Viability assays of intra-erythrocytic organisms using fluorescent dyes

Three intra-erythrocytic tick fever organisms of cattle (Babesia bovis, Babesia bigemina and Anaplasma centrale) were subjected to a range of stressors, including heat, storage over time, specific chemotherapy and cryopreservation. Various stains, both alone and in combination, were used in an attem...

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Bibliographic Details
Published in:	Veterinary parasitology Vol. 163; no. 1-2; pp. 144 - 147
Main Authors:	Fletcher, Taryn I., Wigg, Jacqueline L., Rolls, Peter J., de Vos, Albertus J.
Format:	Journal Article
Language:	English
Published:	Netherlands Elsevier B.V 07-07-2009 Amsterdam; New York: Elsevier
Subjects:	abiotic stress Anaplasma centrale Anaplasma centrale - cytology Anaplasma centrale - drug effects Anaplasma centrale - physiology Anaplasmosis - blood Animals antiprotozoal agents Antiprotozoal Agents - therapeutic use Babesia - cytology Babesia - drug effects Babesia - physiology Babesia bigemina Babesia bovis babesiosis Babesiosis - parasitology Babesiosis - veterinary bioassays bovine anaplasmosis carboxyfluorescein diacetate succinimidyl ester Cattle cattle diseases Cattle Diseases - blood Cattle Diseases - drug therapy CFSE Cryopreservation differential staining erythrocytes Erythrocytes - parasitology Eth-D ethidium homodimer-1 FDA fluorescein diacetate Fluorescent Dyes fluorescent labeling Fluorescent staining Hot Temperature iodides Ixodidae labeling techniques parasitemia Propidium iodide Specimen Handling Staining and Labeling storage time stress tolerance validity viability Viability assay Viability assay Babesia bigemina FDA Eth-D Fluorescent staining Propidium iodide CFSE Babesia bovis Anaplasma centrale
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Summary:	Three intra-erythrocytic tick fever organisms of cattle (Babesia bovis, Babesia bigemina and Anaplasma centrale) were subjected to a range of stressors, including heat, storage over time, specific chemotherapy and cryopreservation. Various stains, both alone and in combination, were used in an attempt to assess viability of these organisms before and after the stressors were applied. Carboxyfluorescein diacetate succinimidyl ester (CFSE) stained live Babesia spp. very well while fluorescein diacetate (FDA) stained A. centrale successfully. Propidium iodide (PI) and ethidium-homodimer-1 (Eth-D) were used as counter stains to identify dead organisms. Stain combinations allowed differentiation between living and dead Babesia organisms after exposure to heat and after chemotherapy. PI and Eth-D as counter stains were of little value after deglycerolisation of cryopreserved organisms. Possible reasons for this limited success in determining death or viability of tick fever organisms after some treatments include the impermeability of red blood cells to PI and Eth-D counter stains or the loss of live and/or dead organisms during sample processing.
Bibliography:	http://dx.doi.org/10.1016/j.vetpar.2009.03.029 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0304-4017 1873-2550
DOI:	10.1016/j.vetpar.2009.03.029