Identification of polymorphisms in the bovine collagenous lectins and their association with infectious diseases in cattle.

Infectious diseases are a significant issue in animal production systems, including both the dairy and beef cattle industries. Understanding and defining the genetics of infectious disease susceptibility in cattle is an important step in the mitigation of their impact. Collagenous lectins are soluble pattern recognition receptors that form an important part of the innate immune system, which serves as the first line of host defense against pathogens. Polymorphisms in the collagenous lectin genes have been shown in previous studies to contribute to infectious disease susceptibility, and in cattle, mutations in two collagenous lectin genes (MBL1 and MBL2) are associated with mastitis. To further characterize the contribution of variation in the bovine collagenous lectins to infectious disease susceptibility, we used a pooled NGS approach to identify short nucleotide variants (SNVs) in the collagenous lectins (and regulatory DNA) of cattle with (n = 80) and without (n = 40) infectious disease. Allele frequency analysis identified 74 variants that were significantly (p < 5 × 10-6) associated with infectious disease, the majority of which were clustered in a 29-kb segment upstream of the collectin locus on chromosome 28. In silico analysis of the functional effects of all the variants predicted 11 SNVs with a deleterious effect on protein structure and/or function, 148 SNVs that occurred within potential transcription factor binding sites, and 31 SNVs occurring within potential miRNA binding elements. This study provides a detailed look at the genetic variation of the bovine collagenous lectins and identifies potential genetic markers for infectious disease susceptibility.

Temporary protection of rainbow trout gill epithelial cells from infection with viral haemorrhagic septicaemia virus IVb.

The branchial epithelium is not only a primary route of entry for viral pathogens, but is also a site of viral replication and subsequent shedding may also occur from the gill epithelium. This study investigated the potential of agents known to stimulate innate immunity to protect rainbow trout epithelial cells (RTgill-W1) from infection with VHSV IVb. RTgill-W1 cells were pretreated with poly I:C, FuGENE(®) HD + poly I:C, lipopolysaccharide (LPS), LPS + poly I:C or heat-killed VHSV IVb and then infected with VHSV IVb 4 days later. Cytopathic effect (CPE) was determined at 2, 3, 4, 7 and 11 days post-infection. Virus in cells and supernatant was detected using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). All of the treatments delayed the onset of CPE (per cent of monolayer destruction), compared with untreated controls; however, killed VHSV or poly I:C combined with LPS was the most effective. Similarly, the detection of viral RNA in the supernatant was delayed, and the quantity was significantly (P < 0.05) reduced by all treatments with the exception of LPS alone (4 days). Unlike many of the other treatments, pretreatment of RTgill-W1 with heat-killed VHSV did not upregulate interferon 1, 2 or MX 1 gene expression.

Pathogenesis of spring viremia of carp virus in emerald shiner Notropis atherinoides Rafinesque, fathead minnow Pimephales promelas Rafinesque and white sucker Catostomus commersonii (Lacepede).

Spring viremia of carp (SVC) is a reportable disease to the World Organization of Animal Health (OIE) as it is known to cause significant international economic impact. In Canada, the first and only isolation of SVC virus (SVCV) was in 2006, from common carp Cyprinus carpio L., at Hamilton Harbour, Lake Ontario. The susceptibility of fathead minnow Pimephales promelas Rafinesque, emerald shiner Notropis atherinoides Rafinesque and white sucker Catostomus commersonii (Lacepede) to intraperitoneal injection of the Canadian isolate (HHOcarp06) was evaluated using experimental infection, virus isolation, quantitative reverse transcription polymerase chain reaction (qRT-PCR), histopathology and immunohistochemistry (IHC). Emerald shiner and fathead minnow were most susceptible with 43 and 53% cumulative mortality, respectively, compared with koi at 33%. Quantitative RT-PCR demonstrated that koi had high viral loads throughout the experiment. At 34 days post-infection, SVCV was detected from sampled emerald shiner and white sucker in very low titre and was not detected from fathead minnow. Koi, fathead minnow and emerald shiner had gross lesions typical of SVC disease. The histopathological picture was mostly dominated by necrotic changes in kidney, spleen, liver, pancreas and intestine. IHC further confirmed SVCV infection, and staining was largely correlated with histological lesions.

Differential viral haemorrhagic septicaemia virus genotype IVb infection in fin fibroblast and epithelial cell lines from walleye, Sander vitreus (Mitchill), at cold temperatures.

