Molecular characterization and Immunodiagnostic potential of various antigenic proteins of Fasciola gigantica species isolated from sheep of North West Himalayan Region

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The control of the digenetic trematode Fasciola gigantica has been the major challenge in both cattle and small ruminants as there is a paucity of an effective and commercial vaccine. Thus, the accurate identification and prepatent diagnosis of F. gigantica is an essential prerequisite for its successful prevention and control. In the present study, the morphologically identified specimens isolated from the liver and bile ducts of sheep (Ovis aries) were validated through molecular data. The sequence analysis of ITS-2 of our isolates showed high degree of similarity with F. gigantica and F. hepatica using BLAST function of NCBI. The phylogenetic analysis of our isolates showed a close relationship with previously described F. gigantica and F. hepatica isolates from different countries. The antigenic profile of somatic and E/S antigens of F. gigantica were revealed by SDS–PAGE and immunoblotting using sera from sheep naturally infected with F. gigantica. By SDS-PAGE, 20 distinct bands were revealed from crude somatic fraction. Immunoblotting analysis of these proteins with positive sera exhibited 8 sero-reactive bands ranging from 14 to 97 kDa. Among these 38 and 44 kDa bands were quite specific with high diagnostic specificity and sensitivity. The E/S fraction comprised 7 distinct bands, as revealed by SDS-PAGE analysis. Immunoblotting analysis of these proteins with positive sera exhibited 6 antigenic bands ranging from 23 – 54 kDa. Among these 27 and 33 kDa were found to be quite specific with high diagnostic specificity and sensitivity. The present study concludes that the protein bands of 38 and 44 kDa in somatic fraction and 27 and 33 kDa in E/S fraction can be used for the immunodiagnostic purpose for this economically important parasite, which may also entice further studies regarding their vaccine potential.

Adlard, R.D., Barker, S.C., Blair, D., Cribb, T.H. (1993). Comparison of the second internal transcribed spacer (ribosomal DNA) from populations and species of Fasciolidae (Digenea). Int. J. Parasitol., 23: 422 – 425. DOI: 10.1016/0020-7519(93)90022-Q

Agatsuma, T., Arakawa, Y., Iwagami, M., Honzako, Y., Cahyaningsih, U., Kang, S.Y., Hong, S.J. (2000). Molecular evidence of natural hybridization between Fasciola hepatica and F. gigantica. Parasitol. Int., 49: 231 – 238. DOI: 10.1016/S1383-5769(00)00051-9

Ali, H., Ai, L., Song, H.Q., Ali, S., Lin, R.Q., Seyni, B., Issa, G., Zhu, X.Q. (2008). Genetic characterisation of Fasciola samples from different host species and geographical localities revealed the existence of F. hepatica and F. gigantica in Niger. Parasitol. Res., 102(5): 1021 – 1024. DOI: 10.1007/s00436-007-0870-7

Allam, A.F., El-Agamy, E-S., Helmy, M.H. (2002). Molecular and immunological characterization of Fasciola species. Br. J. Biomed. Sci., 59: 191 – 195

Amor, N., Halajian, A., Farjallah, S., Merella, P., Said, K., Slimane, B.B. (2011). Molecular characterization of Fasciola spp. From the endemic area of northern Iran based on nuclear ribosomal DNA sequences. Exp. Parasitol., 128(3): 196 – 204. DOI: 10.1016/j. exppara.2011.03.011

Anonymous (1995). Control of foodborne trematode infections. Report of a WHO Study Group, World Health Organ. Tech. Rep. Ser., 849: 1 – 157

Anuracpreeda, P., Wanichanon, C., Sobhon, P. (2009). Fasciola gigantica: immunolocalization of 28.5 kDa antigen in the tegument of metacercaria and juvenile fluke. Exp. Parasitol., 122: 75 – 83. DOI: 10.1016/j.exppara.2009.03.007

Ashrafi, K., Valero, M.A., Panova, M., Masoud, J., Mas-Coma, S. (2006). Phenotypic analysis of adults of Fasciola hepatica, Fasciola gigantica and intermediate forms from the endemic region of Gilan, Iran. Parasitol. Int., 55: 249 – 260. DOI: 10.1016/j. parint.2006.06.003

