Transcriptome analysis of amyloodinium ocellatum tomonts revealed basic information on the major potential virulence factors

The ectoparasite protozoan Amyloodinium ocellatum (AO) is the etiological agent of amyloodiniosis in European seabass (Dicentrarchus labrax) (ESB). There is a lack of information about basic molecular data on AO biology and its interaction with the host. Therefore, de novo transcriptome sequencing of AO tomonts was performed. AO trophonts were detached from infested ESB gills, and quickly becoming early tomonts were purified by Percoll\uae density gradient. Tomont total RNA was processed and quality was assessed immediately. cDNA libraries were constructed using TruSeq\uae Stranded mRNA kit and sequenced using Illumina sequencer. CLC assembly was used to generate the Transcriptome assembly of AO tomonts. Out of 48,188 contigs, 56.12% belong to dinophyceae wherein Symbiodinium microadriaticum had 94.61% similarity among dinophyceae. Functional annotations of contigs indicated that 12,677 had associated GO term, 9005 with KEGG term. The contigs belonging to dinophyceae resulted in the detection of several peptidases. A BLAST search for known virulent factors from the virulence database resulted in hits to Rab proteins, AP120, Ribosomal phosphoprotein, Heat-shock protein70, Casein kinases, Plasmepsin IV, and Brucipain. Hsp70 and casein kinase II alpha were characterized in-silico. Altogether, these results provide a reference database in understanding AO molecular biology, aiding to the development of novel diagnostics and future vaccines

Comparative Therapeutic Effects of Natural Compounds Against Saprolegnia spp. (Oomycota) and Amyloodinium ocellatum (Dinophyceae)

The fish parasites Saprolegnia spp. (Oomycota) and Amyloodinium ocellatum (Dinophyceae) cause important losses in freshwater and marine aquaculture industry, respectively. The possible adverse effects of compounds used to control these parasites in aquaculture resulted in increased interest on the search for natural products with antiparasitic activity. In this work, eighteen plant-derived compounds (2′,4′-Dihydroxychalcone; 7-Hydroxyflavone; Artemisinin; Camphor (1R); Diallyl sulfide; Esculetin; Eucalyptol; Garlicin 80%; Harmalol hydrochloride dihydrate; Palmatine chloride; Piperine; Plumbagin; Resveratrol; Rosmarinic acid; Sclareolide; Tomatine, Umbelliferone, and Usnic Acid) have been tested in vitro. Sixteen of these were used to determine their effects on the gill cell line G1B (ATCC®CRL-2536™) and on the motility of viable dinospores of Amyloodinium ocellatum, and thirteen were screened for inhibitory activity against Saprolegnia spp. The cytotoxicity results on G1B cells determined that only two compounds (2′,4′-Dihydroxychalcone and Tomatine) exhibited dose-dependent toxic effects. The highest surveyed concentrations (0.1 and 0.01 mM) reduced cell viability by 80%. Upon lowering the compound concentration the percentage of dead cells was lower than 20%. The same two compounds revealed to be potential antiparasitics by reducing in a dose-dependent manner the motility of A. ocellatum dinospores up to 100%. With respect to Saprolegnia, a Minimum Inhibitory Concentration was found for Tomatine (0.1 mM), Piperine and Plumbagin (0.25 mM), while 2′,4′-Dihydroxychalcone considerably slowed down mycelial growth for 24 h at a concentration of 0.1 mM. Therefore, this research allowed to identify two compounds, Tomatine and 2′,4′-Dihydroxychalcone, effective against both parasites. These compounds could represent promising candidates for the treatment of amyloodiniosis and saprolegniosis in aquaculture. Nevertheless, further in vitro and in vivo tests are required in order to determine concentrations that are effective against the considered pathogens but at the same time safe for hosts, environment and consumers

Host-Parasite Interaction between Parasitic Cymothoid Ceratothoa Oestroides and Its Host, Farmed European Sea Bass (Dicentrarchus labrax)

Parasitic isopod Ceratothoa oestroides (Cymothoidea, Isopoda) is a common and generalist buccal cavity-dweller in marine fish, recognised for its detrimental effect in fingerling and juvenile farmed European sea bass (Dicentrarchus labrax). Although distributed throughout the Mediterranean, the isopod provokes acute outbreaks mainly limited to particular endemic areas in Croatia (Adriatic Sea) and Greece (Aegean Sea). While numerous studies have previously evidenced its gross effect on farmed fish (i.e. decreased condition index, slower growth rate, lethargy and mortality), details on the host-parasite interaction are still lacking. Therefore, using a multimethodological approach, we closely examined the structure and appearance of isopod body parts acting in the attachment and feeding (stereomicroscopy, scanning and transmission electron microscopy), and the extent of host tissues damage (histology, immunohistochemistry, micro-computational tomography) induced by parasitation. Interestingly, while hematophagous nature of the parasite has been previously postulated we found no unambiguous data to support this; we observed host tissues fragmentation and extensive hyperplasia at the parasitation site, and no structures indicative of heme detoxifying mechanisms in the parasite gut, or other traces of a blood meal. The bacterial biofilm covering C. oestroides mouthparts and pereopods suggests that the isopod may play a role in conveying secondary pathogens to the infected host, or alternatively, it serves the parasite in normal interaction with its environment

