Seroprevalence of fasciolosis by using rapid immunodetection assays in ruminants of Pakistan

Published: Oct 25, 2022
2022-10-25 (1)
Fasciolosis Indirect ELISA Immunoblotting Pakistan Somatic and excretory secretory antigen
Maria Komal
Kiran Afshan
Mazhar Qayyum

Fasciolosis, caused by liver fluke species of the genus Fasciola, are well recognized because of its high veterinary impact. Stool examination for Fasciola eggs is not a sensitive method, and limited efforts to find a reliable and cheaper means of detection are available. The present study aimed to develop rapid diagnostic ELISA test against fasciolosis. The excretory/secretory (ES) and somatic (SA) products of Fasciola helminths were analysed using polyacrylamide gel electrophoresis (PAGE). Immunogenicity was evaluated by immunoblotting using hyperimmune sera raised in rabbits and seroprevalence was determined by indirect ELISA.  The results of SA antigen of Fasciola species showed polypeptide bands ranged from 10kDa-100kDa, while ES antigen of Fasciola showed bands of 15kDa-55kDa. The immunoblotting results showed the most prominent bands against ES antibodies were 25, 35, 55-70, 100 and 250 kDa and SA antigens showed 10, 15-25, 35, 70, 100 and 250 kDa polypeptide bands. The sensitivity and specificity of developed indirect ELISA for SA antigens was 95.45% and 87.1%, while for ES antigens was 100% and 77.42% respectively. The overall seroprevalence recorded for fascioliasis based on SA antigen was 39.8% and 29.8% for ES antigen. The fasciolosis did not show significant association with host type, sex and age groups of examined animals, however significantly higher infection was found in months of September and October. The result provides sensitive in house immunodetection assay for diagnosis of fasciolosis alternative to commercial kits with high import cost.

