Protein Detection of Excretory-Secretory Products and Somatic Extracts from Fasciola hepatica and F. gigantica Using Two-Dimensional Electrophoresis
AbstractBackground: The aim of this research was to compare excretory-secretory and somatic extract materials of Fasciola hepatica and F. gigantica to detect protein maps of two species. Methods: Twenty infected livers were collected from sheep in industrial slaughterhouse in Tehran, 2017-2019. Worms were detached from bile ducts, then recognized according to morphologic and morphometric criteria. After three times washing, worms were incubated in RPMI culture media and excretory-secretory products were collected. Worms were crushed and homogenized for preparation of somatic extract. Two Dimensional Electrophoresis gels were accomplished for both excretory-secretory material and somatic extracts. Gels were scanned with densitometer and analyzed with Same Spots software and protein spots were identified with Expasy database. Results: For both excretory-secretory products and somatic extract, protein spots were appeared with two-dimensional electrophoresis technique. Quantitative analysis showed 40 and 28 protein spots for excretory-secretory of F. hepatica and F. gigantica respectively. For somatic extract 19 and 12 protein spots were recognized for F. hepatica and F. gigantica in that order. Conclusion: The rate of expression of some proteins were more in F. hepatica while expression of other proteins was high in F. gigantica. The expression of protease enzyme was higher in F. gigantica than F. hepatica. These data could be considered for biochemical differentiation of Fasciola species and subsequently to design and prepare of antigens for diagnosis/vaccine development.
Admassu B, Shite A, Kinfe G. A Review on Bovine Fasciolosis. European Journal of Biological Sciences. 2015;7(3):139-46.
Dzik JM. Molecules released by helminth parasites involved in host colonization. Acta Biochimica Polonica. 2006;53(1):33-64.
Faridi A, Farahnak A, Golmohammadi T, Eshraghian M, Sharifi Y, Rad MM. Triclabendazole (Anthelmintic Drug) Effects on the Excretory-Secretory Proteome of Fasciola hepatica in Two Dimension Electrophoresis Gel. Iranian journal of parasitology. 2014;9(2):202.
Davey J, Lord JM. Essential Cell Biology Vol 1: Cell Structure. 2003;1.
Weber K, Pringle J, Osborn M.  Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. Methods in enzymology. 1972;26:3-27.
Meshgi B, Eslami A, Hemmatzadeh F. Determination of somatic and excretory-secretory antigens of Fasciola hepatica and Fasciola gigantica using SDS-PAGE. Iranian Journal of Veterinary Research. 2008;9(1):77-80.
Morphew RM, Wright HA, LaCourse EJ, Woods DJ, Brophy PM. Comparative proteomics of excretory-secretory proteins released by the liver fluke Fasciola hepatica in sheep host bile and during in vitro culture ex host. Molecular & Cellular Proteomics. 2007;6(6):963-72.
Harold W, David L. Basic clinical parasitology. Basic Clinical Parasitology 2nd Edition. 1964.
Intapan PM, Sadee P, Wongkham C, Maleewong W. Development of rapid agglutination test using Fasciola gigantica specific antigen for serodiagnosis of human fascioliasis. Southeast Asian J Trop Med Public Health. 2004;35:313-7.
Alirahmi H, Farahnak A, Golmohamadi T, Esharghian MR. Comparative assay of glutathione S-transferase (GSTs) activity of excretory-secretory materials and somatic extract of Fasciola spp parasites. Acta Medica Iranica. 2010;48(6):367.
Neuhoff V, Arold N, Taube D, Ehrhardt W. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G‐250 and R‐250. Electrophoresis. 1988;9(6):255-62.
Brunelle JL, Green R. Coomassie blue staining. Methods in enzymology. 541: Elsevier; 2014. p. 161-7.
Weber K, Pringle J, Osborn M.  Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. Methods in enzymology. 26: Elsevier; 1972. p. 3-27.
Beckham SA, Piedrafita D, Phillips CI, Samarawickrema N, Law RH, Smooker PM, et al. A major cathepsin B protease from the liver fluke Fasciola hepatica has atypical active site features and a potential role in the digestive tract of newly excysted juvenile parasites. The international journal of biochemistry & cell biology. 2009;41(7):1601-12.
Collins PR, Stack CM, O'Neill SM, Doyle S, Ryan T, Brennan GP, et al. Cathepsin L1, the Major Protease Involved in Liver Fluke (Fasciola hepatica) virulence propeptide cleavage sites and autoactivation of the zymogen secreted from gastrodermal cells. Journal of Biological Chemistry. 2004;279(17):17038-46.
Marcilla A, De la Rubia J, Sotillo J, Bernal D, Carmona C, Villavicencio Z, et al. Leucine aminopeptidase is an immunodominant antigen of Fasciola hepatica excretory and secretory products in human infections. Clinical and Vaccine Immunology. 2008;15(1):95-100.
Morphew RM, Hamilton CM, Wright HA, Dowling DJ, O’Neill SM, Brophy PM. Identification of the major proteins of an immune modulating fraction from adult Fasciola hepatica released by Nonidet P40. Veterinary parasitology. 2013;191(3-4):379-85.
Campos ÉG, Hermes-Lima M, Smith JM, Prichard RK. Characterisation of Fasciola hepatica cytochrome c peroxidase as an enzyme with potential antioxidant activity in vitro. International journal for parasitology. 1999;29(5):655-62.