Attallah, A.M., Karawia, E.A., Ismail, H., Tabll, A.A., Nawar, A.A., Ragab, W.A., Abdel Aziz, M.M., El-Dosoky, I. (2002). Identification and characterization of a 26 – 28 kDa circulating antigen of Fasciola gigantica. Ann. Trop. Med. Parasitol., 96(3): 271 – 282. DOI: 10.1179/000349802125000754

Bradford, M.M. (1976). A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Dye Binding. Anal. Biochem., 72: 248 – 254. DOI: 10.1016/0003-2697(76)90527-3

Cervi, L.A., Rubinstein, H., Masih, D.T. (1992). Serological, electrophoretic and biological properties of Fasciola hepatica antigens. Rev. Inst. Med. Trop. Sao Paulo, 34: 517 – 525. DOI: 10.1590/S0036-46651992000600005

Charlier, J., Meulemeester, L., Claerebout, E., Williams, D., Vercruyse, J. (2008). Qualitative and quantitative evaluation of coprological and serological techniques for the diagnosis of fascioliasis in cattle. Vet. Parasitol., 153: 44 – 51. DOI: 10.1016/j. vetpar.2008.01.035

Choe, S., Nguyen, T., Kang, T., Kweon, C., Kang S. (2011). Genetic analysis of Fasciola isolates from cattle in Korea based on second internal transcribed spacer (ITS-2) sequence of nuclear ribosomal DNA. Parasitol. Res., 109: 833 – 839. DOI: 10.1007/s00436-011-2323-6

Coles, G.C., Rubano, D. (1988). Antigenicity of a proteolytic enzyme of Fasciola hepatica. J. Helminthol., 62: 257 – 260. DOI: 10.1017/S0022149X00011627

Cornelissen, J.B.W.J., Gaasenbeek, C.P.H., Boersma, W., Borgsteede, F.H.M., Van Milligen, F.J. (1999). Use of pre-selected epitope cathepsin–L1 in a highly specific peptide-based immunoassay for the diagnosis of Fasciola hepatica infection in cattle. Int J Parasitol., 29: 685 – 696. DOI: 10.1016/S0020-7519(99)00017-X

Dalton, J., Heffernan, M. (1989). Thiol proteases released in vitroby Fasciola hepatica. Mol. Biochem. Parasitol., 35: 161 – 166. DOI: 10.1016/0166-6851(89)90118-7

Dalton, J.P., McGonigle, S., Rolph, T.P., Andrews, S.J. (1996). Induction of protective immunity in cattle against infection with Fasciola hepatica by vaccination with cathepsin L proteinases and with hemoglobin. Infect. Immun., 64: 5066 – 5074

De Vera, M.E., Sato, K., Oyong, G., Claveria, F.G. (2009). Comparison of protein profile of co-existing Fasciola hepatica and Fasciola gigantica parasite in Bos taurus (cattle) and Bubalus bubalis (Philippine water buffalo). J. Protozool. Res., 19: 1 – 9

Dixit, A.K., Dixit, P., Sharma, R.L. (2008). Immunodiagnostic/protective role of cathepsin L cysteine proteinases secreted by Fasciola species. Vet. Parasitol., 154: 177 – 184. DOI: 10.1016/j.vetpar. 2008.03.017

Dorchies, P. (2007). Comparison of methods for the veterinary diagnosis of liver flukes (Fasciola hepatica) in cattle, Bulletin USAMV-CN, 64(1 – 2): 14 – 19. DOI: 10.15835/buasvmcn-vm:64:1-2:2270

Dowd, A.J., Smith, A.M., McGonigle, S., Dalton, J.P. (1994). Purification and characterisation of a second cathepsin L proteinase secreted by the parasitic trematode Fasciola hepatica. Eur. J. Biochem., 223: 91 – 98. DOI: 10.1111/j.1432-1033.1994.tb18969.x

Galavani, H., Gholizadeh, S. Tapeh, K.H. (2016). Genetic characterization of Fasciola isolates from west Azerbaijan province Iran based on ITS1 and ITS2 sequence of ribosomal DNA. Iran J. Parasitol., 11: 52 – 64. DOI: 10.3923/jp.2016.61.66

