Comparison of Dot-ELISA and ELISA Techniques for Detection of Fasciola hepatica in Sheep Using Excretory-Secretory Antigens

Öz

Fascioliasis is one of the most important zoonosis with worldwide distribution, which causes great losses in livestock production. Serological methods are used for early detection of disease. The current investigation undertook to challenge dot-ELISA and ELISA techniques in diagnosing of Fasciola hepatica in sheep using excretory-secretory (E/S) antigen to select the most appropriate method. The serum samples (n=281) were collected from 50 cases of fascioliasis, 81 animals with parasitic diseases other than fascioliasis, and 150 healthy control cases. The E/S antigen used was obtained from infected livers (F. hepatica) of sheep slaughtered in Hamedan industrial abattoir (Iran). The result of dot-ELISA showed 100% sensitivity and 92.2% specificity. Also, positive and negative predictive values, and accuracy of assay were 73.52%, 100% and 93.59%, respectively. In the case of ELISA, the results were as follows: 98% sensitivity, 100% specificity, 100% positive predictive values, 99.56% negative predictive values, and 99.64% accuracy of assay. In both techniques, cross reaction with fascioliasis was not observed. In conclusion, although these two tests had very similar results, dot-ELISA was more acceptable with respect to its higher sensitivity, simplicity in practice and cheaper than ELISA. Therefore, dot-ELISA can be recommended as a routine test in paraclinical laboratories and epidemiological studies.

Anahtar Kelimeler

• Fasciola hepatica, excretory-secretory

Koyunlarda Fasciola hepatica Enfeksiyonunun Tespitinde Ekskresyon / Sekresyon Antijenleri Kullanılarak Dot-ELISA ve ELISA Tekniklerinin Karşılaştırılması

Öz

Fasiolazis, dünya çapında hayvancılıkta büyük kayıplara neden olan önemli zoonozlardan biridir. Hastalığın erken tanısı için serolojik metotlar kullanılmaktadır. Bu araştırmada, koyunlarda Fasciola hepatica enfeksiyonlarının ekskresyon/sekresyon antijenleri kullanılarak tanısında Dot-ELISA ve ELISA tekniklerinden hangisinin en uygun olduğu karşılaştırılmıştır. Serum numuneleri (n=281) 50 adet fascioliasis olgusundan, 81 adet fasiolasis dışındaki paraziter hastalık olgusundan ve 150 adet sağlıklı kontrol olgularından toplanmıştır. Kullanılan E/S antijeni, Hamedan mezbahasında (İran) kesilen enfekte koyun karaciğerinden (F. hepatica) temin edilmiştir. Dot ELISA sonuçları %100 duyarlılık ve %92,2 belirleyicilik göstermiştir. Ayrıca, pozitif, negatif tahmini değerler ve testin doğruluğu sırasıyla %73,52, %100, %93,59 bulunmuştur. ELISA metodunda sonuçlar, %98 duyarlılık, %100 belirleyicilik, %100 pozitif tahmini sonuç, %99,56 negatif tahmini sonuç ve %99,64 testin doğruluğu olarak belirlenmiştir. Her iki teknikte de fasiyolazis ile çapraz reaksiyon gözlenmemiştir. Sonuç olarak bu iki testin benzer sonuçları olmasına rağmen, Dot ELISA metodu, ELISA metoduna göre daha yüksek duyarlılığa sahip olması, uygulamadaki basitliği ve ucuz olması açısından daha kabul edilebilirdir. Bu nedenle Dot-ELISA, paraklinik laboratuvarlarında ve epidemiyolojik çalışmalarda rutin bir test olarak tavsiye edilebilir.

