A protocol to count Cryptosporidium oocysts by flow cytometry without antibody staining.

Karine Sonzogni-Desautels,Momar Ndao,Marc-André Gascon,Thomas Z Di Lenardo,Axel E Renteria,Timothy G Geary,Alessandra Morassutti

doi:10.1371/journal.pntd.0007259

Karine Sonzogni-Desautels, Momar Ndao + Show 5 more

Open Access

https://doi.org/10.1371/journal.pntd.0007259

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Abstract

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.

Highlights

Cryptosporidiosis is an ubiquitous disease common in young children in developing countries [1]
A prospective case-controlled study conducted in sub-Saharan Africa and South Asia on children with moderate-to-severe diarrhea showed that Cryptosporidium spp. was the second most prevalent pathogen among infants 0–11 months old [2]
Mortality rates of 10–16% have been reported in C. parvuminfected newborn dairy calves co-infected with other enteric pathogens [7, 8]

Summary

Introduction

Cryptosporidiosis is an ubiquitous disease common in young children in developing countries [1]. A prospective case-controlled study conducted in sub-Saharan Africa and South Asia on children with moderate-to-severe diarrhea showed that Cryptosporidium spp. was the second most prevalent pathogen among infants 0–11 months old [2]. Infection with this parasite was associated with an increased risk of death in children 12–23 months old [2]. This disease affects most pre-weaned dairy calves and causes significant economic losses [4,5,6]. Mortality rates of 10–16% have been reported in C. parvuminfected newborn dairy calves co-infected with other enteric pathogens [7, 8]

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