Ciliate Cryptocaryon Irritans Research Articles

Tandem mass tag (TMT) quantitative proteomics and phosphoproteomic of Takifugu rubripes infected with Cryptocaryon irritans

In this study, we identified the differentially expressed proteins in gills stimulated by infected ciliates and analyzed the immune mechanisms of T. rubripes infected with the ciliate Cryptocaryon irritans. Through liquid chromatography analysis, a total of 144 proteins were identified with significant differences, of which 58 were upregulated and 86 were downregulated. Among phosphorylated proteins, we identified a total of 167 significantly different phosphorylated proteins, of which 44 were upregulated, 123 were downregulated, 60 were upregulated, and 208 were downregulated. We analyzed the data of proteomics and Phosphorylated proteome quantification protein omics to finally identify three phosphorylated proteins (RPS27, eNOS and CaM) and two phosphorylated protein kinases(CaMKII and MAPK1) as potential biomarkers for T. rubripes immune responses. We finally identified three phosphorylated proteins (RPS27, eNOS and CaM) and two phosphorylated protein kinases (CaMKII and MAPK1) as potential biomarkers of immune response of T. rubripes. Our research findings provide new insights into the immune mechanism of T. rubripes, which may serve as an effective indicator of C. irritans infection in T. rubripes.

Vibrio harveyi co-infected with Cryptocaryon irritans to orange-spotted groupers Epinephelus coioides.

The orange-spotted grouper (Epinephelus coioides) is a high economic value aquacultural fish in China, however, it often suffers from the outbreak of parasitic ciliate Cryptocaryon irritans as well as bacterium Vibrio harveyi which bring great loss in grouper farming. In the present study, we established a high dose C. irritans local-infected model which caused the mortality of groupers which showed low vitality and histopathological analysis demonstrated inflammatory response and degeneration in infected skin, gill and liver. In addition, gene expression of inflammatory cytokines was detected to assist the estimate of inflammatory response. Furthermore, we also found that the activity of Na+/K+ ATPase in gill was decreased in groupers infected C. irritans and the concentration of Na+/Cl- in blood were varied. Base on the morbidity symptom occurring in noninfected organs, we hypothesized that the result of morbidity and mortality were due to secondary bacterial infection post parasitism of C. irritans. Moreover, four strains of bacteria were isolated from the infected site skin and liver of local-infected groupers which were identified as V. harveyi in accordance of phenotypic traits, biochemical characterization and molecular analysis of 16S rDNA genes, housekeeping genes (gyrB and cpn60) and species-specific gene Vhhp2. Regression tests of injecting the isolated strain V. harveyi has showed high pathogenicity to groupers. In conclusion, these findings provide the evidence of coinfections with C. irritans and V. harveyi in orange-spotted grouper.

Immune response of large yellow croaker Larimichthys crocea towards a recombinant vaccine candidate targeting the parasitic ciliate Cryptocaryon irritans.

Cryptocaryon irritans, a parasitic ciliate, pose a major threat to marine teleost fish aquaculture. So far, no effective and safe control method is available. In this study, the protective efficacy of a recombinant truncated surface antigen of C. irritans (rCiSA32.6t) for large yellow croaker (Larimichthys crocea) against the parasite challenge with a sub-lethal dose of the infective theronts was evaluated by comparing the relative percent survivals (RPS), the specific antibody titers in sera, and the expression levels of the immune-related genes among the negative or adjuvant control fish, fish intraperitoneally immunized with rCiSA32.6t. The results showed that a RPS of 50.1% in rCiSA32.6t-immunized fish was achieved in comparison to negative control fish against C. irritans. A significant increase was noted in the antigen-specific immunoglobulin M (IgM) and immunoglobulin T (IgT) antibody levels in the sera of the rCiSA32.6t-vaccinated fish. Compared to the negative control fish, quantitative real-time PCR analysis indicated that the interleukin-1beta, IgT, and IgM heavy chain mRNA level in the fish head kidney, spleen, gill, and skin tissue were upregulated post-rCiSA32.6t immunization. This study indicates that the rCiSA32.6t can provide a high level of immune protection against C. irritans infection in grouper and is therefore pursued as a candidate C. irritans vaccine.

Host responses against the fish parasitizing ciliate Cryptocaryon irritans.