Gonenc, B., Sarimehmetolu, H.O., Koro, M., Kiracali, F. (2004). Comparison of crude and excretory-secretory antigens for the diagnosis of Fasciola hepatica in sheep by western blotting. Turk. J. Vet. Anim. Sci., 28: 943 – 949

Goreish, I.A., Williams, D.J., McGarry, J., Abdel Salam, E.B., Majid, A.M., Mukhtar, M.M. (2008). Protein Profile of Fasciola gigantica Antigens. Sudan J. Vet. Res., 23: 1 – 9

Gorman, T., Aballay, J., Fredes, F., Silva, M., Aguillon, J.C., Alcaino, H.A. (1997). Immunodiagnosis of fasciolosis in horses and pigs using western blots. Int. J. Parasitol., 27: 1429 – 1432. DOI: 10.1016/S0020-7519(97)00090-8

Gupta, S.C., Ghosh, S., Joseph, D., Singh, B.P. (2003). Diagnosis of experimental Fasciola gigantica in cattle by affinity purified antigen. Indian J. Anim. Sci., 7: 963 – 966

Hall, T.A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser., 41: 95 – 98

Hammami, H., Ayadi, A., Camus, D., Dutoit, E. (1997). Diagnostic value of the demonstration of specific antigen of Fasciola hepatica by western blot technique. Parasite, 4: 291 – 295. DOI: 10.1051/parasite/1997043291

Hashimoto, K., Watanabe, T., Liu, C.X., Init, I., Blair, D., Ohnishi, S. (1997). Mitochondrial DNA and nuclear DNA indicate that the Japanese Fasciola species is F. gigantica. Parasitol. Res., 8: 220 – 225

Hasan, S.E., Ghazy, A.A., Abdel-Rahman, E. (2014). Value of serum and milk ELISAs in lactating buffaloes using Fasciola gigantica partially purified somatic antigen. Glob. Vet., 13(3): 365 – 372. DOI: 10.5829/idosi.gv.2014.13.03.84270

Hillyer, G.V. (1985). Induction of immunity in mice to Fasciola hepatica with a Fasciola/Schistosoma cross-reactive defined immunity antigen. Am. J. Trop. Med. Hyg., 34: 1127 – 1131. DOI: 10.4269/ajtmh.1985.34.1127

Hillyer, G.V., Solerde G alanes, M., Battisti, G. (1992). Fasciola hepatica: host responders and non responders to parasite glutathione S-transferase. Exp. Parasitol., 75(2): 176 – 186. DOI: 10.1016/0014-4894(92)90177-C

Huang, W.Y., He B., Wang, C.R., Zhu, X.Q. (2004). Characterization of Fasciola species from Mainland China by ITS-2 ribosomal DNA sequence. Vet. Parasitol., 120: 75 – 83. DOI: 10.1016/j.vetpar. 2003.12.006

Ichikawa, M., Itagaki, T. (2010). Discrimination of the ITS1 types of Fasciola spp. based on a PCR-RFLP method. Parasitol. Res., 106: 757 – 761. DOI: 10.1007/s00436-010-1724-2

Intapan, P.M., Maleewong, W., Wongkham, C., Tomanakarn, K., Ieamviteevanich, K., Pipitgool, V., Sukolapong, V. (1998). Excretory-secretory antigenic components of adult Fasciola gigantica recognized by infected human sera. Southeast Asian J. Trop. Med. Public Health, 29: 579 – 583

Intapan, P.M., Maleewong, W., Nateeworanart, S., Wongkham, C., Pipitgool, V., Sukolapong, V., Sangmaneedet, S. (2003). Immunodiagnosis of human fasciolosis using an antigen of Fasciola gigantica adult worm with the molecular mass of 27 kDa by a dot ELISA. Southeast Asian J. Trop. Public Health, 34(4): 713 – 717

Itasgaki, T., Tsutsumi, K. (1998). Triploid form of Fasciola in Japan: Genetic relationships between Fasciola hepatica and Fasciola gigantica determined by ITS-2 sequence of the nuclear rDNA. Int. J. Parasitol., 28: 777 – 781. DOI: 10.1016/S0020-7519(98)00037-X

