The development of Cryptocaryon irritans in a less susceptible host rabbitfish, Siganus oramin.

Cryptocaryon irritans is a ciliated protozoan parasite infecting the gills and skin of almost all marine teleosts, resulting in severe disease. Notably, C. irritans is unable to cause significant pathogenesis in rabbitfish, Siganus oramin, a well-known less susceptible host. However, little is known about the development of C. irritans in rabbitfish. Thus, in the present study, rabbitfish were artificially infected with C. irritans at a 1/2 maximal tolerance dose (MTD), using the susceptible host, large yellow croaker, Larimichthys crocea, as a control. The daily food consumption (DFC), survival, and relative infection intensity (RII) of the fish were observed, and the relative number of tomonts (RNT), reproductive rate, and characteristics of C. irritans tomonts were measured. The lethal concentration 50 (LC50) of C. irritans on rabbitfish and large yellow croaker was 2236, and 264 theronts/g fish, respectively. The DFC of rabbitfish decreased by 14.6% at day 1 post-C. irritans infection (PI), but immediately returned to normal DFC levels by day 2 PI, and was not affected by secondary infection. However, large yellow croaker stopped feeding at day 3 PI, and all fish died following secondary infection with C. irritans. The RII of rabbitfish exhibited a significant downward trend at 6 h PI, and the reproductive rate of C. irritans was 8%; in contrast, the RII of large yellow croaker was not significantly different from 0 to 72 h PI, and the reproductive rate of C. irritans reached to 59.05-fold. Over the same time frame, the diameter of tomonts from rabbitfish was smaller compared with those from large yellow croaker. These results indicated that the rabbitfish were somehow able to limit the development of C. irritans, such that most trophonts left the host during early developmental stages, whereas those that survive could only develop into small tomonts.

Evidence of multiple species of Chilodonella (Protozoa, Ciliophora) infecting Australian farmed freshwater fishes.

Parasitic Chilodonella species, Chilodonella piscicola and Chilodonella hexasticha, cause considerable economic losses globally to freshwater farmed fish production. Some genetic studies of Chilodonella spp. have indicated that many species within the genus may form cryptic species complexes. To understand the diversity of Chilodonella spp. infecting Australian freshwater farmed fish, specimens were isolated from infected barramundi (Lates calcarifer) and Murray cod (Maccullochella peelii) from fish farms in tropical north Queensland (QLD), temperate Victoria (Vic) and New South Wales (NSW) for genetic and morphological analysis. Parasites were stained and measured for morphological description and comparative phylogenetic analyses were performed using the mitochondrial small subunit (mtSSU) rDNA marker. Morphological analyses revealed four distinct morphotypes of Chilodonella infecting farmed barramundi and Murray Cod. Three putative species were isolated from barramundi (Chilodonella hexasticha, C. acuta and C. uncinata) and one from Murray cod (C. piscicola). However, phylogenetic analyses detected only three distinct genotypes, with the putative C. hexasticha and C. piscicola sharing 100% sequence identity. This suggests that Australian isolates of C. hexasticha and C. piscicola could represent the same species and may exhibit phenotypic plasticity. Further molecular analysis, including isolates from the type localities, should be performed to support or refute the synonymy of these species.

Effect of potassium ferrate(VI) on survival and reproduction of Ichthyophthirius multifiliis tomonts.

Ichthyophthirius multifiliis is an important freshwater teleost pathogen that can infect most species of freshwater fish worldwide and often leads to significant economic losses to the aquaculture industry. Potassium ferrate(VI), as a potential therapeutic agent for external protozoan parasite infections, has been proven to kill I. multifiliis theronts effectively; however, no information is available on effects of potassium ferrate(VI) exposure to I. multifiliis tomonts. This study evaluated the effects of potassium ferrate(VI) on the survival and reproduction of I. multifiliis tomonts. The results of experiment 1 showed that potassium ferrate(VI) at concentrations of 2.4, 4.8, 9.6, and 19.2 mg/L resulted in tomont survival rates of 82.2%, 34.2%, 14.6%, and 0, respectively, and significant differences were noted in tomont reproduction between the treatments and the control (P < 0.05). Additionally, this study was designed to determine the effect of potassium ferrate(VI) toxicity on age of the tomont. The results indicated that encysted I. multifiliis was more resistant to potassium ferrate(VI) treatments. In addition, this study was designed to investigate I. multifiliis tomont survival and reproduction when collected from infested goldfish in potassium ferrate(VI) solutions at different concentrations (0, 2.4, 4.8, 9.6, and 19.2 mg/L) for 30 min. It was found that the tomont survivals in the treatments were significantly lower than those in the control (P < 0.05). This study demonstrated that a bath with potassium ferrate(VI) possibly was an effective method to treat ichthyophthiriasis.