The parasitic ciliate Cryptocaryon irritans, which infects almost all marine fish species occurring in both tropical and subtropical regions throughout the world. The disease, cryptocaryonosis, accounts for significant economic losses to the aquaculture industry. This review attempts to provide a comprehensive overview of the biology of the parasite, host-parasite interactions and both specific and non-specific host defense mechanisms are responsible for the protection of fish against challenge infections with this ciliate. Also, this article reflects the current interest in this subject area and the quest to develop an available vaccine against the disease. Due to the high frequency of clinical fish cryptocaryonosis, the study of fish immune responses to C. irritans provides an optimal experimental model for understanding immunity against extracellular protozoa.

Molecular characterization of a profilin gene from a parasitic ciliate Cryptocaryon irritans

Profilin, known as one of the core actin-binding proteins, is an integral part of actin-based cytoskeleton involved in cell motility, cytokinesis, neuronal differentiation, and synaptic plasticity. In this study, a putative profilin gene designated as CiProfilin (GenBank accession number: JX987286) was screened out from a cDNA library of Cryptocaryon irritans trophonts. The full-length cDNA of CiProfilin gene is 582 bp, containing an open reading frame (ORF) of 471 bp, which encodes a polypeptide consisting of 156 amino acids with a predicted molecular weight of 17.3 kDa. Quantification of CiProfilin mRNA expression by real-time PCR suggested that CiProfilin was expressed in all stages of C. irritans life cycle with a significantly higher level in trophonts. Five non-universal codons (TAAs) coding glutamines (Gln) were found in the ORF and mutated to CAAs (universal codons for Gln) by site-directed mutagenesis. Then the modified ORF was inserted into the plasmid pGEX-4T-1, the recombinant plasmid was subsequently transformed into Escherichia coli. The bacteria were subsequently induced to express the recombinant CiProfilin protein fused with glutathione S transferase (G-rCiProfilin), which was then purified with glutathione sepharose 4B and thrombin cleavage. The molecular weight and the antigenicity of rCiProfilin were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. The native CiProfilin was found abundant in the peripheral area beneath the cell membrane and around the cytostomes of theronts, suggesting its vital roles in food uptake, stomatogenesis, and parasitic invasion. Co-precipitation assay also revealed the activity of rCiProfilin in actin binding. This study will help further elucidate the specific roles of CiProfilin on the growth of C. irritans and the preliminary mechanism of its invasion to hosts.

Genomic selection for parasitic ciliate Cryptocaryon irritans resistance in large yellow croaker

Large yellow croaker is an important marine aquaculture species in the southeast coast of China. It suffers serious diseases caused by Cryptocaryon irritans, a parasitic cilliate which causes huge economic losses to the aquaculture industry of large yellow croaker. This study aims to apply genomic selection (GS) technology to the selection breeding of large yellow croaker with resistance to C. irritans. The resistant phenotype was measured as a binary trait, survival or death, in challenge experiments using reference population. Double-digest restriction-associated DNA (ddRAD) sequencing approach was used to generate genome-wide single nucleotide polymorphism (SNP) genotypes for reference population and selection candidates. A database of 811 individuals and 16,515 SNPs was constructed and used for GS analysis. The GS model containing the top 600 SNP markers with the smallest P value possessed highest predictability and was used to calculate genetic estimated breeding value (GEBV) in selection candidates. The individuals with high GEBV in selection candidates was selected as broodstocks for the breeding of resistant strain (RS), and the remaining individuals for the control strain (CS). Significant resistance difference between RS and CS was detected in a C. irritans challenge test. After 96-h, the survival rate of RS and CS were 59.2% and 9.9%, respectively. Moreover, 120-h survival rate of RS declined to 40.8%, while CS had no surviving individuals. The results confirmed that GS was effective genetic breeding techniques in resistant breeding against C. irritans in large yellow croaker.

