异育银鲫武汉单极虫病发生、发展、消退和消失的病理变化与PCR分析

为探究武汉单极虫病疾病过程和病理变化等特点,根据患病异育银鲫体表孢囊内有无成熟孢子和成熟孢子的崩溶解程度,将该病划分为发生、发展、消退和消失4个疾病时期,并分别进行病理变化观察和巢式PCR分析。结果发现,发生期的病鱼体表刚形成的孢囊使鳞片和皮肤微微隆起,孢囊乳白色,孢囊内分布着正在繁育逐渐增多的营养体,尚未出现成熟孢子;发展期的病鱼孢囊内已出现成熟孢子,孢囊逐渐增多增大,其表面黑色素细胞增加而呈灰黑色,感染强度高的病鱼出现死亡或畸形;消退期的成熟孢子通过破裂孢囊流入水体或不同步地崩溶解而减少直至全部溶解,孢囊随之逐渐缩小,使得黑色素细胞更为密集,此时期病鱼病情减轻不再出现死亡现象;消失期的病鱼孢囊平坦,内已无成熟孢子,只残留逐渐减少的成熟孢子崩溶解物质,黑色素细胞逐渐减少,最后原孢囊部位被结缔组织取代。巢式PCR分析结果表明,巢式第一轮和第二轮PCR在4个疾病时期都分别能扩增出1 584和853 bp的武汉单极虫目的条带,但在消失期的后期只有巢式第二轮PCR扩增出853 bp目的条带,说明残留的核酸物质含量逐渐减少,10月下旬后原孢囊部位巢式PCR扩增已无条带出现。本研究不仅揭示了武汉单极虫寄生部位在4个疾病时期的病理变化特点,而且明确了疾病消退和消失的2种方式,孢囊内成熟孢子通过破裂孢囊进入水体的方式和首次发现的孢囊内成熟孢子通过崩溶解的方式。

Pathology and PCR assay of Thelohanellus wuhanensis disease in Allogynogenetic crucian carp (Carassius auratus gibelio) in the periods of occurrence, development, fade and disappearance

In order to explore the process and pathology of Thelohanellus wuhanensis diseased Allogynogenetic crucian carp (Carassius auratus gibelio), the disease was divided into four periods of occurrence, development, fade and disappearance according to whether there were mature spores and the degenerated degree of them or not. Pathological observation and analysis of nested PCR were made respectively. Results showed that in the period of occurrence, formed cysts on diseased fish were small and a little uplifted. The cysts were milk white in colour. Trophozoites increased by profilication and no mature spores appeared in the cysts. In the period of development, mature spores appeared in the cysts. Number and size of cysts increased. The cysts were gray black in the colour due to the increase of melanocytes. Diseased fish with higher infection would show death or deformity. In the period of fade, mature spores reduced by flowing out into water through the ruptured gap of cysts or by degenerating gradually until all of them degenerated. At the same time, cysts shrunk and melanocytes were more dense gradually. Diseased fish did not die in the present period due to relieved symptoms. In the period of disappearance, no mature spores and only degenerated materials were left in the flattened cysts. Melanocytes reduced gradually. Finally, the original part of cysts was replaced by connective tissue. Nested PCR analyses showed that the 1 584 bp and 853 bp in the first and second runs of nested PCR were respectively obtained in all of four periods of disease. However, in the late stage of the period of disappearance only 853 bp in the second run of nested PCR was obtained, which illustrated that its quantity reduced. No bands were obtained after late October. Present study not only revealed the pathology of T.wuhanensis infected sides in the four periods of disease, but also defined two ways of the disease fade and disappearance. One way was flow out of mature spores from ruptured cysts into water and another way discovered for the first time was degeneration of mature spores in the cysts.