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Publication Details
AFRICAN RESEARCH NEXUS
SHINING A SPOTLIGHT ON AFRICAN RESEARCH
immunology and microbiology
Generally applicable methods to purify intracellular coccidia from cell cultures and to quantify purification efficacy using quantitative PCR
Veterinary Parasitology, Volume 135, No. 3-4, Year 2006
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Description
The objective of this study was to evaluate the utility of a simple, efficient, and rapid method for the isolation of Sarcocystis neurona merozoites and Besnoitia darlingi tachyzoites from cultured cells. The efficacy of this purification method was assessed by microscopy, SDS-PAGE, Western blotting, immuno-fluorescence, and three novel quantitative PCR assays. Culture medium containing host cell debris and parasites was eluted through PD-10 desalting columns. This purification method was compared to alternatives employing filtration through a cellulose filter pad or filter paper. The estimated recovery of S. neurona merozoites purified by the column method was 82% (±3.7) of the original merozoites with 97.5% purity. In contrast, estimated recovery of S. neurona merozoites purified by filter pad and filter paper was 40% and 30% with 76% and 83% purity, respectively. The same procedures were applied to purify B. darlingi tachyzoites from cultured cells. Of the original cultured B. darlingi tachyzoites, 94% (±2.5) were recovered from the PD-10 column with 96.5%, purity whereas percentage recovery of B. darlingi tachyzoites purified by filter pad and filter paper were 51% and 35% with 84% and 88% purity, respectively. All described methods maintained sterility so that purified parasites could be subsequently cultured in vitro. However, purification using a PD-10 column minimized parasite loss and the loss of viability as determined by the trypan blue dye exclusion assay, the rate of parasite production, and plaque forming efficiency in cell culture. Moreover, column-purified parasites improved the sensitivity of an immuno-fluorescent (IFA) analysis and real-time quantitative PCR assays targeted to parasite 18S ribosomal DNA and hsp70 genes. This technique appears generally applicable for purifying coccidia grown in cell cultures. © 2005 Elsevier B.V. All rights reserved.
Authors & Co-Authors
Elsheikha, Hany M.
United States, East Lansing
Michigan State University
Egypt, Mansoura
Mansoura University
Rosenthal, Benjamin Martin
United States, Beltsville
Usda Ars Beltsville Agricultural Research Center
Murphy, A. J.
United States, East Lansing
Michigan State University
Dunams, D. B.
United States, Beltsville
Usda Ars Beltsville Agricultural Research Center
Neelis, D. A.
United States, East Lansing
Michigan State University
Mansfield, L. S.
United States, East Lansing
Michigan State University
Statistics
Citations: 25
Authors: 6
Affiliations: 3
Identifiers
Doi:
10.1016/j.vetpar.2005.09.014
ISSN:
03044017
Research Areas
Genetics And Genomics
Study Approach
Quantitative