Transcriptomic analysis reveals the key immune-related signalling pathways of Sebastiscus marmoratus in response to infection with the parasitic ciliate Cryptocaryon irritans

BackgroundFalse kelpfish (Sebastiscus marmoratus) is one of the target species in artificial breeding in China, and is susceptible to infection by Cryptocaryon irritans, which is an obligate parasitic ciliate that lives in the epithelium of the fish gills, skin and fins. Here, we sought to understand the mechanisms of molecular immunity of S. marmoratus against C. irritans infection.MethodsWe carried out an extensive analysis of the transcriptome of S. marmoratus immune-related tissues. A paired-end library was constructed from the cDNA synthesized using a Genomic Sample Prep Kit. Five normalized cDNA libraries were constructed using RNA from the control group and the four groups of C. irritans-infected fish. The libraries were sequenced on an Illumina Mi-Seq platform, and functional annotation of the transcriptome was performed using bioinformatics software.ResultsThe data produced a total of 149,983,397 clean reads from five cDNA libraries constructed from S. marmoratus immune-related tissues. A total of 33,291 unigenes were assembled with an average length of 1768 bp. In eggNOG (Evolutionary Genealogy of Genes: non-supervised orthologous groups) categories, 333 unigenes (0.94%) were assigned to defense mechanisms. In the immune system process sub-categories of gene ontology (GO) enrichment analysis, with the passage of time post-infection, the number of differentially expressed genes (DEGs) was reduced from 24 h to 48 h but then increased from 72 h to 96 h. Specifically, the immune-related differentially expressed genes (IRDEGs), which belong to the KEGG (Kyoto encyclopedia of genes and genomes) pathways, such as the complement and coagulation cascades, chemokine signalling pathways and toll-like receptor signalling pathways were mainly observed at 24 h post-infection.ConclusionsInfection with C. irritans resulted in a large number of DEGs in the immune-related tissues of S. marmoratus. The rapid and significant response of the S. marmoratus immune signalling pathways following C. irritans infection may be associated with their involvement in the immune process.

Molecular identification and expression analysis of TLR5M and TLR5S from orange-spotted grouper (Epinepheluscoioides)

Toll-like receptor 5 (TLR5) is an important receptor that interacts with bacterial flagellin and regulates host immune response in mammal. Recent studies demonstrate that piscine contains two types of TLR5, namely membrane form of TLR5 (TLR5M) and soluble form of TLR5 (TLR5S), and both of which perform crucial role in flagellin response. In the present study, a TLR5M and a TLR5S sequence was cloned from orange-spotted grouper (Epinepheluscoioides), and their ORFs are respectively 2466 bp (821 aas) and 1935 bp (644 aas). EcTLR5M has the typical TLR structure of a LRR domain, a transmembrane region and a TIR domain, while EcTLR5S only contains a LRR domain like other species' TLR5S. Both molecules have 23 LRR motifs, a LRR-NT and a LRR-CT in the LRR domain, similar to those of other species. Phylogenetic and sequence alignment indicated that both EcTLR5s respectively displayed closer relationship and higher sequence identity with those in other fish species. In healthy grouper, EcTLR5M was highly expressed in the skin, head kidney and spleen, while EcTLR5S was mainly detected in the liver. Ciliate Cryptocaryon irritans infection could significantly up-regulate the expression level of EcTLR5s in the gill and spleen from day 1 to day 3, and higher expression fold change was observed in the spleen. Taken together, the present studies contributed to understanding the function of piscine TLR5M/S and clarify their possible role in fish immune response against ciliate infection.

Placemat and rotational culturing: A novel method to control Cryptocaryon irritans infection by removing tomonts

