Search

The objective of this study was to evaluate differential expression of innate and adaptive immune genes, including immunoglobulin, immune cell receptor, cytokine, inflammatory protein, toll-like receptors (TLR) and recombination-activating gene (RAG) in skin from channel catfish, Ictalurus punctatus after immunization with live theronts of Ichthyophthirius multifiliis (Ich) by intraperitoneal injection. The immunized catfish showed significantly higher survival rate (95%) than those of mock-immunized control fish (0% survival) after the theront challenge. The gene expression of innate immune system, such as cytokines (IL-1β type a, IL-1β type b, IFN-γ, TGF1-β and TNF-α) and inflammatory proteins (NF-kB and iNOS 2), showed significant upregulation at day 1 (D1) post-immunization. Expression of TLR genes exhibited a rapid increase from hour 4 (h4) to D10 post-immunization. Genes of the adaptive response, such as the cell receptor MHC I, CD8 + , CD4 + and TCR-α, showed upregulation at D1, D6 and D10. The TCR-β expression increased rapidly at h4 and remained upregulated until D10. Immunoglobulin IgM upregulation was detected from h4 until D2 while IgD expression was increased from D1 until D10. Rapid upregulation of innate and adaptive immune genes in skin of catfish following live theront vaccination was demonstrated in this study ultimately resulting in significant protection against Ich infection., (© 2016 John Wiley & Sons Ltd.)

Since the use of malachite green was banned, it is desirable to search for alternative drug to control Ichthyophthirius multifiliis infection. Moreover, route of administration which is easy to implement and less stressful to fish is important to treat infection of fish with this parasite. It is also important to enhance protective effect against Aeromonas hydrophila infection after parasitism by I. multifiliis. The present study evaluated the protective effect of diet supplemented with Artemisia annua against I. multifiliis and A. hydrophila infection after the parasitism. The results showed that oral administration with A. annua at a concentration of 20 g/kg feed for 45 days can provide strong protection against I. multifiliis infection. The incidence of infestation and mean number of trophonts on fish fins on day 3 post-challenge (15,000 theronts/fish) were significantly reduced compared with the control, and the survival rate of fish during 15-day period was also increased by nearly 30 %. But, this diet provided very limited protection against higher infection dose. Besides, this study demonstrated that this diet enhanced protective effect against A. hydrophila after the fish were infected with I. multifiliis. On the basis of the above results, oral administration with A. annua can be used as a potential strategy for control I. multifiliis infection and increasing fish survival after parasitism by I. multifiliis.

Background: Cryptocaryon irritans is an obligate ectoparasitic ciliate pathogen of marine fishes. It can infect most marine teleosts and cause heavy economic losses in aquaculture. There is currently no effective method of controlling this disease, and little information is available regarding the genes involved in its development and virulence. We aimed to investigate the distinct features of the three major life-cycle stages of C. irritans in terms of gene transcription level, and identify candidate vaccines/drug targets. We established a reference transcriptome of C. irritans by RNA-seq., Methods: Three cDNA libraries using total poly(A) + mRNA isolated from trophonts, tomonts, and theronts was constructed and sequenced, respectively. Clean reads from the three stages were de novo assembled to generated unigene. Annotation of unigenes and transcriptomic comparison of three stages was performed., Results: Totals of 73.15, 62.23, and 109.57 million clean reads were generated from trophont, tomont, and theront libraries, respectively. After de novo assembly, 49,104 unigenes were obtained, including 9,253 unigenes with significant similarities to proteins from other ciliates. Transcriptomic comparisons revealed that 2,470 genes were differentially expressed among the three stages, including 2,011, 1,404, and 1,797 genes that were significantly differentially expressed in tomont/theront, tomont/trophont, and theront/trophont pairwise comparisons, respectively. Based on the results of hierarchical clustering, all differentially expressed genes (DEGs) were located in five major clusters. DEGs in clusters 1 and 2 were more highly expressed in tomonts than in other stages, DEGs in cluster 3 were dominant in the tomont and trophont stages, whereas clusters 4 and 5 included genes upregulated in the theront stage. In addition, Immobilization antigens (I-antigens) and proteases have long been considered major targets for vaccine development and potential drug targets in parasites, respectively. In the present study, nine putative I-antigens transcripts and 161 protease transcripts were found in the transcriptome of C. irritans., Conclusion: It was concluded that DEGs enriched in tomonts were involved in cell division, to increase the number of theronts and ensure parasite continuity. DEGs enriched in theronts were associated with response to stimuli, whereas genes enriched in trophonts were related to nutrient accumulation and cell growth. In addition, the I-antigen and protease transcripts in our transcriptome could contribute to the development of vaccines or targeted drugs. Together, the results of the present study provide novel insights into the physiological processes of a marine parasitic ciliate.

The present study describes a new species of Trichodina found in the mucus of the body surface of 137 farmed and wild silver catfish (Rhamdia quelen) located in southern Brazil and in the gills of 99 banded knife fish (Gymnotus sp.) captured in the basin of Pantanal. Smears of the body surface mucus and gills were air dried at room temperature and impregnated with silver nitrate and stained with Giemsa for analysis of the adhesive disc, denticles and macronucleus. Characterized as a medium trichodinid, Trichodina quelenii n. sp. resembles T. heterodentata and T. acuta. It differs from T. heterodentata by the shape of the blade and from T. acuta by the lack of central circle. Furthermore, the distinct shape of the blade of T. quelenii n. sp. differs from all previously described species for the genus, leaving no doubt it is a new species.

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Trichodiniasis of fishes cause economic losses in the fish industry through mortality and decrease growth rate. Very few studies of these ectoparasites have been made in India. A comprehensive icthyoparasotological survey in some ornamental fish farms of different parts of India has been done since March 2014-May 2015. During the study these trichodinid parasites have been identified from the gills of Oranda Gold Fish (Carassius auratus auratus L.). They are Trichodinella epizootica, Tripartiella viz. Tripartiella bulbosa, Tripartiella copiosa and Tripartiella obtusa. All these parasites have been recorded for the first time from the gills of Oranda Gold Fish (Carassius auratus auratus L.) in India i.e. The paper deals with the diversity and description of ciliophorans parasites of ornamental fish.

The parasite Ichthyophthirius multifiliis (Ich) has been reported in various freshwater fishes worldwide and results in severe losses to both food and aquarium fish production. Lactobacillus strains have a number of properties that make them attractive candidates as delivery vehicles for the presentation to the mucosa of compounds with pharmaceutical interest, in particular vaccines. Here, the present study was conducted to evaluate a live recombinant Lactococcus lactis vaccine expressing immobilization antigen (IAG-52X) in protection against I. multifiliis. A 1266 bp gene fragment containing a potential antigenic epitope of the 48 kDa immobilization antigen of I. multifiliis was assembled from six synthetic ohgonucleotides and cloned into pSIP409 and electrotransformed into Lactobacillus plantarum NC8. The recombinant vaccine candidate was then orally fed into goldfish. The expression of immune-related genes: complement component 3 (C3), MHC I, IgM gene in blood from goldfish at different time points after immunization were evaluated. Immunized fish were than challenged with a lethal dose of infectious I. multifiliis. The cumulative mortality and relative percentage survival (RPS) were also determined. Our results showed that the antibody level in the blood and skin of the immunized fish was statistically significant (P < 0.05) in relation to the control groups. Goldfish orally immunized with NC8-pSIP409- IAG-52X had high serum antibody titers that ranged from 32 to 256 after 28d post immunization, while fish fed with NC8-pSIP409 or PBS had no detectable immobilizing antibody response. Expression of IgM, C3, MHC I genes in the group immunized with IAG-52X were significantly (P < 0.05) up regulated as compared with control group, indicating that different immune cells were actively involved in cellular immune response. The results showed that the average survival rate of fish orally immunized with 10 8 and 10 6 NC8-pSIP409-IAG-52X was 60% and 50% respectively. Therefore, NC8-pSIP409-IAG-52X could become a promising oral vaccine candidate against I. multifiliis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

