Title of the thesis: Immunological and nucleic acid based diagnosis of Fasciola gigantica infection

Name of the student: Dr. A.K. Jayraw

Name and address of the advisor: Dr. B.P. Singh, Division of Parasitology, Indian Veterinary Research Institute, Izatnagar-243 122, India

Degree awarded: Ph.D. (Veterinary Parasitology)

Year of award: 2005

Name of the university: Deemed University, Indian Veterinary Research Institute, Izatnagar-243 122, India

The major objective of the present investigation was to study the antibody kinetics, early and sensitive immunological detection of Fasciola gigantica infection in the bovine host and use of PCR as an epidemiological tool for screening of snail intermediate hosts for the parasite infection. Fifteen Holstein-Friesian cross-bred male calves were randomly divided into 3 groups (Gr). Animals of Gr-A and Gr-B were infected with four-month and sixteen-month-old metacercariae (n=400), respectively, whereas animals of Gr-C served as uninfected control. Coprological examination revealed a mean prepatency of 99 and 96 days in animals of Gr-A and Gr-B, respectively. Animals treated with triclabendazole @ 12 mg kg⁻¹ body weight on 28 weeks post-infection (WPI) exhibited hundred per cent efficacy against adult flukes. SDS-PAGE analysis of somatic preparations of F. gigantica, G. explanatum and S. spindale revealed polypeptides in the range of 94–14, 85–29 and 152–27 kDa, respectively. Similarly, SDS-PAGE analysis of DEAE-Sepharose anion exchange purified antigen (0.1M NaCl) revealed polypeptides ranging from 119–21 kDa. The somatic extract of F. gigantica revealed 52 kDa polypeptide as the prominent immunoreactive protein by immunoblot analysis. This antigen was detectable at 2 WPI when probed with sera from animals of 10 days, 2, 3, 4, 5 and 7 weeks PI. IgGl was the predominant immunoglobulin isotype detectable against F. gigantica infection in bovine calves as early as 10–14 DPI, when compared to IgG and IgG2 responses. Whereas, IgG and IgG2 responses were found to be elicited on 2–3 and 4–6 WPI, respectively and IgM response between 10–14 DPI. Specific mucosal immune response in the form of IgA was found to be elicited by 4 WPI, which showed the highest titre at 19 WPI.

Investigations were carried out for detection of F. gigantica circulatory antigen targeting 52 kDa antigen by a sandwich ELISA. Using the assay the antigen could be detected as early as 10 DPI. However, following 3 weeks of triclabendazole therapy no antigen could be detected in circulation by the assay.

Fasciola specific primers amplified a 124 bp fragment in PCR when the genomic DNA preparation from F. gigantica worms and the snail intermediate host (Lymnaea auricularia) infected with the parasite were used in the assay. In addition to the 124 bp amplicon, a ladder of DNA fragments representing amplification of the 124 bp repetitive sequence was also observed.

The present investigation established that Western blot and antibody sandwich ELISA are highly sensitive for detection of F. gigantica infection as early as 14 and 10 DPI in bovine calves, respectively while PCR was sensitive for detection of the parasite infection in snail intermediate host.