框镜鲤肠管单极虫病的组织病理学分析及分子鉴定
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摘要
2017年6月,四川德阳某养殖场的框镜鲤患病且大量死亡。主要症状为腹部膨大,肠管有大小不等的肿物。为明确其死亡病因,进行了细菌学、寄生虫形态学观察,组织病理学和PCR检测。细菌学检查为阴性;病理组织学观察发现肠道的损伤最为严重,表现为有大量寄生虫样孢囊突出于肠管。寄生虫形态学观察可见孢子长约23～30μm,宽10～15μm,极囊长11～15μm,呈长卵圆形,约占孢子长的1/3～1/2,孢子外面被有一层无色透明的薄膜,前端孢子质中有一个大核和两个小核,与单极虫形态一致;针对吉陶单极虫的18S SSUrRNA基因进行巢式PCR检测,扩增出大小为716 bp的目的片段(命名为TKF-1),目的条带的测序和序列分析结果表明其为吉陶单极虫。根据组织病理特点及PCR检测结果确认框镜鲤的死亡是感染吉陶单极虫所致。
In order to identify the pathogen which caused mass mortality in farmed Cyprinus carpio var. specularis in Deyang district of Sichuan Province, China, the pathogen of this event was determined following bacteriology,parasite morphological observation, histopathology and PCR detection. The results showed that bacteriological examination was negative whereas parasite was positive with Thelohanellus kitauei. Histologically, intestinal damage is the most serious, where a large number of parasitoid cysts were noted. Morphological observation showed that the total length and width of the spores were about 23–30 μm and 10–15 μm, respectively. In addition,the polar capsule was 11–15 μm long and ovalellipsoidal, which accounted for about 1/3–1/2 of spores. The outer layer of the spores covered with a thin colorless and transparent film. One pyriform macronucleus was in the upper part and two globular micronuclei near the iodinophilous vacuole. Tissue DNA from infected intestine was identified as T. kitauei(TKF-1) by nest polymerase chain reaction amplification of the 18 S SSUrRNA gene and the sequenced 18 S SSUrRNA gene of TKF-1 has a homology up to 100% with T. kitauei in GenBank. According to the results we confirmed that the death of C. carpio var. specularis in this region was caused by T. kitauei.
In order to identify the pathogen which caused mass mortality in farmed Cyprinus carpio var. specularis in Deyang district of Sichuan Province, China, the pathogen of this event was determined following bacteriology,parasite morphological observation, histopathology and PCR detection. The results showed that bacteriological examination was negative whereas parasite was positive with Thelohanellus kitauei. Histologically, intestinal damage is the most serious, where a large number of parasitoid cysts were noted. Morphological observation showed that the total length and width of the spores were about 23–30 μm and 10–15 μm, respectively. In addition,the polar capsule was 11–15 μm long and ovalellipsoidal, which accounted for about 1/3–1/2 of spores. The outer layer of the spores covered with a thin colorless and transparent film. One pyriform macronucleus was in the upper part and two globular micronuclei near the iodinophilous vacuole. Tissue DNA from infected intestine was identified as T. kitauei(TKF-1) by nest polymerase chain reaction amplification of the 18 S SSUrRNA gene and the sequenced 18 S SSUrRNA gene of TKF-1 has a homology up to 100% with T. kitauei in GenBank. According to the results we confirmed that the death of C. carpio var. specularis in this region was caused by T. kitauei.
引文
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[14]Zhao D D,Borkhanuddin M H,Wang W M,et al.The life cycle of Thelohanellus kitauei(Myxozoa:Myxosporea)infecting common carp(Cyprinus carpio)involves aurantiactinomyxon in Branchiura sowerbyi[J].Parasitology Research,2016,115(11):4317-4325.
[15]Rhee J K,Kim J O,Park B K.Prophylactic and therapeutic studies on intestinal giant-cystic disease of the Israel carp caused by Thelohanellus kitauei.II.Effects of physical and chemical factors on T.kitauei spores in vitro[J].The Korean Journal of Parasitology,1990,28(4):241-252.
