To React or Not to React: The Dilemma of Fish Immune Systems Facing Myxozoan Infections.

Myxozoans are microscopic, metazoan, obligate parasites, belonging to the phylum Cnidaria. In contrast to the free-living lifestyle of most members of this taxon, myxozoans have complex life cycles alternating between vertebrate and invertebrate hosts. Vertebrate hosts are primarily fish, although they are also reported from amphibians, reptiles, trematodes, mollusks, birds and mammals. Invertebrate hosts include annelids and bryozoans. Most myxozoans are not overtly pathogenic to fish hosts, but some are responsible for severe economic losses in fisheries and aquaculture. In both scenarios, the interaction between the parasite and the host immune system is key to explain such different outcomes of this relationship. Innate immune responses contribute to the resistance of certain fish strains and species, and the absence or low levels of some innate and regulatory factors explain the high pathogenicity of some infections. In many cases, immune evasion explains the absence of a host response and allows the parasite to proliferate covertly during the first stages of the infection. In some infections, the lack of an appropriate regulatory response results in an excessive inflammatory response, causing immunopathological consequences that are worse than inflicted by the parasite itself. This review will update the available information about the immune responses against Myxozoa, with special focus on T and B lymphocyte and immunoglobulin responses, how these immune effectors are modulated by different biotic and abiotic factors, and on the mechanisms of immune evasion targeting specific immune effectors. The current and future design of control strategies for myxozoan diseases is based on understanding this myxozoan-fish interaction, and immune-based strategies such as improvement of innate and specific factors through diets and additives, host genetic selection, passive immunization and vaccination, are starting to be considered.

Know your enemy - transcriptome of myxozoan Tetracapsuloides bryosalmonae reveals potential drug targets against proliferative kidney disease in salmonids.

The myxozoan Tetracapsuloides bryosalmonae is a widely spread endoparasite that causes proliferative kidney disease (PKD) in salmonid fish. We developed an in silico pipeline to separate transcripts of T. bryosalmonae from the kidney tissue of its natural vertebrate host, brown trout (Salmo trutta). After stringent filtering, we constructed a partial transcriptome assembly T. bryosalmonae, comprising 3427 transcripts. Based on homology-restricted searches of the assembled parasite transcriptome and Atlantic salmon (Salmo salar) proteome, we identified four protein targets (Endoglycoceramidase, Legumain-like protease, Carbonic anhydrase 2, Pancreatic lipase-related protein 2) for the development of anti-parasitic drugs against T. bryosalmonae. Earlier work of these proteins on parasitic protists and helminths suggests that the identified anti-parasitic drug targets represent promising chemotherapeutic candidates also against T. bryosalmonae, and strengthen the view that the known inhibitors can be effective in evolutionarily distant organisms. In addition, we identified differentially expressed T. bryosalmonae genes between moderately and severely infected fish, indicating an increased abundance of T. bryosalmonae sporogonic stages in fish with low parasite load. In conclusion, this study paves the way for future genomic research in T. bryosalmonae and represents an important step towards the development of effective drugs against PKD.

[Discovery of Putative Anti-Kudoa septempunctata Chemical Agents by Comprehensive Assay].

We screened 360 chemicals and discovered that 71 chemicals had anti-Kudoa septempunctata effect. Especially 19 and seven of 71 chemicals were antibiotics and antibacterial agents/disinfectants, respectively. The other 45 chemicals were pesticides, natural toxins, industrial chemicals and medicines for non-infectious diseases. Nineteen antibiotics that possessed anti-Kudoa effect contained four tetracyclines, one steroid, two macrolides, one aminoglycoside, three ß-lactams, one quinolone, two rifamycines, one polyene, one novobiocine, one sulfonamide and two nitroimidazoles. To use these drugs for prevention of Kudoa infection, the further study is need for the determination of effective dose.

Effect of Epigallocatechin Gallate on Viability of Kudoa septempunctata.

Kudoa septempunctata have been reported as a causative agent for acute transient gastrointestinal troubles after eating raw olive flounder (Paralichthys olivaceus). It raised public health concerns and quarantine control in several countries. Quantitative evaluation on viability of K. septempunctata is crucial to develop effective chemotherapeutics against it. A cytometry using fluorescent stains was employed to assess effect of three compounds on viability of K. septempunctata. Epigallocatechin gallate reduced markedly viability of K. septempunctata at 0.5 mM or more, and damaged K. septempunctata spores by producing cracks.

