Impact of Salmonid alphavirus infection in diploid and triploid Atlantic salmon (Salmo salar L.) fry

Abstract

With increasing interest in the use of triploid salmon in commercial aquaculture, gaining an understanding of how economically important pathogens affect triploid stocks is important. To compare the susceptibility of diploid and triploid Atlantic salmon (Salmo salar L.) to viral pathogens, fry were experimentally infected with Salmonid alphavirus sub-type 1 (SAV1), the aetiological agent of pancreas disease (PD) affecting Atlantic salmon aquaculture in Europe. Three groups of fry were exposed to the virus via different routes of infection: intraperitoneal injection (IP), bath immersion, or cohabitation (co-hab) and untreated fry were used as a control group. Mortalities commenced in the co-hab challenged diploid and triploid fish from 11 days post infection (dpi), and the experiment was terminated at 17 dpi. Both diploid and triploid IP challenged groups had similar levels of cumulative mortality at the end of the experimental period (41.1% and 38.9% respectively), and these were significantly higher (p < 0.01) than for the other challenge routes. A TaqMan-based quantitative PCR was used to assess SAV load in the heart, a main target organ of the virus, and also liver, which does not normally display any pathological changes during clinical infections, but exhibited severe degenerative lesions in the present study. The median viral RNA copy number was higher in diploid fish compared to triploid fish in both the heart and the liver of all three challenged groups. However, a significant statistical difference (p < 0.05) was only apparent in the liver of the co-hab groups. Diploid fry also displayed significantly higher levels of pancreatic and myocardial degeneration than triploids. This study showed that both diploid and triploid fry are susceptible to experimental SAV1 infection. The lower virus load seen in the triploids compared to the diploids may possibly be related to differences in cell metabolism between the two groups, however, further investigation is necessary to confirm this and also to assess the outcome of PD outbreaks in other developmental stages of the fish when maintained in commercial production systems.

Fig 1. Cumulative daily percentage mortality ± standard deveation of diploid and triploid fry infected with Salmonid alphavirus (SAV).

(A) diploid and (B) triploid Atlantic salmon fry (n = 30/tank) exposed to SAV 1, F02-143 Irish isolate via different routes of infection; intraperitoneal (IP) (TCID₅₀ = 2.5x10³/fry), bath (exposed for 2h for TCID₅₀ = 5x10⁴ mL^-1 x 2L) and co-habitation (three IP injected fish mixed into each tank) compared to untreated control fry.

Fig 2. Real time PCR results.

Salmonid alphavirus copy number (copies gm tissue^-1), determined by measuring nSP1 levels by qRT-PCR in triploid and diploid fry infected using either a cohabitation (co-hab), immersion (IM) or intraperioneal (IP) route of infection. No any virus virus was detected in the control fish.

Fig 3. Pancreatic histopathology of SAV-infected Atlantic salmon fry.

(A) Normal pancreatic tissue from control fry. Normal exocrine acinar tissue (EP) seen adjacent to a section of pyloric caeca (PC); (B) Exocrine pancreas (EP) with severe diffuse mononuclear inflammatory cell infiltration (arrow) along with some intact acinar cells (*) observed in co-habitation group. (C) Marked acinar necrosis in diploid cohabitation fry, cell breakdown with leakage of zymogen granules (thick arrow) (D) Pancreas from triploid IP fry showing almost complete destruction and absence of acinar tissue (Arrow).

Fig 4. Histopathology of heart in SAV infected Atlantic salmon fry.

(A) Normal heart of diploid control fry; (B) Normal heart of diploid cohabitation fry showing cell infiltration in myocardium (thick arrow) and epicardium (thick arrow), increased serous fluid accumulation in the ventricle, and hyper-eosinophilia in the compact and spongy cardiomyocytes; (C) Triploid immersion fry: severe diffuse myocardial degeneration affecting spongy myocardium with hyper-esosinophilia and hyalinisation in myocyte; (D) High magnification view of diploid immersion heart. Inflammatory cell infiltrate within epicardium (EC), spongy myocardium (S) and at interface of the compact spongy myocardium (thick arrow), severe diffuse hyalinisation in compact myocardium (blue arrow), nuclear pyknosis and karyorrhexis.

Fig 5. Liver histopathology of SAV infected Atlantic salmon fry.

(A) H & E stain and (B) PAS stained liver from diploid control fry showing normal hepatocyte morphology and intracellular lipid with (C) H & E stain and (D) PAS stained liver in diploid cohabitation fry with diffuse moderate degenerative change and depletion of intra-hepatocyte lipid (E) H & E stain and (F) PAS stained of diploid cohabitation fry with severe diffuse coagulative necrosis hypereosinophilia (thick arrow) and cytoplasmic vacuolisation in liver.

Fig 6. Renal histopathology of SAV infected Atlantic salmon fry.

H & E stained (A) trunk kidney; (B) head kidney of unchallenged fish with high number of melanomacrophages (white circles); (C) trunk and (D) head kidney of cohabitation fry, note depletion of melanomacrophages (white circles), clear, enlarged sinusoidal spaces (si) and relatively large parenchymal cells (thick arrow) compared to control fry.

Fig 7. Interval plots comparing average histological scores between ploidy and treatment.

(a) Liver inflammation, no significant differences detected between ploidy or treatments; (b) Liver degeneration. Significant difference between treatments; (c) Pancreas inflammation. Significant difference between treatments; (d) Pancreas degeneration. Significant difference between ploidy (more severe degeneration in diploids) and treatment; (e) Heart inflammation. Significant difference between treatments; (f) Heart degeneration. Significant difference between ploidy (more severe degeneration in diploids) and treatment (g), Epicarditis. No significant difference between ploidy or treatment.