Severe Natural Outbreak of Cryptocaryon irritans in Gilthead Seabream Produces Leukocyte Mobilization and Innate Immunity at the Gill Tissue.

The protozoan parasite Cryptocaryon irritans causes marine white spot disease in a wide range of fish hosts, including gilthead seabream, a very sensitive species with great economic importance in the Mediterranean area. Thus, we aimed to evaluate the immunity of gilthead seabream after a severe natural outbreak of C. irritans. Morphological alterations and immune cell appearance in the gills were studied by light microscopy and immunohistochemical staining. The expression of several immune-related genes in the gills and head kidney were studied by qPCR, including inflammatory and immune cell markers, antimicrobial peptides (AMP), and cell-mediated cytotoxicity (CMC) molecules. Serum humoral innate immune activities were also assayed. Fish mortality reached 100% 8 days after the appearance of the C. irritans episode. Gill filaments were engrossed and packed without any space between filaments and included parasites and large numbers of undifferentiated and immune cells, namely acidophilic granulocytes. Our data suggest leukocyte mobilization from the head kidney, while the gills show the up-regulated transcription of inflammatory, AMPs, and CMC-related molecules. Meanwhile, only serum bactericidal activity was increased upon infection. A potent local innate immune response in the gills, probably orchestrated by AMPs and CMC, is triggered by a severe natural outbreak of C. irritans.

Characterization and immune regulation role of an immobilization antigen from Cryptocaryon irritans on groupers.

Immobilization antigens (i-antigens) are surface membrane proteins that are widely recognized to be the ideal candidates as vaccines antigens for immunization against Cryptocaryon irritans. In this study, we cloned a putative i-antigen gene from C. irritans, which was expressed in all three stages of the C. irritans life-cycle, and localized primarily to the cell surface. The recombinant GDCI3 i-antigen was expressed and purified using the free-living ciliate, Tetrahymena thermophila as an expression system. The purified recombinant protein was recognized by rabbit anti-C. irritans antiserum and was capable of eliciting immobilizing antibodies in rabbits and fish suggesting that the antigen itself was correctly folded. Following immunization and parasite challenge, groupers vaccinated with, recombinant GDCI3 i-antigen had a 25% cumulative percent survival rate compared to 8.3% for controls. Both non-specific and parasite-specific IgMs were generated in fish following immunization, with the levels of both increasing following challenge. Parasite-specific IgM in mucus could only be elicited after challenge of the GDCI3 i-antigen vaccinated groupers. To our knowledge, this is the first report using the Tetrahymena expression system to generate C. irritans i-antigens and investigate their use for fish vaccination.

Grass carp which survive Dactylogyrus ctenopharyngodonid infection also gain partial immunity against Ichthyophthirius multifiliis.

Dactylogyrus ctenopharyngodonid and Ichthyophthirius multifiliis are 2 important ectoparasites of fish. Both parasites can induce an immune response in fish that leads to a decrease in parasitic infection intensity and the development of resistance against parasitic reinfection. The present study evaluated whether grass carp Ctenopharyngodon idella that survived a D. ctenopharyngodonid infection could develop immunity against infection by D. ctenopharyngodonid and I. multifiliis. The results demonstrated that when grass carp were infected with D. ctenopharyngodonid, the number of red blood cells and the percentages of thrombocytes, monocytes, and neutrophils in the white blood cells increased significantly in the early stage of infection. The percentage of lymphocytes increased over time following parasitic infection. The mean infection intensity of D. ctenopharyngodonid decreased to 0 on Day 28. The activities of serum acid phosphatase, alkaline phosphatase, lysozyme, and superoxide dismutase increased significantly after D. ctenopharyngodonid infection. In addition, the grass carp that survived a previous D. ctenopharyngodonid infection could completely resist D. ctenopharyngodonid reinfection and partially resist I. multifiliis infection.

Analysis of genes encoding high-antigenicity polypeptides in three serotypes of Miamiensis avidus.

The ciliate Miamiensis avidus causes scuticociliatosis in Japanese flounder Paralichthys olivaceus. We previously reported three serotypes of this ciliate distinguishable by serotype-specific antigenic polypeptides (serotype I, 30kDa; serotype II, 38kDa; serotype III, 34kDa). In this study, we determined the localization site of the serotype-specific polypeptides in the ciliate and determined the genes encoding the polypeptides, using the isolates IyoI (serotype I), Nakajima (serotype II), and Mie0301 (serotype III). SDS-PAGE and immunoblot analysis of cilia, membrane proteins, and cytoskeletal elements of the ciliates revealed that the polypeptides were abundant in the former two. Scanning electron microscopy of ciliates immobilized by homologous antiserum showed morphological changes in the cilia. These evidences suggested that the polypeptides were ciliary membrane immobilization antigens. The ciliary genes identified showed low identity scores-<51.5% between serotypes. To differentiate the serotypes, we designed serotype-specific PCR primer sets based on the DNA sequences. The PCR-based serotyping results were completely consistent with conventional serotyping methods (immobilization assay and immunoblot analysis). Twenty of 21 isolates were classified as either serotype I or II, and one isolate was undistinguishable. The combination of species-specific PCR previously reported and three serotype-specific PCR could be useful for identifying, serotyping, and surveillance for occurrences of new serotypes of M. avidus.

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

BACKGROUND: False 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. METHODS: We 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. RESULTS: The 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. CONCLUSIONS: Infection 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.

The expressed TCRß CDR3 repertoire is dominated by conserved DNA sequences in channel catfish.