Sodium chloride treatment effects on rainbow trout suffering from proliferative kidney disease caused by Tetracapsuloides bryosalmonae.

The aim of the present study was to evaluate the effect of a long-term sodium chloride bath on rainbow trout Oncorhynchus mykiss naturally infected by Tetracapsuloides bryosalmonae. A total of 106 infected fish were divided into 2 groups. One group was left untreated and the other was treated with sodium chloride in increasing doses up a concentration of 0.8%. After 14 d, treatment was stopped and for a further 7 d the fish response to the sodium chloride bath was observed. Cumulative mortality was significantly lower in the treated group (19.2%) compared to the untreated group (31.5%) after 21 d. This corresponded to the lower but non-significant parasite intensity in kidney and spleen in the treated group after 14 d of treatment. However, lower prevalence of parasites in both tissues was recorded in the untreated group after 21 d of treatment, but a significant difference was observed only in spleen tissue. Furthermore, significant increases in leukocytes, hemoglobin, haematocrit, ferric reducing ability of plasma, and ceruloplasmin, and significant decreases in alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase activities were noticed in the treated group compared to the untreated group. In contrast, significant decreases in lysozyme concentration in the mucus and phagocyte oxidative burst in the blood were observed in the treated group. Histopathological examination revealed proliferative and reparative changes in parenchymatous tissues in the treated group. The 14- and 21-d salt bath used in rainbow trout with proliferative kidney disease was associated with a reduction in mortality and enhanced the reparative phase in the treated group.

Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets.

BACKGROUND: The constant increase of aquaculture production and wealthy seafood consumption has forced the industry to explore alternative and more sustainable raw aquafeed materials, and plant ingredients have been used to replace marine feedstuffs in many farmed fish. The objective of the present study was to assess whether plant-based diets can induce changes in the intestinal mucus proteome, gut autochthonous microbiota and disease susceptibility of fish, and whether these changes could be reversed by the addition of sodium butyrate to the diets. Three different trials were performed using the teleostean gilthead sea bream (Sparus aurata) as model. In a first preliminary short-term trial, fish were fed with the additive (0.8%) supplementing a basal diet with low vegetable inclusion (D1) and then challenged with a bacteria to detect possible effects on survival. In a second trial, fish were fed with diets with greater vegetable inclusion levels (D2, D3) and the long-term effect of sodium butyrate at a lower dose (0.4%) added to D3 (D4 diet) was tested on the intestinal proteome and microbiome. In a third trial, the long-term effectiveness of sodium butyrate (D4) to prevent disease outcome after an intestinal parasite (Enteromyxum leei) challenge was tested. RESULTS: The results showed that opposed forces were driven by dietary plant ingredients and sodium butyrate supplementation in fish diet. On the one hand, vegetable diets induced high parasite infection levels that provoked drops in growth performance, decreased intestinal microbiota diversity, induced the dominance of the Photobacterium genus, as well as altered the gut mucosal proteome suggesting detrimental effects on intestinal function. On the other hand, butyrate addition slightly decreased cumulative mortality after bacterial challenge, avoided growth retardation in parasitized fish, increased intestinal microbiota diversity with a higher representation of butyrate-producing bacteria and reversed most vegetable diet-induced changes in the gut proteome. CONCLUSIONS: This integrative work gives insights on the pleiotropic effects of a dietary additive on the restoration of intestinal homeostasis and disease resilience, using a multifaceted approach.

In vitro effect of two commercial anti-coccidial drugs against myxospores of Kudoa septempunctata genotype ST3 (Myxozoa, Multivalvulida).

