8B) but no bacterin was observed in the skin or the associated mucus 3 hours post vaccination. Supporting Information files. Abstract is the causative agent of enteric redmouth disease (ERM) in rainbow trout, and the first commercially available fish vaccine was an immersion vaccine against ERM consisting of bacterin. The ERM immersion vaccine has been successfully used in aquaculture farming of salmonids for more than 35 years. The FABP4 gills and the gastrointestinal (GI) tract are believed to be the portals of antigen uptake during waterborne vaccination against ERM; however, the actual sites of bacterin uptake are only partly understood. In order to obtain insight into bacterin uptake during waterborne vaccination, optical projection tomography (OPT) together with immunohistochemistry (IHC) was applied to visualize bacterin uptake and processing in whole rainbow trout fry. Visualization by OPT revealed that the bacterin was initially taken up via gill lamellae from within 30 seconds post vaccination. Later, bacterin uptake was detected on other mucosal surfaces such as skin and olfactory bulb from 5 to 30 minutes post vaccination. The GI tract was found to be filled with a complex of bacterin and mucus at 3 hours post vaccination and the bacterin remained in the GI tract for at least 24 hours. Large amounts of bacterin were present in the blood, and an MK-0359 accumulation of bacterin was found in filtering lymphoid organs such as spleen and trunk kidney where the bacterin accumulates 24 hours post vaccination as demonstrated by OPT and IHC. These results suggest that bacterin is taken up via the gill epithelium in the earliest phases of the bath exposure and from the GI tract in the later phase. The bacterin then enters the blood circulatory system, after which it is filtered by spleen and trunk kidney, MK-0359 before finally accumulating in lymphoid organs where adaptive immunity against ERM is likely to develop. Introduction In fish, even MK-0359 formalin-killed bacterin that can be administered to fish by immersion [3C6]. The effect of the ERM immersion vaccine has been demonstrated MK-0359 in controlled laboratory efficacy tests [6,7], as well as in a large field test [8]. However, even though the efficacy of ERM immersion vaccination of rainbow trout is well documented, very little is known regarding the uptake of the bacterin leading to specific immunity in rainbow trout [1,9,10]. Immersion times from as little as 5 seconds have been reported to be sufficient for induction of protective immunity in rainbow trout, but most commercial vaccine producers recommend a 30 second immersion time in order to ensure sufficient antigen uptake for development of immunity [5,9,11]. Besides the time of exposure, development of immunity from immersion ERM vaccination in rainbow trout depends on the weight of the trout [12], and significant protection has been obtained in trout fry at 325 mg [13]. The uptake of bacterin induces an increase in transcripts of several pro-inflammatory genes in the spleen of rainbow trout fry and development of adaptive immunity [7]. Recently it was shown that immersion immunization of trout with bacterin led to the production of specific antibodies and protection against exposure to a challenge [6]. Furthermore, passive transfer of serum from immersion vaccinated fish to na?ve trout is known to confer immunity [14,15]. The highest level of protection MK-0359 is obtained with transfer of the serum fraction with highest level of specific antibodies indicating that specific antibodies play a protective role against development of ERM disease [15]. The specific antibodies in rainbow trout are secreted from B lymphocytes and plasma cells in the lymphoid organs such as spleen and kidney [16C18]. Although waterborne administration of the bacterin has been shown to induce systemic humoral or mucosal immunity in rainbow trout, it is generally unknown how the bacterin antigens reach the lymphoid organs and activates an.