Itagaki, T., Kikawa, M., Sakaguchi, K., Shimo, J., Terasaki, K., Shibahara, T. (2005a). Genetic characterization of parthenogenetic Fasciola sp. in Japan on the basis of the sequences of ribosomal and mitochondrial DNA. Parasitology, 131: 679 – 685. DOI: 10.1017/S0031182005008292

Itagaki, T., Kikawa, M., Terasaki, K., Shibahara, T., Fukuda, K. (2005b). Molecular characterization of parthenogenic Fasciola sp. in Korea on the basis of DNA sequence of ribosomal ITS1 and mitochondrial NDI gene. J. Vet. Med. Sci., 67: 1115 – 1118. DOI: 10.1292/jvms.67.1115

Kamel, H.H., Saad, G.A., Sarhan, R.M. (2013). Dot-blot immunoassay of Fasciola gigantica infection using 27 kDa and adult worm regurge antigens in Egyptian patients. Korean J. Parasitol., 51: 177 – 182. DOI: 10.3347/kjp.2013.51.2.177.

Karimi, A. (2008). Genetic diagnosis of Fasciola species based on 18S ribosomal DNA sequences. J. Biol. Sci., 7: 1166 – 1173. DOI: 10.3923/jbs.2008.1166.1173

Khabisi, S.A., Sarkari, B. (2016). Detection of Fasciola hepatica and Fasciola gigantica commonand uncommon antigens, using rabbit hyper immune serum raised against their excretory-secretory and somatic antigens. J. Parasit. Dis., 40(4): 1552 – 1557. DOI: 10.1007/s12639-015-0726-5

Khan, M.A.H., Shareef, P.A.A., Rehman, A., Ullah, R., Rehman, L., Abidi, S.M.A. (2017). Genotoxic potential of Fasciola gigantica infection in experimentally infected rabbits. J. Parasit. Dis., 41: 423 – 428. DOI: 10.1007/s12639-016-0820-3

Kumar, S., Stecher, G., Tamura, K. (2016). MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol. Biol. Evol., 33: 1870 – 1874. DOI: 10.1093/molbev/msw054

Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680 – 685. DOI: 10.1038/227680a0

Latchumikanthan, A., Soundararajan, C., Dhinakarraj, G., Basith, S.A. (2012). Purification and Characterization of Excretory/Secretory Antigens of Fasciola gigantica. J. Vet. Anim. Sci., 8(1): 14 – 18

Le, T.H., De, N.V., Agatsuma, T., Nguyen, T.G.T., Nguyen, Q.D., Mc-Manus, D.P. (2008). Human fascioliasis and the presence of hybrid/introgressed forms of Fasciola hepatica and Fasciola gigantica in Vietnam. Int. J. Parasitol., 38: 725 – 730. DOI: 10.1016/j. ijpara.2007.10.003

Maleewong, W., Intapan, P.M., Tomanakan, K., Wongkham, C. (1997). Antigenic components of somatic extract from adult Fasciola gigantica recognized by infected human sera. Asian Pac. J. Allergy Immunol., 15: 213 – 218

Maleewong, W., Wongkhan, C., Intapan, P.M., Pipitgool, V. (1999). Fasciola gigantica specific antigens: purification by a continuous-elusion method and its evaluation for the diagnosis of human Fascioliasis. Am. J. Trop. Med. Hyg., 61: 648 – 651

Marcilla, A., Bargues, M.D, Mas-Coma, S. (2002). A PCR-RFLP assay for the distinction between Fasciola hepatica and Fasciola gigantica. Mol. Cell Probes, 16: 327 – 343. DOI: 10.1006/mcpr.2002.0429

Meshgi, B., Eslami, A., Hemmatzadeh, F. (2008). Determination of somatic and excretory antigens of Fasciola hepatica and Fasciola gigantica using SDS-PAG. Iranian J. Vet. Res., 9: 77 – 80. DOI: 10.22099/IJVR.2008.526

