Urteekstrakter som bæredygtigt bekæmpelsesmiddel mod parasitter i økologisk fiskeopdræt

Opdræt af regnbueørred, særligt økologisk ørred, er udfordret på grund af gælle- og hudparasitinfektioner forårsaget af en række forskellige patogener. Det drejer sig især om mikroskopiske flagellater, fimredyr samt en række amøber. Da flere andre typer parasitter er følsomme over for indholdsstoffer i hvidløg, oregano og timian kunne ekstrakter fra disse krydderurter måske være en del af løsningen også for dambrugsfisk. Vore laboratorieforsøg viste, at fiskeparasitter faktisk var følsomme over for et eller flere indholdsstoffer i hvidløg, oregano og timian. Men det viste sig også, at det kun var timian og en biologisk sæbe fra bakterien Pseudomonas, som blev veltolereret af fiskene, mens ekstrakter af oregano og hvidløg ikke blev vel modtaget af regnbueørrederne. Timian-ekstrakt havde således antiparasitisk effekt, idet snylterne på fiskens hud døde og løsnedes fra både hud, finner og gæller. En andet stof, isoleret fra Pseudomonas H6 bakterien, som er en biologisk sæbe (en surfaktant, et overfladeaktivt molekyle), viste sig også at være effektiv mod flere typer parasitter

A non-lethal method for detection of <i>Bonamia ostreae</i> in flat oyster (<i>Ostrea edulis</i>) using environmental DNA

Surveillance and diagnosis of parasitic Bonamia ostreae infections in flat oysters (Ostrea edulis) are prerequisites for protection and management of wild populations. In addition, reliable and non-lethal detection methods are required for selection of healthy brood oysters in aquaculture productions. Here we present a non-lethal diagnostic technique based on environmental DNA (eDNA) from water samples and demonstrate applications in laboratory trials. Forty oysters originating from Limfjorden, Denmark were kept in 30 ppt sea water in individual tanks. Water was sampled 6 days later, after which all oysters were euthanized and examined for infection, applying PCR. Four oysters (10%) were found to be infected with B. ostreae in gill and mantle tissue. eDNA purified from the water surrounding these oysters contained parasite DNA. A subsequent sampling from the field encompassed 20 oysters and 15 water samples from 5 different locations. Only one oyster turned out positive and all water samples proved negative for B. ostreae eDNA. With this new method B. ostreae may be detected by only sampling water from the environment of isolated oysters or isolated oyster populations. This non-lethal diagnostic eDNA method could have potential for future surveys and oyster breeding programs aiming at producing disease-free oysters.publishedVersio

Comparative In Vitro and In Vivo Effects of Feed Additives on Rainbow Trout Response to Ichthyophthirius multifiliis

Control of the skin parasitic ciliate Ichthyophthirius multifiliis is currently based on laborious chemical and mechanical approaches, thus advocating for alternative control measures. Here, we show that the early development of trophonts (feeding stage residing in the epidermis) in the Rainbow Trout Oncorhynchus mykiss epidermis was inhib-ited at 5 d postinfection after the administration of feed additives, including garlic Allium sativum, oregano Origanum onites, thyme Thymus vulgaris, coriander Coriandrum sativum, and astaxanthin (a carotenoid derived from the alga Haematococcus pluvialis). However, no inhibition was observed at 8 d postinfection. We investigated whether the early inhibition was caused by (1) the feed additives’ direct effect on the parasite and/or (2) stimulation of the Rainbow Trout immune responses. Garlic exhibited the highest in vitro killing capacity toward theronts (the infective stage of the parasite), followed by oregano, thyme, and astaxanthin, whereas coriander had no in vitro parasiticidal effect. Immune reactions were measured by plasma lysozyme activity in Rainbow Trout after feeding and by recording immune gene expression in trout leukocytes that were exposed to feed additives. Oregano-fed fish showed a signifi-cantly (P < 0.05) elevated plasma lysozyme activity. Oregano and thyme—and to a lesser extent, garlic, astaxanthin, and coriander—induced a significant regulation of innate and adaptive immune genes in vitro. The results suggest that the investigated additives inhibit early parasite development directly as well as indirectly (i.e., by elevating the host immune response)

Acute immune responses in zebrafish and evasive behavior of a parasite – who is winning?

