The nasopharynx-associated lymphoid tissue (NALT) in the olfactory organ of teleost fish represents an ancient arm of mucosal immunity that has protected them from waterborne stimuli for millions of years. Many neurotrophic pathogens exploit the olfactory route to access the central nervous system. Despite the significant advances in understanding the neuro-immune interaction in fish in recent years, there is a considerable knowledge gap, especially of how this interaction is influenced by external factors such as temperature.
Atlantic salmon (Salmo salar) is one of the most economically important farmed fish species in the world. However, the industry is facing high mortality rates of up to 20%, largely caused by infectious diseases. In recent years, a significant body of evidence indicates that disease resistance, robustness and performance later in life could be influenced by stimuli, like temperature and oxygen, during the crucial stages of early development (Krasnov et al. 2021; Mateus et al. 2017). However, our understanding of how host immunity is shaped by exposure to these stimuli during early development is limited.
The present study explored the molecular and structural consequences in the brain and olfactory organ of Yersinia ruckeri infection in Atlantic salmon parr. We compared two groups of fish with different embryonic temperature histories to investigate if temperature stress during early development could reshape the responses to Y. ruckeri infection. Previous studies in rainbow trout (Oncorhynchus mykiss) revealed that Y. ruckeri is a neurotropic pathogen.
Materials and methods
Eggs were exposed to two different temperatures – 4ºC and 8ºC, from fertilization until the eyed stage (~320-day degrees). The latter was considered the commonly employed temperature in the industry. Thereafter, the fish were reared at 8ºC until they reached the parr stage. Fish (around 12 cm in length) were then exposed to Y. ruckeri serotype O1 by bath exposure. Disease development was followed for 2 weeks, and organs were sampled the day before infection and 1, 2, 3, 7, and 14 days after infection. Brain and olfactory organs were collected for qPCR, histopathology, and immunohistochemistry.
Results and Discussion
Infected fish showed the classic signs of Y. ruckeri infection, including exophthalmos, skin darkening and splenomegaly (Figure 1). The pathogen was detected in the brain and olfactory organs of infected fish by qPCR. This was further verified by the identification of the pathogen in the optic tectum in the brain and in the basal lamina of the olfactory lamella by immunohistochemistry (Figure 2).
Gene expression analysis in the brain showed an increase in the transcript level of microglia cell markers (e.g., aif1) in infected groups compared to the control group. The expression of microglia cell markers in infected brain increased over time, and there was a temporal delay in immune response tendency in the 8oC group. The expression of cd4 also increased in infected groups, and a significant difference was observed between the two temperature groups at several time points, suggesting that embryonic temperature history affected the CD4-mediated response to infection.
In the olfactory organ, the expression of antimicrobial peptides (e.g., cathelicidin), was elevated in infected groups compared to the control group. This elevation increased over time in both temperature groups. Interleukin 1β (il1β), an important pro-inflammatory cytokine, increased around 3 days after infection.
Preliminary histopathology analysis shows mild signs of infection in both tissues. Significant differences were not found in the nasal morphometries, such as the thickness of mucosa and lamina propria of the olfactory lamella and the number of mucus cells present in the nasal wall and on the tip of the olfactory lamella.
The study confirms the neurotrophic nature of Y. ruckeri and further supports earlier evidence suggesting that it is taking the olfactory route to gain entry to the brain. The results indicate that the olfactory organ and brain could mount immune responses to the pathogenic infection. Both temperature groups were able to respond to infection, however, embryonic temperature history modulated the expression of some immune genes.
The research was supported by the Research Council of Norway (COOLFISH; No. 29685)
Krasnov A, Burgerhout E, Johnsen H, Tveiten H, Bakke AF, Lund H, Afanasyev S, Rebl A, Johansen L-H. 2021. Development of atlantic salmon (salmo salar l.) under hypoxic conditions induced sustained changes in expression of immune genes and reduced resistance to moritella viscosa. Frontiers in Ecology and Evolution. 9.
Mateus AP, Costa RA, Cardoso JCR, Andree KB, Estévez A, Gisbert E, Power DM. 2017. Thermal imprinting modifies adult stress and innate immune responsiveness in the teleost sea bream. Journal of Endocrinology. 233(3):381-394.