B cell antigen receptor (BCR) plays a crucial role in B cell development and antibody production. It comprises membrane immunoglobulin non-covalently associated with CD79a/CD79b heterodimer. After B cell activation, initial extracellular signals are transduced by BCR complex and amplified by two protein tyrosine kinases, LYN and SYK, which then trigger various pathways. In the present study, we cloned grouper genes for BCR accessory molecules, EcCD79a (669 bp) and EcCD79b (639 bp), as well as two protein tyrosine kinases, EcLYN (1482 bp) and EcSYK (1854 bp). Homology analysis showed that all four molecules had a relatively high amino acid identity compared with those in other animals. Among them, they all shared the highest identity with Takifugu rubripes (EcCD79a 49%, EcCD79b 52%, EcLYN 82% and EcSYK 77%). The conserved features and important functional residues were analyzed. Together with IgM and IgT, tissue distribution analysis showed that all six molecules were mainly expressed in immune organs, particularly systematic immune organs. In groupers infected with Cryptocaryon irritans, up-regulation of EcCD79a and b, EcIgM and EcIgT were not seen in the early stage skin and gill until 14-21 days. Up-regulation of EcCD79a was seen in head kidney at most time points, while EcCD79a and b were only significantly up-regulated in day 14 spleen. Significant up-regulation of EcIgT were seen in day 21 head kidney and day 1, day14 spleen. Significant up-regulation of EcIgM were seen in day 1 head kidney and 12 h spleen. In addition, two protein kinase genes, EcLYN and EcSYK, were up-regulated in the skin at most time points, which suggested that B cells may be activated at the skin local infection site., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Ichthyophthirius multifiliis, causing white spot disease, is a serious pathogen in aquaculture as well as for the ornamental fish industry. In carp, channel catfish and rainbow trout the immune responses against the parasite have been partly elucidated and these species are able to acquire a high level of immunity against the disease. Zebrafish are however, known to be more resilient towards the disease than channel catfish but the pathology and the ability to obtain protection is unknown. In this study a primary infection in the gills of zebrafish was described and the subsequent acquirement of immunity was evaluated. The parasites in the gills induced hyperplasia, an increase of mucus cells, adhesion and shortening of the secondary lamellae, narrowing of water channels and proliferation. The parasite burden was significantly lower in survivor fish and where all naive fish died within 6 days following secondary infection (challenge) only one of the survivor fish died. This study demonstrates that zebrafish are susceptible to I. multifiliis and that gill pathology is similar to the situation observed in rainbow trout. Furthermore, zebrafish are able to acquire immunity against white spot disease and may therefore be a suitable model organism to study innate and protective immunology and host/parasite interactions during I. multifiliis infections., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In vitro effect of the Melaleuca alternifolia, Lavandula angustifolia and Mentha piperita essential oils (EOs) against Ichthyophthirius multifiliis and in vivo effect of M. alternifolia for treating ichthyophthiriasis in one of the most important South American fish, Piaractus mesopotamicus (Holmberg), were evaluated. The in vitro test consisted of three EOs, each at concentrations of 57 μL L(-1) , 114 μL L (-1) , 227 μL L(-1) and 455 μL L (-1) , which were assessed once an hour for 4 h in microtitre plates (96 wells). The in vitro results demonstrated that all tested EOs showed a cytotoxic effect against I. multifiliis compared to control groups (P < 0.05). The in vivo treatment for white spot disease was performed in a bath for 2 h day(-1) for 5 days using the M. alternifolia EO (50 μL L (-1) ). In this study, 53.33% of the fish severely infected by I. multifiliis survived after the treatment with M. alternifolia (50 μL L (-1) ) and the parasitological analysis has shown an efficacy of nearly 100% in the skin and gills, while all the fish in the control group died. Furthermore, the potential positive effect of M. alternifolia EO against two emergent opportunistic bacteria in South America Edwardsiella tarda and Citrobacter freundii was discussed., (© 2016 John Wiley & Sons Ltd.)

The scuticociliatosis is a very serious disease that affects the cultured turbot, and whose causal agent is the anphizoic and marine euryhaline ciliate Philasterides dicentrarchi. Several protozoans possess acidic organelles that contain high concentrations of pyrophosphate (PPi), Ca(2+) and other elements with essential roles in vesicular trafficking, pH homeostasis and osmoregulation. P. dicentrarchi possesses a pyrophosphatase (H(+)-PPase) that pumps H(+) through the membranes of vacuolar and alveolar sacs. These compartments share common features with the acidocalcisomes described in other parasitic protozoa (e.g. acid content and Ca(2+) storage). We evaluated the effects of Ca(2+) and ATP on H (+)-PPase activity in this ciliate and analyzed their role in maintaining intracellular pH homeostasis and osmoregulation, by the addition of PPi and inorganic molecules that affect osmolarity. Addition of PPi led to acidification of the intracellular compartments, while the addition of ATP, CaCl2 and bisphosphonates analogous of PPi and Ca(2+) metabolism regulators led to alkalinization and a decrease in H(+)-PPase expression in trophozoites. Addition of NaCl led to proton release, intracellular Ca(2+) accumulation and downregulation of H(+)-PPase expression. We conclude that the regulation of the acidification of intracellular compartments may be essential for maintaining the intracellular pH homeostasis necessary for survival of ciliates and their adaptation to salt stress, which they will presumably face during the endoparasitic phase, in which the salinity levels are lower than in their natural environment., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (© 2016 John Wiley & Sons Ltd.)

Cells of Cryptocaryon irritans at different life history stages were studied using both light and electron microscopy. The characteristics of several organelles were revealed for the first time at the ultrastructural level. It was confirmed that the cytostome of trophonts, protomonts and theronts was surrounded by cilium-palp triplets rather than ciliary triplets. The nematodesmata underlying the circumoral dikinetids were single bundles, whereas these were always paired in Prorodontids. Toxicysts were present in late-stage tomonts and theronts, but were absent in trophonts and protomonts. We posited that toxicysts might play a role in infection and invasion of host-fish tissue by theronts. The adoral brosse was unlike that of any other family of the class Prostomatea based on its location and morphology. Membranous folds were present in trophonts, protomonts and theronts. These folds were longer and more highly developed in C. irritans than in exclusively free-living prostome ciliates suggesting that they might be linked to parasitism in C. irritans. Trophonts, protomonts and theronts had multiple contractile vacuoles. The basic ultrastructure of the contractile vacuole of C. irritans was similar to that of other kinetofragminophoran ciliates. They might play different roles in different stages of the life cycle since their ultrastructure varied among trophonts, protomonts and theronts.

As crucial components of the toll-like receptor (TLR) and interleukin-1 (IL-1) receptor (IL-1R) signaling pathways, interleukin-1 receptor associated kinase (IRAK) family members play essential roles in an animal's immune response. In this study, an IRAK family member, designated EcIRAK-1, was identified in the orange-spotted grouper Epinephelus coioides, and its role in signal transduction investigated. The full-length EcIRAK-1 gene is 2822 bp, encoding a 760-amino-acid protein that has the typical characteristics of mammalian IRAK-1, including an N-terminal death domain, a ProST domain, a central kinase domain, and C-terminal C1 and C2 domains. EcIRAK-1 shares 42%-79% sequence identity with other fish IRAK-1 proteins, and the death and kinase domains are more conserved than the other domains. Several important amino acids and motifs of mammalian IRAK-1 are also conserved in the grouper and other piscine IRAK-1s. In healthy grouper, EcIRAK-1 was broadly expressed in all the tissues tested, with the highest expression in the gill and skin. After infection with Cryptocaryon irritans, EcIRAK-1 expression increased in the gill and spleen. After its exogenous expression in HEK293T cells, EcIRAK-1 significantly activated nuclear factor kappaB (NF-κB). The death domain, ProST domain, and some conserved amino acids, such as T58, T207, K237, and T387, in EcIRAK-1 are required for its signaling function. These data demonstrate that piscine IRAK-1 has the same structural characteristics as its mammalian counterpart and that its function is conserved among vertebrates., (Copyright © 2016. Published by Elsevier Ltd.)

