Localization of Toxoplasma gondii in feline intestinal tissue using PCR.
Gametogony of Toxoplasma gondii occurs only in the epithelial cell layers within the intestine of the definitive feline host. Infected feline intestine is required in order to study the physiology, histology, and molecular biology of the gametogenic stages. Therefore, we set out to devise a rapid, conservative, and reproducible technique to determine which portions of the intestine were infected. Several methods of collecting and processing infected material were assessed for their ability to detect T. gondii. Infected and uninfected intestines from domestic cats were used to produce nitrocellulose lift impressions along the entire small intestine. The nitrocellulose was analyzed for the presence of T. gondii DNA using polymerase chain reaction (PCR) primers specific for the T. gondii alpha-tubulin gene. In addition, mucosal tissue scrapings, derived from segments of intestinal tissue, were used to isolate DNA and RNA for subsequent PCR and reverse transcriptase PCR analysis, respectively. The nitrocellulose impression lift method demonstrated distribution of parasite throughout the intestine. Histological staining and indirect immunofluorescence antibody analysis of sections obtained from the same infected tissue confirmed the presence of T. gondii intraepithelial stages. Comparison among the different techniques indicates that the nitrocellulose impression lift technique proved to be effective for easily and quickly assessing presence of T. gondii in infected tissue. This technique does not require a significant amount of the experimental material.
Feng
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Heska Corporation, Fort Collins, Colorado 80525, USA.
Milhausen
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D000818:Animals; D002371:Cat Diseases / Q000469:parasitology*; D002415:Cats; D016054:DNA, Protozoan / Q000032:analysis; D007150:Immunohistochemistry; D007411:Intestinal Diseases, Parasitic / Q000469:parasitology / Q000662:veterinary*; D007422:Intestines / Q000469:parasitology*; D051379:Mice; D010270:Parasite Egg Count / Q000662:veterinary; D016133:Polymerase Chain Reaction / Q000662:veterinary; D016053:RNA, Protozoan / Q000032:analysis; D014122:Toxoplasma / Q000235:genetics / Q000302:isolation & purification*; D014124:Toxoplasmosis, Animal / Q000469:parasitology*
D016054:DNA, Protozoan; D016053:RNA, Protozoan