Quantitative proteomic analysis in serum of Takifugu rubripes infected with Cryptocaryon irritans.

Cryptocaryon irritans can cause cryptocaryonosis (white spot disease) in marine fish but the pathogenesis of the disease is unclear. In this work, we used high-throughput proteomics to identify differentially expressed proteins in the serum of Takifugu rubripes challenged with C. irritans. By using quantitative proteomic assays combined with Tandem Mass Tag-labeled quantitative proteomic analysis, we identified a total of 2088 differentially abundant proteins (1706 proteins were quantified, p < 0.05, fold-change threshold ≥ 2), including 21 up-regulated and 44 down-regulated. Combined with STRING-based functional analysis, we ultimately obtained eight proteins including glucokinase-like, integrin beta-1-like isoform X2, H4, H2A.V, histone H1-like, histone H2AX-like, histone H2B 1/2-like and myosin-9 isoform X1, which could be considered as potential biomarkers for T. rubripes immune responses. Eight proteins that were selected to validate significant differentially expressed genes at the proteomic level were consistent with qPCR at the transcriptomic level. The proteins identified in our work may serve as candidates for elucidating the molecular mechanism of cryptocaryonosis in T. rubripes. Our collective findings could provide new insights into searching for disease-specific targets and biomarkers, which may be effective indicators of C. irritans infection in T. rubripes.

Serum adenosine deaminase and xanthine oxidase activities in silver catfish naturally infected with Ichthyophthirius multifiliis: The influence of these enzymes on inflammatory and oxidative status.

Adenosine deaminase (ADA) activity, through adenosine (Ado) levels, as well as xanthine oxidase (XO) activity through uric acid levels exerts an essential role on immune and inflammatory responses during infectious diseases. Thus, the aim of this study was to evaluate the involvement of seric ADA and XO activities in the inflammatory and oxidative status of silver catfish naturally infected with Ichthyophthirius multifiliis. Seric ADA activity decreased, while Ado levels increased in infected animals compared to uninfected animals. Moreover, the seric XO activity increased in infected animals compared to uninfected animals, alongside the seric levels of uric acid, metabolites of nitric oxide (NOx) and reactive oxygen species (ROS). Based on this evidence, the downregulation of seric ADA activity exerts an anti-inflammatory profile, contributing to restricting the inflammatory process. The most important finding is that upregulation of seric XO activity leads to an excessive formation of uric acid, which contributes to oxidative and inflammatory processes. Moreover, uric acid induces the release of pro-inflammatory and pro-oxidative mediators, such NOx and ROS, which contribute directly to disease pathogenesis. In summary, the upregulation of XO activity may be considered a pathway involved in NOx and ROS production in silver catfish infected with I. multifiliis.

The Essential Oil of Hyptis mutabilis in Ichthyophthirius multifiliis Infection and its Effect on Hematological, Biochemical, and Immunological Parameters in Silver Catfish, Rhamdia quelen.

This study evaluated the activity of leaf essential oil (EO) from Hyptis mutabilis as well as its major constituent, (-)-globulol, in infections by the parasite Ichthyophthirius multifiliis (ich). Effects on hematological, biochemical, and immunological parameters of silver catfish, Rhamdia quelen, exposed to the same samples also were evaluated. In the first experiment, naturally infected fish were treated with EO (0, 10, and 20 mg · L-1) and ethanol, using several methods of exposure. Fish mortality and the number of trophonts per fish were assessed after 48 and 96 hr. Hour-long daily baths resulted in optimal survival, so this methodology was used for the second experiment, in which infected animals were exposed to (-)-globulol at 2.5 and 5 mg · L-1. The most effective concentrations in Experiments 1 and 2 were chosen for Experiment 3, in which healthy animals were subjected to hour-long daily baths with EO (20 mg · L-1) or (-)-globulol (2.5 mg · L-1). Additionally, an in vitro experiment was performed with EO and globulol at the same concentrations of the in vivo test. EO and (-)-globulol increased the survival of fish infected with ich and altered certain hematological and biochemical parameters. After 4 days, levels of hematocrit, erythrocytes, and leukocytes increased significantly in healthy animals exposed to EO. Exposure to (-)-globulol increased leukocyte number alone. No significant differences in nonspecific immunological parameters were detected when treated groups were compared to controls, but the leukocytosis observed in EO- and globulol-treated healthy animals indicates that EO and (-)-globulol increased innate immunity in these fish. An in vitro antiparasitic effect was observed in both samples.

Hemato-immunological and biochemical parameters of silver catfish Rhamdia quelen immunized with live theronts of Ichthyophthirius multifiliis.

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.