Ichthyophthirius multifiliis is a ciliated protozoan parasite infecting the skin and gills of freshwater fish. Neutrophils are attracted to the infection sites, as a part of the innate immune response. In this study a transgenic line of zebrafish (Tg(MPO:GFP)(i114)) with GFP-tagged neutrophils was infected with I. multifiliis and the neutrophil influx in the caudal fin was quantified. Twenty-four hours post infection (pi) the neutrophil count had gone up with an average of 3.4 fold. Forty-eight h pi the neutrophil count had dropped 12% and 72 h pi it had dropped to 21% compared to 24 h pi. At 72 h pi the neutrophil count was 2.7 times higher than prior to infection. A few dead parasites were observed, which were disintegrated and covered internally and externally with neutrophils. Live parasites, both surrounded by neutrophils and with no neutrophils in the near vicinity, were found during the infection. Neutrophils interacted directly with the parasites with pseudopod formation projecting towards the pathogen. These results indicate a strong innate immune response immediately following infection and/or a subsequent immune evasion by the parasite., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

As fundamental immunologic mechanism, the innate immunity system is more important than the specific immunity system in teleost fishes during pathogens infection. Antimicrobial peptides are integral parts of the innate immune system, and play significant roles against pathogens infection. NK-lysin, the compounds of the natural killer cells and cytotoxic T cells, are potent and effective antimicrobial peptides widely distributed in animals. In this study, we reported the sequence characteristics, expression profiles and antibacterial activities of a NK-lysin gene (Lc-NK-lysin) from a commercially important marine fish, the large yellow croaker (Larimichthys crocea). The open reading frame of Lc-NK-lysin cDNA sequence was 447 bp in length, coding 148 amino acids. The genomic DNA of Lc-NK-lysin has the common features of NK-lysin family, consisting of five exons and four introns, and in its deduced mature peptide, there are six well-conserved cysteine residues and a Saposin B domain. Lc-NK-lysin was expressed in all tested tissues (skin, muscle, gill, brain, head kidney, heart, liver, spleen, stomach and intestine) with different expression patterns. In pathogens infection the expression profiles of Lc-NK-lysin varied significantly in gill, head kidney, spleen and liver, indicating its role in immune response. Two peptides (Lc-NK-lysin-1 and Lc-NK-lysin-2) divided from the core region of the Lc-NK-lysin mature polypeptide were chemically synthesized and their antibacterial activities were examined; the potential function on the inhibition of bacteria propagation was revealed. Our results suggested that Lc-NK-lysin is a typical member of the NK-lysin family and as an immune-related gene it involves in the immune response when pathogens invasion., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Trichodinid ciliophorans are opportunistic parasites of many species of fish, amphibians, and molluscs, but yet never reported in association with lesions in birds. Postmortem and histopathological evaluation of a commercial adult Toulouse gander and female goose, and a wild Mallard drake revealed the presence of severe pathological parasitic colonization of their reproductive tracts. Histopathological findings included moderate to severe granulocytic inflammation, acanthosis, accentuation of the rete pegs, and proliferative hyperplastic squamous metaplasia of the mucosa of the ejaculatory ducts and groove, sulcus spermaticus, glandular part of the phallus (cavum penis), and oviduct in association with large numbers of ciliated protozoa anchored to the tissues or free in the lumen. These protozoa had characteristic morphological features analogous to the family of Trichodinidae. The source of this parasitism could not be determined. To our knowledge, this is the first report of trichodinosis associated with pathology in birds.

We used a microarray approach to examine changes in gene expression in turbot peritoneal cells after injection of the fish with vaccines containing the ciliate parasite Philasterides dicentrarchi as antigen and one of the following adjuvants: chitosan-PVMMA microspheres, Freund́s complete adjuvant, aluminium hydroxide gel or Matrix-Q (Isconova, Sweden). We identified 374 genes that were differentially expressed in all groups of fish. Forty-two genes related to tight junctions and focal adhesions and/or actin cytoskeleton were differentially expressed in free peritoneal cells. The profound changes in gene expression related to cell adherence and cytoskeleton may be associated with cell migration and also with the formation of cell-vaccine masses and their attachment to the peritoneal wall. Thirty-five genes related to apoptosis were differentially expressed. Although most of the proteins coded by these genes have a proapoptotic effect, others are antiapoptotic, indicating that both types of signals occur in peritoneal leukocytes of vaccinated fish. Interestingly, many of the genes related to lymphocytes and lymphocyte activity were downregulated in the groups injected with vaccine. We also observed decreased expression of genes related to antigen presentation, suggesting that macrophages (which were abundant in the peritoneal cavity after vaccination) did not express these during the early inflammatory response in the peritoneal cavity. Finally, several genes that participate in the inflammatory response were differentially expressed, and most participated in resolution of inflammation, indicating that an M2 macrophage response is generated in the peritoneal cavity of fish one day post vaccination., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The parasite Ichthyophthirius multifiliis (Ich) has been reported in various freshwater fishes worldwide and results in severe losses to both food and aquarium fish production. The fish surviving natural infections or immunized with live theronts develop strong specific and non-specific immune responses. Little is known about how these immune genes are induced or how they interact and lead to specific immunity against Ichthyophthirius multifiliis in channel catfish Ictalurus punctatus. This study evaluated the differential expression of immune-related genes, including immunoglobulin, immune cell receptor, cytokine, complement factor and toll-like receptors in head kidney from channel catfish at different time points after immunization with live theronts of I. multifiliis. The immunized fish showed significantly higher anti-Ich antibody expressed as immobilization titer and ELISA titer than those of control fish. The vast majority of immunized fish (95%) survived theront challenge. Expression of IgM and IgD heavy chain genes exhibited a rapid increase from 4 hour (h4) to 2 days (d2) post immunization. Expression of immune cell receptor genes (CD4, CD8-α, MHC I, MHC II β, TcR-α, and TcR-β) showed up-regulation from h4 to d6 post immunization, indicating that different immune cells were actively involved in cellular immune response. Cytokine gene expression (IL-1βa, IL-1βb, IFN-γ and TNF-α) increased rapidly at h4 post immunization and were at an up-regulated level until d2 compared to the bovine serum albumin control. Expression of complement factor and toll-like receptor genes exhibited a rapid increase from h4 to d2 post immunization. Results of this study demonstrated differential expression of genes involved in the specific or non-specific immune response post immunization and that the vaccination against Ich resulted in protection against infection by I. multifiliis., (Published by Elsevier Ltd.)

Inorganic pyrophosphate (PPi) is a key metabolite in cellular bioenergetics under chronic stress conditions in prokaryotes, protists and plants. Inorganic pyrophosphatases (PPases) are essential enzymes controlling the cellular concentration of PPi and mediating intracellular pH and Ca(2+) homeostasis. We report the effects of the antimalarial drugs chloroquine (CQ) and artemisinin (ART) on the in vitro growth of Philasterides dicentrarchi, a scuticociliate parasite of turbot; we also evaluated the action of these drugs on soluble (sPPases) and vacuolar H+-PPases (H+-PPases). CQ and ART inhibited the in vitro growth of ciliates with IC50 values of respectively 74 ± 9 μM and 80 ± 8 μM. CQ inhibits the H+ translocation (with an IC50 of 13.4 ± 0.2 μM), while ART increased translocation of H+ and acidification. However, both drugs caused a decrease in gene expression of H+-PPases. CQ significantly inhibited the enzymatic activity of sPPases, decreasing the consumption of intracellular PPi. ART inhibited intracellular accumulation of Ca(2+) induced by ATP, indicating an effect on the Ca(2+) -ATPase. The results suggest that CQ and ART deregulate enzymes associated with PPi and Ca(2+) metabolism, altering the intracellular pH homeostasis vital for parasite survival and providing a target for the development of new drugs against scuticociliatosis., (© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.)