Transcriptional profiling of stage specific gene expression in the parasitic ciliate Ichthyophthirius multifiliis.

The parasitic ciliate, Ichthyophthirius multifiliis (Ich), is among the most important protozoan pathogens of freshwater fish. Ichthyophthirius cannot be grown in cell culture, and the development of effective prophylactic and therapeutic treatments has been hampered by a lack of information regarding genes involved in virulence, differentiation and growth. To help address this issue, we have generated EST libraries from the two major stages of the parasite life cycle that infect and develop within host tissues. A total of 25,084 ESTs were generated from non-normalized libraries prepared from polyA+ RNA of infective theronts and host-associated trophonts, respectively. Cluster analysis identified 5311 unique transcripts (UniScripts), of which 2091 were contigs and 3220 singletons. Extrapolation of the data based on rates of EST discovery suggests that more than half the expected protein-coding genes of I. multifiliis are represented in this data. BLASTX comparisons against GenBank nr, UniProtKB (SwissProt and TrEMBL), as well as Tetrahymena thermophila, Plasmodium falciparum, and Paramecium tetraurelia protein databases produced 3694 significant (E-value ≤1e(-10)) hits, of which 1178 were annotated using gene ontology (GO) analysis. A high proportion of UniScripts (63%) showed similarity to other ciliate proteins. When combined with expression profiling data, GO ontology analysis of Biological Process, Cellular Component, and Molecular Function revealed interesting differences in gene families expressed in the two stages. Indeed, the most abundant transcripts were highly stage-specific and coincided with the metabolic activities associated with each stage. This work provides an effective genomics resource to further our understanding of Ichthyophthirius biology, and lays the groundwork for the identification of potential drug targets and vaccines candidates for the control of this devastating fish pathogen.

Effect of sanguinarine from the leaves of Macleaya cordata against Ichthyophthirius multifiliis in grass carp (Ctenopharyngodon idella).

The ciliate Ichthyophthirius multifiliis is one of the most pathogenic parasites of fish maintained in captivity. In this study, effects of crude extracts, fractions, and compounds from the leaves of Macleaya cordata against I. multifiliis were investigated under in vitro conditions by bioactivity-guided isolation method. The dried ethanol extract of M. cordata was extracted successively in a separating funnel with petroleum ether, ethyl acetate, chloroform and n-butanol. Among them, only the chloroform extract showed promising activity and therefore, was subjected to further separation and purification using various chromatographic techniques. Four compounds were isolated from chloroform extract, but only one compound showed potent activity. The structure of the active compound was elucidated as sanguinarine by hydrogen, carbon-13 nuclear magnetic resonance spectrum and electron ionization mass spectrometry. In vitro antiparasitic efficacy tests exhibited that sanguinarine was 100% effective against I. multifiliis at a concentration of 0.7 mg l(-1), with LC(50) and LC(90) values of 0.437 and 0.853 mg l(-1) after 4 h of exposure. In vivo antiparasitic efficacy tests showed that the number of I. multifiliis on the gills in the treatment group (in 0.9 mg l(-1) sanguinarine) was reduced by 96.8%, in comparison to untreated group at 25°C for 48 h. Mortality of fish did not occur in the treatment group during the trail, although 40% of untreated fish died. Our results indicate that the studied plant extracts, as well as sanguinarine might be potential sources of new antiparasitic drug for the control of I. multifiliis.

Redescription and neotypification of a poorly known freshwater ectocommensal ciliate, Larvulina variabilis (Penard, 122), and consideration of its systematic position.

The morphology and infraciliature of Larvulina variabilis collected from freshwater Gammarus near Boise, Idaho (USA) were investigated using live observation and silver impregnation. Larvulina variabilis was collected from three sites in Idaho and one site in Michigan, indicating a wide distribution. Larvulina variabilis is a small (21-25 x 14-19microm in vivo) hymenostome ectocommensal on freshwater amphipods. Distinguishing morphologic features include an inflexible cortex with a prominent notch in the left posterior margin, a single globular macronucleus, a single posterior contractile vacuole, a somatic ciliature with 10 kineties all consisting of ciliated dikinetids, a "6"-shaped paroral membrane of the stichodyad type, long (approximately 20microm), immobile cilia arising from three adoral membranelles, a discrete dorsal thigmotactic ciliary field consisting of five kineties, distinctive extrusomes and a coarsely polygonal silverline pattern. The forward-projecting anterior cilium emphasized in the original description is an inconstant finding not essential for identification. An improved diagnosis is provided for the genus and the species. Because no type specimen exists and prior descriptions were conflicting, a specimen from the Idaho population is designated as the neotype. The systematic position of this unusual ciliate remains obscure. Possibly, it belongs to the Pleuronematida or the Thigmotrichida.