Red mark syndrome of trout (Oncorhynchus mykiss; Walbaum, 1792): Histopathological scoring and correlation with gross lesions

Red mark syndrome (RMS) is a skin disorder affecting rainbow trout (Oncorhynchus mykiss). The present work aimed to correlate the gross skin lesions affecting 46 fish sampled from farms surveyed for RMS with their microscopic features, identifying histological parameters that may be suggestive of disease progression. Skin lesions were grossly included in one of three categories (types I, II and III) according to the progressive degree of severity. Histological parameters and anti-proliferating cell nuclear antigen (PCNA) tissue immunoreactivity were semi-quantitatively assessed. In the dermis, PCNA-positive lymphocytes, fibroblasts and endothelial cells were indicative of active phlogosis. A significant increase in PCNA-immunoreactive lymphocytes, from gross type I to type III cases, was found only in the hypodermis. The histological parameters significantly associated with the gross lesion severity were progressive loss of the epithelium and scales, recruitment of inflammatory cells in the stratum compactum, loss of architecture of the stratum compactum, perivascular and perineural granulomatous inflammation and increase in lymphocyte infiltration of the muscular layer. In the type II and type III categories, inflammation in the hypodermis and muscle displayed a granulomatous pattern, reinforcing the hypothesis of an immunopathological mechanism. The morphological diagnosis of “deep chronic dermatitis associated to panniculitis and myositis, characterised by lympho-histiocytic and granulomatous reaction” is suggested

Immune profiling of rainbow trout (Oncorhynchus mykiss) exposed to Lactococcus garvieae: Evidence in asymptomatic versus symptomatic or vaccinated fish

Lactococcosis, caused by the Gram-positive bacterium Lactococcus garvieae, is a major concern in rainbow trout (Oncorhynchus mykiss) farms, which are regularly affected by outbreaks especially during the summer/fall months. In these farms, unvaccinated healthy and symptomatic fish can coexist with vaccinated fish. In the present study, innate (leukogram, serum lysozyme activity, peroxidase activity, antiprotease activity, bactericidal activity, total IgM and total proteins), and specific immune parameters (serum antibodies to L. garvieae) were assessed in unvaccinated adult rainbow trout naturally exposed to the pathogen, with or without evidence of clinical signs, or subjected to vaccination. Blood was drawn from all three groups, and blood smears were prepared. Bacteria were found in the blood smears of 70% of the symptomatic fish but not in any of the asymptomatic fish. Symptomatic fish showed lower blood lymphocytes and higher thrombocytes than asymptomatic fish (p ≤.05). Serum lysozyme and bactericidal activity did not vary substantially among groups; however, serum antiprotease and peroxidase activity were significantly lower in the unvaccinated symptomatic group than in the unvaccinated and vaccinated asymptomatic groups (p ≤.05). Serum total proteins and total immunoglobulin (IgM) levels in vaccinated asymptomatic rainbow trout were significantly higher than in unvaccinated asymptomatic and symptomatic groups (p ≤.05). Similarly, vaccinated asymptomatic fish produced more specific IgM against L. garvieae than unvaccinated asymptomatic and symptomatic fish (p ≤.05). This preliminary study provides basic knowledge on the immunological relationship occurring between the rainbow trout and L. garvieae, potentially predicting health outcomes. The approach we proposed could facilitate infield diagnostics, and several non-specific immunological markers could serve as reliable indicators of the trout's innate ability to fight infection

Variation in the molecular weight of Photobacterium damselae subsp. piscicida antigens when cultured under different conditions in vitro

The antigenicity of Photobacterium damselae (Ph. d.) subsp. piscicida, cultured in four different growth media [tryptone soya broth (TSB), glucose-rich medium (GRM), iron-depleted TSB (TSB + IR-), and iron-depleted GRM (GRM + IR-)] was compared by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis using sera obtained from sea bass (Dicentrarchus labrax) raised against live or heat-killed Ph. d. subsp. piscicida. The antigenic expression of Ph. d. subsp. piscicida was found to differ depending on the culture medium used. A significantly higher antibody response was obtained with iron-depleted bacteria by ELISA compared with non-iron depleted bacteria obtained from the sera of sea bass raised against live Ph. d. subsp. piscicida. The sera from sea bass raised against live bacteria showed a band at 22 kDa in bacteria cultured in TSB + IR- or GRM+ IR- when bacteria that had been freshly isolated from fish were used for the screening, while bands at 24 and 47 kDa were observed with bacteria cultured in TSB or GRM. When bacteria were passaged several times on tryptic soya agar prior to culturing in the four different media, only bands at 24 and 47 kDa were recognized, regardless of the medium used to culture the bacteria. It would appear that the molecular weight of Ph. d. subsp. piscicida antigens change in the presence of iron restriction, and sera from sea bass infected with live bacteria are able to detect epitopes on the antigens after this shift in molecular weight