[2]吕大成,单晓枫.框镜鲤嗜水气单胞菌的分离鉴定[J].畜牧与饲料科学,2011,32(8):15-16.LüD C,Shan X F.Isolation and identification of Aeromonas hydrophila from Cyprinus carpio carpio[J].Animal Husbandry and Feed Science,2011,32(8):15-16(in Chinese).
[3]张剑英,邱兆祉,丁雪娟.鱼类寄生虫与寄生虫病[G].北京:科学出版社,1999.Zhang J Y,Qiu Z Z,Ding X J.Parasites and Parasitic Diseases of Fishes[G].Beijing,China:Science Press,1999(in Chinese).
[4]Lom J,Arthur J R.A guideline for the preparation of species descriptions in Myxosporea[J].Journal of Fish Diseases,1989,12(2):151-156.
[5]Egusa S,Nakajima K.A new myxozoa Thelohanellus kitauei,the cause of intestinal giant cystic disease of carp[J].Fish Pathology,1981,15(3-4):213-218.
[6]Ye L T,Lu M M,Quan K Y,et al.Intestinal disease of scattered mirror carp Cyprinus carpio caused by Thelohanellus kitauei and notes on the morphology and phylogeny of the myxosporean from Sichuan Province,southwest China[J].Chinese Journal of Oceanology and Limnology,2017,35(3):587-596.
[7]Seo J S,Jeon E J,Kim M S,et al.Molecular identification and real-time quantitative PCR(qPCR)for rapid detection of Thelohanellus kitauei,a myxozoan parasite causing intestinal giant cystic disease in the Israel carp[J].The Korean Journal of Parasitology,2012,50(2):103-111.
[8]畑井喜司雄,小川和夫.新鱼病图谱[M].任晓明,译.北京:中国农业大学出版社,2007.Hatakai K,Ogawa K.New Atlas of Fish Diseases[M].Ren X M,Trans.Beijing:China Agricultural University Press,2007(in Chinese).
[9]Eszterbauer E,Marton S,Rácz O Z,et al.Morphological and genetic differences among actinosporean stages of fish-parasitic myxosporeans(Myxozoa):difficulties of species identification[J].Systematic Parasitology,2006,65(2):97-114.
[10]高世同,张仁利,黄晓燕,等.间日疟原虫孢子期SSUrRNA基因扩增、鉴定及其诊断应用[J].中国热带医学,2007,7(2):179-181.Gao S T,Zhang R L,Huang X Y,et al.Amplification of specific SSUrRNA gene sequence and its application in diagnosis of Plasmodium vivax malaria[J].China Tropical Medicine,2007,7(2):179-181(in Chinese).
[11]Yokoyama H.Transmission of Thelohanellus hovorkai Achmerov,1960(Myxosporea:Myxozoa)to common carp Cyprinus carpio through the alternate oligochaete host[J].Systematic Parasitology,1997,36(2):79-84.
[12]Székely C,El-Mansy A,Molnár K,et al.Development of Thelohanellus hovorkai and Thelohanellus nikolskii(Myxosporea:Myxozoa)in oligochaete alternate hosts[J].Fish Pathology,1998,33(3):107-114.
[13]Xi B W,Zhou Z G,Xie J,et al.Morphological and molecular characterization of actinosporeans infecting oligochaete Branchiura sowerbyi from Chinese carp ponds[J].Diseases of Aquatic Organisms,2015,114(3):217-228.
[14]Zhao D D,Borkhanuddin M H,Wang W M,et al.The life cycle of Thelohanellus kitauei(Myxozoa:Myxosporea)infecting common carp(Cyprinus carpio)involves aurantiactinomyxon in Branchiura sowerbyi[J].Parasitology Research,2016,115(11):4317-4325.
[15]Rhee J K,Kim J O,Park B K.Prophylactic and therapeutic studies on intestinal giant-cystic disease of the Israel carp caused by Thelohanellus kitauei.II.Effects of physical and chemical factors on T.kitauei spores in vitro[J].The Korean Journal of Parasitology,1990,28(4):241-252.