| The parasitic ciliate Ichthyophthirius multifiliis, the etiologic agent of "white-spot" disease, is a well-known and widely distributed parasite of freshwater fish species causing significant economic losses to the aquaculture industry. It is well documented on the invasion, prevention and immunology, as well as its cell biology and life cycle of Ⅰ. multifiliis. But it is not clear that the immune protection mechanism and the relationship between Ⅰ. multifiliis and the immune organs of grass carp (Ctenopharyngodon idella). In this study, the pathological changes of the gill, skin, muscle, liver, spleen from the grass carp exposed to Ⅰ. multifiliis were observed experimentally by the light microscopy (LM) and transmission electron microscopy (TEM), respectively. The MHⅡ-DAB1gene polymorphism and the associations between some alleles and disease resistance to Ⅰ. multifiliis were analyzed. Meanwhile, the expression of the DAB1gene was dedected in different times after infection with Ⅰ. multifiliis by real-time quantitative PCR (RT-PCR). The results were as follows:1. The infected grass carp showed emaciation, lethargy, dyspnea, multiple pin-point sized white spots on the body surface. Moreover, swollen spleen and kidneys were found in affected fish. Some diseased fish also had eye cataract, pale gill with liver and gill artery tumefaction. In some cases, a large number of Ⅰ. multifiliis were observed in gill sections; the gill filaments were markedly degenerated and desquamated in superficial cells forming vacuolar by the light microscopy (LM). However, epidermal hyperplasia, disrupted cellular integrity and pyknotic nuclei were observed in infected fish skin. In the muscle, these changes were splitting of muscle, dislocation of nuclei, brown atrophy (pigmentation accumulation), destruction of striation, splitting of longitudinal tissues and necrosis. In addition, vacuolar degeneration and pyknotic nuclei were observed in intestinal cells of infected fish by LM. In the liver, the structure of hepatic palte was loosed with hepatocytes vacuolar degeneration and necrosis. These changes were vacuolar degeneration and necrosis in spleen cells. By the transmission electron microscopy (TEM), the gill capillary epithelial cell and gill cartilage cell structure were destroyed and disappeared respectively. However, their nuclei with rrhexis were faint or not visible; empty spaces were present along with vacuolated and necrotic cells in infected fish skin. In the muscle, these changes were destruction of striation and mitochondria, the mitochondria proceeded to lose cristae with vacuolization observed. In addition, the intestinal villus was disrupted and the electron density distribution of brush border was uneven. In the liver, hepatocytes aligned rarefaction and disorganization, pyknotic nuclei were observed. These changes were lymphocyte disorganization and pyknotic nucle in spleen cells.2. The complete exon2of MHⅡ-DAB1gene of grass carp was amplified by PCR, the length of cloned sequences was276bp, and12new alleles of MHⅡ-DAB1gene were detected. Alleles of Ctid-DAB1*0301, Ctid-DAB1*0402, Ctid-DAB1*0501, Ctid-DAB1*0701, Ctid-DAB1*0801and Ctid-DAB1*0901were occurred in16resistant individuals; while alleles of Ctid-DAB1*0902and Ctid-DAB1*1001were found from14susceptible individuals; Four alleles of Ctid-DAB1*0201, Ctid-DAB1*0401, Ctid-DAB1*0601and Ctid-DAB1*0701were shared in both stocks.3. The expression of MHⅡ-DAB1gene had a significant change in grass carp tissues after challenged with Ⅰ. multifiliis. Compared to the control group, a significant increased expression of MHⅡ-DAB1gene from3h to1day was observed, and reached the maximum of3.6-fold at1day in the head kidney; nevertheless, it was down-regulated from day2to day6in comparison to than in control group. In the spleen, the expression pattern of the tested group showed a significant1.3-fold down-regulation at3h after infection, whereas reached a significant2.2-fold higher at day1. Expression of the DAB1gene in gill showed high variation at different time points after infection; a very early increase of1.5-fold at12h was observed, followed by1.6-fold increase at day1and1.3-fold increase at day6; other times the expression levels of the DAB1gene were significantly down-regulated compared to that in control group. Significant variation of the DAB1gene expression was observed in the skin of infected fish; a progressing significant increase was observed from3h to day2(1.5-fold); after infection2days, the transcription level of the DAB1gene was dramatically decreased. In the liver and intestines samples, the expression of DAB1gene in intestines at each detecting time was significantly down-regulated after infection with Ⅰ. multifiliis. Only low expression levels of the MHⅡ-DAB gene were detected at different time after infection from3h to6days in the blood; however, a significant expression peak with1.8-fold higher was found in case group than that in control group at day10. |
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