Cohabitation of Piscirickettsia salmonis genogroups LF-89 and EM-90 : effects on growth dynamics and SRS infection development in Atlantic salmon

Abstract

In the expanding aquaculture industry, farmed Atlantic salmon (Salmo salar) dominates the commercial value. However, intensive farming practices have increased the vulnerability of fish to potential infections by pathogens, affecting their health and welfare, and causing major economic losses to the sector. In Chile, the second largest producer of salmon after Norway, the Salmon Rickettsial Septicemia (SRS) or Piscirickettsiosis is a major concern for the industry. The biological agent of this disease is Piscirickettsia salmonis, a facultative intracellular Gammaproteobacteria that evades the salmon immune system by replicating within cytoplasmic vacuoles of macrophages, avoiding the respiratory burst in these cells. Also in Chile, two genogroups of this bacterium, LF-89 and EM-90, have been identified, and recent intensive genomic analyses suggest they may be genetically isolated species. These findings are significant, as co-infection by LF-89-like and EM- 90-like genogroups of P. salmonis has already been reported in farmed Atlantic salmon. Thus, this study aims to learn how the cohabitation of these P. salmonis genogroups can be linked to the high fish mortality in Chile by SRS. Specifically, related to the implications of a within-host competitive interaction by the genogroups on SRS development and persistence, host immune response, bacterial virulence, and population dynamics of P. salmonis. To achieve this, co-infection and co-cultures models were tested using isolates from the P. salmonis genogroups LF-89-like (Psal-013) and EM-90-like (Psal-051, Psal-068, Psal-163, and Psal182), which were obtained from natural outbreaks in fish farms and chosen based on their genomic differences. Regarding the methodological approach, the bacteria were co-cultured under in vitro conditions using FN2 broth media, and under an in vivo trial by putting the bacterial cultures alone and mixed inside dialysis bags in the abdominal cavity of Atlantic salmon. Results from in vitro conditions showed that when co-culturing P. salmonis LF-89-like and EM-90-like mixed, EM-90-like isolates overgrew LF-89-like, producing a growth curve similar to the EM-90-like monoculture. In addition, the gene expression of four bacterial biomarkers (detected by qPCR) showed that luxR, related to communication through N-acyl homoserine lactone (AHL) quorum-sensing (QS) signaling, was only being expressed in EM-90-like isolates, while there were significant differences between mono- and co-cultures for flaA (encoding the major subunit of flagellin in flagella) and cheA (chemotaxis). This suggests a response to the co-culture by the bacteria. Also, the data evidenced an enhanced biofilm formation triggered by the synergistic effect of co-culturing P. salmonis genogroups, which could indicate a higher virulence during co-culture. On the other hand, transcriptomic analysis (by RNA-seq) in P. salmonis in vivo culture in Atlantic salmon revealed that co-cultures had significant upregulation of transposases, flagellum-related genes (fliI and flgK), transporters, and permeases that may represent virulence effectors used in the early infection process and activated by the genogroups cohabitation. Additionally, to evaluate if the direct physical contact between the P. salmonis genogroups isolates determines the interaction effect, an in vivo spatially separated co-culture system in Atlantic salmon was used. For this, P. salmonis LF-89-like and EM-90-like isolates (cultured in FN2 broth in two different dialysis bags) were placed together inside the abdominal cavity of the fish. After 6 days, the evaluation of the differential expression of genes (DEGs) by RNA-seq in the spatially separated cocultures, showed that although the LF-89-like isolate and the EM-90-like isolate had a similar DEG profile to the mixed co-cultures, important virulence factors observed in the mixed condition (i.e., flagellar-related genes, CydD, and NCS2), were absent during the spatially separated co-cultures. Therefore, by trying to mimic the initial infectious stage of P. salmonis in Atlantic salmon, a synergistic effect was observed related to increased pathogenicity to the host (e.g., increased expression of virulence factors), which could be driven by the physical co-localization and contact of both P. salmonis genogroups. This improves our biological understanding of P. salmonis and its host-pathogen interactions, which is essential for improving SRS management. These results showed that the cohabitation of P. salmonis genogroups can modulate their growth, biofilm production, and virulence factors expression during in vitro and in vivo co-cultures, indicating a synergistic effect in response to the genogroups interaction. Furthermore, to compare the impact of P. salmonis genogroups coinfection in Atlantic salmon immune response, as well as SRS development and disease dynamic, a fish challenge was performed. Results showed that at 22 days post-challenge (dpc), co-infected fish had a higher presence of clinical lesions compared to any of the single infections. Also, co-infected fish showed a significantly higher cumulative mortality (61.6%), compared to EM-90-like single-infected fish (52.6%), while no mortality was observed in LF-89-like single-infected fish. Regarding, the gene expression of salmon immune-related biomarkers (evaluated by qPCR) in head kidney, spleen, and liver, results showed that bacterial co-infection induced the up-regulation of cytokines (e.g., il-1β, ifnγ, il-8, il-10), antimicrobial peptides (hepdicin) and pattern recognition receptors (PRRs) such as tlr5s. In addition, serum samples from EM-90-like single-infected fish and co-infected fish had an increase in total IgM. Interestingly, specific IgM against P. salmonis showed a higher detection of EM-90-like antigens by LF-89-like single-infected fish serum (cross-reaction). These data provide evidence that P. salmonis LF-89-like and EM-90-like interaction can modulate SRS disease dynamic in Atlantic salmon, causing a synergistic effect that increases disease severity, fish mortality rate, and influences the host immune response. Overall, this study contributes to a better understanding of the P. salmonis population dynamics and host-pathogen interactions, highlighting the critical need to research multi-genotype infections of P. salmonis to guide the development of more targeted and effective control strategies for SRS management.

I den ekspanderende havbruksnæringen dominerer oppdrettslaks (Salmo salar) den kommersielle verdien. Intensiv oppdrettspraksis har imidlertid økt fiskens sårbarhet for potensielle mikrobielle infeksjoner som påvirket deres helse og velferd, og forårsaket store økonomiske tap for sektoren. I Chile, den nest største produsenten av laks etter Norge, er sykdommen Salmon Rickettsial Septicemia (SRS) eller piscirickettsiosis en stor bekymring for næringen. Den kausale agens for denne sykdommen er Piscirickettsia salmonis, en fakultativ intracellulær Gammaproteobakterie som unnslipper laksens immunsystem ved å replikere i cytoplasmatiske vakuoler i makrofager, og dermed unngår oksydativ nedbrytning i disse cellene. Det er til nå identifisert to genogrupper, kaldt LF89 og EM90, for denne bakterien som sameksisterer i Chile. Etter intensive genomiske analyser har det nylig blitt foreslått at disse genotypene bør klassifiseres som to ulike arter. Denne oppgaven har derfor som mål å skaffe mer kunnskap om P. salmonis to genotypers samliv kan relateres til den høye fiskedødeligheten i Chile, og i tilfelle hvordan. Spesifikt assosiert med implikasjonene av denne konkurranseinteraksjonen innen verten i utviklingen og persistensen av SRS er vertens immunrespons, samt P. salmonis virulens og populasjonsdynamikk. For å oppnå dette ble ko-infeksjoner og ko-kulturmodeller benyttet ved bruk av ett isolat fra P. salmonis genogruppe LF-89 og fire isolater fra genogruppe EM-90. Disse isolatene ble valgt basert på deres tidligere identifiserte genomiske forskjeller.