Introduction
Piscine myocarditis virus (PMCV) is the causative agent of Cardiomyopathy Syndrome (CMS) (Haugland et al., 2011), a severe viral disease characterized by distinct cardiac inflammation that results in compromised animal welfare and mortality in post-smolt Atlantic salmon . At the same time, sea lice (Lepeophtheirus salmonis) infestations reduce growth and increase the susceptibility of Atlantic salmon to secondary pathogens due to extensive skin degradation. Mechanical delousing is a common treatment used to physically remove sea lice from fish but often causes increased stress and is associated with CMS outbreaks in PMCV positive populations (Sommerset et al., 2023). Despite these observations the interaction between PMCV infection and sea lice infestation in the same host remains poorly understood. In particular, it is unclear whether sea lice attachment and associated tissue damage influences viral load or the development of cardiac lesions associated with CMS. From an immunological perspective, coinfections may be shaped by trade-offs between antagonistic immune responses. The concept of immunity equilibrium suggests that type 1 responses (to viruses) and type 2 responses (to multicellular parasites) can suppress one another, potentially altering host susceptibility and disease outcomes . Therefore, the objective of this study was to investigate how coinfection with PMCV and sea lice influences viral load, cardiac pathology, and the localized immune response in the heart, as well as whether PMCV infection affects sea lice burden.
Materials and Methods
To assess coinfection, controlled laboratory experiments were performed using post-smolt Atlantic salmon reared in a saltwater recirculation system (RAS). Fish were allocated across four infection groups: a negative control, single PMCV infection, single sea lice (SL) infection, or PMCV + SL co-infection, and sampled biweekly over 14-weeks for tissue sampling or at 3, 8, 22, and 36 days post-infestation (dpi) for sea lice counts. PMCV challenge was administered with an intraperitoneal (IP) injection and sea lice through immersion bath. Total lice counts were conducted to assess sea lice burden during different life-stages on Atlantic salmon in relevant groups. Viral load (PMCV RNA) was quantified via RT-qPCR and cardiac lesion scoring assessed through histopathology. In addition, the immune response in the heart was investigated through gene expression using RT-qPCR targeting anti-viral, cellular-mediated, and anti-inflammatory markers, and cytokine quantification with IL-1β specific ELISAs to evaluate the early pro-inflammatory response.
Results
Sea lice challenge took place one day before the 7 weeks post challenge (wpc) timepoint – the projected peak of viral load in the heart. Sea lice burden was statistically similar on salmon in the single SL infection and PMCV + SL coinfection group at 3, 8, 22, and 36 days post infestation (dpi). While, viral load in the heart was statistically similar between single PMCV infection and PMCV + SL coinfection groups post-sea lice challenge at 8, 10, 12, and 14 wpc. Expression of genes associated with cellular mediated responses (MHC-I, MHC-II, IFNγ, Tbet, CD8α, GrmA) were significantly upregulated in the heart of single and co-infected salmon from 6-12 wpc. Although, the expression of antiviral markers (ISG15α and STAT1) was significantly higher in coinfected salmon compared to single infection at 12wpc. In addition, the expression of the anti-inflammatory cytokine TGF-β was only significantly upregulated the heart of coinfected salmon from 8-12 wpc. IL-1β concentration in the heart was significantly higher in groups experiencing single PMCV and PMCV + SL co-infection at 6 and 8wpc but had reduced to baseline levels in the coinfected salmon by 10wpc.
Discussion
To date, our results demonstrate that sea lice, which parasitize the skin of Atlantic salmon, have the capacity to modulate the expression of select anti-viral and anti-inflammatory cytokine genes in the heart during PMCV infection. Despite this, when sea lice infect salmon at the peak of viral load in the heart (6-7wpc), there is no effect on viral load at subsequent timepoints up to 14wpc. This suggests that innate antiviral responses triggered early in infection are more important for limiting viral replication at later timepoints, thereby reducing the need for prolonged antiviral gene expression. Increased expression of TGF- β in coinfected salmon indicates a stronger anti-inflammatory response is induced in the heart to resolve cytotoxic and pro-inflammatory activity. This could also help explain the early suppression of IL-1β in the heart of coinfected salmon. However, histology results are still pending and will be important for interpreting the transcriptional and cytokine data in relation to tissue-level pathology. To our knowledge, this is the first experimental study describing the effects of sea lice on immune gene expression and IL-1β concentration in the heart of Atlantic salmon during both single sea lice infestation and virus-parasite coinfection. Collectively, these findings highlight the importance of considering co-infection when evaluating localized immune responses, as well as its implications on fish welfare and the development of improved health management strategies.
Acknowledgment
This work was funded through an Alliance Grant (ALLRP) issued by the Natural Sciences and Engineering Research Council of Canada (NSERC) (File: ALLRP 608325 – 25) and Onda.
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