蛇(鱼旬)感染多子小瓜虫病一例

20 0 3年 6月 1 8日从长江重庆木洞段购回十几条活泼蛇SaurogobiodabryiBleeker。均由罾网捕捞 ,健康无损伤。放在室外水泥池饲养。室外水温白天2 6℃左右 ,夜晚 2 4℃左右 ,6月 2 6日在常规检查时又发现蛇身上出现小白点 ,病鱼体色发黑、消瘦、反应迟钝、体表粘液增多、浮头、体表和鳃遍布小白点 ;感染后期表皮有脱落现象。取病鱼体表一个白点镜检 ,可见虫体全身密布短而均匀的纤毛 ;大核呈马蹄形 ,确定为多子小瓜虫IchthyophthiriusmultifiliisFouquet。感染小瓜虫病的蛇 7月 6日死亡。蛇是否能大面积发病还有待进一步研究蛇(鱼…     

蛇鮈感染多子小瓜虫病一例

2003年6月18日从长江重庆术洞段购回十几条活泼蛇鮈Saurogobio dabryi Bleeker。均由罾网捕捞,健康无损伤。放在室外水泥池饲养。室外水温白天26℃左右,夜晚24℃左右,6月26日在常规检查时又发现蛇鮈身上出现小白点,病鱼体色发黑、消瘦、反应迟钝、体表粘液增多、浮头、体表和鳃遍布小白点;感染后期表皮有脱落现象。     

长薄鳅感染多子小瓜虫一例

2004年12月5日从长江重庆木洞段购回活泼长薄鳅(Leptobobitia elongata Bleeker)8尾,健康无伤。放在室内水泥池和圆口铜鱼混合饲养。室内水温白天14℃左右,夜晚9℃左右,2005年1月13日在常规检查时发现3尾长薄鳅身上出现小白点(封3,图版),反应迟钝、体表粘液增多;白点出现在鱼体的头部、鳃片、背鳍、胸鳍、腹鳍、臀鳍、尾鳍,身体两侧和尾柄部白点较多;感染后期表皮有脱落现象。取病鱼体表白点镜检,可见虫体呈圆形或椭圆形,作滚动运动,身体遇障碍物可变形,虫体全身密布短而均匀的纤毛。经虫体固定、染色制片测量和观察,     

多子小瓜虫

多子小瓜虫韩志泉，成双红（首都师范大学生物学系１０００３７）多子小瓜虫（Ｉｃｈｔｈｙｏｐｈｔｈｉｒｉｕｓｍｕｌｔｉｆｌｉｉｓ）属原生动物门、纤毛纲、膜口目、睫杵虫科、小瓜虫属。成虫卵形，周身布满排列有序的纤毛，在近前端腹面有胞口，下连袋形的胞咽，胞肛...     

青海湖裸鲤和黄河裸裂尻鱼感染多子小瓜虫的病理学比较研究

为探讨不同裂腹鱼类感染多子小瓜虫后的病理学差异, 利用多子小瓜虫对青海湖裸鲤指名亚种(Gymnocypris przewalskii przewalskii)和黄河裸裂尻鱼(Schizopygopsis pylzovi Kessler)实施感染实验, 并对2种鱼进行了深入的病理学研究。研究结果显示: (1) 2种鱼的死亡量均呈现先激增后明显回落的趋势, 青海湖裸鲤死亡高峰在感染后第3至第4天, 黄河裸裂尻鱼死亡高峰在第3至第5天, 青海湖裸鲤的死亡比黄河裸裂尻鱼急剧。(2)感染后2种鱼体表均出现大量肉眼可见的白点。青海湖裸鲤皮肤黏液分泌量明显增加, 体表形成胶状黏液层, 黏液层中见不同细胞期小瓜虫包囊。黄河裸裂尻鱼鳍出现蛀鳍现象, 皮肤出现细菌感染样溃烂。(3)解剖发现, 感染组青海湖裸鲤和黄河裸裂尻鱼肝脏发生病理改变呈淡黄色, 胆有不同程度肿大。(4)组织切片和电镜观察显示, 小瓜虫在鳃部位的寄生导致青海湖裸鲤和黄河裸裂尻鱼鳃丝黏连, 鳃小片和鳃丝上皮细胞萎缩、脱落, 鳃丝结构被严重破坏。小瓜虫在2种鱼皮肤的寄生使寄生部位组织突起, 周围组织塌陷。青海湖裸鲤表皮下层出现空隙, 表皮结构被严重破坏。黄河裸裂尻鱼皮肤表皮细胞出现空泡化病理改变, 失去原有紧密结构, 表皮层和固有层间界限变模糊。综上所述, 小瓜虫的感染对青海湖裸鲤和黄河裸裂尻鱼的鳃和皮肤组织造成严重损伤, 但2种鱼表现的症状和造成的组织损伤类型有明显差异, 这与2种鱼长期适应不同水体环境密切相关。     

