Plasma Pattern Recognition Receptors of Walleye (Sander vitreus M.) with an Emphasis on Mannose-binding Lectin-Like Protein and Viral Hemorrhagic Septicemia Virus
Walleye (Sander vitreus M.) are valuable in commercial and recreational fisheries and are affected by bacterial, fungal and viral disease. Pattern recognition receptors (PRRs) are germline-encoded and constitutively expressed and bind non-self or altered-self for immune recognition. Walleye were hypothesised to have circulating PRRs that were capable of binding diverse pathogens. These PRRs were hypothesised to increase with infection, be distributed in immunologically relevant tissues and to be strain and age specific. PRR binding was measured by affinity chromatography, plasma binding assays,SDS-PAGE, Western blots, ELISA, PCR, and immunohistochemistry. ELISA and affinity chromatography assays were developed in rainbow trout (Oncorhynchus mykiss) with known PRRs. Trout ladderlectin was confirmed as a PRR binding viral hemorrhagic septicemia virus (VHSV). These techniques were adapted to walleye using Flavobacterium columnare, chitin, VHSV and Sepharose resin. A 22 kDa protein bound to F. columnare, a 17 kDa protein bound to chitin and a 34 kDa protein bound to VHSV were identified as similar to bass apolipoprotein, carp C3 and rainbow trout intelectin, respectively. PCR and 3'-RACE-PCR were used to generate nucleotide sequence to confirm identity of walleye apolipoprotein and mannose-binding lectin (MBL)-like protein from the intelectin-like sequence. Two rabbit polyclonal antibodies were raised to 34 and 67 kDa MBL amino acid sequences and used to verify MBL-like protein as a PRR for VHSV. Healthy walleye MBL-like protein plasma concentration was 7.5 ng/ml. Significant differences were found between geographically distant strains of walleye. An ELISA demonstrated that MBL-like protein had significant differences in binding affinity between multiple strains of VHSV and different viruses found in Ontario. MBL-like protein plasma levels increased with initial infection of naïve fish with waterborne and IP VHSV (107 pfu) but did not change with IP reinfection. Previous infection with VHSV significantly decreased walleye mortality. IHC of walleye shows MBL-like protein is distributed in epithelial surfaces, primarily skin, oropharynx, gill, gastrointestinal system, renal nephrons, connective tissue of gonads and plasma. There was no qualitative difference in MBL-like protein tissue distribution in healthy and VHSV-infected walleye. This is the first evidence for fish lectins binding viruses.