Immunological, ionic and biochemical responses in blood serum of the marine fish Trachinotus ovatus to poly-infection by Cryptocaryon irritans.

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.

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection.

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non-stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 µg per mg protein) compared with CI-, NI- and I-treated groups (94.5, 64 and 62.3 µg mg(-1), respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re-infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3-75%, higher than in the non-exposed control (40.6% mortality).

Dietary supplementation with chitin and chitosan on haematology and innate immune response in Epinephelus bruneus against Philasterides dicentrarchi.

The present study investigated the effect of 1.0% chitin and chitosan supplementation diets on haematology and immune response in Kelp grouper, Epinephelus bruneus against protozoan parasite, Philasterides dicentrarchi. The red blood cells (RBC), white blood cells (WBC), haemoglobin levels, lymphocytes, monocytes, and neutrophils significantly increased in kelp grouper fed with chitin or chitosan enriched diets against P. dicentrarchi. The mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), and thrombocytes did not significantly change against pathogen. The phagocytic activity, respiratory burst activity, complement activity, antiprotease activity, and α2-macroglobulin were significantly enhanced in fish fed with 1% chitin and chitosan diet on weeks 2 and 4. The lysozyme activity, total protein, and myeloperoxidase activity significantly increased in fish fed with chitin or chitosan supplementation diet from weeks 1 to 4 against pathogen. The cumulative mortality was found low in fish fed with chitin and chitosan enriched diets than those of control against pathogen. The present study suggests that supplementation of 1.0% chitin or chitosan in diets positively enhances immune response and affords disease resistance in kelp grouper, E. bruneus against P. dicentrarchi infection.

A novel protein isolated from the serum of rabbitfish (Siganus oramin) is lethal to Cryptocaryon irritans.

The susceptibility of eight marine fish species cultured in South China were tested for infection by the parasitic ciliate, Cryptocaryon irritans, via a challenge examination and an immobilization assay. All species of fish (representing six different families) that we investigated were infected by C. irritans except the rabbitfish (Siganus oramin), which displayed resistance to C. irritans infection. The infection intensity of rabbitfish (0.92+/-0.97, p<0.05) was significantly lower while the immobilization titres of rabbitfish serum were significantly higher (44.51+/-22.98, p<0.05) than the other seven species of fish. Additionally, the serum of the rabbitfish presented a strong killing effect to C. irritans in vitro. Light microscopy, scanning electron microscopy and fluorescence microscopy confirmed that rabbitfish serum could induce the theront cilia fall off, rupture of the cell membrane because of the swell and rupture of the macronucleus. Rabbitfish serum could also induce the rupture of the trophont membrane and content efflux. Herein a novel antiparasitic protein (APP) was isolated and purified from the serum of rabbitfish (S. oramin) by using a series of salting-out, cation exchange chromatography and two step of reversed phase high performance liquid chromatography (RP-HPLC). Analysis of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that APP was a homogenous polymeric protein with an N-terminal amino acid sequence of SSVEKNLAACLRDND. Its monomeric molecular mass, determined by matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometer (MALDI-TOF-TOF-MS), was found to be 61,739.87 Da. Results of homology analyses indicated that this protein was a newly discovered functional protein in the rabbitfish serum. Laser confocal fluorescence microscopy conformed that the action site of the APP was mainly on the cell membrane and nucleus of theront, which agreed with the results of light microscopy, fluorescence microscopy and scanning electron microscopy. These findings suggest that this protein may contribute considerably to the innate host defence mechanism to combat microbes of the rabbitfish.

Chemotactic responses of the fish-parasitic scuticociliate Philasterides dicentrarchi to blood and blood components of the turbot Scophthalmus maximus, evaluated using a new microplate multiassay.

This study describes a new capillary-type microplate multiassay for characterization of protozoal chemotactic responses, allowing up to 32 assays to be run simultaneously. We used the new multiassay to evaluate the chemoattractant activity of turbot blood components and turbot cells for the facultative parasite Philasterides dicentrarchi, which is responsible for significant losses in turbot farming. Preliminary tests indicated that the assay requires 3-4 h for detection of chemoattractant activity, that it can be performed effectively using the ciliate axenic culture medium, and that it distinguishes clearly between different concentrations of chemoattractant. Application of the assay indicated that whole blood and serum from normal turbot, and especially infected turbot, have strong chemoattractant activity for P. dicentrarchi trophozoites, whereas neither turbot blood cells nor other turbot cells nor bacteria were significant chemoattractants. These results raise the possibility that turbot serum components are involved in host detection and host invasion by P. dicentrarchi, in line with previous findings indicating that turbot with skin lesions show increased susceptibility to P. dicentrarchi infection.