圆口铜鱼耐过小瓜虫感染二例

本实验室于 2 0 0 4年 12月 5日从嘉陵江木洞段购得野生健康圆口铜鱼 (Coreiusguishenoti) 15 0尾 ,圆口铜鱼体长从 5cm到 2 0cm不等。运回实验室后于 2 5 3× 135× 10 7cm鱼池中驯养 ,充氧 ,每日换水喂食。至 19日发现大部分圆口铜鱼体表出现小白点 ,尤以鳍条和头部为多 ,经镜检确定为多子小瓜虫 (Ichthyophthiriusmultifiliis)感染。 2 4日 ,病鱼体表小白点明显增多 ,体表逐渐形成包囊。将患病鱼转移至 8个 35× 2 5× 2 5cm水族箱中分养 ,充氧 ,不喂食。至 30日患病鱼绝大部分死亡 ,余下两尾圆口铜鱼体表产生大量白色粘液 ,活动迟缓。 …     

水温对多子小瓜虫孵化及幼虫活力的影响

多子小瓜虫引起的白点病是严重危害淡水鱼类的重要疾病之一,该寄生虫的发育繁殖受温度影响极大,因此,温度是影响小瓜虫培养及其疾病发生的重要因素.为了阐明温度在多子小瓜虫生活史中的作用,在实验条件下,研究了水温对该寄生虫包囊形成、孵化和幼虫活力的影响.结果表明:水温在4℃左右或28～32℃,多子小瓜虫都不能孵化,但有少部分虫体以包囊形式存活下来;在9～27℃,随水温升高,包囊彤成和孵化时间缩短,孵化率升高至100%,孵出的感染性幼虫活力增强,但活力维持时间缩短至4 h.因此,建议在多子小瓜虫培养及人工感染试验中,维持23℃左右的水温较合适,这时感染性幼虫使用的有效时限是孵出后4 h内.     

多子小瓜虫的形态、生活史及其防治方法和一新种的描述

一、引言 小瓜虫是一种身体比较大,肉眼能见的原生动物,寄生在各种淡水鱼的上皮粗械和鳃系粗械中,剥取寄主的上皮细胞和红血球为生。同时,由于它在粗械中不停的活动和钻营的结果,上皮粗械引起病态的浮肿,形成白色突出的小点,故有“白点”病之称。     

抗菌肽HBβ-C在全雄和杂交黄颡鱼对多子小瓜虫抗性差异中的作用

为研究全雄黄颡鱼(Pelteobagrus fulvidraco)、瓦式黄颡鱼(Pelteobagrus vachelli)和杂交黄颡鱼(黄颡鱼P.fulvidraco♀×瓦氏黄颡鱼P. vachelli♂)对多子小瓜虫(Ichthyophthirius multifiliis)的抗性差异,通过生物信息学分析黄颡鱼皮肤黏液蛋白质组,发现其血红蛋白源抗菌肽(HBβ-C)位于血红蛋白β链HBβ的碳端,共33个氨基酸。利用化学合成的不同浓度的HBβ-C肽段进行体外抗虫实验,研究发现其能有效杀死滋养体、包囊体和掠食体阶段的多子小瓜虫,其中15μg/mL的HBβ-C能在3min内杀死所有滋养体。基因表达量分析显示,在杂交黄颡鱼的鳃和皮肤组织中, HBβ的mRNA表达量高于全雄黄颡鱼;但在应对小瓜虫感染的过程中,全雄黄颡鱼的HBβmRNA转录水平快速提升,其表达水平和上升倍率显著高于杂交黄颡鱼。蛋白表达量分析显示,HBβ在全雄黄颡鱼鳃组织中的蛋白表达量明显高于杂交黄颡鱼。免疫荧光定位结果显示,抗菌肽HBβ-C特异地在红细胞中表达,可以分泌并附着在滋养体上。综上所述,相对于杂交黄颡鱼,全雄黄颡鱼中H...     

