Characterization of the humoral immune response in alpacas (Lama pacos) experimentally infected with Fasciola hepatica against cysteine proteinases Fas1 and Fas2 and histopathological findings

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Abstract

A characterization of the humoral immune response of alpacas to Fasciola hepatica Fas1 and Fas2 antigens, two abundant cysteine proteinases in the excretory/secretory (E/S) products, was performed over the course of 6 months of experimental infection. Six adult alpacas aged 1–2 years old received a single dose of 200 F. hepatica metacercariae; two non-infected alpacas were kept as control group. All infected animals shed eggs 8 weeks post-infection (PI) and the number of flukes recovered at necropsy averaged 41 ± 4. The livers of infected animals showed regions with chronic inflammation, granuloma containing parasite eggs, necrosis and cirrhosis. Peripheral eosinophilia in infected animals was greatly enhanced 6 weeks post-infection and later. A single peak of serum glutamic piruvic transaminase (SGPT) was observed 4 weeks PI and serum glutamic oxalacetic transaminase (SGOT) elevated 3 weeks PI and later. Circulating IgG Abs against Fas1 and Fas2 were measured by enzyme-linked immunosorbent assay (ELISA). Fas2-ELISA detected the infection 10 days PI reaching to highest titer on 7–8 weeks PI and kept elevated, until the end of infection. Fas1-ELISA detected the infection 2 weeks PI and followed the same pattern as Fas2-ELISA. Anti Fas2 IgG Abs were in higher titers and showed stronger avidity than anti Fas1 IgG Abs. In addition, rabbit IgG antibodies raised against cysteine proteinase Fas2 showed infiltration of this parasite antigen associated to the degradation of bile ducts and liver parenchyma of infected alpacas.

In the present study we have established a F. hepatica experimental infection of alpacas, Fas2 appears to have a role in the pathogenesis of the liver damage in alpacas caused by the liver fluke. Infected alpacas elicited a strong humoral immune response against fluke cysteine proteinases Fas1 and Fas2, which might be considered as candidates for immunodiagnosis and vaccine development against fasciolosis in alpacas.

Introduction

Fasciola hepatica infection is a serious problem to the alpaca farming by the loss in animal productivity and by the increased mortality observed in untreated animals (Leguía and Casas, 1999). The disease is highly prevalent along the high steppes of the Andes, where most of alpaca farms in Peru are found (Neyra et al., 2002). The transmission of the infection is facilitated by the heavy contamination of the pastures with the infective form, the climate that favors the parasite cycle and the lack of control programs against the fluke. Despite the importance of this infection by its negative impact in fiber productivity and quality, little is known of the liver pathology caused by F. hepatica in alpacas and the fluke factors associated to the pathogenesis of the disease. A differential susceptibility of the host to the fluke is a feature of the disease, being cattle able to mount a strong immune response that immunoprotects to subsequent infections and sheep develop little or absent immunoprotective response to F. hepatica infection (Mulcahy et al., 1999). It is not known whether alpacas become resistant to F. hepatica after a primary infection. However, herd screening suggests that alpacas are frequently re-infected in endemic areas (Neyra et al., 2002).

F. hepatica cysteine proteinases are abundantly expressed and secreted into the parasite gut lumen by the adult worm (Dalton et al., 2003). Fluke cysteine proteinases are reported as sensitive and specific markers for the serodiagnosis of fasciolosis in humans (Cordova et al., 1997, Cordova et al., 1999, O’Neill et al., 1998, Strauss et al., 1999), in sheep and cattle (Cornelissen et al., 1999, Cornelissen et al., 2001), and recently in goats (Ruiz et al., 2003). The major components of F. hepatica E/S products are cysteine proteinases, which are termed Fas1 and Fas2. Both parasite enzymes were purified by a simple procedure (Cordova et al., 1997) and evaluated as markers of the infection in alpacas that were naturally exposed to liver fluke in endemic areas (Neyra et al., 2002).

In the present study, we report an evaluation of the humoral immune response and liver histopathological findings in alpacas experimentally infected with F. hepatica.

Section snippets

Experimental infection

Alpacas were obtained from SAIS Pachacutec, Junin, and transported to Lima, where they were kept for 2 months for adaptation to the housing conditions. Animals were selected on the field by checking for helminth infection and none of them were positive by coprology and serology to F. hepatica. As a preventive measure, all animals received a single dose of triclabendozole and closantel 1 month before the experimental infection. Animals were housed in an uncovered pen and fed with dry hay and

Experimental infection and blood cell count

All infected alpacas shed F. hepatica eggs 8 weeks PI. Eggs were detected in all faecal samples collected during the remaining 16 weeks of the experimental infection. Mature flukes were recovered from the livers of infected animals at necropsy and the parasite load averaged 41 ± 4 (Table 1). No weight loss was observed in the infected animals during the course of experimental infection (data not shown). Haematocrit count of infected animals was lower than controls 4 weeks PI and later (Fig. 1a).

Discussion

In this work, we established an experimental infection of alpacas with F. hepatica. The primary infection is similar to that observed in other species susceptible to F. hepatica infection: mature flukes shed eggs by the 8 weeks PI and adult worms were recovered from the bile ducts at necropsy (Chauvin et al., 1995, Clery et al., 1996, Ruiz et al., 2003). Hepatic lesions as wall thickening of bile ducts, disorganization of the liver parenchyma, stellate scar with fibrosis and destruction of bile

Acknowledgements

This work was partially funded by a grant B/2856-1 from the International Foundation for Science and INCAGRO CONTRACT 007-2003 to JRE. OT received Red SAREC support to conduct the experimental infection. OT and VN are graduate students supported by the Agreement between Consortium of Francophones Universities of Belgium (CIUF) and Universidad Peruana Cayetano Heredia (UPCH). Thanks to E. Chavarry for providing rabbit anti Fas2 IgGs. The authors wish to acknowledge the assistance and facilities

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