Anahtar Kelimeler

• Fasciola sekresyon antijenleri, Dot-ELISA, ELISA, koyun

Kaynakça

1. Arriaga, C,. Paniagua, R., Ruiz-Navarrete, A., Bautista, C.R., Morilla, A., 1989. Comparison of dot enzyme-linked immunosorbent haemagglutination test (PHT) and thin layer immunoassay (TLA) in the diagnosis of natural or experimental Fasciola hepatica infections in sheep. Veterinary Parasitology 30, 197-204. passive
2. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantitive protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254.
3. Dalimi, A., Haghigi, R., Madani, R., 2004. Partially purified fraction antigen from adult F. gigantica for serodiagnosis of human fascioliasis using Dot-ELISA technique. Annals of Saudi Medicine 24, 18-20.
4. Diab, T., Rabia, I., Komy, W., Amir, A., 2011. Efficacy of sandwich and Dot-ELISA in diagnosis of fascioliasis using a pair of polyclonal antibodies against cathepsin L antigen in naturally infected sheep. Research Journal of Parasitology 6, 90-100.
5. Galen, R.S., 1980. Predictive values and efficiency of laboratory testing. Pediatric Journal of Clinician North America 27, 861-869.
6. Hassan, M.M., Matar-nowafy, M.A., Saleh, A., 2002. Dot ELISA for measuring anti-Fasciola IgG isotype among patients with Fascioliasis. Journal of Egyptian Society of Parasitology 32, 571-578.
7. Hillyer, G.V., Sanchez, Z., De-Leon, D., 1985. Immuno- diagnosis of bovine fascioliasis by enzyme-linked immunosorbent assay and immuneprecipitation methods. Journal of Parasitology 71, 449-454.
8. Intapan, P., Maleewong, W., Nateeworanart, S., Wongkhan, C., Pipitgool, V., Sukolapong, V., Sangmaneedet, S., 2003. Immunodiagnosis of human fascioliasis using an antigen of F. gigantica adult worm with molecular mass of 27 kDa by a Dot-ELISA. Southeast Asian Journal of Tropical Medicin and Public Health 34,713- 717.

9. Kooshan, M., Hashemitabar, G., Naghibi, A., 2010. Use of somatic and Excretory/Secretory antigens of F. hepatica in diagnosis of sheep by ELISA. American
10. Heidari et al., J. Fac. Vet. Med. Istanbul Univ., 41 (1), 21-25, 2015 25 Journal of Agriculture and Environment Science 7, 170-175.
11. 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. American Journal of Tropical Medicine and Hygiene 61, 648-651.
12. Njau, B.C., Kasali, O.B., Scholtens, R.G. Akalework, N., 1989. The influence of watering practices on the transmission of Fasciola among sheep in the Ethiopian Communication 13, 67-74. Veterinary Research
13. Noureldin, M.S., EL-Ganaini, G.A., Abou-Elenin, 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. Journal of Egyptian Society of Parasitology 34, 691-704.
14. Pfister, K., 1990. Serodiagnosis of fasciolosis in ruminants. Revue Scientifique Et Technique De L`Office International Des Epizooties 9, 511-518.
15. Rabia, I., Sabry, H., Nagy, F., 2010. Comparison between different immunological techniques for detection of circulating Fasciola antigen in sheep. New York Science Journal 3, 34-39.
16. Rodriguez, J., Hillyer, G.V., 1995. Detection of excretory- secretory circulating antigens in sheep infected with Fasciola hepatica and with Schistosoma mansoni and F. hepatica. Veterinary Parasitology 56, 57-66.
17. 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 Journal of Public Health 33, 8-13.
18. Rokni, M.B., Samani, A., Massoud, J., Babaei, M., 2006. Evaluation of Dot-ELISA method using excretory- secretory antigens of Fasciola hepatica in laboratory diagnosis of human fasciolosis. Iranian Journal of Parasitology 1, 26-30.
19. Santiago, N., Hillyer, G.V., 1988. Antibody profiles by EITB and ELISA of cattle and sheep infected with Fasciola hepatica. Journal of Parasitology 74, 810-818.
20. Shaheen, H.I., Kamal, K.A., Farid, Z., Mansour, N., Boctor, F.N., Woody, J.N., 1989. Dot- enzyme- linked- immunosorbent assay (Dot-ELISA) for the rapid diagnosis Parasitology 75, 549-552. fascioliasis. Journal of
21. Shaker, Z.A., Demerdash, Z.A., Mansour, W.A., Hassanein, H.I., El-Baz, H.G., El-Gindy, H.I., 1994. Evaluation of specific Fasciola antigen in the immunodiagnosis of human Fascioliasis in Egypt. Journal of Egyptian Society of Parasitology 33, 119-123.
22. Soulsby, E.J.L., 1982. Helminths, arthropods and protozoa of domestic animals. 7th Edition, Bailliere Tindall London, Philadelphia.
23. Van-Tiggele, L., Over, H., 1976. Serological diagnosis of fascioliasis. Veterinary Parasitology 1, 239-248.
24. Yamasaki, H., Aoki, T., Oya, H., 1989. A cystein proteinase from the liver fluke Fasciola spp: purification, characterization, localization and application to immunodiagnosis. Japanese Journal of Parasitology 38, 373-384.
25. Zimmerman, G.L., Jen, L.W., Cerro, J.E., Farnsworth, K.L., Wescott, R.B., 1982. Diagnosis of Fasciola hepatica infestions in sheep by an enzyme linked immunosorbent Veterinary Research 43, 2097-2100. American Journal of