多子小瓜虫的形态发生及其寄生导致翘嘴鳃组织病理变化

为阐明小瓜虫的形态发生及其对鳃组织的危害过程,本文开展了多子小瓜虫的光镜观察和翘嘴鲌鳃组织病理切片方面的研究。光镜形态研究发现小瓜虫运动时呈球形,直径500-800 m;前端具有耳廓状胞口和内陷的袋状胞咽;环境温度为18℃时,滋养体经1h左右发育成包囊,体表形成一层透明胶膜,并以虫体中央的分裂沟为界进行二分裂;子代虫体从8细胞期进入16细胞期时,包囊内部形成内胞膜将子代虫体等分为两个集团。组织切片结果显示虫体呈卵圆形,挤压了周围的包裹细胞,导致组织变形;鳃小片黏连,鳃丝上皮细胞受到破坏,严重区域鳃丝结构被完全破坏。本研究解析了小瓜虫的形态发生与发育过程以及对宿主危害的组织病理结构与机制,为揭示小瓜虫的侵染、发育和成熟规律奠定了理论基础,并为小瓜虫病害的有效防控提供理论资料。     

Ichthyophthirius multifiliis Has Membrane-Associated Immobilization Antigens

Sera from fish that survive infections with the ciliated protozoon, Ichthyophthirius multifiliis , immobilize the parasite in vitro. In order to identify ceil surface antigens involved in the immobilization response, integral membrane proteins were extracted from tomites with Triton X-l14 and used to immunize rabbits. The rabbit antisera immobilized the parasite in vitro and antigens were localized to cell and ciliary plasma membranes by indirect immunofluorescent microscopy. The membrane protein fractions from both whole cells and tomite cilia were characterized by 1 - and 2-dimensiona! SDS-PAGE. A 43,000-dalton (D) glycoprotein with an isoelectric point of 7.0 is the predominant protein in these fractions, comprising 12% and 60% of the total protein of whole cell and ciliary membranes, respectively. Western blot analysis of ciliary proteins with immune rabbit sera indicated that the 43,000-D glycoprotein is the principal antigen.     

The I-antigens of Ichthyophthirius multifiliis are GPI-Anchored Proteins

The parasitic ciliate Ichthyophthirius multifiliis has abundant surface membrane proteins (i-antigens) that when clustered, trigger rapid, premature exit from the host. Similar antigens are present in free-living ciliates and are GPI-anchored in both Paramecium and Tetrahymena. Although transmembrane signalling through GPI-anchored proteins has been well-documented in metazoan cells, comparable phenomena have yet to be described in protists. Since premature exit of Ichthyophthirius is likely to involve a transmembrane signalling event, we sought to determine whether i-antigens are GPI-anchored in these cells as well. Based on their solubility properties in Triton X-114, the i-antigens of Ichthyophthirius are amphiphilic in nature and partition with the detergent phase. Nevertheless, following treatment of detergent lysates with phospholipase C, the same proteins become hydrophilic. Concomitantly, they are recognized by antibodies against a cross-reacting determinant exposed on virtually all GPI-anchored proteins following cleavage with phospholipase C. Finally, when expressed in recombinant form in Tetrahymena thermophila, full-length i-antigens are restricted to the membrane, while those lacking hydrophobic C-termini are secreted from the cell. Taken together, these observations argue strongly that the i-antigens of Ichthyophthirius multifiliis are, in fact, GPI-anchored proteins.     