Moghaddam, A.S., Masoud, J., Mahmoodi, M., Mahvi, A.H., Periago, M.V., Artigas, P., Fuentes, M.V., Bargues, M.D., Mas-Coma, S. (2004). Human and animal fascioliasis in Mazandaran province, northern Iran. Parasitol. Res., 94(1): 61 – 69. DOI: 10.1007/s00436-004-1169-6

Moxon, J.V., LaCourse, E.J., Wright, H.A., Perally, S., Prescott, M.C., Gillard, L.J., Barrett, J., Hamilton, J.V., Brophy, P.M. (2010). Proteomic analysis of embryonic Fasciola hepatica: characterization and antigenic potential of a developmentally regulated heat shock protein. Vet. Parasitol., 169: 62 – 75. DOI: 10.1016/j. vetpar.2009.12.031

Nour Eldin, M.S., EL-Ganaini, G.A., Abou EL-Enin, A.M., Hussein, E.M., Sultan, D.M. (2004). Evaluation of seven assays detecting serum immunoglobulin classes and subclasses and salivary and faecal secretory IgG against Fasciola excretory/secretory (ES) antigen in diagnosing Fascioliasis. J. Egypt. Soc. Parasitol., 34: 691 – 704

Olaechea, F.V., Christensen, N.O., Henriksen, S.A. (1990). A comparison of filtration, concentration and thick smear technique in the diagnosis of Schistosoma boois infection in cattle and goats. Acta Trop., 47: 217 – 221

Parkhouse, R.M., Almond, N.M., Cabrera, Z., Harnett, W. (1987). Nematode antigens in protection, diagnosis and pathology. Vet. Immunol. Immunopath., 17: 313 – 324. DOI: 10.1016/0165-2427(87)90150-4

Periago, M.V., Artigas, P., Khoubbane, M., Moghaddam, A.S., Ashrafi, K., Mansoorian, A.B., Masoud, J., Bargues, Mas-Coma, S. (2004). Genotypic analysis of adult liver flukes from Iran based on the ribosomal M.D. DNA markers ITS-1 and ITS-2. In: Symposium on schistosomiasis and distomatoses. Multidisciplinarity for parasites, vectors and parasitic diseases (eds. B. Mas-Coma, M.D. Bargues, M.A. Valero, V. Esteban and H.K.E. Mott), IX European Multicolloquium of Parasitology, Valencia, Spain. 286 – 287

Peters, P.A., El Alamy, M., Warren, K.S., Mahmoud, A.A. (1980). Quick Kato smear for field quantification of Schistosoma mansoni eggs. Am J Trop Med Hyg., 29: 217 – 219. DOI: 10.4269/ajtmh. 1980.29.217

Phiri, I.K., Phiri, A.M., Harrison, L.J.S. (2006). Serum antibody isotype responses of Fasciola-infected sheep and cattle to excretory and secretory products of Fasciola species. Vet. Parasitol., 141: 234 – 242. DOI: 10.1016/j.vetpar.2006.05.019

Prasad, P.K., Tandon, V., Biswal, D.K., Goswami, L.M., Chatterjee, A. (2008). Molecular identification of the Indian liver fluke, Fasciola (Trematoda: fasciolidae) based on the ribosomal internal transcribed spacer regions. Parasitol. Res., 103: 1247 – 1255

Raina, O.K., Jacob, S.S., Sanker, M., Bhattacharya, D., Bandyopadyay, S., Varghese, A., Chamuah, K., Lalrinkima, H. (2015). Genetic characterization of Fasciola gigantica from different geographical regions of India by ribosomal DNA markers. J. Parasit. Dis., 39(1): 27 – 32. DOI: 10.1007/s12639-013-0276-7

Roeber, F., Jex, A.R., Gaser, R.B. (2013). Impact of gastrointestinal parasitic nematodes of sheep, and the role of advanced molecular tools for exploring epidemiology and drug resistance – an Australian perspective. Parasit. Vectors, 6: 153. DOI: 10.1186/1756-3305-6-153

Rokni, M.B., Baghernejad, A., Mohebali, M., Kia, E.B. (2004). Enzyme linked immunotransfer blot analysis of somatic and excretory-secretory antigens of Fasciola hepatica in diagnosis of human Fascioliasis. Iranian J. Public Health, 33: 8 – 13