Rab GTPases, members of the Ras superfamily, encode monomeric G-proteins. Rab proteins regulate key steps in membrane traffic transport and endocytic pathway of host immune responses. Rab5A is involved in immune regulation, particularly in T cell migration and macrophage endocytosis in higher vertebrates. However, little is known of the molecular structure of Rab5A gene in marine teleost fish species and its expression profile during the parasite infection. In this study, the full-length cDNA sequence and genomic structure of Rab5A gene of the large yellow croaker (Larimichthys crocea) (LycRab5A), one of the most economical marine fishes, were identified and characterized. The LycRab5A protein, containing the ATPase/GTPase binding motifs and the effector molecules binding motifs, was highly homologous to that of other animals. The expression plasmid containing LycRab5A cDNA fused with GST was engineered and transformed into Escherichia coli to produce recombinant protein GST-LycRab5A, which was purified to prepare a polyclonal antibody specifically against LycRab5A. Subcellular localization revealed that LycRab5A expressed in the membrane and cytoplasm. Based on real-time PCR and Western blot analysis, we found that both mRNA and protein of LycRab5A were expressed in all tissues we examined; especially it was highly expressed in blood and gill. Interestingly, both mRNA and protein of LycRab5A were substantially up-regulated when parasitic ciliate protozoan (Cryptocaryon irritans) was infected. The expression of LycRab5A was reached to the maximal level at 24 h after infection. The line of evidence suggested that LycRab5A might play an important role in large yellow croaker defense against parasite infection. Moreover, on the basis of protein interaction, it was found that the LycRab5A interacted with myosin light chain (designated as LycMLC), a crucial protein in the process of phagocytosis. This discovery might contribute better understanding to the molecular events involved in fish immune responses., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Since malachite green was banned for using in food fish due to its carcinogenic and teratogenic effects on human, the search of alternative drug to treat Ichthyophthirius multifiliis becomes urgent. This study aimed to (1) evaluate the ethanol extracts of medicinal plants Cynanchum atratum, Zingiber officinale, Cynanchum paniculatum, immunostimulant (A), and immunostimulant (B) for their efficacy against I. multifiliis, and (2) determine effects of medicated feeds with C. atratum, Z. officinale, C. paniculatum, and immunostimulant (A) to treat I. multifiliis in grass carp. The results in this study showed that the minimum concentrations of C. atratum, Z. officinale, and C. paniculatum extracts for killing all theronts were 16, 8, and 16 mg/L, respectively. In vivo experiments, fish fed with medicated feeds of C. atratum for 10 days, or Z. officinale for 3 days, or combination of three plants for 10 days resulted in a significant reduction in the I. multifiliis infective intensity on grass carp after theronts exposure. Grass carp fed with medicated feeds of immunostimulant (A) for 21 days showed no infection and 100 % of survival 15 days post theronts exposure. Therefore, immunostimulant (A) is a promising feed supplement to treated I. multifiliis with good antiparasitic efficacy.

During routine histological examination of oysters (Crassostrea virginica) from Great Bay, New Hampshire, USA, a high prevalence and intensity of ciliate xenomas has been noted since sampling began in 1997. Xenomas are hypertrophic lesions on the gills of bivalve molluscs caused by intracellular ciliates, likely Sphenophrya sp. Although not known to cause mortality in oysters, xenomas have not previously been reported at this high abundance. The objectives of this study were to characterize the xenomas, describe the ciliates, and gather baseline epizootiological data with correlations to environmental and biological parameters. Upon gross examination, xenomas appeared as white nodules, up to 3mm in diameter, located in the gill tissue and occasionally fusing into large masses along the gill filaments. Light microscopy of histological sections revealed xenomas located in the gill water tubes, which they often completely blocked. Higher magnification revealed dual nuclei, eight kineties, and conjugation of the ciliates. Transmission electron microscopy revealed dual nuclei that varied in density, a maximum of twenty cilia in each kinety radiating from the oral apparatus to the posterior, and a 9+2 axoneme structure within the cilia. These traits place the ciliates into the Order Rhynchodida, but insufficient molecular data exist to confirm classification of this ciliate to the Genus Sphenophrya. Since 1997, xenoma prevalence has fluctuated with peaks in 2000, 2004, and 2011. Infected oysters generally contained <30 xenomas, but 2.1% contained >100, sharply contrasting the rare prevalence and low intensity reported elsewhere. Prevalence increased with oyster size, leveling off near 50% in oysters >60mm. Infection intensity peaked in 70-90mm oysters and declined in larger oysters. Individual oyster condition was not associated with xenoma intensity, but sites with oysters in higher condition generally had a greater prevalence and intensity of xenoma infections. Seasonal data indicated an infection cycle increasing from summer to fall, peaking at 55-65% in November and dropping to <10% by spring. The oyster population in Great Bay, NH warrants further examination to understand the mechanisms and conditions controlling xenoma formation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Cryptocaryon irritans, a species of obligatory ciliate ectoparasite, can infect various species of marine teleost fish. Cryptocaryon irritans that fall to the seabed or aquarium bottom in winter can form "dormant tomonts" and wake up when the temperature rises the next year. Abundant studies and analyses on the dormant tomonts were carried out at the transcriptome level, in order to investigate the molecular mechanism of C. irritans tomonts entering the dormant state under low-temperature conditions., Methods: The paired-end sequencing strategy was used to better assemble the entire transcriptome de novo. All clean sequencing reads from each of the three libraries (Group A: untreated blank control; Group B: treated for 24 h at 12 °C; and Group C: developed for 24 h at 25 °C) were respectively mapped back to the transcriptome assembly using the bioinformatics software., Results: In this study, 25,695,034, 21,944,467, and 28,722,875 paired-end clean reads were obtained respectively from the three cDNA libraries of the C. irritans tomont by Illumina paired-end sequencing technology. A total of 25,925 unique transcript fragments (unigenes) were assembled, with an average length of 839 bp. Differentially expressed genes (DEGs) were scrutinized; in Group B/A pairwise comparison, 343 genes presented differential expression, including 265 up-regulated genes and 78 down-regulated genes in Group B; in Group C/A pairwise comparison, there were 567 DEGs, including 548 up-regulated genes and 19 down-regulated genes in Group C; and in Group B/C pairwise comparison, 185 genes showed differential expression, including 145 up-regulated genes and 40 down-regulated genes in Group B., Conclusions: This is the first transcriptomic analytical study of the C. irritans tomonts under low temperature. It can be concluded that most of the genes required for its cell survival under low temperature, or for cell entry into a deeper dormancy state were discovered, and that they might be considered as candidate genes to develop the diagnostic and control measures for cryptocaryoniasis.