Differences in virulence between two serotypes of Ichthyophthirius multifiliis

Naive channel catfish Ictalurus punctatus were infected by 2 isolates of the parasitic ciliate Ichthyophthirius multifiliis that differed in virulence. The isolates, NY1 and G5, Serotypes A and D, respectively, express different surface immobilization-antigens. The virulence of the 2 isolates was compared using tail-fin infections to quantitate parasite numbers and by analysis of the survival of infected fish. Although NY1 infected fish at a lower level than G5, all NY1-infected fish died, but 51% of G5-infected fish survived. The greater virulence of NY1 is apparently a consequence of its shorter life cycle, which results in overwhelming reinfection of fish before they can develop a protective immune response. This report represents the first experimental evidence for differences in virulence between serotypes of I. multifiliis.     

Endosymbiotic Bacteria in the Parasitic Ciliate Ichthyophthirius multifiliis

Endosymbiotic bacteria were identified in the parasitic ciliate Ichthyophthirius multifiliis, a common pathogen of freshwater fish. PCR amplification of DNA prepared from two isolates of I. multifiliis, using primers that bind conserved sequences in bacterial 16S rRNA genes, generated an ∼1,460-bp DNA product, which was cloned and sequenced. Sequence analysis demonstrated that 16S rRNA gene sequences from three classes of bacteria were present in the PCR product. These included Alphaproteobacteria (Rickettsiales), Sphingobacteria, and Flavobacterium columnare. DAPI (4′,6-diamidino-2-phenylindole) staining showed endosymbionts dispersed throughout the cytoplasm of trophonts and, in most, but not all theronts. Endosymbionts were observed by transmission electron microscopy in the cytoplasm, surrounded by a prominent, electron-translucent halo characteristic of Rickettsia. Fluorescence in situ hybridization demonstrated that bacteria from the Rickettsiales and Sphingobacteriales classes are endosymbionts of I. multifiliis, found in the cytoplasm, but not in the macronucleus or micronucleus. In contrast, F. columnare was not detected by fluorescence in situ hybridization. It likely adheres to I. multifiliis through association with cilia. The role that endosymbiotic bacteria play in the life history of I. multifiliis is not known.The ciliate Ichthyophthirius multifiliis is an obligate parasite of freshwater fish that infects epithelia of the skin and gills. The life cycle of I. multifiliis consists of three stages: an infective theront, a parasitic trophont, and a reproductive tomont. Infection is initiated by invasion of the skin and gills by free-swimming, 40-μm-long, pyriform-shaped theronts that burrow several cell layers deep into epithelial tissue of the skin and gills and rapidly differentiate into trophonts. Trophonts feed on epithelial cells and grow into 500- to 800-μm-diameter cells, causing extensive damage to skin and gills, which in severe infections results in mortality (10-12). After feeding for 5 to 7 days, trophonts leave the host, form encysted tomonts, and undergo up to 10 cell divisions over 18 to 24 h, producing as many as 10³ daughter cells, which exit the cyst as infective theronts to reinitiate the life cycle. I. multifiliis is ciliated at all stages (9).DNA sequencing of the I. multifiliis genome at the J. Craig Venter Institute unexpectedly revealed that bacterial DNA sequences, including sequences with homology to Rickettsia, were present in the DNA preparations (R. S. Coyne, 2009 [http://www.jcvi.org/cms/research/projects/ich/overview]). The origin of these sequences was unclear, but they represented evidence for either horizontal gene transfer into the I. multifiliis genome (17, 27) or the presence of intracellular bacteria. No previous evidence suggested the presence of intracellular bacteria in I. multifiliis, even though the fine structure of I. multifiliis theronts and trophonts has been examined by transmission electron microscopy (10-12). Intracellular or endosymbiotic bacteria, however, are commonly found in protists, and about 200 ciliate species are known to harbor intracellular bacteria (13, 15). Sonneborn and Preer in their classic studies on endosymbionts in Paramecium characterized a number of different endosymbionts, including “killers,” named for their ability to kill uninfected strains of Paramecium. Cytoplasmic endosymbionts in Paramecium now include Caedibacter taeniospiralis (Gammaproteobacteria), and Pseudocaedibacter conjugates, Tectibacter vulgaris, and Lyticum flagellatum (Alphaproteobacteria). Macronuclear endosymbionts include the Alphaproteobacteria, Holospora caryophila, and Caedibacter caryophila, which can also infect the cytoplasm (4, 16, 22, 26). The roles these endosymbionts play in protists are not well understood.The presence of sequences with homology to bacterial genomes prompted us to determine if I. multifiliis contained endosymbionts, or if these sequences represented evidence for horizontal gene transfer into the I. multifiliis genome. Our identification of the same two endosymbionts, in two different isolates of I. multifiliis, suggests that endosymbionts are common in I. multifiliis. However, the physiological relationships between I. multifiliis and its resident endosymbionts are unclear. It is not known if the endosymbionts contribute to the growth of I. multifiliis, if they contribute to the severity or pathogenicity of infection, or if they provide their host with any selective advantage, as occurs with Paramecium containing killer particles (4). It has not been determined if they influence the immune response of fish infected with I. multifiliis. It is possible that they may simply be parasites of this parasitic ciliate.     

