Introduction
Transmission electron microscopy (TEM) is a suitable tool to detect virus in the gastrointestinal (GI) tract of teleost fish. In a study conducted by Nofima in collaboration with BioMar, Atlantic salmon were fed functional diets during parr-smolt transformation followed by challenge with Infectious pancreatic necrosis virus (IPNV). Infectious pancreatic necrosis (IPN) is caused by IPNV which is an archetypal birnavirus - a distinctive, small, non-membrane double-stranded RNA virus (Roberts and Pearson 2005). IPN was first reported in brook trout (Salvelinus fontinalis) in North America by Wood et al. (1955). IPNV is known to infect several fish species in both freshwater and seawater as well as other aquatic organisms (Biering 2002). Since 2009, the numbers of IPN outbreaks in Norway have declined from 223 to 56 in 2013. Histologically the disease manifests in the liver and the exocrine cells of pancreas. In addition typical findings are eosinophilic casts in the lumen of pyloric caeca (PC) and nests with necrotic cells in gut epithelium (Lumsden 2006).
Materials and methods
Atlantic salmon (Salmo salar L.) parr were fed either a control basal feed or a feed supplemented with a combination of functional ingredients including yeast cell walls, nucleotides, prebiotic, β-glucans and boosted vitamins (C and E). The fish were fed six weeks in fresh water during parr-smolt transformation. Randomly selected smolts from the pre-fed parr groups were transferred to seawater and challenged with IPNV to assess disease resistance.
PC and distal intestine (DI) were sampled from six moribund fish from the IPNV challenge. The tissue sections (10 mm long), were fixed paraformaldehyde, embedded in paraffin wax and stained with Harris hematoxylin and eosin. The pathological effect of IPNV on the GI tract morphology was observed using light microscopy evaluations. Two paraffin wax embedded tissue samples, from PC and DI, were chosen for immunocytochemistry and TEM analyses. Virus was labeled with monoclonal mouse anti IPNV antibody specific for virus protein 3 and further with Protein A-gold. A detailed description of the method is presented by Webster and Webster (2007).
Results
The IPNV challenge trial revealed a significant difference in mortality between the tested diets. Six PC and DI tissue samples observed by light microscopy revealed pathological changes associated with infection. Two PC and DI samples were further used for analyses using TEM and immunolabeling on cryo-embedded tissue. IPNV was tagged with antibodies and gold particles (10 nm) to enable identification of the virus by TEM. Low numbers of virus were detected in PC and DI enterocytes 19 days post IPNV challenge. IPNV infected Atlantic salmon showed several apoptotic enterocytes (McKnight cells) in PC and DI mucosa layer. Eosinophilic granular cells (EGCs) were identified between base of folds and stratum compactum in DI. Virus was not detected in the negative control samples; none VP3 labeled samples.
Discussion and conclusion
In a previous study, Sundh et al. (2011) using an ex vivo method; Ussing chamber, and real time PCR analyses suggested that the intestine is a port of entry for IPNV. In the present study, morphological examination of the GI tract revealed pathological changes associated with IPN; eosinophilic casts and necrotic enterocytes (McKnight cells), in addition to necrotic pancreatic tissue. The presence of virus in enterocytes of the GI tract was further supported by TEM analysis; detection of IPNV in PC and DI. However, the few IPNV detected in DI may be due to the time period between infection and sampling, thus the IPNV may have migrated to the liver or pancreas which is the major target organ (Ellis et al. 2010). Low virus counts were also observed in PC which correlates with a previous immunohistochemistry study by Ellis et al. (2010) where PC showed one single cell with presence of IPNV. If the GI tract is an infection route for IPNV and dietary supplementation reduces the susceptibility to IPNV an important issue arises; can further dietary manipulation or probiotic supplement improve the susceptibility to the disease? This merits future investigations.
The results of the present study revealed the presence of IPNV in GI tract enterocytes in moribund fish 19 days post cohabitation challenge, and supports that TEM can be used to generate further knowledge on GI tract as infection route for IPNV.
References
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Ellis, A.E., Cavaco, A., Petrie, A., Lockhart, K., Snow, M. & Collet, B. (2010) Histology, immunocytochemistry and qRT-PCR analysis of Atlantic salmon, Salmo salar L., post-smolts following infection with infectious pancreatic necrosis virus (IPNV). Journal of Fish Diseases, 33, 803-818.
Lumsden, J.S. (2006) Gastrointestinal tract, swimbladder, pancreas and peritoneum. In: Systemic pathology of fish: a text and atlas of normal tissue in teleosts and their responses in disease (ed. by H.W. Ferguson), pp. 169-196. Scotian Press. ISBN 13: 978-0-9553037-0-8.
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