Kudoa septempunctata (Myxozoa: Multivalvulida) myxospores infect the trunk muscles of olive flounder (Paralichthys olivaceus). In this study, two popular commercially formulated anti-coccidial drugs (amprolium hydrochloride and toltrazuril) were serially diluted and incubated with purified mature Kudoa septempunctata myxospores. The viability of K. septempunctata spores was determined after a 2-day incubation followed by Hoechst 33342 and propidium iodide staining, and scanning electron microscopy. Amprolium hydrochloride significantly decreased spore viability (18% of control) at a concentration of 920 µg/mL, whereas toltrazuril showed almost no effect (83% of control). Viability of the control (untreated spores) was 90%. In vivo studies are required to confirm the efficacy of amprolium hydrochloride in fish infected with K. septempunctata myxospores on their growth and immune system performance.

Inosine-arginine salt as a promising agent for in vitro activation of waterborne fish-pathogenic myxozoan actinospores.

Mucus-derived nucleosides serve as key host cues for myxozoan actinospore fish host recognition, but to date their use for experimental actinospore activation in the laboratory or application in disease prevention has not progressed very far. One obstacle has been the low solubility of pure inosine and guanosine. To overcome this, we used inosine-arginine salt (ino-arg), which incorporates both high activation properties and high solubility. We tested its efficacy both in microassays directly observing reactions of actinospores of 2 distantly related myxozoan species, Myxobolus cerebralis and M. pseudodispar in comparison to inosine, as well as its actinospore-inactivation properties by premature polar capsule discharge in an infection experiment. Ino-arg was considerably more effective in eliciting polar capsule discharge and sporoplasm emission at much lower concentrations than pure inosine and, in contrast to the latter, remained dissolved in aqueous solution. Ino-arg exposure of M. pseudodispar actinospores resulted in polar capsule discharge and sporoplasm emission before host contact and subsequently in a lower infection rate in roach Rutilus rutilus.

A Chitinase from Aeromonas veronii CD3 with the potential to control myxozoan disease.

BACKGROUND: The class Myxosporea encompasses about 2,400 species, most of which are parasites of fish and cause serious damage in aquaculture. Due to the concerns about food safety issues and limited knowledge of Myxozoa life cycle and fish immune system, no chemicals, antibiotics or immune modulators are available to control myxozoa infection. Therefore, little can be done once Myxozoa establishment has occurred. METHODOLOGY/PRINCIPAL FINDINGS: In this paper we isolated Aeromonas veronii CD3 with significant myxospore shell valve-degrading ability from pond sediment. A 3,057-bp full-length chitinase gene was consequently cloned, and the corresponding mature, recombinant chitinase (ChiCD3) produced by Escherichia coli had substantial chitinase activity. The deduced sequence of ChiCD3 contained one catalytic domain, two chitin-binding domains, and one putative signal peptide. ChiCD3 had an optimal activity at 50°C and pH 6.0, and retained more than 50% of its optimal activity under warm water aquaculture conditions (∼30°C and pH ∼7.0). After incubation with ChiCD3, 38.0±4.8% of the myxospores had damaged shell valves, whereas myxospores incubated with commercially available chitinases remained intact. CONCLUSION/SIGNIFICANCE: This study reveals a new strategy to control myxozoan disease. ChiCD3 that has capacity to damage the shell valve of myxospores can be supplemented into fish feed and used to control Myxozoa-induced diseases specifically.

The effect of lectins on the attachment and invasion of Enteromyxum scophthalmi (Myxozoa) in turbot (Psetta maxima L.) intestinal epithelium in vitro.

The involvement of the lectin/carbohydrate interaction in the invasion of the turbot intestinal epithelium by Enteromyxum scophthalmi was studied in vitro using explants of turbot intestine and pre-treatment of parasite stages with the plant lectins of Canavalia ensiformis (Con A) and Glycine max (SBA). Both lectins inhibited the attachment and invasion of E. scophthalmi stages to the intestinal epithelium, though the inhibitory effect was higher for SBA than for Con A. Such results point to the involvement of N-acetyl-galactosamine (GalNAc) and galactose (Gal) residues and also of mannose/glucose residues in the E. scophthalmi-intestinal epithelium interaction. The inhibitory effect of both lectins on the parasite adhesion and penetration points to the interest of further studies to confirm the presence of putative lectins recognising GalNAc-Gal and mannose/glucose residues in turbot intestine. The obtained results demonstrated also the adequacy of turbot intestinal explants as an in vitro model to study the interaction with E. scophthalmi.