It is interesting to note that there was decline in antibodies 90 days after treatment, when tested with recombinant proteins as compared with WCL where antibodies persisted even after 469 days post treatment Fernández-Villegas et al. (2011) also reported a drop in reactivity against HSP70 in an antigen-specific manner shortly after treatment of chagasic patients with benznidazole. These observations suggest that HSP antibodies are short lived and declined due to the elimination of parasites by drug treatment. Similarly, Boulangé et al. (2002) reported the persistence of antibodies with a crude antigen against T. congolense infection in two cattle for 456 days after treatment with a trypanocidal drug.
The recombinant HSP70 fragments were evaluated using field sero-positive and sero-negative serum samples. A minimum sensitivity was observed by HSP-1 fragment (41.03%), which revealed poor antigenic nature of N-terminal region of HSP70. This was consistent with the previous reports of poor antigenic nature of HSP70 N-terminal region in L. braziliensis and T. congolense (Amorim et al., 1996; Boulangé et al., 2002). Highest sensitivity was observed with HSP-2 fragment among C-terminal fragments and lowest sensitivity was observed with the HSP-4 fragment. However, among the C-terminal fragments lowest specificity was observed by HSP-2 fragment (333–612 amino acids) (66.67%) of HSP70 protein. Flechas et al. (2009) also reported that healthy donor’s sera samples were recognised by an internal fragment of the T. cruzi HSP70 protein covering 192–433 raas inhibitors
showing lowest specificity with absorbance values ranging from 0.161 to 0.922. In the present investigation, ELISA specificity increases in general, when HSP70 fragments shift from N-terminal to C-terminal. Zurita et al. (2003) also showed 70% sensitivity and 100% specificity with
C-terminal fragment of rLb70 (513–663) in diagnosis of cutaneous leishmaniasis and mucocutaneous leishmaniasis caused by L. braziliensis, but HSP-4 fragment (514–690 amino acids of HSP70) of T. evansi showed 91.3% specificity with lower sensitivity of 48.72% among C-terminal regions.
Conflict of interest statement
We are grateful to Director, National Research Centre on Equines, Hisar for providing necessary facilities to conduct the work. The authors also wish to acknowledge Department of Biotechnology (DBT), Govt. of India for funding the research through Grant no. BT/PR14499/ADV/57/107/2010 and University Grants Commission (UGC), New Delhi for providing Junior research fellowship to the first author. The technical support provided by R.K. Dayal is also acknowledged.
The flukes of Eurytrema genus, Looss 1907, belonging to the family Dicrocoeliidae, are parasites of pancreatic ducts of animals and occasionally humans. Among the most important species are Eurytrema coelomaticum and Eurytrema pancreaticum, the first specie being found in Brazil (Mattos Júnior and Vianna, 1987; Bassani et al., 2007). The parasite has two intermediate hosts, the land snail (Bradybaena similares) and arthropods of the genus Conocephalus, commonly known in Brazil as “esperanças” (Mattos Júnior and Vianna, 1987).
Eurytrema spp. is considered a low pathogenic parasite, but may be associated with mortality and loss of productive performance of animals due to chronic lesions in pancreas. Clinical cases of eurytrematosis in cattle have been reported in Brazil (Ilha et al., 2005; Rachid et al., 2011; Quevedo et al., 2013), and the clinical and pathological pattern observed was chronic and progressive weight loss related to interstitial pancreatitis. The prevalence of parasitism in Brazil varies between regions: Lucca et al. (2015) reported 69% in the west of Santa Catarina State, however, Azevedo et al. (2004) reported that prevalence varies between 8.3 and 40.5% in different regions in Paraná state.
The main pathological changes described in the pancreas of cattle naturally infected by Eurytrema spp. are characterized by thickening of the pancreatic ducts due to hyperplasia and fibrosis, presence of the parasite inside the ducts and inflammatory infiltrate consisting of lymphocytes, macrophages and plasma