Using the polymerase chain reaction (PCR) and two primers for conserved regions of the small subunit ribosomal RNA (SSU-rRNA) of Microsporidia, a DNA segment about 1,195 base pairs long was amplified from a DNA template prepared from purified spores of the microsporidian species Pleistophora anguillarum. These spores had been isolated from adult eels (Anguilla japonica) with "Beko Disease." A comparison of sequence data from other microsporidian species showed P. anguillarum SSU-rRNA to be most similar to Vavraia oncoperae. When juvenile eels were artificially infected with P. anguillarum, enzyme-linked immunosorbent assay could detect a positive infection only 12 days post-infection. However, when suitable PCR primers were used, a DNA fragment of about 0.8 kb was detected from these juvenile eels after only 3 days post infection. No PCR product was obtained with templates prepared from clinically healthy control animals.
A humoral response of Japanese eel, Anguilla japonica Temminck & Schlegel, to the microsporean Pleistophora anguillarum Hoshina was demonstrated using immunoblotting and an enzyme-linked immunosorbent assay (ELISA). Japanese eel immunoglobulin was purified by afifinity chromatography. The immunoglobulin was composed of 25-kDa light chains and 72-kDa heavy chains. The ELISA values of P. anguillarum antibodies in naturally infected fish sera were significantly higher than those of clinically healthy fish. Spore proteins from the microsporean were separated by electrophoresis and subjected to analysis by Western blot. Sera from naturally infected fish showed different reaction patterns to the spore proteins. While the sera randomly selected from naturally infected eels all showed a significant positive reaction to P anguillarum antigens, the mucus from only three out of the nine infected eels reacted positively in the ELISA test. Subsequent analyses indicated that there was no significant difference in the amount of mucus immunoglobufin among the tested eels. Therefore, the generally lower ELISA values of mucosal anti-/? anguillarum antibodies from the infected eels tested were evidently not caused by a lack of immunoglobulin per se, but seem to be the result of a lack of anti-7? anguillarum antibodies in the mucus and/or a lower afFmity in the anti-T? anguillarum antibodies that were present.
Adult Japanese eels, Anguilla japonica Temminck & Schlegel, (200–250 g, 45–55 cm) were immunized by intramuscular injection with goat IgG. After 5 weeks, eel immunoglobulin (Ig) was purified using affinity chromatography. The purified eel Ig was used to immunize rabbits to produce anti‐eel Ig antibody. The highest antibody ELISA value in eels was reached 3 weeks after initial immunization with goat IgG, and then gradually decreased. The antibody could still be detected at 140 days post‐immunization. The optimal temperature for antibody production was 30°C. Freund’s complete adjuvant and secondary immunization both increased antibody production in eels.
Pleistophora anguillarum Hoshina, a common from four to five fish was pooled for analysis. Serum microsporean parasite of farmed eels in Taiwan, was separated by centrifugation at 700 g for 30 min infects the skeletal muscle of the Japanese eel, and the anti-P. anguillarum antibodies were assayed Anguilla japonica Temminck & Schlegel, and causes by an ELISA method (Hung, Lo, Tseng & Kou structural changes which include concavities of 1996). A significantly (ANOVA, F ϭ 22.7; the body surface (Hoshina 1951). Glass eels are P Ͻ 0.01) higher quantity of anti-P. anguillarum vulnerable to infection and this can lead to antibody was detected at 6 weeks post-infection. The significant economic loss (T'sui & Wang 1988; optimal anti-P. anguillarum ELISA value occurred at Wang, Shia, Chang, Lo & Kou 1990; Kou, Wang, 9 weeks post-infection, after which the ELISA values Hung, Jan, Chou & Lo 1995). subsequently decreased (Fig. 2). In order to evaluate the effects of P. anguillarum Salati, Ono & Kusuda (1991) reported that glass spore dosage on artificial infection, 180 glass eels eels achieve a protective immune response to the (average weight 0.1 g and average body length 6 cm) bacterium Edwardsiella tarda. They found that glass were separated into six groups for exposure to eels immunized with sonicated products of E. tarda different spore concentrations (2 ϫ 10 7 , 2 ϫ 10 6 , were better protected than the control. In the 2 ϫ 10 5 , 2 ϫ 10 4 , 2 ϫ 10 3 spores ml-1 and control). present study, we provide evidence for the first time Each group was immersed in 200 ml of spore that glass eels can produce specific antibodies after suspension at 28°C for 8 h. Subsequently, the groups microsporean infection, although these antibodies of glass eels were transferred to 20-l plastic tanks do not constitute an effective protection against containing 5 l of water. The experimental eels in all the parasite at the high dose levels administered. groups were examined for white spots on the body However, the presence of antibodies suggests that surface as a clinical sign of parasite infection 14 protective immunity against P. anguillarum is at days post-immersion (T'sui, Wang & Lo 1988). A least possible; further experiments will be required 100% infection rate occurred at spore concentrations to investigate this possibility. at or above 2 ϫ 10 6 ml-1 (Fig. 1). Sixty further Salati et al. (1991) and Kusuda, Ono & Salati healthy glass eels were then immersed in 200 ml of (1988) found that glass eels and adult eels both spore suspension solution (2 ϫ 10 6 spores ml-1) at need about 3 weeks to achieve a protective immune 28°C for 8 h. Subsequently, these eels were also response to E. tarda. In another study, in which transferred to a 20 l plastic tank containing 5 l of antiserum was induced by the immunization of water at 28 Ϯ 2°C. Every 3 weeks after infection, adult Japanese eel with goat IgG, the optimal antiseveral glass eels were anaesthetized by ice and bled goat IgG ELISA value also occurred at 3 weeks by severing the tail. Blood was co...
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