Spencer Russell

MASS MORTALITY ASSOCIATED WITH KOI HERPESVIRUS IN WILD COMMON CARP IN CANADA

Koi herpesvirus (KHV) was identified as being associated with more than one mortality event affecting common carp in Canada. The first was an extensive mortality event that occurred in 2007 in the Kawartha Lakes region, Ontario, affecting Lakes Scugog and Pigeon. Fish had branchial necrosis and hepatic vasculitis with an equivocal interstitial nephritis. Several fish also had branchial columnaris. Subsequent mortality events occurred in 2008 in additional bodies of water in south-central Ontario, such as Lake Katchewanooka and outside of Ontario in Lake Manitoba, Manitoba. Koi herpesvirus was detected in fish submitted for examination from all of these lakes by polymerase chain reaction (PCR), and sequence of the PCR product revealed 100% homology to KHV strains U and I. Real-time PCR analysis of KHV-infected wild carp revealed viral loads ranging from 6.02×101 to 2.4×106 copies μg−1 host DNA. This is the first report of KHV in Canada.

Viral haemorrhagic septicaemia virus IVb experimental infection of rainbow trout, Oncorhynchus mykiss (Walbaum), and fathead minnow, Pimphales promelas (Rafinesque)

Viral haemorrhagic septicaemia virus (VHSV) in the Great Lakes has had a dramatic impact on fish husbandry because of the implications of the presence of a reportable disease. Experimental infections with VHSV IVb were conducted in rainbow trout, Oncorhynchus mykiss (Walbaum), and fathead minnows, Pimphales promelas (Rafinesque), to examine their susceptibility and the clinical impact of infection. Triplicate groups of rainbow trout (n = 40) were injected intraperitoneally (i.p.) with 100 microL 10(6.5)50% tissue culture infective doses (TCID(50)) or waterborne exposed to graded doses (10(4.5), 10(6.5), and 10(8.5) TCID(50) mL(-1)) of VHSV IVb. Duplicate groups of fathead minnows (n = 15) were i.p. injected with (10(6.5) TCID(50) 100 microL) or waterborne exposed (10(6.5) TCID(50) mL(-1)). All experiments were performed with single-pass well water maintained at 12 degrees C. Following either i.p. or waterborne exposure, VHSV RNA was detectable in both rainbow trout and fathead minnows by nested reverse transcription polymerase chain reaction (nRT-PCR) as early as 4-7 days post-infection (p.i.). Infected fathead minnow and rainbow trout exhibited lesions characteristic of VHS at 9 and 15 days p.i., respectively. Route of exposure had little effect on the onset of clinical signs. Cumulative mean mortality in rainbow trout was 4.4%, 2.6%, 2.6% and less than 1% in the i.p., high, medium and low dose waterborne exposures, respectively. Cumulative average mortality of 50% and 13% occurred in i.p. and waterborne-exposed fathead minnows, respectively. VHSV was detected from pooled rainbow trout tissue by RT-PCR and virus isolation at 38 days p.i., but not at 74 days p.i., regardless of the exposure route. Immunohistochemistry (IHC) with a rabbit antibody to VHSV IVb revealed the viral tissue tropisms following infection, with the identification of viral antigen in myocardium and necrotic branchial epithelium of both species and in gonadal tissue of fathead minnows. Rainbow trout, but not fathead minnows, are relatively refractory to experimental infection with VHSV IVb.

Identification, cloning and tissue localization of a rainbow trout (Oncorhynchus mykiss) intelectin-like protein that binds bacteria and chitin.

Intelectins are a recently identified group of animal lectins involved in innate immune surveillance. This paper describes the primary structure, expression and immunohistochemical localization of a rainbow trout plasma intelectin (RTInt). RTInt exhibited calcium-dependent binding to N-acetylglucosamine (GlcNAc) and mannose conjugated Toyopearl Amino 650 M matrices. When GlcNAc eluates from chromatography matrices were analyzed by reducing 1D PAGE and Western blots, the lectin appeared as approximately 37 kDa and approximately 72 kDa bands. Similar analysis of plasma revealed a single 72 kDa band under reducing conditions. MALDI-TOF MS demonstrated five, approximately 37 kDa isoforms (pI 5.3-6.1) separated by 2D-PAGE. A 975 bp cDNA sequence obtained by RT-PCR from liver and spleen tissue encoded a 325 amino acid secretory protein with homology to human and murine intelectins, which bind bacterial components and are induced during parasitic infections. Gene expression and immunohistochemistry detected RTInt in gill, spleen, hepatic sinusoid, renal interstitium, intestine, skin, swim bladder and within leukocytes. Direct binding assays demonstrated the ability of RTInt to bind relevant bacterial and chitinous targets. These findings suggest that RTInt plays a role in innate immune defense against bacterial and chitinous microbial organisms.