Article Details
  • Section
  • Research Articles
Download data is not yet available.
Author Biographies
Kiran Afshan, Department of Zoology, Faculty of Biological Sciences, Quaid-i-Azam University Islamabad, 45320, Pakistan
Assistant Professor
Mazhar Qayyum, Department of Zoology and Biology, Faculty of Sciences, PMAS-Arid Agriculture University, Rawalpindi-46300, Pakistan.
Professor, Dean,  Faculty of Sciences, PMAS-Arid Agriculture University, Rawalpindi-46300, Pakistan.
Abbas A, Iqbal Z, Abbas RZ (2017) In vivo anticoccidial effects of Beta vulgaris (sugar beet) in broiler chickens. Microb Path 111: 139-44.
Acici M, Buyuktanir O, Bolukbas CS, Pekmezci GZ, Gurler AT, Umur S (2017) Serologic detection of antibodies against Fasciola hepatica in sheep in the middle Black Sea region of Turkey. J Microbiol Immunol Infec 50 (3): 377-381.
Afshan K, Valero MA, Qayyum M, Peixoto RV, Magraner A, Mas-Coma S (2013) Phenotypic description of Fasciola hepatica/Fasciola gigantica intermediate forms infecting buffaloes from Central Punjab, Pakistan. J Helminthol 4: 1-10.
Anjum R, Khan MS, Sajid MS (2014) Frequency distribution of fasciolosis in small ruminants population at District Sargodha. Glob Vet 12: 26-32.
Anuracpreeda P, Chawengkirtikul R, Tinikul Y, Poljaroen J, Chotwiwatthanakun C, Sobhon P (2013) Diagnosis of Fasciola gigantica infection using a monoclonal antibody-based sandwich ELISA for detection of circulating cathepsin B3 protease. Acta Trop 127: 38-45.
Awad WS, Ibrahim AK, Salib FA (2009) Using indirect ELISA to assess different antigens for the serodiagnosis of Fasciola gigantica infection in cattle, sheep and donkeys. Res Vet Sci 86 (3): 466-471.
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72 (1-2): 248-254.
Brockwell YM, Spithill TW, Anderson GR, Grillo V, Sangster NC (2013) Comparative kinetics of serological and coproantigen ELISA and faecal egg count in cattle experimentally infected with Fasciola hepatica and following treatment with triclabendazole. Vet Parasitol 196: 417-26.
Dar JS, Ganai BA, Shahardar RA, Zargar UR (2019) Molecular characterization and Immunodiagnostic potential of various antigenic proteins of Fasciola gigantica species isolated from sheep of North West Himalayan Region. Helminthologia 56 (2): 93-107.
De Almeida MA, Ferreira MB, Planchart S, Terashima A, Maco V, Marcos L, Gotuzzo E, Sánchez E, Náquira C, Scorza JV, Incani RN (2007) Preliminary antigenic characterisation of an adult worm vomit preparation of Fasciola hepatica by infected human sera. Rev Inst Med Trop São Paulo 49 (1): 31-35.
El-Tahawy AS, Bazh EK, Khalafalla RE (2017) Epidemiology of bovine fascioliasis in the Nile Delta region of Egypt: Its prevalence, evaluation of risk factors, and its economic significance. Vet World 10 (10): 1241.
Gönenç B, Sarimehmetoğlu HO, Kara M, Kircali F (2004) Comparison of crude and excretory/secretory antigens for the diagnosis of Fasciola hepatica in sheep by western blotting. Turk J Vet Ani Sci 28 (5): 943-949.
Guobadia EE, Fabemi BO (1997) The isolation of Fasciola gigantica-specific antigens and their use in the serodiagnosis of fasciolosis in sheep by the detection of circulating antigens. Vet Parasitol 68 (3): 269-282.
Hongbao Ma (2006) Western Blotting Method. Am J Sci 2 (2): 23-27.
Irfan Ullah MFN, Jadoon AAK, Tabassum S (2016) Prevalence of Fasciola hepatica in domesticated cattle of district Karak, Khyber Pakhtunkhwa, Pakistan. Asian J Ani Sci 10: 85-91.
Isah H (2019) Spatial species distribution models to predict liver fluke disease (Fasciola gigantica) In Cattle: a case study of Sokoto State, Nigeria (Doctoral dissertation, Department of Geography).
Kamel HH, Saad GA, Sarhan RM (2013) Dot-blot immunoassay of Fasciola gigantic infection using 27 kDa and adult worm regurge antigens in Egyptian patients. Korean J Parasitol 51: 177–182.
Khan MH, Shareef PAA, Rehman A, Ullah R, Rehman L, Abidi SMA (2017) Genotoxic potential of Fasciola gigantica infection in experimentally infected rabbits. J Parasit Dis 41: 423-28.
Khan YA, Khan MAH, Abidi SM (2014) 2D-PAGE analysis of the soluble proteins of the tropical liver fluke, Fasciola gigantica and biliary amphistome, Gigantocotyle explanatum, concurrently infecting Bubalus bubalis. J Parasit Dis.
Kumar N, Ghosh S, Gupta SC (2008) Detection of Fasciola gigantica infection in buffaloes by enzyme-linked immunosorbent assay. Parasitol Res 104 (1): 155-161.
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (5259): 680.
Markakpo US, Armah GE, Fobil JN, Asmah RH, Anim-Baidoo I, Dodoo AK, Madjitey P, Essuman EE, Kojima S, Bosompem KM (2015) Immunolocalization of the 29 kDa Schistosoma haematobium species-specific antigen: a potential diagnostic marker for urinary schistosomiasis. BMC Infect Dis 15: 198.
Mehmood K, Zhang H, Sabir AJ (2017) A review on epidemiology, global prevalence and economical losses of fasciolosis in ruminants. Microb Path 109: 253-62.
Meshgi B, Eslami A, Hemmatzadeh F (2008) Determination of somatic and excretory-secretory antigens of Fasciola hepatica and Fasciola gigantica using SDS-PAGE. Iranian J Vet Res 9 (1): 77-80.
Mezo M, González-Warleta M, Castro-Hermida JA, Muiño L, Ubeira FM (2010) Field evaluation of the MM3-SERO ELISA for detection of anti-Fasciola IgG antibodies in milk samples from individual cows and bulk milk tanks. Parasitol Int 59 (4): 610-615.
Morales A, Espino AM (2012) Evaluation and characterization of Fasciola hepatica tegument protein extract for serodiagnosis of human fascioliasis. Clin Vaccine Immunol 19 (11): 1870-1878.
Mufti S, Afshan K, Khan IA, Irum S, Qureshi IZ, Rizvi SSR, Mukhtar M, Mushtaq M, Iqbal Z, Qayyum M (2015) Serological and coprological studies of bovine fasciolosis in the Pothwar Region, Pakistan. Pak Vet J 35 (2): 178-182.
Saifullah MK, Ahmad G, Abidi SMA (2013) Immunodetection of coproantigens for the diagnosis of amphistomosis in naturally infected Indian Water Buffalo, Bubalus bubalis. Vet Parasitol 191: 66-72.
Shafiei R, Sarkari B, Moshfe A (2013) Consistent PCR-RFLP assay based on ITS-2 ribosomal DNA for differentiation of Fasciola species. Iran J Basic Med Sci 16: 1266–1269.
Shafiei R, Sarkari B, Sadjjadi SM (2015) Performance of a 27 kDa Fasciola hepatica antigen in the diagnosis of human fascioliasis. J Lab Physicians 7: 17–20.
Shafiei R, Sarkari B, Sadjjadi SM, Mowlavi GR, 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.
Shareef PA, Brennan GP, McVeigh P, Khan MH, Morphew RM, Mousley A, Marks NJ, Saifullah MK, Brophy PM, Maule AG, Abidi SMA (2014) Time-dependent tegumental surface changes in juvenile Fasciola gigantica in response to triclabendazole treatment in goat. Acta Trop 136: 108-17.
Shareef PPA (2014) Biochemical, Pharmacological and Pathological Studies on some developmental stages of Fasciola gigantica. PhD Thesis.
Soulsby EJL (1982) Helminths: Arthropods and Protozoan of domesticated animals. PP: 291.
Ullah R, Rehman A, Zafeer MF, Rehman L, Khan YA, Khan MH, Khan SN, Khan AU, Abidi SMA (2017) Anthelmintic potential of thymoquinone and curcumin on Fasciola gigantica. PLoS ONE 12: 2.
Zafar A, Khan MK, Sindhu D, Abbas RZ, Masood S, Abbas Z, Mahmood MS, Saleemi MK, Khan JA, Hussain R, Naseer MU (2019) Seroprevalence of Fasciola hepatica in small ruminants of district Chakwal, Punjab, Pakistan. Pak Vet J 39 (1): 96-100.
Most read articles by the same author(s)