Galectins are a family of endogenous lectins with β-galactosides affinity, playing significant roles in the innate immunity of vertebrates and invertebrates. In this report, a new galectin-9 cDNA was identified and characterized in large yellow croaker Larimichthys crocea (designated as LcGal-9). The complete cDNA sequence of LcGal-9 was 1795 bp, with an open reading frame (ORF) of 1032 bp encoding 343 amino acids. The putative LcGal-9 protein contained two carbohydrate recognition domains (CRDs) connected by a linker peptide, with each carrying two conserved β-galactoside binding motifs H-NPR and WG-EE-, and it possessed neither a signal peptide nor a transmembrane domain. LcGal-9 protein shared 43-74% identity with galectin-9 sequences from other species. The qRT-PCR analysis revealed that LcGal-9 mRNA was constitutively expressed in all tissues examined, predominately expressed in liver, spleen, gill, kidney, head-kidney and intestine. Western blot analysis showed that LcGal-9 protein was highly expressed in liver, spleen, intestine, kidney, head-kidney, skin, gill, and heart, but not detected in muscle and plasma. LcGal-9 mRNA transcripts were induced by poly I:C in the liver (from 6 h to 48 h), spleen (at 12 h) and head-kidney (at 12 h and 24 h). In contrast, Vibrio parahaemolyticus caused a significant down-regulation in these three tissues, except for in spleen of 48 h and head-kidney of 3 h. Post-infection with Cryptocaryon irritans, the transcripts were dramatically up-regulated in gill, skin, spleen and head-kidney during initial infection period, while significant down-regulation afterward was also observed both in spleen and head-kidney. The recombinant LcGal-9 (named as rLcGal-9) purified from Escherichia coli BL21 (DE3) demonstrated hemagglutination against human, rabbit and L. crocea in a Ca(2+)-independent manner, which was inhibited by α-Lactose and LPS. The results of bacterial agglutination assays showed that rLcGal-9 was able to agglutinate Gram-negative bacteria V. alginolyticus and Aeromonas hydrophila in a Ca(2+)-independent manner. By immunohistochemistry assay, significant increases of LcGal-9 protein appeared in the spleen stimulated with poly I:C (for 12 h) and V. parahaemolyticus (for 48 h) compared with the control. Based on the collective data, LcGal-9 might play an important role in innate immune responses, especially defense against Gram-negative bacteria in L. crocea., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Trichodina heterodentata Duncan, 1977 and Ichthyophthirius multifiliis Fouquet, 1876 obtained from gills during a parasitological survey conducted for the protozoan parasitic fauna of Schizothorax niger a snow trout in Dal Lake, Kashmir, India during the period October 2013 and March 2015. Thirty out of 180 fish were found infected with protozoan parasites. During the study of their pathogenecity the most common deteriorating signs observed in gill tissue were necrosis, hypertrophy, hyperplasia and fusion of secondary lamellae. Prevalence of infection was found to be 16.66%. This is the first record of the protozoan fauna of the schizothoracines from Kashmir valley, India., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Water-soluble chlorophyll (chlorophyllin) exerts pronounced photodynamic activity on fish parasites. In order to determine its potential as a remedy against ectoparasites in fish carps were incubated in water with defined concentrations of chlorophyllin. The main focus of the experiments was on the ciliate Ichthyophthirius multifiliis (Fouquet) which is responsible for considerable losses in livestock in aquaculture. As malachite green, which in the past efficiently cured infected fishes, is banned because of its possible carcinogenicity; no effective remedy is presently available in aquaculture to treat ichthyophthiriasis. Using chlorophyllin, the number of trophonts was significantly reduced (more than 50 %) after 3 h incubation of infested fish at 2 and 4 mg/L and subsequent irradiation with simulated solar radiation. The lack of reinfection after light treatment indicates that also the remaining parasites have lost their multiplication capacity. In the controls (no chlorophyllin and no light, light but no chlorophyllin, or chlorophyllin but no light), no reduction of the I. multifiliis infection was observed. We propose that chlorophyllin (or other photodynamic substances) is a possible effective countermeasure against I. multifiliis and other ectoparasites in aquaculture.

H+-pyrophosphatases (H+-PPases) are integral membrane proteins that couple pyrophosphate energy to an electrochemical gradient across biological membranes and promote the acidification of cellular compartments. Eukaryotic organisms, essentially plants and protozoan parasites, contain various types of H+-PPases associated with vacuoles, plasma membrane and acidic Ca+2 storage organelles called acidocalcisomes. We used Lysotracker Red DND-99 staining to identify two acidic cellular compartments in trophozoites of the marine scuticociliate parasite Philasterides dicentrarchi: the phagocytic vacuoles and the alveolar sacs. The membranes of these compartments also contain H+-PPase, which may promote acidification of these cell structures. We also demonstrated for the first time that the P. dicentrarchi H+-PPase has two isoforms: H+-PPase 1 and 2. Isoform 2, which is probably generated by splicing, is located in the membranes of the alveolar sacs and has an amino acid motif recognized by the H+-PPase-specific antibody PABHK. The amino acid sequences of different isolates of this ciliate are highly conserved. Gene and protein expression in this isoform are significantly regulated by variations in salinity, indicating a possible physiological role of this enzyme and the alveolar sacs in osmoregulation and salt tolerance in P. dicentrarchi.

Large yellow croaker (Larimichthys crocea) is one of the most valuable marine fish in southern China. Given to the rapid development of aquaculture industry, the L. crocea was subjected to ciliate ectoparasite Cryptocaryon irritans. It therefore is indispensable and urgent to understand the mechanism of L. crocea host defense against C. irritans infection. In the present study, the extensively analysis at the transcriptome level for Cryptocaryoniasis in L. crocea was carried out. These results showed that 15,826,911, 16,462,921, and 15,625,433 paired-end clean reads were obtained from three cDNA libraries (A: 0 theronts/fish, B: 12,000 theronts/fish, and C: 24,000 theronts/fish) of the L. crocea immune-related tissues by Illumina paired-end sequencing technology. Totally, 30,509 unique transcript fragments (unigenes) were assembled, with an average length of 1715 bp. In B/A, C/A, and C/B pairwise comparison, 972, 900, and 1126 genes showed differential expression respectively. Differently expressed immune-related genes (DEIGs) were scrutinized, in B/A pairwise comparison, 48 genes showed differential expression, including 26 up-regulated genes and 22 down-regulated genes in B; in C/A pairwise comparison, there were 39 DEIGs, including 7 up-regulated genes and 32 down-regulated genes in C; in C/B pairwise comparison, 40 genes showed differential expression, including 11 up-regulated genes and 29 down-regulated genes in C. There were 16 DEIGs enriched KEGG pathways, in which the complement and coagulation cascades pathway was the top most DEIGs enriched pathway (B:A = 42; C:A = 28; C:B = 42). The coagulation and fibrinolytic system was in a highly active state after infected by C. irritans with non-lethal concentration; the alternative complement pathway may play an important role in the early stages of C. irritans infection. These results demonstrated that low-concentration infection can significantly induce the immunological response in fishes, however, when fishes were in fatal conditions, the immunity was suppressed., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

C-type lectins (CTLs) are generally recognized as a superfamily of Ca(2+)-dependent carbohydrate-binding proteins, which serve as pattern recognition receptors (PRRs) in innate immunity of vertebrates. In this study, the molecular characterization and immune roles of a novel CTL from Larimichthys crocea (designated as LcNTC) were investigated. LcNTC is a novel protein that shared 33%-49% homology with other teleosts CTLs. The full-length cDNA of LcNTC was composed of 859 bp with a 465 bp open reading frame encoding a putative protein of 154 residues. LcNTC contained a single CRD with four conserved disulfide-bonded cysteine residues (Cys(57)-Cys(148), Cys(126)-Cys(140)) and EPN/AND motifs instead of invariant EPN/WND motifs required for carbohydrate-binding specificity and constructing Ca(2+)-binding sites. LcNTC mRNA was detected in all examined tissues with the most abundant in the gill. After challenged with poly I:C and Vibrio parahaemolyticus, the temporal expression of LcNTC was significantly up-regulated in the liver, spleen and head-kidney. LcNTC transcripts were also induced in the gill, skin, spleen and head-kidney post-infection with Cryptocaryon irritans. The recombinant LcNTC (rLcNTC) purified from Escherichia coli BL21 (DE3) exhibited strong agglutination activity against erythrocytes from human, rabbit and large yellow croaker in a Ca(2+)-dependent manner, and the agglutination could be inhibited by D-Mannose, D-Glucose, D-Fructose, α-Lactose, D-Maltose and LPS. Positive microbial agglutination activities of rLcNTC were observed against all tested bacteria in the presence of Ca(2+), including Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus and Micrococcus lysoleikticus) and Gram-negative bacteria (E. coli, V. parahaemolyticus, Vibrio alginolyticus and Aeromonas hydrophila). These findings collectively indicated that LcNTC might be involved in the innate immunity of L. crocea as a PRR., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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 10 min-, 1 h-, 2 h-, 3 h- and 4 h-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) mg L(-1) against theronts of C. irritans and the 6h-LC50 (95% CI) of 3.0 (2.8-3.2) mg L(-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 3.1 mg L(-1) and 6.3 mg L(-1), respectively. In the test of exposing fish to 40 mg L(-1) compound SFrD for 24h, no visible effects were observed affecting the normal behavior or any macroscopic changes. By spectrum analysis (EI-MS, (1)H NMR and (13)C 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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The aim of this study was to compare the parasite communities in two sympatric host populations, Trachelyopterus coriaceus andTrachelyopterus galeatus, which were caught in tributary of the Amazon River in Brazil. All the specimens of T. galeatusand T. coriaceus were infected by one or more parasites, such as Ichthyophthirius multifiliis, Tripartiella tetramerii, Trichodina nobilis, Cosmetocleithrum striatuli, Contracaecumsp., Cystidicoloides sp., Dadaytremoides parauchenipteri and Gorytocephalus spectabilis. Seven species were common to both host fish, and there were 1-5 parasite species per host. In both hosts, trichodinids were dominant. Aggregate dispersion of ectoparasites and endoparasites was observed, with greater aggregation among endoparasites. Only the ectoparasites species showed differences in intensity and/or abundance. However, the parasite communities of the two hosts were taxonomically similar (99%) and characterized by high prevalence and high abundance of ectoparasites, but with low diversity, prevalence and abundance of endoparasites. Trachelyopterus galeatus, the host with the larger body size, presented greater variation of Brillouin diversity and evenness, while T. coriaceus had higher Berger-Parker dominance values and total numbers of parasites. This first study on these parasites ofT. galeatus and T. coriaceus showed that the life mode, size of the hosts and the availability of infective forms of the parasites were the main factors that influenced the parasite communities structure.