In vivo morphological and antigenic characteristics of Photobacterium damselae subsp. piscicida

The present study was conducted to examine the morphology and antigenicity of Photobacterium damselae subsp. piscicida by culturing the bacterium in vivo in the peritoneal cavity of sea bass (Dicentrarchus labrax) within dialysis bags with either a low molecular weight (LMW) cut-off of 25 kDa or a high molecular weight (HMW) cut-off of 300 kDa. Differences were observed in the growth rate between the bacteria cultured in vivo or in vitro. Bacteria cultured in vivo were smaller and produced a capsular layer, which was more prominent in bacteria cultured in the HMW bag. Antigenicity was examined by Western blot analysis using sera from sea bass injected with live Ph. d. subsp. piscicida. The sera recognised bands at 45 and 20 kDa in bacteria cultured in vivo in the LMW bag. Bacteria cultured in vivo in the HMW bag did not express the 45 kDa band when whole cell extracts were examined, although the antigen was present in their extracellular products. In addition, these bacteria had a band at 18 kDa rather than 20 kDa. Differences in glycoprotein were also evident between bacteria cultured in vitro and in vivo. Bacteria cultured in vitro in LMW and HMW bags displayed a single 26 kDa band. Bacteria cultured in the LMW bag in vivo displayed bands at 26 and 27 kDa, while bacteria cultured in vivo in the HMW bag possessed only the 27 kDa band. These bands may represent sialic acid. The significance of the changes observed in the bacterium's structure and antigenicity when cultured in vivo is discussed

Time and concentration dependency of MacroGard® induced apoptosis

In previous studies an effect of β-glucan on apoptosis in fish was noted and in this investigation we determine the time and concentration dependency of this effect. Primary cell cultures of pronephric carp cells were incubated for 6, 24, 48 h with various concentrations ranging from 0 to 1000 μg/ml of MacroGard® β-glucan. Apoptosis was monitored via acridine orange staining. Results indicate a clear effect of time and concentration on the induction of apoptosis in vitro, with only concentration ≥500 μg/ml causing significantly higher percentages of apoptotic cells. Apoptosis was detected after 6 h. This concentration dependent effect has to be considered when studying apoptosis in relation to immunostimulation

X-linked myotubular myopathy is associated with epigenetic alterations and is ameliorated by HDAC inhibition

X-linked myotubular myopathy (XLMTM) is a fatal neuromuscular disorder caused by loss of function mutations in MTM1. At present, there are no directed therapies for XLMTM, and incomplete understanding of disease pathomechanisms. To address these knowledge gaps, we performed a drug screen in mtm1 mutant zebrafish and identified four positive hits, including valproic acid, which functions as a potent suppressor of the mtm1 zebrafish phenotype via HDAC inhibition. We translated these findings to a mouse XLMTM model, and showed that valproic acid ameliorates the murine phenotype. These observations led us to interrogate the epigenome in Mtm1 knockout mice; we found increased DNA methylation, which is normalized with valproic acid, and likely mediated through aberrant 1-carbon metabolism. Finally, we made the unexpected observation that XLMTM patients share a distinct DNA methylation signature, suggesting that epigenetic alteration is a conserved disease feature amenable to therapeutic intervention

Egg-laying and locomotory screens with C. elegans yield a nematode-selective small molecule stimulator of neurotransmitter release

Nematode parasites of humans, livestock and crops dramatically impact human health and welfare. Alarmingly, parasitic nematodes of animals have rapidly evolved resistance to anthelmintic drugs, and traditional nematicides that protect crops are facing increasing restrictions because of poor phylogenetic selectivity. Here, we exploit multiple motor outputs of the model nematode C. elegans towards nematicide discovery. This work yielded multiple compounds that selectively kill and/or immobilize diverse nematode parasites. We focus on one compound that induces violent convulsions and paralysis that we call nementin. We find that nementin stimulates neuronal dense core vesicle release, which in turn enhances cholinergic signaling. Consequently, nementin synergistically enhances the potency of widely-used non-selective acetylcholinesterase (AChE) inhibitors, but in a nematode-selective manner. Nementin therefore has the potential to reduce the environmental impact of toxic AChE inhibitors that are used to control nematode infections and infestations