Acute toxicity of peracetic acid (PAA) formulations to Ichthyophthirius multifiliis theronts.

Peracetic acid (PAA) is an antimicrobial disinfectant used in agriculture, food processing, and medical facilities. It has recently been suggested as a means to control infestations of Ichthyophthirius multifiliis. The purpose of this study was to determine the acute toxicity of two products containing 4.5% and 40% PAA to I. multifiliis theronts from two geographically separate isolates. Theronts were exposed to concentrations of PAA in 96-well plates containing groundwater at 23 degrees C. Acute toxicity was observed over a 4-h period. No significant difference in the median lethal concentration (LC(50)) estimates was evident between the two isolates at 4 h with the 4.5% PAA product (0.146 versus 0.108 mg/l PAA), while there was a statistical difference between the 4 h LC(50) with the 40% PAA product (0.274 versus 0.158 mg/l PAA). These results suggest that PAA is toxic to I. multifiliis theronts at low concentrations and that one of the isolates was more resistant to this compound.

Ichthyophthirius multifiliis Fouquet and Ichthyophthiriosis in Freshwater Teleosts.

The ciliate Ichthyophthirius multifiliis is an important pathogen of freshwater teleosts occurring in both temperate and tropical regions throughout the world. The disease, ichthyophthiriosis, accounts for significant economic losses to the aquaculture industry, including the ornamental fish trade, and epizootics in wild fish populations can result in mass kills. This review attempts to provide a comprehensive overview of the biology of the parasite, covering the free-living and parasitic stages in the life cycle, host-parasite interactions, and the immune response of host and immune evasion strategies by the parasite. Emphasis on the immunological aspects of infection within the fish host, including molecular studies of i-antigens, reflects the current interest in this subject area and the quest to develop a recombinant vaccine against the disease. The current status of methods for the control of ichthyophthiriosis is discussed, together with new approaches in combating this important disease.

Partial cross protection against Ichthyophthirius multifiliis in Gyrodactylus derjavini immunized rainbow trout.

Partial cross protection against a skin-parasitic ciliate has been recorded in rainbow trout previously immunized with an ectoparasitic platyhelminth. The susceptibility to infection by Ichthyophthirius multifiliis differed significantly between naive and Gyrodactylus derjavini immunized rainbow trout. Fish partly immune to the ectoparasitic monogenean G. derjavini became less infected and experienced lower mortality than naive fish when exposed to I. multifiliis infections. In vitro studies on immobilization of theronts using decomplemented (heat-inactivated) serum from G. derjavini immune or non-immune hosts showed no immobilization. However, untreated serum from both immune and non-immune fish containing intact complement immobilized theronts (titre 128-256). In addition, non-specific priming of the host response with interleukin (IL-1), bacterial lipopolysaccharide (LPS), concanavalin A (Con A) or mannan did confer a partial resistance to I. multifiliis infection. This will suggest that non-specific factors including complement could be partly responsible for the host response against infections with this ciliate.

Surface antigen cross-linking triggers forced exit of a protozoan parasite from its host.

We used the common fish pathogen Ichthyophthirius multifiliis as a model for studying interactions between parasitic ciliates and their vertebrate hosts. Although highly pathogenic, Ichthyophthirius can elicit a strong protective immune response in fish after exposure to controlled infections. To investigate the mechanisms underlying host resistance, a series of passive immunization experiments were carried out using mouse monoclonal antibodies against a class of surface membrane proteins, known as immobilization antigens (or i-antigens), thought to play a role in the protective response. Such antibodies bind to cilia and immobilize I. multifiliis in vitro. Surprisingly, we found that passive antibody transfer in vivo caused rapid exit of parasites from the host. The effect was highly specific for a given I. multifiliis serotype. F(ab)2 subfragments had the same effect as intact antibody, whereas monovalent Fab fragments failed to protect. The activity of Fab could, nevertheless, be restored after subsequent i.p. injection of bivalent goat anti-mouse IgG. Parasites that exit the host had detectable antibody on their surface and appeared viable in all respects. These findings represent a novel instance among protists in which protective immunity (and evasion of the host response) result from an effect of antibody on parasite behavior.

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.

[The population level and distribution of ectoparasites in carp]. / Uroven' chislennosti i raspredelenie éktoparazitov u karpa.

The abundance and the distribution of Dactylogyrus extensus and Ichthyophthirius multifiliis on gills of two-years old carps have been investigated. It has been established that at least there are five groups, which differs from each other on the asymmetry of infection.