The large yellow croaker (Larimichthys crocea) is an economically important marine fish cultured in China and East Asian countries and is facing a serious threat from Cryptocaryon irritans, which is a protozoan ectoparasite that infects most reared marine fish species. To understand the molecular immune mechanisms underlying the response to C. irritans, we first performed a comparative gene transcription analysis using livers from C. irritans-immunized L. croceas and from a control group through RNA-Seq technology. After the removal of low-quality sequences and assembly, 51360 contigs were obtained, with an average length of 1066.93 bp. Further, a blast analysis indicates that 30747 contigs can be annotated based on homology with matches in the NT, NR, gene, and string databases. A gene ontology analysis was used to classify 21598 genes according to three major functional categories: molecular function, cellular component, and biological process. Moreover, 14470 genes were found in 303 KEGG pathways. We used RSEM and EdgeR to determine that 3841 genes were significantly differentially expressed (FDR < 0.001), including 2129 up-regulated genes and 1712 down-regulated genes. A significant enrichment analysis of these differentially expressed genes and isogenes revealed major immune-related pathways, including the toll-like receptor, complement and coagulation cascades, and chemokine signaling pathways. In addition, 28748 potential simple sequence repeats (SSRs) were detected from 12776 transcripts, and 62992 candidate single nucleotide polymorphisms (SNPs) were identified in the L. croceas liver transcriptome. This study characterized a gene expression pattern for normal and C. irritans-immunized L. croceas for the first time and not only sheds new light on the molecular mechanisms underlying the host-C. irritans interaction but also facilitates future studies on L. croceas gene expression and functional genomics., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

This paper describes cultivation methods for ciliates from the digestive tract of horses. Members of three different genera were successfully grown in vitro for short periods of time. However, only cells belonging to the genus Blepharocorys, which resides in the horse's large intestine, were maintained for longer periods. This Blepharocorys culture was successfully grown in vitro after inoculation of freshly excreted horse faeces in culture medium containing a population of bacteria. The ciliates survived for over six months, and the density of their population varied between 1.7 × 10(3) and 2.4 × 10(3) cells mL(-1). Favourable conditions for the prolonged cultivation of this ciliate were observed when the medium was prepared by mixing horse faeces and 'caudatum' salt solution in a 1:1 V/V ratio together with food (60% powdered meadow hay, 16% wheat gluten, 12% barley flour and 12% microcrystalline cellulose) supplied as 0.20 mg mL(-1) culture per day., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Infection by Ichthyophthirius multifiliis, a ciliated protozoan parasite, results in high fish mortality and causes severe economic losses in aquaculture. To find new, efficient anti-I. multifiliis agents, cynatratoside-C was isolated from Cynanchum atratum by bioassay-guided fractionation in a previous study. The present study investigated the anti-theront activity, determined the toxicity of cynatratoside-C to grass carp Ctenopharyngodon idellus and mammalian blood cells, and evaluated the protection of cynatratoside-C against I. multifiliis theront infection in grass carp. Results showed that all theronts were killed by 0.25 mg l-1 of cynatratoside-C in 186.7 ± 5.8 min. Cynatratoside-C at 0.25 mg l-1 was effective in treating infected grass carp and protecting naive fish from I. multifiliis infestation. The 96 h median lethal concentration (LC50) of cynatratoside-C to grass carp and 4 h median effective concentration (EC50) of cynatratoside-C to theront were 46.8 and 0.088 mg l-1, respectively. In addition, the hemolysis assay demonstrated that cynatratoside-C had no cytotoxicity to rabbit red blood cells. Therefore, cynatratoside-C could be a safe and effective potential parasiticide for controlling I. multifiliis.

To clarify the effects of a Cryptocaryon irritans infection on the stress, antioxidant and mucosal immune response of the large yellow croaker Pseudosciaena crocea, this study utilized C. irritans at dose of 12,000 (group I); 24,000 (group II); and 36,000 (group III) theronts/fish to infect large yellow croaker weighing 100 ± 10 g. The food intake, survival and relative infection intensity (RII); levels of reactive oxygen species (ROS), malondialdehyde (MDA) and vitamin C (VC), activities of super oxide dismutase (SOD) and catalase (CAT) in liver; variation patterns of lysozyme (LZM), alkaline phosphatase (AKP), complement component 3 (C3) and immunoglobulin M (IgM) levels in the body surface mucus at different time points after infection were compared. These results showed that with the increase of the infection dose and the passage of time, the food intake and survival of the fish gradually decreased. The final survival of the control group (0 theronts/fish), group I, group II, and group III was 100, 100, 96.67 ± 5.77, and 48.33 ± 7.64. Group I, II, and III stopped feeding respectively on the third, third and second days after infection. RII increased significantly with increased infection dose. The RII of the control group, group I, group II, and group III was 0, 0.73 ± 0.06, 1.30 ± 0.26, and 1.84 ± 0.02. With the infection dose increased, ROS contents showed an overall upward trend; MDA contents of the group I, group II and group III did not show significant changes at any timepoint compared with the control group; Activities of SOD and CAT and the overall VC levels in the liver of P. crocea dropped; LZM activity showed an overall upward trend; AKP activity increased first then dropped at each timepoint with its highest level appearing at group II; Complement C3 and IgM levels in body surface mucus were significantly increased. In conclusion, P. crocea has a strong ability to resist oxidative stress caused by the infection of C. irritans. The body surface mucus of P. crocea contains high levels of immune factors, which presented a rapid and significant response to the infection of C. irritans., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

CCR6 have been demonstrated playing an important role in immune cells homing to mucosal tissues, mediating antigen presentation and immune response in mammals. CCR6 in lower vertebrate leukocyte homing has not yet been revealed. Cryptocaryon irritans is believed to be a good pathogen model for skin and gill mucosal immunity. In this study, we identified two CCR6s and their three possible ligands CCL20 like cDNA sequences, designated as grouper EcCCR6A, EcCCR6B, EcCCL20L1, EcCCL20L2 and EcCCL20L3. It is interesting to find that EcCCR6A has a longer second extracellular loop than EcCCR6B, which is more similar to mammalian CCR6. Tissue distribution analysis showed that EcCCR6A pronouncedly dominates in gill and brain while EcCCR6B dominates in head kidney, trunk kidney and thymus. Three chemokine ligands have their own distinct expression pattern in health grouper tissues. EcCCL20L1 dominates in spleen and head kidney, EcCCL20L2 dominates in gill and thymus, whereas EcCCL20L3 dominates in skin and brain. The expression patterns of these chemokines and chemokine receptors were detected in C. irritans infected grouper and the results showed that EcCCR6A, EcCCR6B and EcCCL20L1 were significantly up-regulated in the skin of C. irritans infected fish, which indicated these two chemokine receptors and their ligand may play important role in immune cells' homing to skin mucosal immune tissues under pathogen caused inflammation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A database with all available published accounts of the ciliate parasite species of Mexican fishes was assembled. This information, along with records derived from own recent research, allow generating a checklist containing all the records, which is a necessary first step to address future questions in the areas of ecology, evolutionary biology and biogeography of these host-parasite associations. The checklist is presented as a parasite-host list, and a host-parasite list. The checklist contains 30 nominal species, from 9 genera and 8 families of ciliate parasites. Most of the primary records were done for exotic fish species, artificially introduced to Mexico for aquaculture purposes; however, recent works have been conducted in diverse species of native fishes. Excepting one, all the ciliate species listed here have been previously recorded for diverse fish species from different localities around the world. Based on the amount of information contained in this checklist, much more effort is necessary to accurately know the diversity of species of this type of parasites in fish fauna of Mexico.

