Development of Fasciola Vaccine in an Animal Model
Fasciola hepatica and F. gigantica are the parasites that cause the zoonotic parasitic disease called fasciolosis. Although several anthelmintic drugs have been used to treat these parasitic infections, recombinant protein vaccines have been developed to
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Introduction Fasciolosis (also called fascioliasis) is a zoonotic parasitic disease that is caused by the infection of trematodes Fasciola hepatica (F. hepatica) and Fasciola gigantica (F. gigantica), found in temperate and tropical regions, respectively. The disease causes reductions in the meat and milk production and decreased fertility in animals as well as afflicting the health of infected animals and humans. The World Health Organization (WHO) has estimated that 2.4 million people are infected with Fasciola, and a further 180 million are at risk of infection [1]. Triclabendazole is the most effective anthelmintic drug for controlling the disease [2]. However, resistance to this drug has been reported in many countries including Argentina [3], Australia [4], the Netherlands [5], and Scotland [6]. Vaccine becomes an alternative approach because of its safety, environmental friendliness, and cost-effectiveness and is acceptable by consumers [7]. Several vaccine candidates have been identified and tested for their efficacies against Fasciola infection in both experimental and economic animals. These include cathepsin B, cathepsin L, glutathione S-transferase, leucine aminopeptidase, and fatty acid-binding protein in F. hepatica [8–12] and cathepsin B, cathepsin L, glutathione S-transferase, leucine aminopeptidase, and saposin-like protein 2 in F. gigantica [13–17]. High percentages of protection have been detected using the recombinant protein immunizations suggesting that these could be developed into vaccines for preventing Fasciola infection in the future. A few DNA vaccines related to these candidates have also been shown to induce high levels of immune responses against the Fasciola infection [18–20], but they will not be described in this chapter.
Sunil Thomas (ed.), Vaccine Design: Methods and Protocols, Volume 2: Vaccines for Veterinary Diseases, Methods in Molecular Biology, vol. 1404, DOI 10.1007/978-1-4939-3389-1_8, © Springer Science+Business Media New York 2016
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Krai Meemon and Prasert Sobhon
In this chapter, optimized methods for vaccination using the recombinant proteins against Fasciola infection in mice are described. The production and purification of recombinant proteins in the yeast Pichia pastoris expression system are detailed. The detection of antibody levels is also described to determine the levels of IgG by ELISA technique.
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Materials
2.1 Recombinant Protein Production in the Yeast Pichia pastoris Expression System
1. pPICZαA, B, C vectors (Invitrogen). 2. Selected restriction enzymes and appropriate 10× buffers. 3. Gel documentation system. 4. QIAquick gel extraction kit (QIAGEN). 5. 0.2 mL thin-walled PCR tube. 6. T4 DNA ligase and ligation buffer. 7. 16 and 42 °C water baths or temperature blocks. 8. E. coli (DH5α) competent cells. 9. Zeocin™. 10. Low-salt LB broth containing 50 μg/mL Zeocin™: To 1 L of distilled H2O, add 10 g tryptone, 5 g yeast extract, and 5 g NaCl. Mix thoroughly and sterilize by autoclaving. Cool the solution to approximately 60 °C; add Zeoci
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