Diagnosis of bovine cryptosporidiosis by indirect immunofluorescence assay using monoclonal antibody

, Wee, S H; , Lee, C G; , Joo, H D

doi:1995.33.2.107

Korean J Parasito > Volume 33(2):1995 > Article

Original Article


Korean J Parasitol. 1995 Jun;33(2):107-115. English. Published online Jun 20, 1995. http://dx.doi.org/10.3347/kjp.1995.33.2.107
Copyright © 1995 by The Korean Society for Parasitology


Diagnosis of bovine cryptosporidiosis by indirect immunofluorescence assay using monoclonal antibody
Sung-Hwan Wee,^*¹Chung-Gil Lee,² and Hoo-Don Joo¹
¹National Veterinary Research Institute, RDA, Anyang 430-016, Korea.
²College of Veterinary Medicine, Chonnam National University, Kwangju 500-757, Korea.

Received February 09, 1995; Accepted May 09, 1995.
Abstract
Two hybridoma cell lines against Cryptosporidium parvum oocysts (VRI- CN91) were produced. The isotype of these 2 monoclonal antibodies (mAbs) was IgG2b (1E7.2) and and IgM (C6). Enzyme immuno-transfer blotting analysis showed that 1E7.2 reacted specifically to 36 kDa protein and C6 reacted to 67 and 70 kDa proteins. C. parvum was bound specifically to the surface region of oocysts by these mAbs. No cross- reactivity was observed with tachyzoites of Toxoplasma gondii and oocysts of Eimeria zuernii, E. bovis and E. canadensis of bovine origin. The indirect immunofluorescence assay (IIF) using mAb C6 was successful with counterstain. With the IIF using mAb C6, oocysts appeared as 3 to 5 µm spherical objects fluorescing bright apple green against a reddish dark background. The IIF using mAb C6 was agreed in specificity and sensitivity with those of a commercial diagnostic kit. These results demonstrated that the produced mAbs were specific to C. parvum and that the mAb C6 could be used for diagnosis of cryptosporidiosis.

Figures


	Fig. 1 The proteins of Cryptosporidium parvum shown on 5-15% gradient SDS-PAGE using Coomassie brilliant blue G staining technique; standard molecular weights are indicated on the left.


	Fig. 2 Antigenic profiles of Cryptosporidium parvum oocysts detected by enzyme immuno-transfer blotting. Lane 1: polyclonal anti-C. parvum rabbit serum. Lane 2: hybridoma supernatants containing monoclonal antibody 1E7.2 followed by a goat anti-mouse horseradish peroxidase-conjugated IgG; standard molecular weights ard indicated on th left.


	Fig. 3 Enzyme immuno-transfer blotting of 5-15% gradients SDS-PAGE of oocyst antigens detected with hybridoma supernatants containing monoclonal antibody C6 followed by a goat anti-mouse horseradish peroxidase-conjugated IgM; standard molecular weights are indicated on the left.


	Fig. 4 Cryptosporidium parvum oocysts labeled by monoclonal antibody 1E7.2, showing the antibody reacted specifically with the surface region of the oocysts (× 500).


	Fig. 5 Cryptosporidium parvum oocysts in calf diarrheal fecal specimens diagnosed by indirect immunofluorescent assay using monoclonal antibody C6 (× 250).

Tables


	Table 1 Comparison of diagnostic efficacy between modified acid-fast stain and immunofluorescent assay for detection of Cryptosporidium parvum oocysts in diarrheal feces of an experimentally infected calf


	Table 2 Comparison of th relative specificity and sensitivity between modified acid-fast stain and immunofluorescent assay for detection of Cryptosporidium parvum oocysts in diarrheal feces of an experimentally infected calf

References


1.	Anderson BC. Patterns of shedding of cryptosporidial oocysts in Idaho calves. J Am Vet Med Assoc 1981;178(9):982–984.

2.	Angus KW. In Practice 1987;9:47–49.

3.	Anusz KZ, Mason PH, Riggs MW, Perryman LE. Detection of Cryptosporidium parvum oocysts in bovine feces by monoclonal antibody capture enzyme-linked immunosorbent assay. J Clin Microbiol 1990;28(12):2770–2774.

4.	Arrowood MJ, Sterling CR. Comparison of conventional staining methods and monoclonal antibody-based methods for Cryptosporidium oocyst detection. J Clin Microbiol 1989;27(7):1490–1495.

5.	Baron EJ, Schenone C, Tanenbaum B. Comparison of three methods for detection of Cryptosporidium oocysts in a low-prevalence population. J Clin Microbiol 1989;27(1):223–224.

6.	Bronsdon MA. Rapid dimethyl sulfoxide-modified acid-fast stain of Cryptosporidium oocysts in stool specimens. J Clin Microbiol 1984;19(6):952–953.