A cell line, WE-cfin11e, with an epithelial-like morphology was developed from a caudal fin of walleye, Sander vitreus (Mitchill), characterized as distinct from the established walleye caudal fin fibroblast-like cell line, WE-cfin11f, and compared with WE-cfin11f for susceptibility to VHSV IVb. Immunocytochemistry and confocal microscopy were used to localize the intermediate filament protein, vimentin, the tight junction protein, zonula occludens-1 (ZO-1), the extracellular matrix protein, collagen I, and the viral protein, G. Although both cell lines contained vimentin, only WE-cfin11e stained for ZO-1 and only WE-cfin11f stained for collagen I. Ascorbic acid increased the accumulation of collagen I and caused the appearance of collagen fibres only in WE-cfin11f cultures. At 14 °C, both cell lines produced VHSV IVb, but the infection developed more rapidly in WE-cfin11f. At 4 °C, both cell lines became infected with VHSV IVb as judged by the expression of viral proteins, N and G, but only WE-cfin11f produced virus. The results suggest that cold temperatures can modulate viral tropism.

Epitheliocystis in lake trout Salvelinus namaycush (Walbaum) is associated with a ß-proteobacteria.

Lake trout Salvelinus namaycush (Walbaum) raised for stocking experienced yearly (2011-13) winter epizootics of epitheliocystis. Affected fish were dispersed on the bottom of the tank, had decreased feed and fright response, and mortality often reached 40%. Peak mortality occurred within 3 weeks of the appearance of clinical signs, and outbreaks typically lasted 6 weeks. Affected fish had no gross lesions but histologically had branchial epithelial necrosis and lamellar hyperplasia, with small to large numbers of scattered epithelial cells containing 10- to 20-µm inclusions. A longitudinal study was undertaken of one annual outbreak, and lamellar hyperplasia was most closely associated with mortality. The number of inclusions was statistically greater (P < 0.05) before and during peak mortality, but inclusions were present in low numbers before clinical signs occurred. Results of histochemical staining, immunohistochemistry and transmission electron microscopy supported the presence of a ß-proteobacteria rather than a Chlamydiales bacterium within inclusions. PCR primers to identify Chlamydiales did not give consistent results. However, the use of universal 16S rDNA bacterial primers in conjunction with laser capture microdissection of inclusions demonstrated that a ß-proteobacteria was consistently associated with affected gills and is more likely the cause of the disease in lake trout.

Development of a walleye cell line and use to study the effects of temperature on infection by viral haemorrhagic septicaemia virus group IVb.

A cell line, WE-cfin11f, with a fibroblast-like morphology was developed from a walleye caudal fin and used to study the intersection of thermobiology of walleye, Sander vitreus (Mitchill), with the thermal requirements for replication of viral haemorrhagic septicaemia virus (VHSV) IVb. WE-cfin11f proliferated from 10 to 32 °C and endured as a monolayer for at least a week at 1-34 °C. WE-cfin11f adopted an epithelial shape and did not proliferate at 4 °C. Adding VHSV IVb to cultures at 4 and 14 °C but not 26 °C led to cytopathic effects (CPE) and virus production. At 4 °C, virus production developed more slowly, but Western blotting showed more N protein accumulation. Infecting monolayer cultures at 4 °C for 7 days and then shifting them to 26 °C resulted in the monolayers being broken in small areas by CPE, but with time at 26 °C, the monolayers were restored. These results suggest that at 26 °C, the VHSV IVb life cycle stages responsible for CPE can be completed, but the production of virus and the initiation of infections cannot be accomplished.

Purified deoxynivalenol or feed restriction reduces mortality in rainbow trout, Oncorhynchus mykiss (Walbaum), with experimental bacterial coldwater disease but biologically relevant concentrations of deoxynivalenol do not impair the growth of Flavobacterium psychrophilum.

