Biochemical Characterization and Differential Expression of a 16.5-Kilodalton Tegument-Associated Antigen from the Liver Fluke Fasciola hepatica

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>A cDNA encoding a 16.5-kDa protein termed FhTP16.5 was identified by immunoscreening of a cDNA library from<jats:named-content content-type="genus-species">Fasciola hepatica</jats:named-content>adult flukes using pooled sera from rabbits infected with<jats:named-content content-type="genus-species">F. hepatica</jats:named-content>for 4 weeks. Quantitative reverse transcriptase PCR (qPCR) analysis revealed that FhTP16.5 is not expressed in unembryonated eggs. It is poorly expressed in miracidia and highly expressed at the juvenile and adult stages; however, significant differences were found between the expression levels of FhTP16.5 in juveniles versus adult flukes. Recombinant FhTP16.5 was expressed at high levels in<jats:named-content content-type="genus-species">Escherichia coli</jats:named-content>, purified by affinity chromatography, and used to raise anti-FhTP16.5 polyclonal antibodies in rabbits. Immunoblot analysis using the anti-FhTP16.5 IgG antibody identified FhTP16.5 in crude and tegumental extracts and in excretory-secretory products of<jats:named-content content-type="genus-species">F. hepatica</jats:named-content>. The protein was not detected in crude extracts of<jats:named-content content-type="genus-species">Schistosoma mansoni</jats:named-content>or<jats:named-content content-type="genus-species">Schistosoma</jats:named-content><jats:named-content content-type="genus-species">japonicum</jats:named-content>. Antibodies to FhTP16.5 were detected in the sera of rabbits at 3 to 12 weeks of<jats:named-content content-type="genus-species">F. hepatica</jats:named-content>infection as well as in the sera of humans with chronic fascioliasis; these findings suggest that FhTP16.5 could be a good antigen for serodiagnosis of fascioliasis. Immunohistochemistry demonstrated that FhTP16.5 localizes to the surface of the tegument of various developmental stages and in parenchymal tissues of the adult fluke. Such specific localization makes FhTP16.5 an attractive target for immunoprophylaxis or chemotherapy.</jats:p>

Development of Two Antibody Detection Enzyme-Linked Immunosorbent Assays for Serodiagnosis of Human Chronic Fascioliasis

“…This finding supports our previous studies using experimentally infected rabbits, in which antibodies were detected between 2 to 4 weeks after infection using recombinant FhFtn-1 and FhTP16.5 proteins (29,30). Unfortunately, in the present study we were not able to obtain serum a Sensitivity ϭ 96.6% (95% CI, 88.5% to 99.1%), calculated as true-positive number/ (true-positive number ϩ false-negative number) ϫ 100; specificity ϭ 95.7% (95% CI, 89.5% to 98.3%), calculated as true-negative number/(true-negative number ϩ falsepositive number) ϫ 100.…”

“…AY851159) was cloned into the pGEX4T-1 plasmid and expressed in the same bacterial system. The glutathione S-transferase (GST)-tagged recombinant protein was purified by affinity chromatography using a GST-Trap HR 5/5 column (GE Healthcare) and was cleaved from the GST tag as described by Gaudier et al (29).…”

“…One of these antigens is a 16.5-kDa tegument-associated protein of unknown function, termed FhTP16.5 (29), and the other is a protein with ferroxidase activity classified as a member of the F. hepatica ferritin protein family (FhFtn-1) (30). Both molecules are differentially expressed during parasite development and have been shown to be highly reactive with sera from experimental animals with acute or chronic infections.…”

Liver fluke vaccines in ruminants: strategies, progress and future opportunities

“…These studies 518 revealed few differences between immature and adult flukes, 519 implying that many surface proteins are shared between these 520 developmental stages and that the analysis of adult surface pro-521 teins would be informative (Dalton and Joyce, 1987). cDNA 522 sequences encoding two surface tegument proteins in F. hepatica 523 have been cloned (Trudgett et al, 2000;Gaudier et al, 2012). (Davies et al, 1989;Bodian et al, 1997).…”

Evaluation and Characterization of Fasciola hepatica Tegument Protein Extract for Serodiagnosis of Human Fascioliasis

“…The surface protein fraction was isolated from adult F. hepatica flukes as previously described but with minor modifications (21). Briefly, F. hepatica adult flukes obtained from freshly killed cattle were washed several times with cold PBS to minimize the expression and release of ES products, parasite enzymes, and muscle constriction of the oral and ventral suckers to avoid possible ES products becoming attached to the surface.…”