7.	Chai JY, Shin SM, Yun CK, Yu JR, Lee SH. [Experimental activation of cryptosporidiosis in mice by immunosuppression]. Korean J Parasitol 1990;28(1):31–37.

8.	Cross RF, Moorhead PD. A rapid staining technic for cryptosporidia. Mod Vet Pract 1984;65(4):307.

9.	Current WL, Haynes TB. Complete development of Cryptosporidium in cell culture. Science 1984;224(4649):603–605.

*10.*	Garcia LS, Bruckner DA, Brewer TC, Shimizu RY. Techniques for the recovery and identification of Cryptosporidium oocysts from stool specimens. J Clin Microbiol 1983;18(1):185–190.

*11.*	Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227(5259):680–685.

*12.*	Lazo A, Barriga OO, Redman DR, Bech-Nielsen S. Identification by transfer blot of antigens reactive in the enzyme-linked immunosorbent assay (ELISA) in rabbits immunized and a calf infected with Cryptosporidium sp. Vet Parasitol 1986;21(3):151–163.

*13.*	Lumb R, et al. Immunol Cell Biol 1989;67:267–270.

*14.*	Mead JR, Arrowood MJ, Sterling CR. Antigens of Cryptosporidium sporozoites recognized by immune sera of infected animals and humans. J Parasitol 1988;74(1):135–143.

*15.*	Miller RA, Wasserheit JN, Kirihara J, Coyle MB. Detection of Cryptosporidium oocysts in sputum during screening for mycobacteria. J Clin Microbiol 1984;20(6):1192–1193.

*16.*	Ortega-Mora LM, Troncoso JM, Rojo-Vazquez FA, Gomez-Bautista M. Cross-reactivity of polyclonal serum antibodies generated against Cryptosporidium parvum oocysts. Infect Immun 1992;60(8):3442–3445.

*17.*	Peeters JE, Villacorta I, Vanopdenbosch E, Vandergheynst D, Naciri M, Ares-Mazas E, Yvore P. Cryptosporidium parvum in calves: kinetics and immunoblot analysis of specific serum and local antibody responses (immunoglobulin A [IgA., IgG, and IgM) after natural and experimental infections. Infect Immun 1992;60(6):2309–2316.

*18.*	Pohjola S. Negative staining method with nigrosin for the detection of cryptosporidial oocysts: a comparative study. Res Vet Sci 1984;36(2):217–219.

*19.*	Robert B, Ginter A, Antoine H, Collard A, Coppe P. Diagnosis of bovine cryptosporidiosis by an enzyme-linked immunosorbent assay. Vet Parasitol 1990;37(1):1–8.

*20.*	Rusnak J, Hadfield TL, Rhodes MM, Gaines JK. Detection of Cryptosporidium oocysts in human fecal specimens by an indirect immunofluorescence assay with monoclonal antibodies. J Clin Microbiol 1989;27(5):1135–1136.

*21.*	Smith HV, McDiarmid A, Smith AL, Hinson AR, Gilmour RA. An analysis of staining methods for the detection of Cryptosporidium spp. oocysts in water-related samples. Parasitology 1989;99(Pt 3):323–327.

*22.*	Tilley M, Upton SJ, Fayer R, Barta JR, Chrisp CE, Freed PS, Blagburn BL, Anderson BC, Barnard SM. Identification of a 15-kilodalton surface glycoprotein on sporozoites of Cryptosporidium parvum. Infect Immun 1991;59(3):1002–1007.

*23.*	Tyzzer EE. Arch Protistenke 1912;26:394–412.

*24.*	Ungar BL, Nash TE. Quantification of specific antibody response to Cryptosporidium antigens by laser densitometry. Infect Immun 1986;53(1):124–128.

*25.*	Wagner ED, et al. Jpn J Parasitol 1986:273–278.

*26.*	Wee SH, et al. Korean J Wet Res 1990;230:103–106.

*27.*	Wee SH, et al. Res Rept RDA (γ) 1992;34:43–48.

*28.*	Wee SH, Lee CG, Kim BS, Joo HD, Kang SW. [Isolation of Cryptosporidium parvum oocysts from fecal samples--the combination of ether extraction and discontinuous sucrose gradients]. Korean J Parasitol 1994;32(1):7–12.

*29.*	Whitmire WM, Harp JA. Characterization of bovine cellular and serum antibody responses during infection by Cryptosporidium parvum. Infect Immun 1991;59(3):990–995.

*30.*	Zierdt WS. Concentration and identification of Cryptosporidium sp. by use of a parasite concentrator. J Clin Microbiol 1984;20(5):860–861.