Effects of antiparasitic treatment for argulosis on innate immune system of a cyprinid fish (Fathead Minnow; Pimephales promelas, Rafinesque 1820)

Parasitic diseases in European aquaculture continue to pose economic and ecological threats to farmed and wild fish populations. The primary infection with ectoparasites can open the door to secondary or super- infections caused by bacterial and viral pathogens. Specifically, the infection with Argulus foliaceus (fish louse) in freshwater cyprinid fish such as common carp (Cyprinuscarpio) can increase production losses in affected aquaculture operations, or stocked water bodies. However, current veterinary drug use regulations in Germany limit the range of medications approved as treatment against ectoparasites in freshwater fish. A comparable parasite Lepeoptheirussalmonis (salmon louse) treatment with Diflubenzuron, Ivermectin and Doramectin (pesticides), has been approved in Salmon aquaculture in various countries. In order to control parasite populations, fish farmers frequently reach for non-approved but available treatments without veterinary supervision, possibly leading to consequences such as environmental damage, emergence of drug resistance or unwanted side effects on cultured and wild fish. The focus of this study is to investigate possible side effects of Diflubenzuron, Ivermectin, and Doramectin treatments on the innate immune system of a cyprinide fish. The overall research objective is to determine which antiparasitic drug and in what concentration and dose would present an effective treatment of the A. foliaceus without causing measurable side effects on fish neutrophil function. The first specific aim was to determine in vitro effects of commercial formulations of diflubenzuron, ivermectin and doramectin on fathead minnow (Pimephales promelas) neutrophil function. Treatment effects in vitro on oxidative burst, degranulation, and neutrophil extracellular trap (NETs) release were studied. Application of ivermectin and doramectin (in estimated plasma concentrations of 308 and 125 ng/ml respectively) caused no significant stimulation of oxidative burst, degranulation of primary granules and NETs release. However, application of diflubenzuron (in estimated plasma concentrations of 200 ng/ml) caused a significant stimulation of oxidative burst. Diflubenzuron treated neutrophils showed up to three time’s higher activity than the non-treated control. The second specific aim of the study was establishing a cell culture protocol of fathead minnow liver, spleen and kidney cells. In order to examine whether an incubation of the cells in culture of 48 hours with Diflubenzuron would alter the RNA expression in these organs, no significant change in expression of the tested genes (glutathione reductase, glutathione synthase, catalase, glutathione-s-transferase, catalase and CYP A1) compared to a control could be proven. The observed effect indicates that the tested antiparasitic compounds have the potential to interfere with disease resistance in fish populations by modulating immune responses during treatment. Therefore, further study is required to find optimal therapeutic dose for effective and safe treatment against ectoparasites in cyprinide fish. The following conclusions can be drawn from the presented findings: In vitro studies on the innate immune system as the here presented neutrophil studies can give reliable first answers to questions concerning in vivo diseases and treatments. With studies on the gene expression, far-reaching and profound negative effects can be ruled out, when such investigations remain without significant results, as shown here.