Cryptocaryoniasis is a severe parasitic disease caused by ciliate Cryptocaryon irritans in farmed marine fish. To control this disease, we established a method to interrupt the life cycle of C. irritans via the removal of its tomonts. Marine fish Plectorhinchus cinctus were divided randomly into four groups, including placemat removal group (Group I), rotational culturing group (Group II), infection control group (IF group) and blank control group (Control); the former three groups were infected with C. irritans at low dose. A daily food consumption (DFC), relative infection intensity (RII) and survival rate of each group were observed. At 4-week post infection, some fish were challenged with C. irritans theronts at a lethal dose. Results showed that the fish' DFC in IF Group, Group I and II decreased significantly on 2nd day post infection, and gradually restored food intake when the trophonts exited the fish on the 5th day. However, their DFC in IF Group decreased significantly on 7th day, and eventually stopped feeding. Trophonts on fish' body of the three groups began to reduce from the 4th day and those of Group I and II almost disappeared on the 7th day, conversely the trophonts of IF group increased ten times. At 2-week post infection, the survival rates of Group I, II, IF and Control were 97%, 98%, 0%, and 100%, respectively. The survival rates of Group I, II and Control were 88.3%, 93.3%, 66.7%, respectively after challenge. Results demonstrate that placemat and rotational culturing method to remove tomonts can effectively control C. irritans infection. Statement of relevanceCryptocaryoniasis is a severe parasitic disease caused by ciliate Cryptocaryon irritans in farmed marine fish. To control this disease, we established a method to interrupt the life cycle of C. irritans via the removal of its tomonts. Marine fish P. cinctus were divided randomly into four groups, including placemat removal group (Group I), rotational culturing group (Group II), infection control group (IF group) and blank control group (Control); the former three groups were infected with C. irritans at low dose. A daily food consumption (DFC), relative infection intensity (RII) and survival rate of each group were observed. At 4-week post infection, some fish were challenged with C. irritans theronts at a lethal dose. Results showed that the fish' DFC in IF Group, Group I and II decreased significantly on 2nd day post infection, and gradually restored food intake when the trophonts exited the fish on the 5th day. However, their DFC in IF Group decreased significantly on 7th day, and eventually stopped feeding. Trophonts on fish' body of the three groups began to reduce from the 4th day and those of Group I and II almost disappeared on the 7th day, conversely the trophonts of IF group increased ten times. At 2-week post infection, the survival rates of Group I, II, IF and Control were 97%, 98%, 0%, and 100%, respectively. The survival rates of Group I, II and Control were 88.3%, 93.3%, 66.7%, respectively after challenge. Results demonstrate that placemat and rotational culturing method to remove tomonts can effectively control C. irritans infection. This method is an mechanical preventive one, not only provides an ideal means for controlling C. irritans in small scale culture systems, but also solves food safety problems.

Anti-parasitic effects of Leptomycin B isolated from Streptomyces sp. CJK17 on marine fish ciliate Cryptocaryon irritans

The present study was conducted aiming to evaluate the in vitro and in vivo anti-parasitic efficacy of an isolated compound against the ciliate Cryptocaryon irritans. The compound was previously isolated from fermentation products of Streptomyces sp. CJK17 and designated as SFrD. Toxicity of the compound SFrD against the fish hosts (Larimichthys crocea) was also tested and its chemical structure was elucidated. The obtained results showed that the compound has potent anti-parasitic efficacy with the 10min-, 1h-, 2h-, 3h- and 4h-LC50 (95% Confidence Intervals) of 6.8 (6.5–7.1), 3.9 (2.8–5.0), 3.3 (2.6–4.0), 2.7 (2.3–3.1) and 2.5 (2.2–2.8)mgL−1 against theronts of C. irritans and the 6h-LC50 (95% CI) of 3.0 (2.8–3.2)mgL−1 against the tomonts, respectively. Exposure of the compound SFrD remarkably reduced the mortality of fish infected with C. Irritans, from 100% in the control group to 61.7% and 38.3% in groups of 3.1mgL−1 and 6.3mgL−1, respectively. In the test of exposing fish to 40mgL−1 compound SFrD for 24h, no visible effects were observed affecting the normal behavior or any macroscopic changes. By spectrum analysis (EI-MS, 1H NMR and 13C NMR), the compound SFrD was identified as Leptomycin B. This study firstly demonstrated that Leptomycin B has potent anti-parasitic efficacy against ciliates in cultured marine fish.

Proteomic analysis of differentially expressed proteins in the marine fish parasitic ciliate Cryptocaryon irritans