Diets containing deoxynivalenol (DON) were fed to rainbow trout Oncorhynchus mykiss (Walbaum) for 4 weeks followed by experimental infection (intraperitoneal) with Flavobacterium psychrophilum (4.1 × 10(6) colony-forming units [CFU] mL(-1) ). Mortality of rainbow trout fed either 6.4 mg kg(-1) DON or trout pair-fed the control diet was significantly reduced (P < 0.05) in comparison with trout fed the control diet to apparent satiation (<0.1 mg kg(-1) DON). In a second experiment, trout were fed one of three experimental diets; a control diet, a diet produced with corn naturally contaminated with DON (3.3 mg kg(-1) DON) or a diet containing purified DON (3.8 mg kg(-1) ); however, these fish were not experimentally infected. The presence of DON resulted in significant reduction (P < 0.0001) in feed intake as well as weight gain after 4 weeks. Respiratory burst of head-kidney leucocytes isolated from rainbow trout fed diets containing purified DON (3.8 mg kg(-1) ) was significantly higher (P < 0.05) at 35 day post-exposure compared with controls. The antimicrobial activity of DON was examined by subjecting F. psychrophilum in vitro to serial dilutions of the chemical. Complete inhibition occurred at a concentration of 75 mg L(-1) DON, but no effect was observed below this concentration (0-30 mg L(-1) ).

Walleye Sander vitreus (Mitchill) are relatively resistant to experimental infection with VHSV IVb and extant walleye strains vary in susceptibility.

Compared to fathead minnow, walleye demonstrate low susceptibility to experimental infection with VHSV IVb, regardless of route of exposure or water temperature at time of infection. In triplicate and duplicate groups, walleye were intraperitoneally (i.p.) injected (102 -108  pfu/fish) or waterborne-exposed (w; 1.4 × 107  pfu mL-1 ) with VHSV IVb. High cumulative mortality (64-100%) and severe gross lesions associated with VHSV IVb infection were evident only in fish i.p. injected with 108  pfu at 12 °C. These fish had multifocal necrosis of several tissues including the gill and heart. There was no difference in mortality between walleye infected (w or i.p.) at 12 °C (spring stocking) compared with a declining temperature profile from 18 to 12 °C (fall stocking). There were significant differences (P < 0.05) in mortality between four extant walleye strains following i.p. infection, indicating that the choice of walleye strain for stocking might be an important consideration. Viral antigen was found in both i.p. and w-exposed walleye using immunohistochemistry, mostly within the gill and skin of w-exposed fish and most prominently in dermal fibrocytes. VHSV IVb was detected in multiple tissues from 6 to 21 days post-infection using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR).

Amyloid associated with neoplasia in two captive tricolour sharkminnows Balantiocheilus melanopterus Bleeker.

Amyloid associated with pancreatic adenocarcinoma was discovered in two captive adult tricolour sharkminnows Balantiocheilus melanopterus Bleeker found dead in a freshwater display. Enlarged abdomens expanded by bloody ascitic fluid and grossly visible masses of abnormal tissue were present surrounding sections of the stomach and intestine. Histologically, the masses were composed of areas of well-organized exocrine pancreatic acini interspersed with cords of poorly differentiated, spindle-shaped cells that compressed and effaced normal parenchyma. These cells possessed small numbers of cytoplasmic zymogen granules; the exocrine nature of these cells was confirmed using transmission electron microscopy (TEM). Fibrovascular connective tissue of the hepatopancreas and mesenteries was expanded by lightly eosinophilic, hyaline, homogeneous acellular material. Similar material greatly expanded the tunica media of large blood vessels in the hepatopancreas. After staining with Congo red or thioflavin T, this material exhibited red-green dichroism under polarized light or bright green fluorescence under ultraviolet light (255 nm), respectively. The non-branching fibrils, of indeterminate length, had an approximate diameter of 10-20 nm using TEM. Although exocrine pancreatic neoplasia is relatively common in fish, the presence of amyloid is not. To our current knowledge, the latter has not yet been described in association with a neoplastic lesion in fish.

Diseases of captive yellow seahorse Hippocampus kuda Bleeker, pot-bellied seahorse Hippocampus abdominalis Lesson and weedy seadragon Phyllopteryx taeniolatus (Lacépède).

Seahorses, pipefish and seadragons are fish of the Family Syngnathidae. From 1998 to 2010, 172 syngnathid cases from the Toronto Zoo were submitted for post-mortem diagnostics and retrospectively examined. Among the submitted species were yellow seahorses Hippocampus kuda Bleeker (n=133), pot-bellied seahorses Hippocampus abdominalis Lesson (n=35) and weedy seadragons Phyllopteryx taeniolatus (Lacépède; n=4). The three most common causes of morbidity and mortality in this population were bacterial dermatitis, bilaterally symmetrical myopathy and mycobacteriosis, accounting for 24%, 17% and 15% of cases, respectively. Inflammatory processes were the most common diagnoses, present in 117 cases. Seven neoplasms were diagnosed, environmental aetiologies were identified in 46 cases, and two congenital defects were identified.

