Immunization and challenge shown by hamsters infected with Opisthorchis viverrini following exposure to gamma-irradiated metacercariae of this carcinogenic liver fluke

Abstract

Here we report findings to optimize and standardize conditions to attenuate metacercariae of Opisthorchis viverrini by ionizing radiation to elicit protective immune responses to challenge infection. Metacercariae were gamma-irradiated and the ability of irradiated metacercariae to prevent patent infection of challenge metacercariae in hamsters was determined, as well as their ability to induce a host antibody response. Metacercariae irradiated in a dose-dependent manner, with 3, 5, 10, 12, 20, 25 and 50 Gray, were used to infect Syrian golden hamsters by stomach gavage to ascertain the effect of irradiation on ability of the worms to establish infection. In addition, other hamsters were infected with metacercariae irradiated with 20-50 Gray, followed by challenge with intact/wild-type (non-irradiated) metacercariae to determine the protective effect as established by the numbers of adult flukes, eggs of O. viverrini in hamster faeces and anti-O. viverrini antibody titres. Significantly fewer worms were recovered from hamsters immunized with metacercariae irradiated at 20, 25 and 50 Gray than from control hamsters infected with intact metacercariae or 0 Gray, and the worms showed damaged reproductive organs. Faecal egg numbers were decreased significantly in hamsters immunized with 25 and 50 Gray metacercariae of O. viverrini. Moreover, hamsters administered metacercariae that were protected elicited a robust, specific anti-fluke immunoglobulin G response compared to control hamsters, suggesting a role for antibody in protection elicited by radiation-attenuated metacercariae.

Fig. 1

Experimental design and the timelines of immunization with irradiated O. viverrini metacercariae, challenge infection and necropsy. Upper panel: infection of hamsters with radiation-attenuated metacercariae. Lower panel: vaccine study involving immunization with radiation-attenuated metacercariae, followed by challenge with intact parasites. Ov-MC, Opisthorchis viverrini metacercariae; Gy, Gray; EPG, worm eggs per gram of hamster faeces; W, weeks.

Fig. 2

The number of adult worms of O. viverrini in the bile ducts, and eggs recovered in pooled faeces, from hamsters infected with O. viverrini metacercariae subjected to γ-radiation. (A) The number of worms recovered 6 weeks after infection with 50 irradiated Ov-MC subjected to different doses of γ-radiation. (B) Faecal egg counts of hamsters infected with irradiated and wild-type metacercariae (0 Gy). *** Denotes significant differences between worms treated compared with control groups infected with wild-type parasites (P ≤ 0.001).

Fig. 3

IgG responses to a lysate of adult O. viverrini worms in the sera of hamsters 6 weeks after infection with irradiated metacercariae. C: uninfected hamsters.

Fig. 4

The number of adult worms of Opisthorchis viverrini recovered from hamsters after immunization with irradiated metacercariae and subsequent challenge with 50 wild-type metacercariae. Control: hamsters infected with 50 intact Ov-MC. Levels of significant differences: ** P ≤ 0.01 and *** P ≤ 0.001.

Fig. 5

The number of eggs per gram of faeces (EPG) in hamsters exposed to irradiated metacercariae at 4, 5 and 6 weeks after infection and challenged with 50 wild-type metacercariae. Levels of significant differences: * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001.

Fig. 6

IgG responses against soluble lysate of adult O. viverrini as determined by ELISA in sera of hamsters 6 weeks after challenge infection with wild-type O. viverrini metacercariae following immunization with radiation-attenuated metacercariae at 20, 25 and 50 Gy. The term ‘ − ve control’ (C−) refers to uninfected hamsters whereas ‘control’ (C+) refers to non-vaccinated but infected hamsters. Levels of significant differences: ** P ≤ 0.01 and *** P ≤ 0.001.

Fig. 7

Immunoblot analysis of IgG in hamster sera against a soluble lysate of adult O. viverrini worms. The terms 20, 25 and 50 Gy refer to hamsters immunized with radiation-attenuated O. viverrini metacercariae that had been exposed to 20, 25 and 50 Gy. The term 0 Gy refers to hamsters vaccinated with non-irradiated metacercariae and infected with wild-type metacercariae; C− refers to a pool of pre-immunized serum. C+ refers to non-vaccinated hamsters infected with 50 wild-type metacercariae.