Detection of Cryptosporidium parvum oocysts in calf fecal samples by direct immunofluorescence assay

Weslen Fabricio Pires Teixeira,Willian Marinho Dourado Coelho,Cáris Maroni Nunes,Marcelo Vasconcelos Meireles

doi:10.1590/s1984-29612011000400003

Weslen Fabricio Pires Teixeira, Willian Marinho Dourado Coelho + Show 2 more

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https://doi.org/10.1590/s1984-29612011000400003

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Abstract

The aim of this study was to produce a conjugate containing anti-Cryptosporidium parvum polyclonal antibodies and standardize a Direct Immunofluorescence Assay (DIF) for detecting C. parvum oocysts in fecal samples from calves. In order to obtain anti-C. parvum polyclonal antibodies, two New Zealand rabbits were immunized with a purified solution of C. parvum oocysts and Freund's adjuvant. Purification of the immunoglobulin G (IgG) fraction was performed by means of precipitation in ammonium sulfate and chromatography using a DEAE-cellulose column. The anti-C. parvum polyclonal antibody titer was determined by means of the enzyme-linked immunosorbent assay (ELISA). The rabbit anti-C. parvum IgG fraction was conjugated with fluorescein isothiocyanate and standardization of the DIF was performed using various dilutions of conjugate on slides positive for C. parvum oocysts. The cross-reactivity of the anti-C. parvum conjugate was tested using oocysts of Cryptosporidium serpentis, Cryptosporidium andersoni, Escherichia coli, Eimeria sp., and Candida sp. An anti-C. parvum conjugate was successfully produced, thus allowing standardization of DIF for detection of Cryptosporidium oocysts in fecal samples. Cross-reactivity of anti-C. parvum polyclonal antibodies with C. andersoni and C. serpentis was also observed.

Highlights

Cryptosporidiosis in neonate calves is generally caused by Cryptosporidium parvum, with reports of infections in which up to 6×1011 oocysts per animal are eliminated during the first month of life (UGA et al, 2000)
(IgG) in serum of rabbits inoculated with oocysts of C. parvum
Stibbs and Ongerth (1986) standardized the indirect immunofluorescence assay for detecting Cryptosporidium sp. oocysts in fecal samples from monkeys and humans, and they achieved the best results in relation to oocyst fluorescence at conjugate dilutions of 1:20, 1:40 and 1:60 in PBS, with a considerable decrease at the dilution of 1:80

Summary

Introduction

Cryptosporidiosis in neonate calves is generally caused by Cryptosporidium parvum, with reports of infections in which up to 6×1011 oocysts per animal are eliminated during the first month of life (UGA et al, 2000). This represents a risk of transmission to other animals and to humans, given that C. parvum presents high zoonotic potential (XIAO; FAYER, 2008). Among the techniques used to detect Cryptosporidium oocysts in fecal samples, the direct immunofluorescence assay (DIF) is prominent (FAYER et al, 2000; BIALEK et al, 2002; JEX et al, 2008) It has been recognized as presenting better sensitivity and specificity than shown by traditional staining techniques (ARROWOOD; STERLING, 1989; JOHNSTON et al, 2003). The acquisition cost of these products makes it practically impossible to use them in routine laboratory analyses (ALLES et al, 1995; CARVALHO, 2009)

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