We analyzed by high-throughput sequencing T cell receptor beta CDR3 repertoires expressed by αß T cells in outbred channel catfish before and after an immunizing infection with the parasitic protozoan Ichthyophthirius multifiliis. We compared CDR3 repertoires in caudal fin before infection and at three weeks after infection, and in skin, PBL, spleen and head kidney at seven and twenty-one weeks after infection. Public clonotypes with the same CDR3 amino acid sequence were expressed by αß T cells that underwent clonal expansion following development of immunity. These clonally expanded αß T cells were primarily located in spleen and skin, which is a site of infection. Although multiple DNA sequences were expected to code for each public clonotype, each public clonotype was predominately coded by an identical CDR3 DNA sequence in combination with the same J gene in all fish. The processes underlying this shared use of CDR3 DNA sequences are not clear.

Analysis of liver and gill miRNAs of Larimichthys crocea against Cryptocryon irritans challenge.

The white-spot disease caused by marine ciliate Cryptocryon irritans hindered the sustainable development of large yellow croaker Larimichthys crocea industry. Better understandings about the parasite-host interactions in the molecular level will facilitate the prevention of mass mortality of the L. crocea caused by white-spot disease. MicroRNAs (miRNAs) are a class of small RNA molecules about 20-22 nucleotides which post-transcriptionally regulated many protein-coding genes and involved in many biological processes, especially in host-pathogen responses. In this study, we identified known and novel miRNAs in the gill and liver of L. crocea challenged by C. irritans by high throughput sequencing using Solexa technology. The data were further studied to screen differentially expressed miRNAs, and predict their target genes. The differential expression (p < 0.05) between libraries was observed in 103 miRNAs in liver tissue, among which 65 and 38 were conserved and novel miRNAs, 67 and 36 were up- and down-regulated miRNAs. While in gill tissue, 122 significant differentially expressed miRNAs were identified, among which 83 and 39 were conserved and novel miRNAs, 79 and 43 were up- and down-regulated miRNAs. In addition, these differentially expressed miRNAs target a series of genes which involved in many important biological processes including immune response. Here via deep sequencing, we for the first time characterize L. crocea miRNAs in response to C. irritans challenge, the results should help for better understandings about the immune response of the L. crocea under C. irritans challenge.

Cryptocaryon irritans recombinant proteins as potential antigens for sero-surveillance of cryptocaryonosis.

Cryptocaryonosis is a major problem for mariculture, and the absence of suitable sero-surveillance tools for the detection of cryptocaryonosis makes it difficult to screen Cryptocaryon irritans-infected fish, particularly asymptomatic fish. In this study, we proposed a serum-based assay using selected C. irritans proteins to screen infected and asymptomatic fish. Eight highly expressed genes were chosen from an earlier study on C. irritans expressed sequence tags and ciliate glutamine codons were converted to universal glutamine codons. The chemically synthesized C. irritans genes were then expressed in an Escherichia coli expression host under optimized conditions. Five C. irritans proteins were successfully expressed in E. coli and purified by affinity chromatography. These proteins were used as antigens in an enzyme-linked immunosorbent assay (ELISA) to screen sera from experimentally immunized fish and naturally infected fish. Sera from both categories of fish reacted equally well with the expressed C. irritans recombinant proteins as well as with sonicated theronts. This study demonstrated the utility of producing ciliate recombinant proteins in a heterologous expression host. An ELISA was successfully developed to diagnose infected and asymptomatic fish using the recombinant proteins as antigens.

Immobilization antigen vaccine adjuvanted by parasitic heat shock protein 70C confers high protection in fish against cryptocaryonosis.

The immobilization antigen (iAg) has been demonstrated as a protective immunogen against Cryptocaryon irritans infection. In this study, C-terminal domain of heat shock protein 70 cloned from C. irritans (Hsp70C) was tested for its immuno-stimulatory effects. The iAg and Hsp70C cDNAs were constructed independently in secretory forms and were encapsulated in chitosan nanoparticles. In the first immunization trial, grouper fingerlings orally intubated with iAg and iAg:Hsp70C presented 96% and 100% relative percent survival (RPS), respectively, after a lethal challenge. In the second trial, both iAg and iAg:Hsp70C groups showed 100% RPS and the skin trophont burden was significantly lowered. The iAg:Hsp70C still provides a significantly high protection of 51% RPS at 49 days post immunization, when an even more serious lethal infection occurs. RT-qPCR results showed that Hsp70C could up-regulate the expression of i) T cell markers: Cluster of Differentiation 8 alpha (CD8α) and CD4, ii) cytokine genes: Interferon gamma (IFNγ), Tumor Necrosis Factor alpha (TNFα) and Interleukin 12 p40 (IL-12/P40), iii) antibody genes: Immunoglobulin M heavy chain (IgMH) and IgTH, and iv) major histocompatibility complex (MHC-I & MHC-II), in the spleen of iAg:Hsp70C group. Furthermore, significantly high levels of iAg-specific IgM was detected in skin mucus which efficiently immobilized live theronts in iAg- and iAg:Hsp70C-immunized fish at 5 weeks post immunization. Hsp70C significantly increased the number of nonspecific CD8(+) skin leucocytes which exerted cytotoxicity against theronts, although cytotoxic activity showed no difference among the various groups. Because of this complementary cooperation of cellular and humoral immune responses, Hsp70C enhances the efficacy of iAg vaccine and constrains C. irritans infection. In view of the severe loss caused by cryptocaryonosis, application of this parasitic vaccine in farmed and ornamental fish, is worthy to be considered.