. mutans. Serotype d was next most frequently isolated from subjects, comprising 23.2%. 5. Serotype c strain was found in 64.7% of CAS, 77.4% of CFS. 6. Of the isolates from CAS and CFS, serotype c was most commonly found, comprising 48.8%, serotype d was found in 16.3%, serotypes f. e, and g comprising 13.2%, 9.3%, and 7.8% respectively. Serotypes a and b were also found but in far lower frequencies(2.3%, 0.8%). 7. Serotype c strains were more found in CFS than in CAS, but serotypes d and e were more found in CAS.

• 한국동물위생학회지
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• 제18권1호
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• pp.1-21
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• 1995

To elucidate pathogenesis of bovine leukemia virus(BLV) in Korean native goats, the goats experimentally infected with BLV were studied especially for the aspects of infectivity and hematological changes. The experimental goats were examined for 27 months by agar-gel immunodiffusion(AGID) test and syncytium formation assay. During this period, changes of total leucocyte, absolute Iymphocyte and atypical Iymphocyte were examined, and the distribution of surface immunoglobulin ( sIg ) -bearing cells and rosette forming cell (RFC) in the peripheral Iymphocyte were also investigated. By indirect immunofluorescence (IFA) and complement dependent antibody cytotoxicity (CDAC) assay using monoclonal antibody(Mab) against bovine leukosis tumor-associated anti-gen(BL-TAA), changes of BL-TAA positive Iymphocyte in peripheral blood were measured. The results obtained through the experiment were summarized as follows. 1. Antibody titers were measured by AGID using gP51 and P24 antigens. The animals were serologically converted at 2 months post-inoculation(pi) in gP51 antigen, whereas sero-converted at 4 months pi in P24 antigen. In comparison with antibody titers for gP51, P24 antigen showed lower titers throughout the trial period. 2. The peripheral lymphocytes from all of the infected goats, as co-cultivated with F8l cells manifested syncytial formation at 4 months pi. 3. On counting total leucocyte, Iymphocyte and atypical Iymphocyte, two out of four infected goats showed normal distribution, while No 2 of the remaining two revealed temporal and No 3, Persistant increasing number of the cells. 4. The optimal condition of rosette formation of the peripheral Iymphocyte of normal Korean native goats was shown in the sheep erythrocyte treated with 0.1M AET for 30 nun at $37^{\circ}C$. When the Iymphocytes were treated in nylon wool column, the number of sIg-bear-ing cell were increased in the nylon wool adherent cells, but RFC was increased in the non-adherent cells. Of the infected goats, No 2 and No 3 showed significantly increasing number of sIg-bearing cells at 18 months pi. 5. The Iymphocytes of No 2 and No 3 goats reacted positively in IFA using Mab against BL-TAA at 12 months pi and 18 months pi, respectively. In CDAC test, all of four infected goats revealed positive reaction at 24 months pi. The higher positive rates were observed in No 2 and No 3 as compared with the remainders.

• 한국동물위생학회지
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• 제35권1호
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• pp.9-17
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• 2012

The objective of the present study was to isolate and identify infectious bursal disease viruses (IBDVs) from broiler and layer chickens of outbreaks of infectious bursal disease (IBD) in three districts of Bangladesh. A total of 70 bursal samples were collected from dead broiler (n=40) and layer (n=30) chickens showing specific lesions of IBD from seven commercial poultry farms of three different districts (Mymensingh, Chittagong and Tangail) of Bangladesh during the year 2007. Five representative bursal samples from each farm were used for the isolation of IBDVs using 9-day-old embryonated eggs of seronegative flock of layer birds and for identification the samples were subjected to agar gel immunodiffusion test (AGIDT), immunohistochemistry (IHC) and reverse transcription-polymerase chain reaction (RT-PCR). Out of 35 bursal samples, IBDVs were successfully isolated from 28 (80%) samples. By AGIDT, 32 (91.4%) samples were found positive for IBDV antigen. Results of AGIDT clearly indicated that IBDVs detected in 29 bursal samples of six affected farms were identical to each other but not to IBDVs present in the remaining three samples of another farm. Indirect immunoperoxidase staining of the bursal sections revealed the presence of IBDV antigen in 32 (91.4%) samples and the IBDV antigen was detected mainly in the cortex of the lymphoid follicles of the bursal tissues. In histopathology, cell depletion, atrophy and necrosis were observed in many bursal follicles with severe edema of interfollicular septa. Of the 35 bursal samples, 34 (97.1%) samples generated 254 bp product by RT-PCR. In conclusion, the results of virus isolation and identification by AGIDT, IHC and the analysis of viral genome by RT-PCR confirmed the outbreaks of acute IBD in commercial poultry of Bangladesh. Moreover, histopathological findings and results of AGIDT gave a clear indication that the isolates from six outbreaks were different from classical strain and it seems to be of very virulent strain. On the other hand, the isolates from the other outbreak were similar to the classical strain.