A protocol to count Cryptosporidium oocysts by flow cytometry without antibody staining.
Cryptosporidiosis caused by the protozoan parasites Cryptosporidium hominis and C. parvum, threatens the lives of young children in developing countries. In veterinary medicine, C. parvum causes life-threatening diarrhea and dehydration in newborn dairy calves. Protocols to detect Cryptosporidium sp...
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2019-03-01
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Series: | PLoS Neglected Tropical Diseases |
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doaj-0c0d857e3d734151acb196e548867e4b2020-11-25T02:08:33ZengPublic Library of Science (PLoS)PLoS Neglected Tropical Diseases1935-27271935-27352019-03-01133e000725910.1371/journal.pntd.0007259A protocol to count Cryptosporidium oocysts by flow cytometry without antibody staining.Karine Sonzogni-DesautelsThomas Z Di LenardoAxel E RenteriaMarc-André GasconTimothy G GearyMomar NdaoCryptosporidiosis caused by the protozoan parasites Cryptosporidium hominis and C. parvum, threatens the lives of young children in developing countries. In veterinary medicine, C. parvum causes life-threatening diarrhea and dehydration in newborn dairy calves. Protocols to detect Cryptosporidium spp. oocysts using flow cytometry have been reported; however, these protocols use antibodies against the parasite and typically focus on detection of oocysts, not quantification. These techniques are not well-suited for studies that generate large variations in oocyst burdens because the amount of antibody required is proportional to the number of oocysts expected in samples. Also, oocysts are lost in washes in the staining protocol, reducing accuracy of oocyst counts. Moreover, these protocols require costly fluorochrome-conjugated monoclonal antibodies and are not optimal for studies involving large numbers of samples. Here we present an optimized protocol for purifying oocysts from mouse stool and intestine samples combined with a reliable method to quantify oocysts in a relatively pure population without the need for antibody staining. We used morphology (SSC-A vs FSC-A) and the innate characteristics of C. parvum oocysts compared to fecal and intestinal contaminants to develop a two-step gating strategy that can differentiate oocysts from debris. This method is a fast, reliable, and high-throughput technique to promote research projects on C. parvum infections in mice and potentially other animal hosts.http://europepmc.org/articles/PMC6443187?pdf=render |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Karine Sonzogni-Desautels Thomas Z Di Lenardo Axel E Renteria Marc-André Gascon Timothy G Geary Momar Ndao |
spellingShingle |
Karine Sonzogni-Desautels Thomas Z Di Lenardo Axel E Renteria Marc-André Gascon Timothy G Geary Momar Ndao A protocol to count Cryptosporidium oocysts by flow cytometry without antibody staining. PLoS Neglected Tropical Diseases |
author_facet |
Karine Sonzogni-Desautels Thomas Z Di Lenardo Axel E Renteria Marc-André Gascon Timothy G Geary Momar Ndao |
author_sort |
Karine Sonzogni-Desautels |
title |
A protocol to count Cryptosporidium oocysts by flow cytometry without antibody staining. |
title_short |
A protocol to count Cryptosporidium oocysts by flow cytometry without antibody staining. |
title_full |
A protocol to count Cryptosporidium oocysts by flow cytometry without antibody staining. |
title_fullStr |
A protocol to count Cryptosporidium oocysts by flow cytometry without antibody staining. |
title_full_unstemmed |
A protocol to count Cryptosporidium oocysts by flow cytometry without antibody staining. |
title_sort |
protocol to count cryptosporidium oocysts by flow cytometry without antibody staining. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS Neglected Tropical Diseases |
issn |
1935-2727 1935-2735 |
publishDate |
2019-03-01 |
description |
Cryptosporidiosis caused by the protozoan parasites Cryptosporidium hominis and C. parvum, threatens the lives of young children in developing countries. In veterinary medicine, C. parvum causes life-threatening diarrhea and dehydration in newborn dairy calves. Protocols to detect Cryptosporidium spp. oocysts using flow cytometry have been reported; however, these protocols use antibodies against the parasite and typically focus on detection of oocysts, not quantification. These techniques are not well-suited for studies that generate large variations in oocyst burdens because the amount of antibody required is proportional to the number of oocysts expected in samples. Also, oocysts are lost in washes in the staining protocol, reducing accuracy of oocyst counts. Moreover, these protocols require costly fluorochrome-conjugated monoclonal antibodies and are not optimal for studies involving large numbers of samples. Here we present an optimized protocol for purifying oocysts from mouse stool and intestine samples combined with a reliable method to quantify oocysts in a relatively pure population without the need for antibody staining. We used morphology (SSC-A vs FSC-A) and the innate characteristics of C. parvum oocysts compared to fecal and intestinal contaminants to develop a two-step gating strategy that can differentiate oocysts from debris. This method is a fast, reliable, and high-throughput technique to promote research projects on C. parvum infections in mice and potentially other animal hosts. |
url |
http://europepmc.org/articles/PMC6443187?pdf=render |
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