The development and application of an antigen capture ELISA for detecting Fasciola infections of cattle in South-east Asia (PBF 01-11 )

PBF 01-11 | Amount: $ 75,000 | Project Leader: N Anderson | Project Period: Apr 2001 – Sep 2004

A project undertaken at Monash University, Melbourne, and supervised by T Spithill and N Anderson

Infection of cattle and buffaloes with Fasciola parasites is widespread throughout the world and overall losses in productivity due to these infections has been estimated to be US$ 3 billion annually. In south-east Asia most households engaged in agriculture keep only 1 or 2 ruminants for draught purposes or for the production of milk and meat. These animals become infected with Fasciola when grazing grassed verges or rice stubbles that have the infective stage released from aquatic snails. Sequential infections destroy tissue and cause fibrosis that impairs liver function and reduces productivity.

Detection of infection relies on finding small numbers of Fasciola eggs in samples of voluminous faeces. Eggs are not present in faeces until flukes reach maturity, usually 10 to 14 weeks after infection and about one third of infected cattle have zero egg counts. Various immunological methods, including ELISA, have been devised to measure serum antibodies produced after infection. Positive tests indicate previous infection but provide no indication that an animal is currently infected or, like egg counts, the number of flukes present.

The objectives of this project are to develop and characterise an ELISA to detect specific antigens released by Fasciola in cattle and to determine the quantitative relationship between the amount of antigen in blood and faeces and the number of flukes present in the liver. This ELISA will then be applied in the field, to determine when cattle first become infected and to measure the disappearance of antigen after treatment for fluke infections. Researchers in Melbourne and at the National Institute for Veterinary Research in Hanoi, Vietnam are collaborating to achieve these objectives. .

During the first year of the project, 3 attempts were made in Melbourne to prepare monoclonal antibodies to a purified cathepsin L antigen that comprises about 70% of the excretory/secretory antigens produced by Fasciola. IgM antibodies were produced but were found unsuitable in the ELISA. When the excretory/secretory  antigen was used, 6 clones producing IgG antibodies were identified for further evaluation now currently underway. Polyclonal antibodies to the same antigens were also prepared, in Melbourne and in Hanoi. Immunoglobulin containing these antibodies was used to capture antigen by ELISA but non-specific binding precluded differentiation of samples with and without antigen. To overcome non-specific binding, it will be necessary to separate out the specific anti-Fasciola antibody using affinity chromatography.  The first attempt at this in Hanoi was not successful and fresh reagents are needed before the next attempt is made.

Concurrently with the development of the antigen capture ELISA, a standard has been prepared for determining the operating characteristics (sensitivity and specificity) of all tests. The number of fluke in each of 150 cattle killed at abattoirs in Hanoi were counted (range 0–200) and samples of blood and faeces, from the same animals, stored for analysis by both ELISA methods.

Anderson N, Luong T T, Vo N G, Bui K L, Smooker P M and Spithill T W. 1999. The sensitivity and specificity of two methods for detecting Fasciola infections in cattle. Veterinary Parasitology, 83:15–24.