Comparative Proteomics of Excretory- Secretory Proteins Released by the Liver Fluke Fasciola hepatica in Sheep Host Bile and during in Vitro Culture ex Host*□S

Livestock infection by the parasitic fluke Fasciola hepatica causes major economic losses worldwide. The excretory-secretory (ES) products produced by F. hepatica are key players in understanding the host-parasite interaction and offer targets for chemoand immunotherapy. For the first time, subproteomics has been used to compare ES products produced by adult F. hepatica in vivo, within ovine host bile, with classical ex host in vitro ES methods. Only cathepsin L proteases from F. hepatica were identified in our ovine host bile preparations. Several host proteins were also identified including albumin and enolase with host trypsin inhibitor complex identified as a potential biomarker for F. hepatica infection. Time course in vitro analysis confirmed cathepsin L proteases as the major constituents of the in vitro ES proteome. In addition, detoxification proteins (glutathione transferase and fatty acid-binding protein), actin, and the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase were all identified in vitro. Western blotting of in vitro and in vivo ES proteins showed only cathepsin L proteases were recognized by serum pooled from F. hepatica-infected animals. Other liver fluke proteins released during in vitro culture may be released into the host bile environment via natural shedding of the adult fluke tegument. These proteins may not have been detected during our in vivo analysis because of an increased bile turnover rate and may not be recognized by pooled liver fluke infection sera as they are only produced in adults. This study highlights the difficulties identifying authentic ES proteins ex host, and further confirms the potential of the cathepsin L proteases as therapy candidates. Molecular & Cellular Proteomics 6:963–972, 2007. The parasitic fluke Fasciola hepatica infects humans and ruminant livestock worldwide. An estimated 2.4 million people are infected with Fasciola species, and a further 180 million are at risk (1). In addition, F. hepatica causes an estimated loss of $3 billion worldwide per annum through livestock mortality, especially in sheep, and by decreased productivity via reduction of milk and meat yields in cattle (2). Presently the rate of fluke infection in parts of northwest Europe has been estimated as greater than 30%. In the absence of commercial vaccines, the benzimidazole derivative triclabendazole (TCBZ; Fasinex ) is the drug most extensively used against Fasciola. Unlike other fasciolicides, TCBZ shows activity against both juvenile flukes, which are responsible for the damage to the liver of acute fasciolosis, and the mature flukes, which cause the debilitation of chronic fasciolosis. Recently resistance to TCBZ has been reported in several countries suggesting that chemotherapeutic control of this infection may soon be compromised (3–5). The most common diagnostic field method used to detect Fasciola infection requires counting fluke eggs in fecal samples. However, egg counts are time-consuming and expensive and have limited ability to diagnose early acute stages of infection. New immunological diagnostic methods with commercially protected antigen identification are available in the form of a rapid on-site field test, DriDot (Biotechnology Ireland) based on latex agglutination and a companion ELISA for laboratory veterinary applications. Further diagnosticsbased research is required to freely validate current tests and to provide alternative molecular approaches for liver fluke populations. In well characterized cattle models, the immune response to F. hepatica infection is proinflammatory lasting for about 4–6 weeks postinfection. This immune response appears to