Cryptocaryoniasis is a severe disease of farmed marine fish caused by the parasitic ciliate Cryptocaryon irritans. This disease can lead to considerable economic loss, but studies on proteins linked to disease development and antigenic proteins for vaccine development have been relatively scarce to date. In this study, 53 protein spots with differential abundance, representing 12 proteins, were identified based on a pair-wise comparison among theronts, trophonts, and tomonts. Meanwhile, 33 protein spots that elicited serological responses in rabbits were identified, representing 9 proteins. In addition, 27 common antigenic protein spots reacted with grouper anti-sera, representing 10 proteins. Most of the identified proteins were involved in cytoskeletal and metabolic pathways. Among these proteins, actin and α-tubulin appeared in all three developmental stages with differences in molecular weights and isoelectric points; 4 proteins (vacuolar ATP synthase catalytic subunit α, mcm2-3-5 family protein, 26S proteasome subunit P45 family protein and dnaK protein) were highly expressed only in theronts; while protein kinase domain containing protein and heat shock protein 70 showed high levels of expression only in trophonts and tomonts, respectively. Moreover, actin was co-detected with 3 rabbit anti-sera while β-tubulin, V-type ATPase α subunit family protein, heat shock protein 70, mitochondrial-type hsp70, and dnaK proteins showed immunoreactivity with corresponding rabbit anti-sera in theronts, trophonts, and tomonts. Furthermore, β-tubulin, the metabolic-related protein enolase, NADH-ubiquinone oxidoreductase 75kDa subunit, malate dehydrogenase, as well as polypyrimidine tract-binding protein, glutamine synthetase, protein kinase domain containing protein, TNFR/NGFR cysteine-rich region family protein, and vacuolar ATP synthase catalytic subunit α, were commonly detected by grouper anti-sera. Therefore, these findings could contribute to an understanding of the differences in gene expression and phenotypes among the different stages of parasitic infection, and might be considered as a source of candidate proteins for disease diagnosis and vaccine development.

Corrigendum to “In vitro and in vivo evaluation of quinine in gilthead sea bream, Sparus aurata naturally infected with the ciliate Cryptocaryon irritans” [Aquaculture 416–417 (2013) 185–191

Corrigendum to “In vitro and in vivo evaluation of quinine in gilthead sea bream, Sparus aurata naturally infected with the ciliate Cryptocaryon irritans” [Aquaculture 416–417 (2013) 185–191

Dynamics and distribution properties of theronts of the parasitic ciliate Cryptocaryon irritans

Abstract Cryptocaryon irritans is a parasitic ciliate that causes major economic losses in marine fish aquaculture globally. Despite the wide array of treatment methods, control of cryptocaryoniasis is still very challenging, especially in food fish culture. Thus, additional control methods against this parasite might be required to further reduce the occurrence of this disease. In this study, the swimming ability, excystment sequence, and distribution of theronts, the infective stage of C. irritans , were investigated in our effort to develop a physical control strategy. A video analysis for assessing the swimming ability of theronts showed diminishing mobility over time. The excystment of theronts primarily occurred during the dark period. Examination of the vertical distribution of theronts showed that they were mostly distributed at the 5 cm sampling point measured from the substrate, indicating that they have low upward swimming ability. From these results, we conclude that theronts possess limited mobility. Theronts also displayed an excystment pattern that might be influenced by photoperiod, since most theronts were released during the dark period of the day. Control strategies can be developed from these properties, such as increasing the water flow in a culture tank during the release period. Further, combined treatment methods against multiple stages of C. irritans can help minimize the occurrence of cryptocaryoniasis in culture facilities.

In vitro and in vivo evaluation of quinine in gilthead sea bream, Sparus aurata naturally infected with the ciliate Cryptocaryon irritans

The use of quinine as a potential antiparasitic compound was evaluated at cellular level and in naturally infected gilthead sea bream, Sparus aurata with the ciliate Cryptocaryon irritans. Quinine concentrations up to 10μg/ml (30μΜ) did not exhibit any in vitro hemolytic or pro-apoptotic effects on red blood cells or head kidney macrophages, respectively. Dietary administration of quinine in gilthead sea bream at a concentration of 100mg/kg fish for 10days was incomplete due to palatability problems after the fifth day post treatment. A decreasing distribution profile of quinine was evident in the analyzed tissues even during the period where quinine consumption was supposedly ensured. The highest concentrations were measured on day 1 reaching 8.98±3.85μg/ml (27μΜ), 1.33±0.45μg/g and 0.75±0.48μg/g in plasma, skin and gills respectively. Quinine was rapidly removed from the tissue compartment while it remained at low concentration in fish circulation after therapy. A 100% C. irritans prevalence was accompanied with no mortalities during the experiment. Parasitic intensity, measured as trophont number, was significantly reduced in quinine-treated fish at the first sampling point (3rd day: 8±0.8 vs 11±2.6), while no statistical differences were observed thereafter. A significant reduction in plasma glucose level was also observed at the same time point. Organoleptic evaluation in fillets, showed no difference in the flesh bitterness between quinine-treated and control fish.In conclusion, quinine has no cytotoxic effect on the assessed fish cells and is adequately absorbed in circulation, affecting the glycemic level but its distribution to the targeted tissues by the ciliate tissues is relatively small. Quinine significantly reduced C. irritans' intensity in lightly-infected gilthead sea bream. The sensory attributes of quinine-treated fish are not affected by the treatment.

