Tracing the Olfactory Circuit of Bacteria and Virus in Rainbow Trout (Oncorhynchus mykiss)
Authors: Mancha Fabiola , Huertas Mar 
Published: April 2020

The olfactory system detects odorants in the environment and sends signals to the brain, which in turn integrate the information and trigger vital physiological processes including reproduction, predator evasion and food detection. The olfactory sensory system is comprised of olfactory sensory neurons (OSNs) which express unique receptors that discriminate a vast array of odorants. Direct exposure to the external environment makes OSNs susceptible to infection by microorganisms. Crypt cells, a specialized type of OSN found only in fish, have been shown to act as a portal of infection for pathogens such as Infectious Hematopoietic Necrosis Virus (IHNV). Intranasal exposure to IHNV results in activation of crypt cells which propagates an action potential that activates the brain immune system and results in apoptosis of crypt cells. It is unclear if fish can also smell bacteria and what the neural route of integration for this signal is. We hypothesize that virus and bacteria are detected by a different subset of crypt cells and the information is integrated in different areas of the brain. The objective of this study is to visualize the olfactory pathway of detection of pathogens, and the concomitant brain areas of integration of information. To investigate this, rainbow trout were intranasally exposed to two vaccine models: IHNV live attenuated vaccine and formalin‐killed Yersenia Ruckeri vaccine mixed with Alexa Fluor 568 10000 MW. Rainbow trout were intranasally exposed to the vaccine solution for 3 minutes and subsequently placed in water tanks after exposure. We expected that the specific saturated olfactory receptors would be endocytosed together with the dye. Then, the anterograde dye will migrate through the olfactory axons to specific areas in the olfactory bulb and brain. Fish were kept in water tanks for periods of 24 hours, 4 days, 7 days, and 14 days. Following the tracing period, fish were anesthetized and decapitated. The olfactory organ and brain were dissected out and fixed in 4% PFA for 24 hours and then transferred to 30% sucrose for cryopreservation. 10μm cryosections were cut and viewed using a fluorescent microscope and confocal microscope. Results from each vaccine model showed fluorescent olfactory sensory neurons throughout the lamellae of the olfactory epithelium, indicating internalization of the molecular receptor in response to vaccination. The IHNV vaccine treated samples showed fluorescence predominantly in globular‐like cells localized in the olfactory lamellae. In contrast, formalin killed Y. Ruckeri treated samples showed fluorescence in oblong shaped cells. Comparatively, no fluorescence was noted in control samples, indicating that the olfactory response is activated when exposed to the IHNV and formalin‐killed Y.Ruckeri vaccines. Future research will focus on nasal neuro‐immune responses following exposure to formalin‐killed Y. RuckeriSupport or Funding InformationThis material is based upon work supported by the National Science Foundation under Grant N. 1755348