Introduction
Intensive aquaculture production has led to an increased occurrence of infectious diseases. In addition, co-infections in fish are natural events that should be considered when studying the biology of the pathogens for control measures, since infection with multiple pathogens may increase the severity of disease in fish. The SRS (Salmon Rickettsial Syndrome) produces a systemic disease in Atlantic salmon that was responsible for the mortality of 54.2% (of total infectious diseases) during first semester 2022 in Chile, resulting in annual economic losses of USD $700 million for this industry.
T he biological agent of SRS, Piscirickettsia salmonis (a facultative intracellular bacterium) has been found in all the others important salmon farming countries like Norway, Canada, Scotland, and Ireland, where the disease can be considered as a potential emergence disease (Long, A, et al. 2021). P. salmonis evades the salmon immune system by replicating within cytoplasmic vacuoles of macrophages, avoiding the respiratory burst in these cells. That is why is hard to control by the host innate and adaptive immune responses and it could also be one of the reasons why vaccines still do not provide efficient protection over time (Rozas-Serri, 2022) . There are currently two genogroups (LF-89 and EM-90) identified for P. salmonis with different virulence levels that cohabit in Chile (Saavedra, J, et al. 2017) . Thus, our study focuses how this cohabitation can be related to high fish mortality, as the within-host competitive interactions are linked to virulence, changing the development and persistence of diseases. This, also modulate the population dynamics of pathogens by cooperative or competitive interactions with different antigenic epitopes triggering a cross-reactive immune response in the host, altering its efficiency and the health and welfare of fish (Kotob et al. 2016).
Materials and Methods
I n this study, we evaluated the initial effect of co-infection in Atlantic salmon with the two different genogroups LF-89 and EM-90 in an intraperitoneal challenge model to compare pathogenicity and host immune response. Fish (average weight of 60.4 g) were smoltified at VESO Vikan (Namsos, Norway ) in brackish water (25‰ ± 2‰) at 15ºC and continuous 24 h light exposure (24:0). Then, f ish were starved for 48 h prior to challenge and divided into groups of 80 fish each ( in three identical tanks with a stocking density of 40 kg m3). To perform the challenge, fish were sedated and intraperitoneally (i.p .) injected with 0.1 mL of a 1.0 × 107 cfu /fish (with one of the P. salmonis strain or in a ratio of 1:1 for co-infections) . Fish were fed ad libitum and monitored daily. At day 0, 12 fish were sampled as negative control . Moreover, at 7, 14 and 21 days post challenge (dpc ), 12 fish per tank were randomly selected for sampling of head kidney, liver and spleen for total RNA extraction and qPCR gene expression analyses of immune-related biomarkers . S erum samples were evaluated by ELISA after 14- and 21-dpc for total and specific IgM detection.
Results and discussion
The results revealed a variation in mortality between single- and co-infection. The LF-89 strain alone did not induce mortality, while the EM-90 strain produced 52.6% mortality at 22 dpc . However, during the co-infection challenge, mortality started earlier and reached 61.6% mortality after 22 dpc in a steeper curve. The gene expression of immune-related biomarkers at 14-dpc showed the up-regulation of il-10 (anti-inflammatory cytokine) during the co-infection compared to EM-90 alone in spleen and in head-kidney. At the same time, il- 8 (chemokine ) was significantly up-regulated in co-infection compared with EM-90 alone in spleen and liver, and il-1b in the liver . In EM-90 challenges this is already described (Rozas-Serri, 2022) but never with higher levels of expression in a co-infection study.
ELISA analyses in serum showed an increase in the protein levels of total IgM (in EM-90 infections and in co-infections) at 14- and 21-dpc, being significative different to the control at day 0 and to LF-89 infection. Moreover, specific IgM against P. salmonis showed a peak production at 14-dpc for the three infected groups (EM-90, LF-89 and co-infection). Interestingly , serum from fish infected LF-89 had a higher cross-reaction using EM-90 as antigen than serum from fish infected EM-90 against LF-89.
Conclusions
Our study proposes that there is a synergistic interaction effect among the P. salmonis genogroups (LF-89 and EM-90) during the infection in Atlantic salmon that could modulate the host immune response , triggering higher mortality . This novel approach to SRS characterization as a multiple-genotype infection may offer better insights into the development and further control of the disease.
References
Kotob MH, et al. 2017. The impact of co-infections on fish: a review. BMC Vet. Res. 47(1 ): 1-12.
Long, A, et al. 2021. Piscirickettsia salmonis shedding and tissue burden, and hematological responses during cohabitation infections in chum Oncorhynchus keta , pink O. gorbuscha and Atlantic salmon Salmo salar. PLoS One, 16, e0248098.
Rozas-Serri M. 2022. Why does Piscirickettsia salmonis break the immunological paradigm in farmed salmon? biological context to understand the relative control of piscirickettsiosis. Front. Immunol. 13:856896.
Saavedra J, et al. 2017 . Prevalence, geographic distribution and phenotypic differences of Piscirickettsia salmonis EM-90-like isolates. J Fish Dis. 40(8):1055-1063.