Biology ETDs

Linda L. Cole

Publication Date

5-5-1977

Abstract

Recent investigations have revealed the widespread involvement of Chlamydia trachomatis in nongonococcal genital infections in man. In an effort to develop the necessary methodology for isolating this agent, several different cell culture techniques were evaluated.

Additionally, a fluorescent antibody technique and neutralization test were developed for possible diagnostic use.

Quantitation of Chlamydia in cell culture was accomplished using inclusion counts which showed linear dose response curves. Inclusions were stained by the PAS, iodine, and Giemsa methods, and these stains were found to be equal in their sensitivity in detecting chlamydia inclusions. Using inclusion counts as a quantitative assay, a variety of enhancement techniques were studied which included centrifugation and pretreatment of cell monolayers with 5-iodo-2-deoxyuridine (IUDR), DEAE-dextran, and sucrose. The optimal centrifugal force for inoculation of a chlamydia stock was found to be 2000 x g when forces of 500 x g to 10,000 x g were employed, and centrifugation was consistently observed to be superior to any other single method of enhancement. Further enhancement of chlamydia infection was observed with UDR and sucrose pretreatment of McCoy cells and DEAE-dextran pretreatment of HeLa 229 cells when these methods were used in combination with centrifugation. In a comparison of these methods for the primary isolation of chlamydia from genital specimens, IUDR pretreatment was found to be superior.

Using centrifugation as the only method of enhancement, a variety of different cell types were compared for their sensitivity to infection with a genital and an ocular chlamydial strain. The McCoy cell line was found to be significantly more susceptible than a variety of other cell types including HeLa 229, KIT, CCl-131, BSC-1, NHP, RK-13, BHK-21, FHM, Aedes albopictus, primary mouse macrophages, and Raji cells, The McCoy, RK-13, BHK-21, and HeLa 229 cell lines were also compared for susceptibility to type 2 herpes simplex infection. Only McCoy cells failed to support the replication of herpes simplex virus as evidence by lack of cytopathic effect, intranuclear inclusions, or increase in virus titers. Although not sensitive for the isolation of herpes simplex virus, preliminary observations indicated that inoculation of McCoy cells using centrifugation may prove to be a useful system for isolation of another venereal agent, Trichomonas.

Rapid detection of chlamydia inclusions in urethral and cervical smears was also evaluated using an indirect fluorescent antibody technique. These studies failed to demonstrate any inclusion bodies even though many of the patients from whom the smears were taken were culture positive for Chlamydia trachomatis. Finally, evidence is presented supporting the frequent association of Chlamydia with certain genital infections in man.

Language

English

Document Type

Thesis

Degree Name

Microbiology

Level of Degree

Masters

Department Name

UNM Biology Department

First Committee Member (Chair)

Leroy Clarence McLaren

Second Committee Member

John August Ulrich

Third Committee Member

Scott Wilson Jordan

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