Immunohistochemical localization of rainbow trout ladderlectin and intelectin in healthy and infected rainbow trout (Oncorhynchus mykiss).

In the present study, the pattern of immuno-reactive ladderlectin and intelectin in healthy rainbow trout is compared to rainbow trout infected with a variety of infectious agents. In healthy rainbow trout, both proteins were localized to individual epithelial cells of the gill and intestine and both proteins were clearly demonstrated within cytoplasmic granules of polymorphonuclear leucocytes and macrophages/monocytes found in blood vessels, hepatic sinusoids, renal interstitium, mucosal epithelium and submucosa of normal intestine. In tissue from infected rainbow trout, there was an overall relative increase in both lectins compared to healthy fish and both proteins were detected in extra-cellular spaces surrounding bacteria, fungi and protozoa. Increased distribution and density of both RTLL and RTInt was demonstrated along mucosal surfaces and within inflammatory leucocytes in infected tissues and immune related organs. These findings represent one of the few examples of in vivo association of defence lectins and infectious agents.

The influence of oocyte cortisol on the early ontogeny of intelectin and TLR-5, and changes in lysozyme activity in rainbow trout (Oncorhynchus mykiss) embryos

The ontogeny of lysozyme activity, intelectin, TLR-5M and TLR-5S gene expression and intelectin localization was examined in rainbow trout (Oncorhynchus mykiss) reared from oocytes immersed for 3h prior to fertilization in either ovarian fluid alone (CC) or cortisol-enriched ovarian fluid at either 100 ng mL(-1) (C1) or 1000 ng mL(-1) (C2) [final oocyte cortisol concentrations were ~3, ~5, and ~7.5 ng oocyte(-1) for the CC, C1 and C2 treatment groups, respectively]. Lysozyme activity was elevated in the cortisol-treated groups from the zygote until 13-days post fertilization (dpf), but was not affected at 21-dpf. Intelectin levels were elevated in both cortisol treatment groups at 12-hpf (2-cell stage) and then suppressed between 36- and 48-hpf. Intelectin mRNA transcript levels were elevated in both cortisol treatment groups in oocytes; there were no differences among treatment groups at 1- and 5-dpf, and suppressed in the C2 treatment group in 13-dpf and 26-dpf. TLR-5 mRNA transcripts were higher in cortisol-treated oocytes prior to fertilization; TLR-5S mRNA was more abundant than TLR-5M mRNA. The ontogeny of the gene expression patterns, and the gene, lectin and lysozyme responses to oocyte cortisol adjustments suggest an important role of innate immune systems in the early cleavage stages of embryonic cells.

Cloning, binding properties, and tissue localization of rainbow trout (Oncorhynchus mykiss) ladderlectin

The present paper describes the primary structure, expression and immunohistochemical localization of rainbow trout ladderlectin (RTLL), a multimeric serum lectin that binds Sepharose and LPS of Aeromonas salmonicida. Two rainbow trout cDNAs (504 and 546bp) and a genomic sequence (2kb) were amplified using ladderlectin-specific primers. The sequences were identified as group VII mannose-binding C-type lectins from predicted amino acid sequences and showed highest identity with the Atlantic salmon mannose-binding lectin. The two cDNA sequences (RTLL-1 and RTLL-2) had 92% identity and encoded 173 and 187 amino acids, respectively. The genomic sequence of RTLL, obtained by PCR, was found to encompass six exons and five introns, with exon 2 encoding 14 amino acids which were exclusive to RTLL-2. The relative expression of both transcripts was highest in the renal kidney, while the intestine, gill and skin exhibited higher relative RTLL-2 expression than RTLL-1. RTLL was immunohistochemically present within cells of the branchial epithelium, hepatic sinusoids, biliary epithelium, renal interstitium, skin, and sub-mucosal granular layer of the intestine. RTLL bound galactan-based Sepharose 6B and Sepharose CL-6B matrices but did not bind unmodified acrylic resin base Toyopearl AF-Epoxy 650M, Toyopearl AF-Amino 650M matrices or N-acetylated Toyopearl AF-Amino 650M acrylic matrices. Two-dimensional SDS-PAGE and Western blots of whole plasma and plasma proteins which bound chitin and intact bacteria demonstrated multiple electrophoretic isoforms of RTLL ranging in size from 16 to 18kDa and isoelectric points between pH 4.9 and 6.3. These findings show that RTLL is a group VII C-type lectin with multiple isoforms that bind pathogen-associated molecular patterns such as chitin and microbial surfaces.

