When diseases in fish are discovered, the fish show either clinical signs or behavioural changes. At this timepoint fish are morbid and decreased welfare and economic loss may be a consequence for production facilities. Therefore, prophylaxis and early detection of the pathogens before clinical disease signs occur could present the facility managers with the possibility to intervene or treat the disease before clinical outcome. For the first time, environmental DNA/RNA (collectively eNA) has been used to detect the salmonid pathogen Aeromonas salmonicida subsp. salmonicida in the water hosting rainbow trout (Oncorhynchus mykiss) under controlled experimental conditions before clinical outcome and mortality occurred. Using a standard curve, the concentration of bacteria in the water was determined at the early stage of disease and when fish became morbid to determine the window of possible intervention. Furthermore, the acute phase protein, serum amyloid a, produced by the rainbow trout was detected in the water and was found to be up-regulated four days post challenge. It was thereby shown that it is possible to detect strong immune responses in the fish in a non-invasive manner. The detection of eNA from water can be used as a tool to detect pathogens in the early phase before disease becomes severe facilitating interventions to limit disease and increase welfare of the fish, which is becoming a requirement for sustainable animal production.
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.
The host-pathogen interactions are tripartite and include the host, the pathogen, and the environment. For a long time, attention has mainly been addressed to the host's immune mechanisms and the pathogen's virulence factors while the environmental factor has received less interest. About a decade ago, studies on environmental DNA (eDNA) started to emerge as this approach is versatile for numerous research fields and interest is persistently growing. In the present study, the relationship between disease progression and pathogen water environmental DNA level was investigated. To perform this investigation, an infection model was setup using zebrafish and the gram-negative bacteria Vibrio anguillarum. Two different fish densities and bacterial concentrations were used to determine different patterns of pathogen eDNA in the fish tanks. Water sampling to collect eDNA was conducted alongside with observation of the fish Clinical Signs, mortalities, and immune responses. Water eDNA relative abundance of the pathogen could be determined directly after fish challenge, when Clinical Signs occurred, and when mortality started. Moreover, immune-relevant gene expression in zebrafish gills showed that il-1β, il-8, il-10, nf-κb and saa correlated to the pathogen eDNA signal, depending on fish density and bacterial concentration. In conclusion, this study demonstrated that water eDNA is a promising non-invasive tool to state disease progression and immune response in zebrafish infected with V. anguillarum.
Abstract Aquaculture industries face challenges in fish production, primarily due to disease occurrence. Clinical manifestations of these diseases often indicate a considerable advancement in the pathological process. Infected fish often experience morbidity, leading to compromised welfare and substantial economic losses for production facilities. Therefore, prophylaxis and early detection of the pathogens could allow the facility managers to intervene or treat the disease before clinical disease signs occur. For the first time, environmental DNA/RNA (collectively eNA) was used to detect the salmonid pathogen Aeromonas salmonicida subsp. salmonicida in the water hosting rainbow trout ( Oncorhynchus mykiss ). This took place under controlled experimental conditions, before the occurrence of clinical outcome and mortality. The concentration of bacteria in the water was determined by using a standard curve. To determine the window of possible intervention, bacterial concentration was measured from an early stage of disease and throughout when fish became moribund. Furthermore, the acute phase protein, serum amyloid A produced by rainbow trout, was detected in the water and was found to be up‐regulated 4 days post challenge. It was thereby shown that it is possible to detect strong immune responses in the fish in a non‐invasive manner. The detection of eNA from the water can be used as a tool to detect pathogens in the early phase of infection before the disease becomes severe. This will facilitate interventions to limit the disease and increase the welfare of the fish, which is becoming a requirement for a more sustainable and ethical animal production.
Farmed fish are regularly subjected to various stressors due to farming practices, and their effect in the context of a disease outbreak is uncertain. This research evaluated the effects of unpredictable repeated stress in rainbow trout challenged with the ciliate Ichthyophthirius multifiliis, known to cause white spot disease in freshwater fish. Before and after the pathogen exposure, fish were handled with a random rotation of three procedures. At 7 days post-infection (dpi), the parasite burden was evaluated in fish and in the tank’s water, and the local and systemic immune responses were investigated in the gill and spleen, respectively. The fish mortality was recorded until 12 dpi, when all the fish from the infected groups died. There was no statistical difference in parasite burden (fish and tank’s water) and infection severity between the two infected fish groups. The immune gene expression analysis suggested a differential immune response between the gill and the spleen. In gills, a T helper cell type 2 immune response was initiated, whereas in spleen, a T helper cell type 1 immune response was observed. The stress has induced mainly upregulations of immune genes in the gill (cat-1, hep, il-10) and downregulations in the spleen (il-2, il-4/13a, il-8). Our results suggested that the unpredictable repeated stress protocol employed did not impair the fish immune system.
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