The large yellow croaker Larimichthys crocea is an important mariculture fish species in China, and the bacterium Vibrio harveyi (V. harveyi) and the ciliate protozoan Cryptocaryon irritans (C. irritans) are the two major pathogens in its aquaculture sector. Interferon-gamma (IFN-γ) plays important roles in regulating both innate and cell mediated immune responses as an inflammatory cytokine. In this study, we obtained the nucleotide sequence of IFN-γ from the large yellow croaker (LcIFN-γ). The phylogenetic relationship tree of 18 available IFN-γ genes was constructed based on their sequences. Expression analyses in 10 various tissues were conducted after the croaker challenged with V. harveyi and C. irritans, respectively. Real time PCR analysis showed that the expression of LcIFN-γ was observed broadly in health individuals. After injected with V. harveyi, the 10 tissues had a higher expression of IFN-γ at the first day (1 d); only spleen, muscle, intestine, heart and skin had higher expressions after infected with C. irritans at 1 d. Major immune tissues (skin, gill, head kidney and spleen) and detoxification tissue (liver) were sampled at 0 h, 6 h, 1 d, 2 d, 3 d, 4 d, 5 d and 7 d to understand the expression trends of LcIFN-γ after challenged with C. irritans. The expressions of LcIFN-γ in skin and gill (the primary immune organs) showed a clear correlative relationship with the life cycle of C. irritans., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLR) are key components of innate immunity that play significant roles in immune defense against pathogens invasion. Recent frequent outbreaks of the "white spot disease" caused by parasitic infection in farmed Tibetan fishes had resulted in great economic losses. However, to our knowledge, the roles of TLRs in mediating immune response to parasitic infection in Tibetan fishes remain to be determined. Here, we performed data-mining on a widely-farmed Tibetan fish (Gymnocypris przewalskii or Gp) transcriptome to determine the genetic variation and expression pattern of TLRs. We totally obtained 14 GpTLRs and identified 5 with a complete coding sequence. Phylogenetic analysis verified their identities and supported the classification of TLRs into six families as in other vertebrates. The TLR family motifs, such as leucine rich repeat (LRR) and Toll/interleukin (IL)-1 receptor (TIR) domain, are conserved in GpTLR1-5. Selective pressure test demonstrated that all known GpTLRs are under purifying selection, except GpTLR4 underwent positive selection. Further, site model analysis suggested that 11 positively selected sites are found in LRR domain of GpTLR4. Three positively selected sites are located on outside surface of TLR4 3D structure, indicating that function of GpTLR4 may be affected. Tissue specific expression analysis showed all GpTLRs are present in gill, head-kidney and spleen but the relative abundance varied among tissues. In response to parasite Ichthyophthirius multifiliis infection, 5 GpTLR (GpTLR1, -2, -4, -9 and -20) expressions were induced. Intriguingly, GpTLR4 was significantly up-regulated in gills, while GpTLR19 and GpTLR21 unexpectedly showed no any change. In summary, these results revealed the first genomic resources of TLR family and several parasitic infection responsive TLRs in Tibetan fish. These findings provide key information for future studies aiming to understand the molecular mechanisms underlying the immune response to pathogen invasion in Tibetan fishes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

BCA2, as an E3 ubiquitin ligase, is an important anti-virus immune factor in mammals. Up to date, there are not any related reports on BCA2 protein in fishes yet. In the present investigation, BCA2 in large yellow croaker Larimichthys crocea (named as LcBCA2) was identified and characterized. The full-length cDNA of LcBCA2 was 1571 bp, including an ORF of 888 bp encoding a polypeptide of 295 amino acids. The putative LcBCA2 protein contained a RING-H2 motif at C terminal. The LcBCA2 transcripts were broadly distributed in all detected tissues, with high expression in muscle, moderate in blood, skin, heart, liver and spleen, weak in other tissue as indicated by qPCR analysis. Significant increases were observed in skin, gill and spleen after infection of Cryptocaryon irritans, and in spleen and head-kidney after inactivated Vibrio. parahaemolyticus, LPS and Poly I:C stimulations. Tissue localization by in-situ hybridization showed that LcBCA2 mainly expressed in the spleen of the fish in the test group. Our findings showed that LcBCA2 inclined to sharply increase in immune organs, especially in head-kidney after bacterial and viral stimulations, while in locations (skin and gill) of parasites infections, suggesting that BCA2 may play an important role in fish defense against bacteria, virus and parasites infections, but the immune mechanisms is are different., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

TBK1 has been extensively studied in mammals because of its important roles as a molecular bridge, linking the TLRs (TLR3 and TLR4) and RLRs signals to activate transcriptional factors IRF3 and IRF7 for IFN-I production. However, the information on molecular and functional characteristics of TBK1 in teleosts is limited. In this study, the molecular characterization and immune response of TBK1 in Larimichthys crocea (named as LcTBK1) as well as its interaction with Nrdp1 were investigated. Sequence analysis demonstrated that LcTBK1 included four functional motifs, the N-terminal protein kinase domain and ATP-binding site, middle ULD and C-terminal coiled-coil domain. The tissue expression profiles indicated that LcTBK1 gene was constitutively expressed in the twelve tissues examined, with high expression in brain. Temporal expression analysis showed that LcTBK1 mRNA was obviously increased in the liver after injection of LPS, Poly I:C and inactive Vibrio parahaemolyticus, however, declined at some time points in spleen and head-kidney. Furthermore, we found that LcTBK1 can interact with LcNrdp1, an E3 ubiquitin ligase that involved in immune response to Cryptocaryon irritans infection in L. crocea. The qPCR showed that LcNrdp1 was also significantly up-regulated in liver, down-regualted at some time points in spleen and head-kidney after LPS, Poly I:C and inactive V. parahaemolyticus injection, although the expression patterns of the two genes after the three treatments were different in change magnitude and up-regulation timespan. These results suggested that LcTBK1 was involved in L. crocea defense against the pathogen infection and can be regulated by Nrdp1 in PPRs signaling pathway of fishes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

