Evaluation of recombinant fructose-1,6-bisphosphate aldolase ELISA test for the diagnosis of Schistosoma japonicum in water buffaloes
Introduction
Schistosoma japonicum is one of the most important parasites in the world. It is endemic in the marsh and lake regions of southern China, islands without a definite dry seasons in the Philippines, and the island of Sulawesi (Celebes) in Indonesia (McGarvey et al., 1999). In addition to human, this parasite also infects cattle, pigs, and water buffaloes (He et al., 2001). It has been estimated that more than 100 million people in 380 counties of 12 provinces in China and are exposed to the infection and about 1.2 million cattle acquired the infection (Minggang and Zheng, 1999, Ross et al., 2001).
The fructose-1,6-bisphosphate aldolase (FBPA) is an ubiquitous enzyme essential for glycolysis, gluconeogenesis and the Calvin cycle. This enzyme catalyzes the reversible cleavage of fructose 1,6-phosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate in the glycolytic pathway during gluconeogenesis (Patron et al., 2004). FBPAs can be divided into two classes according to the reaction mechanism and taxonomic distribution. Class I FBPA utilizes a Schiff base reaction mechanism and has been found in animals, plants, protozoa, algae, bacteria and archaea (Ahmed et al., 2005). Class II FBPA uses divalent metal ions for catalysis and has been found only in bacteria and fungi (Marsh and Lebherz, 1992).
In addition to its catalytic activities, immunization with FBPA may also induce immune responses in the host. FBPA has been identified as an immunoreactive protein in a murine model with systemic Candida albicans infection (Pitarch et al., 2001). Antibodies against Plasmodium falciparum FBPA have been detected in serum samples from human with partial immunity to malaria (Srivastava et al., 1990). This enzyme may also induce immunity to P. falciparum in monkeys (Perrin et al., 1985, Certa et al., 1988, Herrera et al., 1990), Trypanosoma brucei in mice (Balaban et al., 1995) and Giardia lamblia (Palm et al., 2003) or Onchocerca volvulus (McCarthy et al., 2002) in human. Moreover, recombinant FBPA has been reported to be useful in the immunodiagnosis of malaria (Moody, 2002).
FBPA has been cloned from the cercariae and lung-stage larvae of S. mansoni. The recombinant protein not only shows enzymatic activities but induces humoral and cellular immune responses in mice (El-Dabaa et al., 1998, Harrop et al., 1999). In this paper, we reported the expression and purification of the recombinant FBPA from the adult worms of S. japonicum. Moreover, we also evaluated the performance of recombinant FBPA enzyme-linked immunoabsorbent assay (ELISA) for the diagnosis of S. japonicum infection in water buffaloes.
Section snippets
Parasite
A Chinese strain of S. japonicum was obtained from the Hunan Institute of Parasite Disease, Hunan Province, People’s Republic of China (PRC) (He et al., 2002). Schistosome-infected snails were air-mailed to our laboratory in Taipei. The life cycle was then maintained in male 8–10-week C57BL/6J mice through Oncomelania hupensis hupensis snails. Eight weeks after infection, adult worms were collected from the mesenteric vasculature by the perfusion technique (Duvall and DeWitt, 1967) and then
Molecular characterization of FBPA
The gene encoding the FBPA of S. japonicum was successfully amplified by PCR from cDNA collected from adult worms. The FBPA amplicon was sequenced and the amino acid sequence was deduced. The full length sequence of the FBPA clone was determined to be 1092 bp encoding a protein of 363 amino acid residues with a molecular mass of 39.6 kDa. This nucleotide sequence has been submitted to the GenBank data bank under the accession number DQ497791. S. japonicum FBPA showed 95% (348/363) amino acid
Discussion
Schistosoma infections may be directly diagnosed by demonstrating parasites eggs in stool, urine or tissue samples or detecting circulating schistosome-derived antigens. Egg microscopy is a traditional and widely used method. Although this method is lowest in cost and technical assistance is readily available (Doenhoff et al., 2004), it has the disadvantages of lacking sensitivity (Bierman et al., 2005), labour-intensive and time-consuming (Zhu, 2005). The Kato–Katz thick smear technique is an
Acknowledgments
We thank the Institute of Parasite Disease Control and Prevention and the Chinese Center for Disease Control for providing the sera for this study.
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