The protozoan parasite Ichthyophthirius multifiliis is an economically important parasite for the aquaculture- and ornamental fish industry. The parasite is abundant worldwide and infects the skin, gills and fins of freshwater fish species. For approximately the last fifty years the innate and protective immune mechanisms induced by I. multifiliis have been in focus in different fish hosts. By utilizing transgenic zebrafish, new tools to investigate this have emerged. The aim of this study was therefore to elucidate early immune responses in zebrafish larvae by using gene expression and in vivo imaging of neutrophil and macrophage behavior during infection. For the first time, zebrafish larvae were infected with the parasite and infection dynamics, parasite size and host-parasite interactions were investigated. Results showed that the larvae responded with mild inflammation and that the 12 compared to 5 days post fertilization larvae were significantly less susceptible. It was furthermore observed that neutrophils and macrophages were attracted to the parasites and that neutrophils reacted with neutrophil extracellular traps (NETs) when fighting the parasite. The parasite was rotating vigorously, presumably to impede the neutrophils and macrophages from attaching to it but on rare occasions, neutrophils and macrophages were able to kill the parasite. Based on these observations, we concluded that the parasite uses the rotation as an immune evasive strategy and that the zebrafish larvae respond with high activity from neutrophils and macrophages locally but systemically only with mild inflammation

Lipopeptide surfactant killing of Ichthyophthirius multifiliis: Mode of action

The lipopeptide SPH6 isolated from the bacterial strain Pseudomonas H6 has surfactant properties and shows a lethal effect on all external stages of the problematic gill parasitic ciliate Ichthyopthirius multifiliis. The mode of action has remained enigmatic, but in the present study we have elucidated the mechanisms. I. multifiliis tomonts were exposed to a range of SPH6 concentrations (from 2.5 to 160 µg/mL), until the total motility inhibition of cells, cytoplasm and cilia. The parasites were subsequently examined by histology, light and scanning electron microscopy and enzyme activity assays (19 enzymes). The surfactant did not interfere with enzyme reactivity at any concentration, but at SPH6 concentrations 40 µg/mL and above the parasite cells became de-ciliated. Perforations were observed in the plasma membrane, and their origin and function may be associated with surfactant damage of the cell membrane or areas for extrusome release. Additional killing mechanisms may be involved, because lower concentrations were lethal as well, although the effect on density of cilia was not as pronounced. The study suggests that one mode of action mechanism is the surfactant attachment to and penetration of the parasite plasma membrane followed by detachment of cilia

Gill amoebae from freshwater rainbow trout (Oncorhynchus mykiss): In vitro evaluation of antiparasitic compounds against Vannella sp.

Gill parasitic infections challenge farming of rainbow trout (Oncorhynchus mykiss, Walbaum) in freshwater facilities. Apart from flagellates (Ichthyobodo, (Pinto) and ciliates (Ichthyophthirius (Fouquet), Ambiphrya (Raabe), Apiosoma (Blanchard), Trichodinella (Sramek-Husek) and Trichodina (Ehrenberg)), we have shown that amoebae are prevalent in Danish trout farms. Gills were isolated from farmed rainbow trout in six fish farms (conventional and organic earth pond and recirculated systems) and placed on non-nutrient agar (NNA) moistened with modified Neff's amoeba saline (AS) (15°C). Gill amoebae from all examined fish colonized the agar and were identified based on morphological criteria showing species within the genera Trinema (Dujardin) (family Trinematidae), Vannella (Bovee) (family Vannellidae). In addition, hartmannellid amoebae were recorded. We established a monoculture of Vannella sp., confirmed the genus identity by PCR and sequencing and performed an in vitro determination of antiparasitic effects (dose–response studies) of various compounds including sodium chloride (NaCl), hydrogen peroxide, peracetic acid, formalin, aqueous garlic and oregano extracts and a Pseudomonas H6 surfactant. All amoebae were killed in concentrations of 16.90 mg/ml (garlic), 17.90 mg/ml (oregano), NaCl (7.5 mg/ml), hydrogen peroxide (100 µg/ml), peracetic acid (0.03 µg/ml), formaldehyde (25 µg/ml) and the Pseudomonas H6 surfactant (250 µg/ml)