Development of an Antibody-Based Capture Enzyme-Linked Immunosorbent Assay for Detecting Echinostoma Caproni (Trematoda) in Experimentally Infected Rats: Kinetics of Coproantigen Excretion

Abstract

The present study reports on the development of a coproantigen capture enzyme-linked immunosorbent assay (ELISA) for detecting Echinostoma caproni in experimentally infected rats. The capture ELISA was based on polyclonal rabbit antibodies that recognize excretory-secretory (ES) antigens. The detection limit of pure ES was 3 ng/ml in sample buffer and 60 ng/ml in fecal samples. The test was evaluated using a follow-up of 10 rats experimentally infected with 100 metacercariae of E. caproni, and the results were compared with those of other diagnostic methods such as parasitological examination and antibody titers determined by indirect ELISA. Coproantigens were detected in all the infected rats from the first day postinfection (DPI). The period of maximal coproantigen excretion was between 7 and 21 DPI. The values remained positive until 49-56 DPI, coinciding with the disappearance of the eggs in the stool samples of the infected rats. The kinetics of coproantigen detection were correlated with those of egg output. The present assay provides an alternative tool for the diagnosis of the echinostome infections. The proposed capture ELISA makes possible an earlier diagnosis than that provided by parasitological examination and indirect ELISA and also allows for the differentiation of past and current infections. Our results show that this assay can also be used to monitor the course of echinostome infections.Echinostomes are intestinal trematodes with no tissue phase in the final host. Several vertebrates, such as aquatic birds and mammals, including humans (Graczyk and Fried, 1998), have been recorded as definitive hosts of echinostomes. Echinostomiasis in vertebrate hosts has been usually diagnosed by the presence of eggs in stool samples. However, this procedure is tedious, and eggs are not always present in feces because the worms may be preovigerous or mature worms may not be voiding eggs. Moreover, parasite maturation and egg production patterns may vary depending on different factors such as worm crowding or host species (Huffman and Fried, 1990;Toledo et al., 2003). Earlier studies have demonstrated that immunodiagnosis of echinostome infections is feasible by indirect enzyme-linked immunosorbent assay (ELISA) using different antigenic extracts. Agger et al. (1993) showed that anti-Echinostoma caproni antibodies are detectable in sera from experimentally infected NMRI mice by the 14th day postinfection (DPI) by using crude adult antigen. Graczyk and Fried (1994, 1995) detected anti-E. caproni immunoglobulins by 8 DPI in ICR mice by using glycocalyx membrane crude antigen from adult worms. However, the application of serologic diagnosis has major limitations, particularly in laboratory animals. For example, antibody titers persist after worm loss and also depend on the host species (Simonsen et al., 1991;Toledo et al., 2003). Moreover, animal handling to obtain a sufficient amount of serum samples is often difficult. These factors justify the need for alternative tools for the diagnosis of echinos...

Kinetics of Echinostoma Caproni (Trematoda: Echinostomatidae) Antigens in Feces and Serum of Experimentally Infected Hamsters and Rats

“…Compared with hamsters, rats developed weaker antibody responses. Our results are in agreement with previous studies and only low levels of antibodies were detected from 49 to 84 DPI (Toledo, Espert, Muñoz-Antoli et al, 2003;Toledo et al, 2004). It has been suggested that weak antibody responses to E. caproni infections occur because worm products are not recognized as antigenic by the host or, alternatively, that released parasite products may be immunosuppressive to the host.…”

“…This may be related to a period of high activity of the newly excysted metacercariae and juvenile adult worms that may be of importance in parasite establishment. In this sense, Toledo et al (2004) demonstrated that a number of polypeptides in the ES products are specifically expressed in the juvenile phases of E. caproni.…”

“…All the infected animals were maintained under conventional conditions with food and water ad libitum. The parasite egg release was investigated weekly in each of the infected animals as described by Toledo, Espert, Carpena et al (2003).…”

Leucine Aminopeptidase Is an Immunodominant Antigen of Fasciola hepatica Excretory and Secretory Products in Human Infections

“…Negative sera corresponded to individuals from the same area with no presence of F. hepatica eggs in feces and also negative by ELISA assays using ESP. ELISA tests using rLAP Fh were performed essentially as described previously (61). An aliquot of 0.5 g of the recombinant LAP Fh protein in coating buffer (5 g/ml) was added to the wells (100 l) of flat-bottomed 96-well microtiter plates (Nunc).…”

Immunology and pathology of echinostome infections in the definitive host.

“…There are three lines of evidence for this: (1) the survival of echinostome infections is prolonged by immunosuppression; (2) expulsion of E. caproni adult worms is impaired by concurrent infection with Schistosoma mansoni (Christensen et al 1985) ; and (3) significant differences in the immunological and pathological responses between hosts in which the parasites are rapidly rejected and those in which chronic infections occur have been observed (Toledo et al 2003b(Toledo et al , 2004b(Toledo et al , 2006bMuñoz-Antoli et al 2007) .…”