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.     

Immunisation of channel catfish,Ictalurus punctatus,with Ichthyophthirius multifiliis immobilisation antigens elicits serotype-specific protection

Surface immobilisation antigens (i-antigens) were purified from two strains of Ichthyophthirius multifiliis (NY1 and G5) that represent different i-antigen serotypes, namely A and D, respectively. The efficacy of the purified antigens as subunit vaccines was then tested in challenge studies using parasites of the homologous or heterologous serotype. Three groups of juvenile channel catfish (70 animals per group) were immunised with i-antigens from either the G5 or NY1 isolates, or with bovine serum albumin (BSA) as a control. Proteins were injected intraperitoneally (i.p.) at a dose of 10 microg/fish with complete Freund's adjuvant on day 1, followed by a second injection in incomplete Freund's adjuvant on day 15. Fish immunised with the purified i-antigens developed high titres of serum immobilising antibodies whereas sera from BSA-injected control fish did not. Fish antisera immobilised parasites of the homologous, but not the heterologous strain, and recognised the corresponding i-antigens on Western blots run under non-reducing conditions. On day 36, each group was divided into two subgroups (n=30). One subgroup was challenged with G5 parasites, and the other was challenged with NY1 parasites. When challenged with G5 parasites, 70% of fish immunised with the G5 i-antigens survived. When challenged with NY1 parasites, 33.3% of fish immunised with the NY1 i-antigens survived. All BSA-injected control fish died, as did all fish injected with the purified antigens and challenged with the non-homologous parasite strain. Statistical analyses indicated significant differences among test and control groups with regard to the mean days to death (MDD). While the results of these studies clearly support a role for i-antigens in protection, active immunity in response to natural infection is not serotype-specific. The utility of i-antigens, as well as the existence of other potential vaccine candidates for the prevention of 'white-spot' disease, are discussed.     

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.     

Heterosis in resistance to Ichthyophthirius multifiliis infections in poeciliid fish

Resistance to experimental infections of Ichthyophthirius multifiliis was investigated in the poeciliid fish Xiphophorus maculatus, X. variatus and their two reciprocal hybrids. A third species, the goodeid Ameca splendens , was infected for comparative purposes. Infection data were analysed using a generalized linear interactive modelling package (GLIM). Infection levels were not significantly influenced by the sex of the fish, temperature, or speciestemperature interactions, but body area and time of infection did affect susceptibility. After accounting for variation due to these factors, it was shown that the two reciprocal hybrids of X. maculatus and X. variatus had significantly lower infection levels than either parental stock. Heterosis, H , was calculated from both raw data (16.2%) and from modelled values (31.3%) after removal of environmental factors. This confirms the existence of genetic factors determining resistance to /. multifiliis infection and has shown that gains in resistance can be made through hybridization.     

Chemoattraction of Ichthyophthirius multifiliis (Ciliophora) theronts to host molecules

Mechanisms in the host-finding process of Ichthyophthirius multifiliis were studied in vitro by a novel bioassay using 24-well multidishes supplied with bottom layers of agar with chemoattractants. It was shown that low molecular weight molecules (carbohydrates, amino acids, fatty acids, urea) did not attract theronts. In contrast, sera and mucus from a range of teleosts (including marine fish) were effective attractants. Fractionation by gel filtration of fish serum allowed determination of the molecular size of the attracting proteins. Further biochemical studies suggested the chemoattractants to be present in fractions with host immunoglobulin and some still undetermined proteins. No clear association between enzyme activity and chemotactic potential was seen. The high chemoattractive effect of serum from various unrelated teleosts corresponds to the low host specificity of I. multifiliis and suggests that serum factors in mucus could be involved in host finding of the parasite. Society for Parasitology Inc.     

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.     

Differences in initial and acquired resistance to Ichthyophthirius multifiliis between populations of rainbowfish

Wild-caught rainbowfish Melanotaenia spp. originating from three isolated populations were infected with a quantified dosage of parasites Ichthyophthirius multifiliis in a controlled environment. The Melanotaenia eachamensis from Dirran Creek were much more susceptible to ichthyophthiriasis than were M. splendida from the Lake Tinaroo or Bluewater Creek populations. When the highly susceptible Dirran Creek rainbowfish were crossed with rainbowfish from a fourth population, Lake Eacham M. eachamensis , they produced hybrids with significantly higher resistance than pure-bred Dirran Creek, but not higher than pure-bred Lake Eacham fish. Hence, intraspecific hybridization increased resistance to I. multifiliis infection in M. eachamensis. Hosts from all three populations were much less susceptible to infection on their second exposure to the parasite. However, the Bluewater Creek population was better able to acquire immunity to I. multifiliis than either the Dirran Creek or Lake Tinaroo populations. It is tentatively suggested that there may be a link between the heterozygosity of populations of rainbowfish and their initial ability to resist infection by Ichthyophthirius multifiliis.     

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。 …     

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,     

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.     

蛇(鱼句)感染多子小瓜虫病一例

2003年6月18日从长江重庆木洞段购回十几条活泼蛇(鱼句)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(鱼句)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Ichthyophthirius multifiliis Fouquet.感染小瓜虫病的蛇(鱼句)7月6日死亡.蛇(鱼句)是否能大面积发病还有待进一步研究.     