An immune-complex glomerulonephritis of Chinook salmon, Oncorhynchus tshawytscha (Walbaum)

Chinook salmon from New Zealand were shown to have a generalized membranous glomerulonephritis that was most severe in large fish. Marked thickening of the glomerular basement membrane was the most consistent lesion, with the presence of an electron-dense deposit beneath the capillary endothelium.Severely affected glomeruli also had expansion of the mesangium and loss of capillaries,synechiae of the visceral and parietal epithelium and mild fibrosis of Bowmans capsule. Chinook salmon from British Columbia, Canada with bacterial kidney disease caused by Renibacterium salmoninarum had similar histological lesions. They also had thickened glomerular basement membranes that were recognized by rabbit antiserum to rainbow trout immunoglobulin. This was true only when frozen sections of kidney were used and not formalin-fixed tissue. An attempt to experimentally produce a glomerulopathy in rainbow trout by repeated immunization with killed R. salmoninarum was not successful. Case records from the Fish Pathology Laboratory at the University of Guelph over a 10-year period revealed that a range of species were diagnosed with glomerulopathies similar to those seen in Chinook salmon. The majority of these cases were determined to have chronic inflammatory disease. This report has identified the presence of immunoglobulin within thickened basement membranes of Chinook salmon with glomerulonephritis and supports the existence of type III hypersensitivity in fish.

Effects of anionic polyacrylamide products on gill histopathology in juvenile rainbow trout (Oncorhynchus mykiss)

Anionic polyacrylamide (PAM) products are commonly used to remove suspended materials from turbid waters and to help mitigate soil erosion. In the present study, juvenile rainbow trout (Oncorhynchus mykiss) were exposed to 3 mg/L to 300 mg/L of 10 commercially available PAM products (Clearflow Water Lynx Polymer Log and Clearflow Soil Lynx Granular Polymer; Clearflow Enviro Systems Group), and gill histological parameters were measured following either 7 d or 30 d of polymer exposure. A cationic polymer product (≤0.38 mg/L MagnaFloc 368; Ciba Specialty Chemical) was also tested for comparison. Mild gill lesions were observed in fish exposed to polymer products. Lamellar fusion, interlamellar hyperplasia, epithelial lifting, mucous cell metaplasia, and cell counts of epithelial swelling and necrosis/apoptosis were minimal in fish exposed to environmentally relevant concentrations of anionic polymer (≤30 mg/L). Gill morphology was largely unaffected by exposure to concentrations up to 300 mg/L of many PAM products. Several anionic polymer products noticeably affected gill tissue by increasing epithelial hypertrophy, interlamellar hyperplasia, mucous cell metaplasia, and the frequency of necrotic cells. The severity of the lesions lessened with time, suggesting that fish may have experienced a short-term irritant effect. Similar levels of gill pathology were frequently observed in fish exposed to cationic polymer MagnaFloc 368 despite the concentration being 1000-fold lower than that of the PAM products. These observations highlight the increased toxicity of cationic polymers to aquatic life compared with anionic PAMs.

Plasma ladderlectin concentration following sterile inflammation and Aeromonas salmonicida subsp. salmonicida infection

Plasma samples obtained from rainbow trout either experimentally infected with Aeromonas salmonicida or injected with either A. salmonicida lipopolysaccharide (LPS) or a commercial A. salmonicida vaccine (Lipogen) were analysed by enzyme immunoassay to evaluate changes in rainbow trout ladderlectin (RTLL) concentrations during the acute phase response (APR). Plasma RTLL concentrations in fish injected with A. salmonicida LPS, vaccine or live A. salmonicida varied over a 10 day period, but did not significantly increase. In contrast, fish experimentally infected with A. salmonicida exhibited a modest, but statistically significant (P < 0.05), decrease in RTLL concentration. These studies demonstrate that RTLL is not detectably induced during the trout APR to sterile inflammation or A. salmonicida infection, but plasma concentration of this protein may be reduced during bacterial infection.