Application of micro-ELISA in serodiagnosis of fascioliasis in cattle

, Rhee, Jae Ku; , Baek, Byeong Kirl; , Lee, Sang Bork

doi:1985.23.1.95

Korean J Parasito > Volume 23(1):1985 > Article

Original Article


Korean J Parasitol. 1985 Jun;23(1):95-101. English. Published online Mar 20, 1994. http://dx.doi.org/10.3347/kjp.1985.23.1.95
Copyright © 1985 by The Korean Society for Parasitology


Application of micro-ELISA in serodiagnosis of fascioliasis in cattle
Jae Ku Rhee,Byeong Kirl Baek and Sang Bork Lee
Department of Veterinary Parasitology, Jeonbuk National University, Jeonju 520, Korea.


Abstract
Fascioliasis in cattle is one of the most common and very serious trematode diseases in Korea. In the present study, the enzyme linked immunosorbent assay (ELISA) was applied in the diagnosis of fascioliasis using antigen of Fasciola hepatica, peroxidase of conjugate anti-cattle IgG and orthophenylenediamine as a substrate by micro-method technique of Voller et al. (1976b) and MacLaren (1978) with a slight modification. Results obtained from the present study are as follows: In assay for optimal dilution of stock antigen, the antigen (protein contents; 0.8 mg/ml) was diluted from 1/50 to 1/600 with carbonate buffer (pH 9.6), and then absorbance values were measured with 1/100 diluted sera. The regression equations between the OD values of ELISA and dilution of antigen were log Y=-0.181-0.00127X in infected sera, and log Y= -0.578-0.000879X in normal sera. The significantly higher (p<0.05) OD value was observed in the former. In assay for optimal dilution of sera, the sera were diluted from 1/25 to 1/400 with in PBS/ Tween 20(pH 7.4), and absorbance values were measured with 1/200 diluted antigen. The regression equation between the OD values of ELISA and dilution of sera were log Y=-0.1540-0.0007238X in infected sera and log Y=-0.4834-0.00116X in normal sera. The former was higher than the latter (p<0.05). In the 27 cases of negative intradermal test, OD values of the ELISA are 0.447 ± 0.144, the 95 percent confidence interval (Mean+2 × SD) of the values was 0.735, and there was no case over the values. Therefore, the sensitivity of the antigen to diagnose fascioliasis was 100 percent in the negative case. The OD value 0.7 which is designed as a criterion (detection level of positive one) is useful for the performance of the ELISA in fascioliasis. According to the OD value of criterion in the regression equations, the optimal dilutions of stock antigen and serum were 1/250 and 1/100, respectively. In the 58 cases of fascioliasis from which the adult could be found in the bile ducts, the OD value was 0.846+0.224(-0.224). The 75 percent (44 cattle) among them had higher value with compared to the criterion, and the 60 percent (20 cattle) of the cases of proliferative cholangitis of 33 cattle which had been infected previousely with Fasciola sp. is higher than the criterion. Prevalence of fascioliasis was 43.4 percent in the application of the ELISA to 272 cattle which were reared in Jeonbug district.

Figures


	Fig. 1 Optimal dilution of antigen.


	Fig. 2 Optimal dilution of serum.


	Fig. 3 Distribution of absorbance values from the 272 heads in field survey.

Tables


	Table 1 Subjected cases tested by ELISA

References


1.	Cho SY, Hong ST, Rho YH, Choi SY, Han YC. Application of micro-ELISA in serodiagnosis of Human paragonimiasis. Korean J Parasitol 1981;19(2):151–156.

2.	Hillyer GV, Santiago de Weil N. Use of immunologic techniques to detect chemotherapeutic success in infections with Fasciola hepatica. II. The enzyme linked immunosorbent assay in infected rats and rabbits. J Parasitol 1979;65(5):680–684.

3.	Ito M, Lujan-T A, Fukumoto S, Kamiya M. Enzyme-linked immunosorbent assay (ELISA) as a diagnostic tool for Guatemalan onchocerciasis using a bovine filaria (Onchocerca gutturosa) antigen and blood samples collected on filter paper. Jpn J Vet Res 1983;31(3-4):141–150.

4.	Kim H, Kim SI, Cho SY. Serological Diagnosis Of Human Sparganosis By Means Of Micro-ELISA. Korean J Parasitol 1984;22(2):222–228.

5.	Long EG, McLaren M, Goddard MJ, Bartholomew RK, Peters P, Goodgame R. Comparison of ELISA, radioimmunoassay and stool examination for Schistosoma mansoni infection. Trans R Soc Trop Med Hyg 1981;75(3):365–371.

6.	McLaren M, Draper CC, Roberts JM, Minter-Goedbloed E, Ligthart GS, Teesdale CH, Amin MA, Omer AH, Bartlett A, Voller A. Studies on the enzyme linked immunosorbent assay (ELISA) test for Schistosoma mansoni infections. Ann Trop Med Parasitol 1978;72(3):243–253.

7.	Moorhouse PD, et al. The Vet Bull 1981;51(5):277–287.

8.	Oldham G. Antibodies to Fasciola hepatica antigens during experimental infections in cattle measured by ELISA. Vet Parasitol 1983;13(2):151–158.

9.	Richardson MD, Turner A, Warnock DW, Llewellyn PA. Computer-assisted rapid enzyme-linked immunosorbent assay (ELISA) in the serological diagnosis of aspergillosis. J Immunol Methods 1983;56(2):201–207.

*10.*	Rotmans JP, Delwel HR. Cross-linking of Schistosoma mansoni antigens and their covalent binding on the surface of polystyrene microtitration trays for use in the ELISA. J Immunol Methods 1983;57(1-3):87–98.

*11.*	Rotmans JP, Van der Voort MJ, Looze M, Mooij GW, Deelder AM. Schistosoma mansoni: use of antigens from excretions and secretions in immunodiagnosis. Exp Parasitol 1981;52(3):319–330.

*12.*	Ruitenberg EJ, van Knapen F. The enzyme-linked immunosorbent assay and its application to parasitic infections. J Infect Dis 1977;136 Suppl:S267–S273.

*13.*	Voller A, Bidwell DE, Bartlett A, Fleck DG, Perkins M, Oladehin B. A microplate enzyme-immunoassay for toxoplasma antibody. J Clin Pathol 1976;29(2):150–153.

*14.*	Voller A, Bidwell DE, Bartlett A. Enzyme immunoassays in diagnostic medicine. Theory and practice. Bull World Health Organ 1976;53(1):55–65.

*15.*	Yogore MG Jr, Lewert RM, Blas BL. Schistosomiasis japonica in Barrio San Antonio, Basey, Samar, in the Philippines. V. The enzyme-linked immunosorbent assay (ELISA) compared with quantitative stool examination and the circumoval precipitin (COP) test. Am J Trop Med Hyg 1981;30(6):1252–1262.