This study investigated the immunization by intraperitoneal injection (i.p.) (assay I) and immersion bath (assay II) with live theronts of Ichthyophthirius multifillis in Rhamdia quelen and its influence on the hemato-immunological and biochemical parameters. Fish were divided in control (non immunized no challenged); non immunized and challenged with 12,000 theronts/fish; non immunized and challenged with 22,000 theronts/fish; immunized and challenged with 12,000 theronts/fish; immunized and challenged with 22,000 theronts/fish. Six days after challenge, either in the assay I or in the assay II the prevalence of I. multifillis in the gills was higher in non immunized fish (33.33% and 27.77%, respectively). In the assay I showed higher numbers of thrombocytes, leukocytes, lymphocytes, neutrophils and monocytes 20 days after injection and lower numbers after challenge. The immunoglobulin values were higher in fish non immunized. Fish immunized by immersion bath (assay II) showed greater values of catalase (CAT) in the liver (1245.49 U/mgprt) when compared to i.p. (198.79 U/mgprt). The levels of CAT in the liver of fish from the assay II were greater (1738.47 U/mgprt) 14 days after immunization than that observed 21 days after (1114.26 U/mgprt). The vaccination by i.p method showed influence on the hematological parameters. On the other hand, the immersion bath vaccination showed greater influence on the catalase activity in the liver. The results showed that new parameters like total protein, immunoglobulin and antioxidant enzymes could be considered in evaluating the host response to infection., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In this study, molecular characteristics of march5b and co-expression of march5b and tlr7 in response to the infection of Cryptocaryon irritans in the large yellow croaker Larimichthys crocea were investigated. The full-length complementary (c)DNA of march5b was 1314 bp, including an open reading frame of 846 bp encoding a polypeptide of 281 amino acids, and the full-length genomic sequence was composed of 23,577 nucleotides, including six exons and five introns. The putative March5b protein contained a RINGv motif and four transmembrane domains. The march5b transcripts were broadly distributed in all detected tissues, with a strong expression in blood, brain and gills, and a weak expression in kidney by quantitative PCR analysis. The expression of march5b and tlr7 in the skin, gills, spleen and head kidney changed in the same manner at most time points post-primary infection with C. irritans. Significant increase was observed in the skin with march5b at days 2 and 3 by 26.10 and 6.88 fold, respectively, and with tlr7 at day 3 by 57.68 fold, when compared with the control. Their expressions, however, were decreased in the gills, especially at day 3 (march5b by 8.9%, tlr7 by 22.06%). In the spleen and head kidney, march5b and tlr7 transcripts were up-regulated early, then noticeably declined at day 3. These results suggested that march5b and tlr7 are co-expressed in response to parasite infection and March5b probably catalyses ubiquitination of some proteins of TLR7 signalling pathway., (© 2015 The Fisheries Society of the British Isles.)

Case Report: Mortality of northern corroboree frog tadpoles and eggs occurred in association with Tetrahymena-like ciliates. The predominant lesions in the tadpoles were inflammation and necrosis of the dermis and skeletal muscle. Some of the egg capsules also contained ciliates, but were overgrown with bacteria and fungi., Conclusion: Disease occurred, secondary to underlying husbandry issues, and resolved following their correction., (© 2015 Australian Veterinary Association.)

Schizothorax o'connori and Oxygymnocypris stewartii are two endangered endemic Tibetan fishes that thrive in the Lhasa River at an average altitude over 4000 m. During artificial reproduction of endemic Tibetan fishes, the juvenile fish of S. o'connori and O. stewartii experienced mass mortality event. The causative agent is diagnosed to be a ciliate parasite, Chilodonella piscicola (syn. C. cyprini), which is common in various fishes. Here, we supplemented its description based on the morphological and molecular data. The body of C. piscicola is oval, 30-60 × 25-40 μm in vivo. Cyrtos is hook-like, composed of 9-10 toothed nematodesmal rods. Somatic kineties usually contain seven right kineties and nine left kineties. Two parallel circumoral kineties revolve round the cyrtos, and one preoral kinety extends to the anterior end of the fourth left-most kinety. Terminal fragment kinety is linear and on the top left of dorsal side. Sequence alignments revealed that the present SSU rDNA and ITS1-5.8S-ITS2 sequences are both most similar to the sequences of C. uncinata with the similarities of 98.2 and 99.5%. The phylogenetic analyses showed that C. piscicola is sister to other Chilodonella species, whereas C. cyprini (FJ873805) cluster with Tetrahymena species. Molecular analysis shows that the ITS1-5.8S-ITS2 sequence of C. cyprini in GenBank is unreliable. Our study extended the host range of C. piscicola and supplemented and revised the molecular data. Besides, as far as we know, this is the first record of C. piscicola in Tibetan plateau.

In this study we describe the anti-Trichodina effects of teflubenzuron (TFB) for Oreochromis niloticus and for Piaractus mesopotamicus. We also evaluated the acute toxicity, for both species, by using TFB in the concentrations of 700.0, 800.0, 900.0 and 1000.0 mg L(-1) and a control, without the drug. To assess the efficacy of TFB against Trichodina spp., we used the concentrations of 30.0 or 50.0 mg L(-1) for one hour exposure in tilapia, and the concentration of 30.0, 50.0 and 80.0 mg L(-1) for one hour and 50 mg L(-1) for two hours exposures in pacu. Teflubenzuron did not present significant toxicity in either species, with LC50;48h > 1000.0 mg L(-1). The drug effectiveness was observed against four identified Trichodina species: T. magna, T. heterodentata, T. compacta and T. centrostrigeata, with 87.9% parasite reduction with one hour exposure to 50.0 mg L(-1) TFB on O. niloticus and 96.1% with two hours exposure to 50.0 mg L(-1) TFB on P. mesopotamicus. Teflubenzuron is a drug with potential to be used in Brazilian aquaculture; it attends to important requirements, such as low toxicity and high efficacy in controlling Trichodina spp. infection in O. niloticus and P. mesopotamicus., (Copyright © 2015 Elsevier Inc. All rights reserved.)

To investigate the response of pompano fish (Trachinotus ovatus) to white spot disease, we used the protozoan Cryptocaryon irritans to infect live 450-g specimens at concentrations of 40,000 theronts/fish. We assessed the relative infection intensity (RII), serum immobilizing titer, and immunity-related enzyme activities (ACP, AKP, LZM), and assessed feeding, serum ion concentrations (Na(+), Cl(-), Ca(2+) and K(+)) and blood biochemistry (ALT, AST, LDH) of pompano. The fish were then treated with a lethal dose of C. irritans (70,000 theronts/fish) and the number of deaths was recorded. We found that the relative infection intensities of the control group, group I, and group II were 0, 0.630 ± 0.179, and 0.014 ± 0.006. Poly-infection induced a significant increase in the serum immobilizing titer (853.33 ± 295.60) of group II. In terms of the biochemical assessment, group II had significantly higher alkaline phosphatase and acid phosphatase activities than the other groups, and the lowest lysozyme activity (P < 0.05), compared to higher activity in the control group and the highest level in group I. Only the fishes of group I had stopped feeding after treatment. The concentrations of Na(+), Cl(-), and Ca(2+) in blood serum did not differ significantly among the three groups, but K(+) concentration increased with the increasing infection frequency. Alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase activities in fish of group II were significantly higher than those of the other groups. Survival of the fish subjected to the lethal dose of C. irritans was 0, 0, and 100 in groups control, I, and II, respectively. In conclusions, based on the food intake of group II, along with the results of relative infection intensity, serum immobilizing titer, and survival, we speculate that the fish in that group acquired high protective immunity following poly-infection by C. irritans, experiencing limited harm for pompano., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The present study was conducted to evaluate the anti-parasitic activity of a pure compound from Streptomyces sp. HL-2-14 against fish parasite Ichthyophthirius multifiliis, and elucidate its chemical structure. By electron ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance spectrum (1H NMR and 13C NMR), the compound was identified as amphotericin B (AmB). The in vitro trials revealed that AmB can effectively kill the theronts and tomonts of I. multifiliis with the median lethal concentration (LC50) of 0·8 mg L-1 at 30 min for the theronts and 4·3 mg L-1 at 2 h for the tomonts, respectively. AmB at 5 mg L-1 significantly reduced I. multifiliis infectivity prevalence and intensity on grass carp (Ctenopharyngodon idella), and consequently decreased fish mortality, from 100% in control group to 30% in treated group. The 72 h acute toxicity (LC50) of AmB on grass carp was 20·6 mg L-1, but fish mortality was occurred when exposure to 13·0 mg L-1. These results indicated that AmB was effective in the therapy of I. multifiliis infection, but the safety concentration margin is relatively narrow. Further efforts aiming to decrease the toxicity and improve the therapeutic profile remain to be needed.