Rzhetsky, A. and Nei, M. (1992). A simple method for estimating and testing minimum evolution trees. Mol. Biol. Evol., 9: 945 – 967. DOI: 10.1093/oxfordjournals.molbev.a040454

Sampaio-Silva, M.L., Da Costa, J.M., Da Costa, A.M., Pires, M.A., Lopes, S.A., Castro, A.M., Monjour, L. (1996). Antigenic components of excretory–secretory products of adult Fasciola hepatica recognized in human infections. Am. J. Trop. Med. Hyg., 54: 146 – 148. DOI: 10.4269/ajtmh.1996.54.146

Santiago, N., Hillyer, G.V., Garcia-Rosa, M., Morales, M.H. (1986). Identification of functional Fasciola hepatica antigens in experimental infections in rabbits. Am. J. Trop. Med. Hyg., 35: 135 – 140. DOI: 10.4269/ajtmh.1986.35.135

Santiago, N., Hillyer, G.V. (1988). Antibody profiles by EITB and ELISA of cattle and sheep infected with Fasciola hepatica. J. Parasitol., 74: 810 – 818. DOI: 10.2307/3282259

Shafiei, R., Sarkari, B., Sadjjadi, S.M., Mowlavi, G.R., Moshfe, A. (2014). Molecular and morphological characterization of Fasciola spp. isolated from different host species in a newly emerging focus of human fascioliasis in Iran. Vet. Med. Int., 405740. DOI: 10.1155/2014/405740

Sharma, R.L., Dhar, D.N., Raina, O.K. (1989). Studies on the prevalence and laboratory transmission of fascioliasis in animals in the Kashmir valley. Br. Vet. J., 145: 57

Shoriki, T., Ichikawa-Seki, M., Suganuma, K., Naito, I., Hayashi, K., Nakao, M., Aita, J., Mohnta, U,K., Inoue, N., Murakami, K., Itagaki, T. (2015). Novel methods for the molecular discrimination of Fasciola spp. on the basis of nuclear protein coding genes. Parasitol. Int., 65: 180 – 183

Smith, A.M., Dowd, A.J., McGonigle, S., Keegan, P.S., Brennan, G., Trudgett, A., Dalton, J.P. (1993). Purification of a cathepsin L-like proteinase secreted by adult Fasciola hepatica. Mol. Biochem. Parasitol., 62: 1 – 8. DOI: 10.1016/0166-6851(93)90171-S

Sobhon, P., Anantavara, S., Dangprasert, T. (1996). Fasciola gigantica: Identification of adult antigens, their tissue sources and possible origins. J. Sci. Soc. Thai., 22: 143 – 162

Soulsby, E.J.L. (1982). Helminths, arthropods and protozoa of domesticated animals. seventh ed. Bailliere Tindall, London Great Britain, 64 – 65. DOI: 10.1016/0035-9203(84)90110-X

Thienpont, D., Rochetle, F., Vanparijis, O.F.J. (1979). Sedimentation. In: Diagnosing Helminthiasis through Coprological Examination. Janssen Research Foundation, Beerse, Belgium, 34 – 36

Thompson, J.D., Higgins, D.G., Gibson, T.J. (1994). CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res., 22: 4673 – 4680

Toledo, R., Bernal, M.D., Marcilla, A. (2011). Proteomics of foodborne trematodes, J. Proteomics, 74: 1485 – 1503. DOI: 10.1016/j. jprot.2011.03.029

Upadhyay, A.K., Kumar, M. (2002). SDS-PAGE analysis of Fasciola gigantica antigen. J. Immunol. Immunopathol., 4: 91 – 92

Vercruyse, J., Claerebout, E. (2001): Anthelmintic treatment vs. non treatment in cattle: defining the threshold. Vet. Parasitol., 98: 195 – 214. DOI: 10.1016/S0304-4017(01)00431-9

Yokananth, S., Ghosh, S., Gupta, S.C., Suresh, M.G., Saravanan, D. (2005). Characterization of specific and cross-reacting antigens of Fasciola gigantica by Immunoblotting. Parasitol. Res., 7(1): 41 – 48. DOI: 10.1007/S00436-005-1371-1

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