Antiparasitic effect of dietary Romet®30 (SDMX–OMP) against ciliate Cryptocaryon irritans infection in the red sea bream Pagrus major and tiger puffer Takifugu rubripes

We investigated the effects of dietary Romet®30 (sulfadimethoxine–ormetoprim (SDMX–OMP)) on Cryptocaryon irritans infection in red sea bream Pagrus major and tiger puffer Takifugu rubripes. In Experiment I, 100% mortality of P. major due to C. irritans infection was observed at 10days and 21days after exposure to theronts (infective stage) in the a control group without Romet®30 and the group treated with 50mg Romet®30/kg body weight (BW)/day for 14days, respectively. Thus, mortality in the treated group was markedly delayed compared with that in the control group. In Experiment II, 100% mortality of P. major in the control group due to C. irritans infection was recorded at 11days after exposure to theronts. In contrast, mortality due to parasite infection was not observed in the group treated with Romet®30 at 50mg/kg BW/day for 14days, and no parasites were found in any surviving fish after 33days exposure. In addition, the number of parasites on the gills of T. rubripes treated with 50mg Romet®30/kg BW/day for 14days was significantly lower than that in the control group after 16days exposure. These results show that in-feed Romet®30 at 50mg/kg BW/day for 14days had antiparasitic and therapeutic effects against C. irritans in both P. major and T. rubripes. Thus, dietary Romet®30 could be useful for controlling C. irritans infection.

Antiparasitic effect of in-feed inhibitors of folic acid synthesis and dihydrofolate reductase against ciliate Cryptocaryon irritans infection in the red sea bream Pagrus major and against ciliate Ichthyophthirius multifiliis infection in black pop-eyed goldfish Carassius auratus

We investigated the effects of feeding with sulfonamides, dihydrofolate reductase inhibitors or their combinations on Cryptocaryon irritans infection in the red sea bream Pagrus major. We also investigated the effects of an in-feed sulfonamide–diaminopyrimidine combination on Ichthyophthirius multifiliis infection in the black pop-eyed goldfish Carassius auratus. In P. major treated with in-feed sulfonamides (300mg/kg body weight (BW)), dihydrofolate reductase inhibitors (60mg/kg BW) and their combination (Sulfadimethoxine (SDMX) 216mg and Trimethoprim (TMP) 24mg/kg BW) for 3days, significantly smaller C. irritans trophonts were found compared to the control group. The smallest trophonts were found in the group treated with the SDMX–TMP combination. In P. major fed with SDMX–TMP (216 and 24mg/kg BW) or Sulfamethoxazole (SMX)–TMP (429 and 86mg/kg BW) for long-term, the number of C. irritans cysts was markedly lower compared to the control group at 5days after exposure to theronts. The cysts in groups treated with these combinations were significantly smaller than those from the control group, and hatching was delayed compared to that of the control group. Further, in the control group, 100% mortality was observed owing to heavy C. irritans infection at 9days after the exposure. On the other hand, mortality owing to parasite infection was not observed in the groups treated with the combinations, and no parasites were found in any surviving fish at the end of the experiment. In addition, Sulfamonomethoxine (SMMX)–Ormetoprim (OMP) (112.5 and 37.5mg/kg BW) combination also had a therapeutic effect against I. multifiliis infection in C. auratus. Thus, in-feed sulfonamides, dihydrofolate reductase inhibitors and their combinations will be useful in aquaculture for controlling C. irritans and I. multifiliis infections.