Reproduction of Ichthyophthirius multifiliis (Ciliata, Ophryoglenidae)

A case of reproduction of Ichthyophthirius multifiliis in the superficial tissues of larvae and fry of Amur wild carp is described. At a temperature of water from 28 to 29 degrees trophonts of Ichthyophthirius encysted on fishes. Inside cysts repeated cell division occurred but this process did not result in swarm spores formation. Later on with the increase of temperature to 29.5--31.5 degrees cysts degenerated.     

Purification and Partial Characterization of Immobilization Antigens from Ichthyophthirius multifiliis

ABSTRACT Ichthyophthirius multifiliis , a parasitic ciliate of freshwater fishes, was found to have surface antigens (Ag) which elieited immobilizing antibodies (Ab) when injected into rabbits. An effort was made to purify and characterize these Ag (referred to as immobilization Ag) because of their potential role in protective immunity in fishes. Mice immunized with theront cilia were used for production of immobilizing monoclonal antibodies (MAb). Hybridomas were screened by indirect immunofluorescent light microscopy and immobilization of live parasites. Six hybridomas producing immobilizing MAb were cloned. Immobilizing MAb were used to affinity purify Ag solubilized with Triton X-114 and Na deoxycholate. Two membrane protein Ag of approximately 48 and 60 kDa were identified. Immobilizing MAb failed to react with these Ag on Western blots and, conversely, MAb that reacted with the Ag on Western blots did not immobilize live organisms. These results suggest that immobilization required native conformational epitopes which were altered by Western blotting procedures. Individual MAb reactive on Western blots recognized both the 48- and 60-kDa proteins indicating the presence of common epitopes. Affinity purified Ag elicited immobilizing antisera when injected into rabbits, mice, and channel catfish.     

Ichthyophthirius multifiliis (Ciliophora) exit from gill epithelium

The first change in the sequence of morphological events occurring as fully developed Ichthyophthirius multifiliis trophonts spontaneously left gill epithelium or as younger trophonts departed, following experimentally induced death of the fish, was the separation of parasites from overlying host cells. Discharge of contractile vacuoles may have played a role in this process. Spaces then appeared between host cells, and individual epithelial cells became vacuolated. Finally, the epithelium ruptured and the parasites swam free. In induced exit after three days residence in the host, departure of the trophont was evident only after autolysis of epithelium had occurred. Induced departure of trophonts after four days residence was more rapid, suggesting an active role for the parasite in exit. Changes in parasite and epithelium observed in induced exit were similar to those in spontaneous departure after five days residence.     

Ichthyophthirius multifiliis (Ciliophora) Invasion of Gill Epithelium1

The sequence of morphologic events associated with Ichthyophthirius rnultifliis invasion of gill epithelium began in the theront with differentiation of secretory mucocysts and the perforatorium. After escaping from the cyst the theront, which stained intensely with Mallory' stain, sought a host. As it approached the host epithelium, the contents of the mucocysts were discharged, enveloping the ciliate in sticky material, which made initial contact with the host epithelium. Rapid penetration by the theront caused disruption of one or more host cells and resulted in a focal necrosis associated with the anterior margin of the ciliate. Within five minutes postexposure, the parasite completed its invasion of the epithelial layer and stained less intensely. The remnants of host cells disrupted during its entry surrounded the trophont until they were ingested by the parasite. Within 40 min postexposure, synthetic activity of the parasite appeared to increase as evidenced by an abundance of organelles, particularly ribosomes and crystalline mucocysts. At this point, the overlying host epithelium appeared normal.