In vitro response of Ichthyophthirius multifiliis to sera from immune channel catfish

Sera from channel catfish rendered immune to the protozoan pathogen Ichthyophthirius multifiliis were screened for activity against live parasites. Cells in the infective stage (tomites) were incubated in doubling dilutions of immune and pre-immune sera from fish that had been immunized by exposure to sublethal infections. When examined by light microscopy, tomites were found to agglutinate in the presence of immune sera. While the stength of individual sera varied, agglutination of cells occurred at dilutions as high as 1:128. Cells showed little tendency to agglutinate in pre-immune sera, and virtually no effects were seen with dilutions of pre-immune sera greater than 1:16. Agglutination was usually accompanied by release of mucus from cells, and while tomites appeared to be immobilized, their cilia continued to beat. Low dilutions of immune sera appeared to be toxic. Similar effects on tomites were seen with rabbit antisera prepared against Ichthyophthirius cilia. The involvement of humoral antibodies in agglutination and protective immunity is discussed.     

Analysis of the Soluble and Membrane-bound Immobilization Antigens of Ichthyophthirius multifiliis

ABSTRACT. The immobilization antigens (i-antigens) are a class of highly abundant surface membrane proteins found on a number of holotrich ciliates. In Ichthyophthirius multifiliis (an obligate parasite of fish) these antigens appear to be targets of the host immune response. While the i-antigens of Ichthyophthirius are predominantly membrane-associated proteins, we now find that they are released into the water surrounding the parasite in a highly enriched form. The membrane-associated and water soluble proteins appear indistinguishable by antigenic means, as well as by several biochemical criteria including peptide mapping, mobility in reducing and non-reducing SDS-polyacrylamide gels, and relative glycosylation. Antibodies raised against the membrane-associated antigens react with the water soluble proteins on Western blots. Not surprisingly, immunocytochemical localization studies show binding of these antibodies to surface membranes of the cell. In addition, however, antibody binding is also detectible on the membranes of a secretory organelle (that is, mucocysts) present in the cortical cytoplasm. The significance of these findings with regard to the potential role of the i-antigens in infection and immunity is discussed.     

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.     

Ultrastructural and Cytochemical Identification of Peroxisomes in the Ciliate Ichthyophthirius multifiliis

ABSTRACT. The peroxisomes of the parasitic ciliate Ichthyophthirius multifiliis were studied, using ultrastructural and cytochemical techniques. In this ciliate most peroxisomes possess a circular or oval section less than 0.6 μm in diameter. However, some dumbbell-shaped and elongated peroxisomes could also be observed. These organelles were frequently associated with the mitochondria and were more abundant in the cell cortex than in the center of the ciliate. Small vesicles and dense nucleoids were usually present in the ultrathin sections of these peroxisomes. Peroxisomal vesicles and tubular structures were selectively impregnated with osmium tetroxide. Catalase was detected by cytochemical techniques in I. multifiliis peroxisomes.     

Purification and partial characterization of immobilization antigens from Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis, a parasitic ciliate of freshwater fishes, was found to have surface antigens (Ag) which elicited 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.     

Serotypic Variation Among Isolates of Ichthyophthirius multifiliis Based on Immobilization

Efforts have been made to determine whether surface antigens could be used as biochemical markers to define strain differences in the parasitic ciliate Ichthyophthirius multifiliis. In previous studies, a wild-type isolate designated G1 was found to have surface proteins analogous to the immobilization antigens of Paramecium and Tetrahymena; rabbit antiserum against this strain immobilizes homologous cells in vitro. It has now been shown for two additional Ichthyophthirius isolates (designated G1.1 and G2) that immobilization antigens are both present and serologically distinct. Proteins of similar size, which cross-react in Western blots with rabbit antisera against immobilization antigens of the G1 strain, are nevertheless found in the G1.1 and G2 isolates. As shown by Southern blotting analysis, the G1.1 and G2 strains also contain genomic DNA sequences which hybridize with an immobilization antigen cDNA from G1 when probed under conditions of reduced stringency. The serotypic differences in immobilization between I. multifiliis isolates appear to be stable over time and provide a means of discriminating strains. In addition to providing a basis for comparative studies, the work described here has implications for the development of vaccines against this important fish parasite.     

Comparing tolerance of Ichthyophthirius multifiliis and Tetrahymena thermophila for new cryopreservation methods

Ichthyophthirius multifiliis is an obligate protozoan parasite of freshwater fishes that has a complex developmental cycle. It has not been successfully cryopreserved, so management studies are restricted to parasites obtained during outbreaks or perpetuated by passage in live fishes. To overcome this serious limitation, free-swimming I. multifiliis parasites were tested in a cryopreservation protocol routinely used for a related ciliate, Tetrahymena. In this protocol, I. multifiliis theronts retained infectivity for 3 days, although the protocol itself was ultimately lethal. Exposure of I. multifiliis and Tetrahymena thermophila to a battery of media and cryopreservative reagents showed that I. multifiliis was less hardy than T. thermophila and likely had significant biological and cytoskeletal differences. No combination of reagents, media, freezing rates, or dilution media permitted cryopreservation of I. multifiliis parasites that could then undergo development or infect fish. However, a vitrification protocol was formulated using Ficoll, 1,2-propanediol, and N,N-dimethylacetamide from which intact cryopreserved theronts with some motility were recovered. Understanding the effects of these reagents may lead to both a cryopreservation method for I. multifiliis and to improved understanding of the biology of ciliates.