Quantitative PCR assay for the detection of the parasitic ciliate Cryptocaryon irritans

We developed a quantitative PCR assay for detecting the parasitic ciliate Cryptocaryon irritans, which causes “white spot disease” in marine fishes, from the natural environment. A specific primer set for C. irritans was designed and its high specificity was confirmed in silico: almost all of the sequences deposited in the GenBank nucleotide database were covered, 22/23 for the forward primer and 7/7 for the reverse primer. We estimated that there were 3,415.9 rRNA gene copies per genome of C. irritans. In artificial mixture experiments to validate whether the qPCR assay is applicable to natural samples, the estimated copy numbers showed significantly positive correlations with the number of theronts added (p < 0.001). When we applied this qPCR assay to natural samples collected bimonthly from surface and bottom seawaters at an aquaculture site (water depth, 10 m) from May 2009 to March 2010, we only detected C. irritans (112.0 ± 6.3 cells/l) in the surface seawater sample in November. This qPCR assay is a useful tool for detecting C. irritans rapidly and quantitatively in natural environments; it could also help advance our understanding of the ecology of C. irritans, as well as facilitate the diagnosis of the disease.

A technique for the preservation of Cryptocaryon irritans at low temperatures

Applied and basic research to evaluate the ciliate Cryptocaryon irritans requires serial passage in fish hosts to maintain the laboratory stock. However, continual repeated passage is labour-intensive and time-consuming and results in the senescence of parasite clones over time. To address these issues, this study was conducted to investigate the preservation of C. irritans in vitro at low temperatures. Trophonts and tomonts of C. irritans were collected and preserved at 5–25 °C. The excystation rate of preserved parasites and the infectivity of the theronts produced by the preserved parasites were then determined every other month for six months. The results revealed that trophonts and tomonts of C. irritans could survive at 12.0 °C for 5 and 4 months, respectively. Additionally, the excystation rate of the preserved parasites and the infectivity of theronts produced by the preserved parasites decreased as the preservation period increased. Specifically, trophonts preserved for 4 months and tomonts preserved for 3 months could produce theronts with infectivity, and these theronts were capable of generating a large number of descendants after two serial passages in the fish. Moreover, the virulence of the descendants did not decrease. Overall, the results of this study confirmed the practicality of preserving C. irritans in vitro at low temperatures. Furthermore, the findings presented here suggest that C. irritans can survive during winter in a dormant status and can resume infectivity during spring.

Some characteristics of host–parasite relationship for Cryptocaryon irritans isolated from South China

A survey on the host range for the parasitic ciliate Cryptocaryon irritans was carried out among the major maricultured fish species in the Huizhou region of Guangdong Province in South China, and some characteristics of its host-parasite relationship were described. The survey showed that all ten investigated species of fish (representing six different families) were infected with C. irritans with similar susceptibility. In chemoattraction assays, sera and mucus collected from investigated fish strongly attracts C. irritans theronts. Sera collected from infected orange-spotted groupers and yellow spotted grunts (Plectorhynchus cinctus) could immobilize C. irritans theronts, and their immobilization titers were 1:40 and 1:6.7, respectively. The surface antigens of C. irritans were demonstrated by indirect immunofluorescence and immunostaining assays using immune orange-spotted grouper serum and a monoclonal antibody against grouper IgM.

Killing effect of various treatments on the monogenean Heterobothrium okamotoi eggs and oncomiracidia and the ciliate Cryptocaryon irritans cysts and theronts

The effect of chlorine (12, 60, and 120 ppm available chlorine concentrations), benzalkonium chloride (100 ppm), freshwater, heat (40 °C), and drying on the hatching ability of Heterobothrium okamotoi eggs and Cryptocaryon irritans cysts was examined. The eggs and cysts at an early stage (1–16 h after spawning or leaving the host for H. okamotoi and C. irritans , respectively) and at a late stage (72–87 h) were exposed to each treatment for 1 or 24 h. Hatching of H. okamotoi eggs and C. irritans cysts was completely suppressed at both early and late stages when they were exposed to heat for 1 h. Exposure to chlorine at a concentration of more than 60 ppm available chlorine or to drying for 24 h was also effective in preventing hatching completely. However, freshwater had limited effect on preventing hatching of both parasites. Although benzalkonium chloride treatment for 1 h prevented hatching of C. irritans cysts completely, it was not effective against H. okamotoi eggs. One-hour treatments with chlorine (2.4 and 12 ppm) or benzalkonium chloride (100 ppm),or heat (40 °C) killed hatched H. okamotoi oncomiracidia and C. irritans theronts. These methods would be useful to prevent the horizontal infection with these parasites from used equipment, tanks, and wastewater in fish-rearing facilities.