Antigen preparation from ornithosis agents grown in cell culture : a dissertation /
ABSTRACT: The purpose of this study was to prepare antigens from cell culture grown ornithosis agents. Special emphasis was placed on the development of an agglutination antigen for possible use in the detection of ornithosis infections in turkeys. McCoy cell monolayers were used for propagation o...
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| Format: | Thesis Book |
| Language: | English |
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College Station, TX :
Texas A&M University,
1967.
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| Summary: | ABSTRACT: The purpose of this study was to prepare antigens from cell culture grown ornithosis agents. Special emphasis was placed on the development of an agglutination antigen for possible use in the detection of ornithosis infections in turkeys. McCoy cell monolayers were used for propagation of ornithosis agents which were maintained in the allantoic cavity of embryonated chicken eggs for seed stock. High yield of particles were obtained from cell cultures inoculated with the Texas turkey (TT) ornithosis agent or a white-winged dove isolate. The Jo strain did not produce usable quantities of particles. In the preparation of agglutination antigens, fluids from inoculated cell cultures were pooled, boiled, and treated with phenol. After cell culture debris was removed by centrifugation, particles were sedimented by centrifugation, re-suspended in distilled water, and re-sedimented. Washed particles were suspended in phosphate-buffered saline or borate-buffered saline (BBS) containing phenol. These antigens became inactive after prolonged storage. Some harvests of washed particles were suspended in BBS containing formaldehyde and, after storage, were re-sedimented then suspended in BBS containing phenol. The latter type of antigen reacted better in agglutination tests and results were more easily interpreted than were those using antigens prepared by the first method. Turkey serums were obtained prior to inoculation and at various intervals after the birds were inoculated with either the TT or Jo strain of ornithosis agent. Serums were tested by microagglutination tests conducted in Boerner microflocculation slides and by complement fixation inhibition (CFI) tests. In 74 [percent] of the serums tested titers were demonstrated by both tests. Agglutination titers were generally lower than CFI titers. In agglutination tests with some postinoculation serums, prozones, or lack of agglutination, and sedimentation of clumped antigen occurred in the lower serum dilutions. Freshly collected serums exhibited higher titers than did those tested after having been stored frozen. Some agglutination reactions occurred with lower dilutions of some preinoculation serums but the agglutination was not as intense as reactions with postinoculation serums. Various treatments of serums did not prevent nonspecific reactions. Antigens reactive with postinoculation turkey serums in the modified direct complement fixation (CF) test were prepared using boiled and phenolized particles which were suspended in saline containing glycerol and phenol. An ether-soluble CF antigen was obtained from cell culture fluid after the particles were removed. The antigen lost most of its reactivity after prolonged storage. Another type of CF antigen was prepared from particles which were boiled, sedimented, re-suspended in veronal-buffered saline, then phenolized. These antigens were reactive in direct CF tests with human, bovine, and ovine serums that reacted with other antigens made from agents antigenically related to ornithosis agents. Titers were generally lower with the cell culture antigens than they were with the other antigens. The latter type of antigen was also usable in CFI tests with turkey serums. A hemagglutination antigen that agglutinated mouse erythrocytes was made from boiled and phenolized particles which were further treated with formaldehyde and sonified. In hemagglutination-inhibition tests with turkey serums, preinoculation serums contained inhibitors which could not be removed by various treatments of serums and postinoculation serums had titers similar to those of preinoculation serums. Results of these studies show that high yields of ornithosis agent particles can be obtained by propagation of these agents in McCoy cell monolayers. Although agglutination reactions were obtained with postinoculation serums, improvements should be attempted in an effort to obtain more stable antigens and circumvent nonspecific reactions. Suitable antigens were made for use in CF tests although the practicality of such methods may be questioned because of the low yields. |
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| Item Description: | "Submitted to the Graduate College of the Texas A&M University in partial fulfillment of the requirements for the degree of Doctor of Philosophy August 1967". "Major Subject: Veterinary Microbiology". Vita. |
| Physical Description: | 66 leaves : illustrations ; 28 cm. |
| Bibliography: | Includes bibliographical references (leaves 59-63). |