Indirect fluorescent antibody test for the diagnosis of clonorchiasis in rabbit and human
A number of serodiagnostic methods have been introduced to diagnose the infection of Clenorchis sinensis. The most commonly used has been the intradermal test with veronal buffered saline antigen prepared from the adult worms. It is recognized as a convenient tool to screen the infection, though the reliability has not yet been recognized. The positiveness lasts 10 to 20 years even after the elimination of the parasite, but shows cross retraction with other related parasitic infections (Sawada et al., 1964: Sadun et al., 1959). The complement fixation test (CFT) has also been tried for diagnosis of the trematodes infections (Sadun et al., 1959; Yokogawa et al., 1962; Sawada et al., 1965). The antibody for CFT is demonstrable during the infestation and short after elimination of the parasite. Yet the
procedure is so complicated for mass survey that it is limited mostly in laboratory for particular purposes. The other serodiagnostic methods are immunodiffusion test (Sun and Gibson, 1969 a: Yogore et al., 1965), indirect hemagglutination test (Pacheco et al., 1960), circum-adult precipitin test (Sun, 1969b) and circum-oval precipitin test (Sun, 1969a). But these methods have also been recognized not suitable for mass survery. For the reason a more simplified and reliable diagnostic method has been looked for to screen the infection during mass survey.
Recently, fluorescent antibody test has been used widely in research for demonstration of parasite and detection of antibody in parasitic infections. The test was applied for the diagnosis of fellowing parasitic infections: ascariasis (Taff and Voller, 1963), hookworm infection (Zaman and Singh, 1965), trichinosis
(Sadun et al., 1962b), filariasis (Duxbury and Sadun, 1967), schistosomiasis (Sadun and Gore, 1977: Hoahino et al., 1970), as well as amoebiasis (Gore and Sadun, 1968) and trypanosomiasis (Toussaint et al. 1965; Wery et al.. 1970).
The present study was designed to know whether this method is applicable as a more suitable method to diagnose the Clonorchis sinensis infestation.
Adult flukes were collected from the liver of the experimentally infected rabbits. washed in saline solution and distilled water, lyophilized and kept at -30℃. Metabolic products were obtained from TC Medium 199 (Difco Laboratory) in
which adult fluke were cultured at 4℃. The metabolites with complete Freund's adjuvant were injected intracutaneously into footpad of rabbit, five times at the 7 day interval.
Preparation of particle antigen was based on a few modification of the method used for serological diagnosis of schistosomiasis reported by Hoshino et al. (1970). Sera from rabbits and human infected or non-infected with Clonorchis sinensis were inactivated at 56℃ for 30 minutes before use.
As conjugate Anti-Rabbit Immunoglobulin (sheep) fluorescein labelled(Wellcome Laboratory) and Anti-Human IgG (sheep) fluorescein labelled(Wellcome Laboratory) were prepared at a 1:32 dilution. Fluorescent microscopy was performed by the methods described by Camargo et al. (1965), and Sadun and Gore(1967). Reactions were examined in dark field under oil immersion with fluorescent microscope (A.0.), No. 702 (A.0., Schott BG-12) as exciting filter arid No. 724 (A,0., Schott GG-1) as barrier filter.
The results obtained in the present study are summarized.
In the anti-metabolites sera of rabbit, the titer of fluorescent antibody did net correlate with the increase of nitrogen contents of antigen and period of the immunization.
In the Clonorchis sinensis infected rabbit sera, the titer increased rapidly from 4-5 weeks after the infection and maintained high titers for 2-5 weeks. The high titer continued until 62th week of investigation.
The indirect fluorescent antibody (IFA) titers of clonorchiasis patients were from 1:16 to 1:512, whereas the control sera were less than 1:8. The control sera wore from those infected with other parasites (paragonimiasis, ascariasis, trichuriasis, ancylostomiasis, trichostrongyliasis and amoebiasis), from nonparasitic malignancy and from parasite negatives.
Of 20 clonorchiasis sera, 9 cases showed weak reaction (1:16-1:32), 4 cases were moderate (1:64-1:128) and 7 eases were high (1:256-1:512).
From the above results, it may be concluded that the test seemed to be promising in terms of sensitivity, specificity and reproducibility, and it could be a valuable diagnostic method as well as in epidemiological mass survey.