Differential effects of viral hemorrhagic septicaemia virus (VHSV) genotypes IVa and IVb on gill epithelial and spleen macrophage cell lines from rainbow trout (Oncorhynchus mykiss).

The two most prominent genotypes of viral hemorrhagic septicemia virus (VHSV) are -I in the Northeastern Atlantic region and -IV in North America, but much more is known about the cellular pathogenesis of genotype -I than -IV. VHSV genotype -IV is divided into -IVa from the Northeast Pacific Ocean and -IVb from the Great Lakes and both of which are less virulent to rainbow trout than genotype -I. In this work, infections of VHSV-IVa and -IVb have been studied in two rainbow trout cell lines, RTgill-W1 from the gill epithelium, and RTS11 from spleen macrophages. RTgill-W1 produced infectious progeny of both VHSV-IVa and -IVb. However, VHSV-IVa was more infectious than -IVb toward RTgill-W1: -IVa caused cytopathic effect (CPE) at a lower viral titre, elicited CPE earlier, and yielded higher titres. By contrast, no CPE and no increase in viral titre were observed in RTS11 cultures infected with either genotype. Yet in RTS11 all six VHSV genes were expressed and antiviral genes, Mx2 and Mx3, were up regulated by VHSV-IVb and -IVa. However, replication appeared to terminate at the translational stage as viral N protein, presumably the most abundant of the VSHV proteins, was not detected in either infected RTS11 cultures. In RTgill-W1, Mx2 and Mx3 were up regulated to similar levels by both viral genotypes, while VHSV-IVa induced higher levels of IFN1, IFN2 and LGP2A than VHSV-IVb.

Rainbow trout Oncorhynchus mykiss ladderlectin, but not intelectin, binds viral hemorrhagic septicemia virus IVb.

The innate immune system of fish is critical for rapid detection and immediate response to infection, as well as to orchestrate the adaptive branch of the immune system. Rainbow trout Oncorhynchus mykiss ladderlectin and intelectin are plasma pattern recognition receptors (PRR) for bacterial and fungal pathogens of rainbow trout, but their role as PRRs for virus is unknown. Viral hemorrhagic septicemia virus (VHSV) IVb is a recently described fish pathogen in the Great Lakes, and rainbow trout can be experimentally infected. Using an indirect enzyme-linked immunosorbent assay, rainbow trout plasma ladderlectin significantly (p < 0.05) bound purified VHSV while intelectin did not. In addition, plasma ladderlectin but not intelectin was eluted from a VHSV-conjugated Toyopearl column using EDTA. Protein identification was confirmed with polyclonal antiserum used with slot immunoblot, 1-dimensional sodium dodecyl sulphate polyacrylamide electrophoresis, and Western immunoblot.

Detection of VHSV IVb within the gonads of Great Lakes fish using in situ hybridization.

Viral haemorrhagic septicaemia virus (VHSV) genotype IVb was recently detected as the cause of numerous mortality events in Great Lakes fish. In situ hybridization was used to examine the gonads from 13 fish, including freshwater drum Aplodinotus grunniens and muskellunge Esox masquinongy that were infected naturally, as well as rainbow trout Oncorhynchus mykiss and fathead minnows Pimphales promelas, which were experimentally infected. Although the ovaries and testes of fish infected by VHSV IVb had few lesions, viral RNA was present in the ovaries of the rainbow trout and fathead minnow and was abundant in the gonads of muskellunge and in the ovaries of freshwater drum. Viral RNA was present mainly surrounding yolk vacuoles/granules or adjacent to the germinal vesicle, with lesser amounts found within the germinal vesicle, in the mesovarium and/or tunica albuginea and blood vessels of the ovary. Viral RNA was also found in and surrounding primary and secondary spermatocytes of the muskellunge.

Immunohistochemistry and pathology of multiple Great Lakes fish from mortality events associated with viral hemorrhagic septicemia virus type IVb.

