The recent development of reliable in vitro methods swii as the fluorescent antibcdy WA), exaltation of Newcastle disease virus (END) and interference (119 rnethcds for detecting and titrating the hog cliolera(HQ virus and its antibody has fxilitatl the study of the infection and thogenesis of HC. For this purpose, the FA eohnirpae combined with a cell culture method ?fluorescent antibody-cell culture test (FAOJI) ‘7, is now applia:l by most investigators.
Lithe present study, the evaluation of the FAOJI’ was made by comparison with the END methcd to determine it reliability as a prccedure for detecting and titating HC virus. The exterimental data are summari as follows:
1 .To obtain a hyerimmune serum as the source of HC fluorescent antibody, the following immunition prccedure is to he recommended. 1) Asingle of HC live virusisgiventoaSPFpigoftwomnonths ofage;2)Twomlofserumofswine infect! with virulent HC virus is given on the 7th day after vaccination; 3) Approximately one hundred and fifty ml of the virulent virus is incculaterl interitoneally twice at 9-14 day intervals ; and 4) The pigs are bled two weeks after the last challenge, and the sea harvest!.
During the pixxtssoflakefling the antibody with fluceescein dye, almost no decrease in antibody titers was found. However, after passing the serum through a DEAB cellulose column, a dase of 1i-1/l6 was oheerved. The final product of the conjugation shower! a staining titer of 1:8. The conjugate thus obtained was very srthfic to HC virus, and considered satisfactory.
2,A comparison of culture-cells to he used for detoting and titrating HC Virus was made in order to estabilish a ketter prcxedure for the FAOJI’. Among several kinds ofthecells examined, thePK-15 cellwas foundtol:ethekest
On the PK-15 cell sheet HCvirus ap easedtoformaso-called ‘plaque” which consist of many infected cells in mass. A dose response reaction was obierved in plaquecount as thenumkeroftheplacpres always changedinparalleltothevirus dilutions.
The numher of plaques obierved after 48-hour incuhetion for the virulent virus obtained directly from infected pigs was not increased over the 24-hour count. On the other hand, the fluorescent cells were diffusely scattered throughout the cell sheetnfect! with cell culture-adapted virus after 48-hour incuketion, The optimum time for counting plaques then concluded to he 24-48 hours incubation for the virulent virus and 24-hours for the cell-adapt! virus.
3.The FAQJI’ and the END method were compared as methods of detecting and titnting HC virus, There were no remarkable differences between the two methods in their detection ra and infection titers of the virus. Both methods proved very useful, eseoially the FAOJI’ which has several additional advantages such as rapidity, simplicity et, in deteoting the virus content of HC infected tissi. In arklition, by theuse of theFAOJI’ usingPK-lS cells, thevirus couldke isolatedfrom infect! serum which contained interferon and showed negative result by the END method.
4 Careful examination of the appearance of the fluorescent ntigen of PK-15 cells were conducte-i with reference to the growth curve of virus, hi the case of the virulent virus obine-I diroily from infai pigs, the fluorescent antigens could If detected on the cells after 9-hour incubution after which the virus was released into the fluid, The placpies observed at 24-hour incubution increased only in si but in nunkers. For the cell- adapted virus, the fluorescence appeased on the cells and release of virus into the fluid cccurred almcGt at the same time after virus incculation. The fluorescent cells at 24-hour incukation formed plaque but were diffusely distribu throughout the cell sht thereafter.
fluorescent antigens formed on PK-l5 cells by the two viruses originati from pigs or cell cultures were distinctly different from each other, Such differences in the apreaearances of the fluorescent antigens keta’en the two viruses onPK-l5 cells indicated the degree of adaptation to the cells. Although it is not easy to differentiate the viruses of different virulence by the FAOJI’, it may If po€sible to distinguish the tissneculture morlified virus from the virulent field isolates. fluorescence was observedonlyinthecytoplasmofinfecalcells butnotinthenucleus.
From these data, both the FA(XIE and the END methcd have been found to If reliable and sensitive for the deeztion and titration of the HC virus. Using the FAOJr, the virulent HC virus of field isolates may be differentiated from the virus used as a live vaccine. (Note: This rerer was a part of his dcctoral thesis submittei to the Abu VeryCollegejaenbythewriterinl%8)