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Aquaculture is the fastest growing animal food sector in the global marketplace. However, disease outbreaks are a major hurdle for the growth of the industry. In order to prevent and/or treat fish diseases, the industry relies on a significant arsenal of fish vaccines as well as chemo- and non-chemotherapeutic options, including probiotics. Vaccines are not always cost effective for a number of aquaculture species, or they simply do not exist for a growing number of fish pathogens, especially parasites. Chemotherapeutics (e.g., antibiotics, formaldehyde, malachite green) are often expensive and have a significant negative ecological impact on the aquatic environment and its surroundings. Thus there is an urgent need to find alternative cost-effective, and eco-friendly therapeutic strategies for the prevention and treatment of fish diseases. Consequently, the use of probiotics has emerged as a powerful therapeutic alternative in the aquaculture industry, although they comprise a very minor part, thus far, of the therapeutics currently being used by the industry. This is in part due to the limited availability of effective probiotic therapies. To improve such therapies, several key areas in this field require much research, particularly, the development of targeted strategies of probiotic discovery and delivery to fish. Critically, little is known on the roles of probiotics in promoting fish mucosal immune and protective responses thus preventing the development of probiotics that enhance mucosal health. Overall fish probiotics have been selected to improve a wide range of fish physiologic processes including growth, digestion, gut inflammation, immunity, pathogen resistance, among others. Here we propose for the first time in fish the identification and characterization of rationally selected fish immunobiotics. The term immunobiotic (IMB) was coined in 2003, and it refers to microbes that are specifically selected to enhance health through activation or potentiation of the mucosal immune system. In our preliminary data we show the development of a novel strategy to select potential fish IMBs based on the detection and isolation of fish microbiota (MB) highly coated with IgT (IgThi-MB). After isolating and growing individual IgThi-MB species, here we show that they function as IMBs based on their capacity to greatly induce mucosal IgT (sIgT) levels in treated fish (IgT is the key mucosal immunoglobulin (Ig) in fish). Critically, the induced skin and mucus sIgT recognizes a variety of fish pathogens, and the IMB-treated fish become significantly more resistant to pathogen challenge. Here we refer to these isolated IgThi-MB species enhancing mucosal immunity as IMBs.Based on the preliminary data presented here, the overarching hypothesis of this proposal is that isolated IMBs of skin and gill mucus induce the elevated production of polyreactive mucosal sIgT in bath-treated fish, and the induced sIgT has a high capacity to bind a wide range of pathogens, and confer protection to fish upon infection. To test this hypothesis we propose the following objectives:OBJECTIVE 1. Identification, isolation and selection IgT-inducing IMB candidates (IMBc). Our pdata shows for the first time a novel selection strategy designed to isolate host-derived (indigenous) IgT-inducing IMBc that when used as bath treatment, they increase by several fold the levels of skin and gill mucus sIgT in treated fish. Importantly, the induced sIgT are able to recognize and bind several fish pathogens in a polyreactive manner. Based on this pdata we hypothesize that the isolation of IgThi-MB with high IgT-inducing capacity is a novel approach to identify fish IMBs. To further substantiate this hypothesis, here we will isolate additional IgT-inducing IMBc via FACS-sorting IgThi-MB from gills and skin mucus. Sorted IMBc will be cultured, and single colonies will be isolated, grown and identified. Top IMBc will be selected based on : 1) Their high capacity to induce mucus sIgT in vivo upon bath treatment of the fish; 2) The ability of the induced sIgT to bind with a high capacity and in a polyreactive manner to a battery of important fish mucosal pathogens. As part of the selection process, we will also test the degree of skin and gill IMB colonization upon treatment and the potential deleterious effects of these IMBs on fish health and microbiome homeostasis. OBJECTIVE 2. Capacity of IMBs to promote in vivo protective responses against mucosal pathogens. The capability of IMB-induced sIgT to highly recognize and coat a variety of fish pathogens lead us to hypothesize a potential protective role of the induced sIgT against these pathogens. This hypothesis is supported by pdata showing the protective effects of our isolated IMBs against Ich, a mucosal parasite. To further substantiate this hypothesis, here we will evaluate the capacity of IMBs isolated from Objective 1 to protect fish from bacterial, viral and parasitic mucosal pathogens. To this end, fish will be treated by bath with each individual IMB, and after treatment fish will be challenged with individual pathogens at different time points post treatment. The IMBs inducing the best protective responses will then be evaluated in combination to ascertain whether these protective responses are enhanced.OBJECTIVE 3. Mechanisms of protection elicited by the selected IMBs. Here we hypothesize that the protection induced by IMBs is mediated by sIgT coating of the pathogen. However, we cannot rule out that IMB-induced protection is sIgT independent, or that sIgT contributes to protection in cooperation with other immune mechanisms. To evaluate these possibilities, here we will assess the specific contribution of sIgT in IMB-mediated protective responses by treating IgT-depleted fish with the IMBs and evaluating whether fish remain or not resistant to the tested pathogens. To evaluate further sIgT-dependent and independent immune mechanisms elicited by the IMBs, the following immune capacities will be assessed upon treatment of fish with IMBs: 1) The agglutination, bactericidal and neutralizing capacity of the skin and gill mucus; 2) The phagocytic and bactericidal capacities of skin and gill phagocytes; 3) The percentages and proliferative status of key immune leukocytes of the skin and gills; 4) The antimicrobial capacity of IMBs against fish pathogens. Moreover, to detect additional pathways and molecules that may be up- or down-regulated upon IMB treatment, transcriptome profiling will be performed on the skin and gill tissues as well as on the spleen of treated fish. In addition to providing a more complete understanding on the immune and/or non-mechanisms involved in IMB-mediated protection, these data may identify critical transcriptomic signatures that can enable a more efficient selection of effective IMBs in future studies.

Sunyer, O.
University of Pennsylvania
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