A novel viral hemorrhagic septicemia virus (VHSV) (genotype IVb) has been isolated from mortality events in a range of wild freshwater fish from the Great Lakes since 2005. In 2005 and 2006, numerous new freshwater host species (approximately 90 fish from 12 different species) were confirmed to have VHSV by cell culture and reverse transcriptase polymerase chain reaction. A prominent feature observed in infected fish were the petechial and ecchymotic haemorrhages on the body surface and in visceral organs, as well as serosanguinous ascites; however, many fish had few and subtle, gross lesions. Histologically, virtually all fish had a vasculitis and multifocal necrosis of numerous tissues. Excellent correlation was found between the presence of VHSV IVb antigen detected by immunohistochemistry and the pathological changes noted by light microscopy. Intact and degenerate leukocytes, including cells resembling lymphocytes and macrophages, also had cytoplasmic viral antigen. By contrast, renal tubules and gonadal tissues (ovary and testis), were strongly immunopositive for VHSV IVb, but no lesions were noted.

Gastric dilation and air sacculitis in Chinook salmon, Oncorhynchus tshawytscha (Walbaum); correlation of macroscopic and microscopic lesions, and relationship of the syndrome to glomerulonephritis and serum biochemistry.

Macroscopic and microscopic assessment procedures were developed to evaluate the severity and enable diagnosis from histological samples, of gastric dilation and air sacculitis (GDAS) in Chinook salmon, Oncorhynchus tshawytscha. Stomachs and swim bladders were examined from young fish with experimentally induced GDAS and from larger fish with the syndrome held in commercial saltwater net-pens. Fish fed a diet previously known to induce GDAS had significantly wider stomachs with decreased prominence of longitudinal stomach folds that contained larger amounts of fluid (P < 0.001), and thinner stomach walls with greater inter-nerve distances (P < 0.001), than fish fed an alternative commercial diet not associated with the syndrome. These fish also had swim bladders that were more likely to be opaque and contain more fluid (P < 0.001). These observations correlated well with selected criteria for stomach tissue (P < 0.002) and swim bladder (P < 0.04) that could be evaluated microscopically. Four stomach measurements, primarily measurements of wall or partial wall thickness and inter-nerve distances, were suitable for discriminating between affected and non-affected fish. A stomach width ratio, that was independent of fish weight and highly correlated with macroscopic stomach measurements (P < 0.0001), was particularly useful; this ratio was derived from the distances between both the outer border of the muscularis mucosa and mesothelium of the serosal surface to the stratum compactum. Serum biochemistry parameters (osmolality, calcium and magnesium) did not differ between fish fed different diets, but serum creatinine concentration was correlated with the microscopic thickness of the muscularis externa of the stomach wall and the total stomach thickness (P < 0.001 and P < 0.003, respectively). A glomerulonephritis was also noted in these fish. The severity of the lesion was not significantly related to GDAS nor to any serum biochemistry parameter assayed however.

Nodular gill disease causing proliferative branchitis and mortality in Chinook salmon (Oncorhynchus tshawytscha).

CASE HISTORY: A period of escalating mortality occurred among Chinook salmon (1-5 g) held in freshwater raceways at a commercial culture facility. The mortality rate peaked at 1.5% of the population per day, water temperature was 9-10 degrees C, and water quality had recently deteriorated due to an influx of suspended solids. The affected fish did not respond to several chloramine-T bath treatments (10 ppm for 1 hour). CLINICAL FINDINGS: The dead fish did not exhibit flared opercula typical of infection with bacterial gill disease (BGD) nor did the remaining fish show obvious signs of respiratory distress. Clinical lesions were limited to the gills of the fish, and were consistent with a proliferative branchitis, including substantial hyperplasia and fusion of lamellae. DIAGNOSIS: Numerous angular amoebic organisms (approximately 10 x 8 microm), with a central, large vesicular nucleus and faintly acidophilic cytoplasm, visible on H&E-stained sections, were located on the hyperplastic epithelia of the lamellae, filaments, and remaining lining of the branchial cavity. Morphologically, the amoebae were similar to representatives of the genus Cochliopodia spp. described from North American salmonids, that presented similar lesions and clinical signs; consequently, the disease was diagnosed as nodular gill disease (NGD). CLINICAL RELEVANCE: Grossly, NGD can be mistaken for BGD; the distinction is important because chloramine-T, routinely used in the treatment of BGD, is ineffective for the treatment of NGD. Static formalin baths of 170 ppm (37% formaldehyde) for 1 hour, supplemented with oxygen, may reduce amoebic infections but should be used cautiously as treatment can precipitate mortality in fish with gill disease. Cues for the onset of NGD are not completely understood, although poor water quality and elevations in temperature are likely to exacerbate infections. Similarly, the prevalence of this disease is not accurately known, but it is possible that it may be more common among salmonids in New Zealand than is presently recognised.

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.