Assessment of Sarcocystis neurona sporocyst viability and differentiation between viable and nonviable sporocysts using propidium iodide stain.
Abstract: Sarcocystis neurona has become recognized as the major causative agent of equine protozoal myeloencephalitis (EPM) in the Americas. At least 3 pathogenic species of Sarcocystis, including S. neurona, can be isolated from opossums. Methods are needed to ascertain whether these isolates are viable and capable of causing infections. In this study, the nuclear stain propidium iodide (PI) was used to differentiate between live (viable) and heat-killed (nonviable) S. neurona sporocysts. PI was excluded by live sporocysts but penetrated compromised sporocyst membrane and stained sporozoite nuclei of dead sporocysts. After live and dead sporocysts were mixed at various ratios, the number of unstained sporocysts detected after the staining procedure correlated significantly (r2 = 0.9978) with the expected numbers of live sporocysts. Sporocyst mixtures were also assayed for in vitro excystation and development in tissue cultures. The correlation between the percentage of plaques formed in tissue cultures and the percentage of expected infectious (live) sporocysts in each mixture was r2 = 0.6712. By analysis of variance, no statistically significant difference was measured between the percentage of viable sporocysts and the percentage of infectious sporocysts (P = 0.3902) in each mixture. In addition, there was evidence of a relation between PI impermeability of sporocysts and animal infectivity. These results suggest that the PI dye-exclusion technique can be a useful tool in identifying viability and potential infectivity of S. neurona sporocysts and in differentiating between viable and nonviable sporocysts.
Publication Date: 2004-09-11 PubMed ID: 15357088DOI: 10.1645/GE-262RGoogle Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
- Animal Health
- Cell Viability
- Clinical Study
- Diagnosis
- Disease Diagnosis
- Disease Treatment
- Epidemiology
- Equine Diseases
- Equine Health
- Equine Protozoal Myeloencephalitis
- Horses
- Immunology
- In Vitro Research
- Infectious Disease
- Laboratory Methods
- Pathogenicity
- Pathogens
- Protozoa
- Sarcocystis
- Veterinary Medicine
- Veterinary Research
Summary
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The research article investigates the use of a nuclear stain called propidium iodide (PI) to distinguish between live and dead sporocysts of Sarcocystis neurona, a parasitic organism causing equine protozoal myeloencephalitis in horses. The results indicate that PI staining can be a useful method for assessing the vitality and potential infectivity of these sporocysts.
Background
- The study focuses on Sarcocystis neurona, a major cause of equine protozoal myeloencephalitis (EPM) in the Americas.
- At least 3 pathogenic species of Sarcocystis, including S. neurona, can be isolated from opossums.
- Given the health issues these parasites can cause, there is a need for reliable methods for determining whether these isolated sporocysts are viable and capable of causing infection.
Use of Propidium Iodide Stain
- The researchers used the nuclear stain propidium iodide (PI) to differentiate between the live (viable) and the heat-killed (nonviable) S. neurona sporocysts.
- PI was found to be excluded by live sporocysts, but it penetrated compromised sporocyst membranes and stained the nuclei of dead sporocysts.
- A high correlation was found between the number of unstained sporocysts detected after PI staining and the expected numbers of live sporocysts when live and dead sporocysts were mixed in various ratios.
Use of Sporocyst Mixtures for In Vitro Testing
- The sporocyst mixtures were also tested for their capability to excyst and develop in tissue cultures.
- A significant correlation was discovered between the percentage of plaques formed in the tissue cultures and the predicted percentage of infectious (live) sporocysts present in each mixture.
- No significant difference was measured between the percentage of viable sporocysts and the percentage of infectious sporocysts in each mixture, as indicated by analysis of variance.
Conclusions
- Findings suggest a relationship between PI impermeability of sporocysts and their infectivity in animals.
- The results imply that the PI dye-exclusion technique can be a useful tool in identifying the viability and potential infectivity of S. neurona sporocysts, in addition to differentiating between viable and nonviable sporocysts.
Cite This Article
APA
Elsheikha HM, Mansfield LS.
(2004).
Assessment of Sarcocystis neurona sporocyst viability and differentiation between viable and nonviable sporocysts using propidium iodide stain.
J Parasitol, 90(4), 872-875.
https://doi.org/10.1645/GE-262R Publication
Researcher Affiliations
- Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan 48824, USA. elsheik2@msu.edu
MeSH Terms
- Analysis of Variance
- Animals
- Biological Assay
- Brain / parasitology
- Coloring Agents
- Encephalomyelitis / parasitology
- Encephalomyelitis / veterinary
- Female
- Horse Diseases / parasitology
- Horses
- Interferon-gamma / genetics
- Intestine, Small / parasitology
- Mice
- Mice, Knockout
- Microscopy, Confocal / veterinary
- Microscopy, Fluorescence / veterinary
- Microscopy, Phase-Contrast / veterinary
- Oocysts / physiology
- Opossums / parasitology
- Propidium
- Sarcocystis / physiology
- Sarcocystosis / parasitology
- Sarcocystosis / veterinary
- Staining and Labeling / veterinary
Citations
This article has been cited 2 times.- Dubey JP, Howe DK, Furr M, Saville WJ, Marsh AE, Reed SM, Grigg ME. An update on Sarcocystis neurona infections in animals and equine protozoal myeloencephalitis (EPM).. Vet Parasitol 2015 Apr 15;209(1-2):1-42.
- Kopper JJ, Mansfield LS. Development of improved methods for delivery of Trichuris muris to the laboratory mouse.. Parasitol Res 2010 Oct;107(5):1103-13.
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