The Detection of Circulating Antigen Glutathione S-Transferase in Sheep Infected with Fasciola hepatica with Double-Antibody Sandwich Signal Amplification Enzyme-Linked Immunosorbent Assay

TL;DR

A new ELISA method detects Fasciola hepatica infection in sheep by identifying circulating glutathione S-transferase antigens, enabling early diagnosis.

Contribution

A novel biotin–streptavidin signal amplification ELISA method for early detection of F. hepatica using circulating GST antigens.

Findings

The SA-ELISA method detected GST antigens in sheep serum with no cross-reaction to other parasites.

The SA-ELISA showed a 17.5% positive rate in tested sheep serum samples from Nong’an County.

The detection results matched 100% with commercial ELISA kits.

Abstract

As a global zoonotic parasitic disease, fasciolosis can cause serious economic losses to animal husbandry. The timely detection of Fasciola hepatica (F. hepatica)-infected animals and the adoption of epidemic prevention measures are of great significance. This manuscript provides a new technical method, a biotin–streptavidin signal amplification ELISA (SA-ELISA), based on anti-rFhGST monoclonal and polyclonal antibodies, which can detect circulating antigens in the serum of sheep infected with F. hepatica and achieve early detection. This study explores its application value in immune diagnosis, laying the foundation for the development of serum detection preparations for F. hepatica infection.

Fasciolosis is a global zoonotic parasitic disease caused by F. hepatica infection that is particularly harmful to cattle and sheep. A biotin–streptavidin signal amplification ELISA (streptavidin-ELISA/SA-ELISA) based on circulating antigens can allow for the early detection of F. hepatica-infected animals and is suitable for batch detection. It is considered to be a better means of detecting F. hepatica infection than traditional detection methods. In this study, using the serum of sheep artificially infected with F. hepatica, the cDNA expression library of F. hepatica was screened, 17 immunodominant antigen genes of F. hepatica were obtained, and glutathione s-transferase (GST) was selected as the candidate detection antigen. Firstly, the GST cDNA sequence was amplified from F. hepatica, followed by the preparation of recombinant protein GST (rFhGST). Then, monoclonal and polyclonal antibodies against rFhGST were prepared using the GST protein. Afterward, the immunolocalization of the target protein in the worm was observed via confocal microscopy, and it was found that the GST protein was localized in the uterus, intestinal tract, and body surface of F. hepatica. Finally, a double-antibody sandwich SA-ELISA based on the detection of circulating antigens was established. There was no cross-reaction with positive sera infected with Dicrocoelium lanceatum (D. lanceatum), Haemonchus contortus (H. contortus), Neospora caninum (N. caninum), or Schistosoma japonicum (S. japonicum). Forty serum and fecal samples from the same batch of sheep in Nong’an County, Changchun City, Jilin Province, China were analyzed using the established detection method and fecal detection method. The positive rate of the SA-ELISA was 17.5%, and the positive rate of the fecal detection method was 15%. The detection results of this method were 100% consistent with commercial ELISA kits. A total of 152 sheep serum samples were tested in Nong’an County, Changchun City, Jilin Province, and the positive rate was 5.92%. This study laid the foundation for the development of serological detection preparations for F. hepatica infection based on the detection of circulating antigens.

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