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

Abstract

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.

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

“…While multiple tissues were affected, including most dramatically hematopoietic tissues and the meninges, IHC revealed viral antigen in the endothelium confirming this tropism of VHSV IVb. Similarly, vasculitis was a dominant lesion in natural infections of freshwater drum (Lumsden et al 2007), and endothelial viral antigen has been noted in experimentally infected rainbow trout using more than one genotype of VHSV (Evensen et al 1994, Al-Hussinee et al 2010. The often extensive, gross hemorrhages observed on serosal surfaces correlated with the presence of viral antigen in blood vessels and tissues, but there were exceptions.…”

“…Virus isolation from fish processed by Cornell was performed as described (USFWS 2004). Fish examined at Guelph were processed according to Al-Hussinee et al (2010). The nRT-PCR assay was performed as described (AlHussinee et al 2010).…”

“…VHSV IVb isolated from freshwater drum (U13653, Lumsden et al 2007) was purified as described (Al-Hussinee et al 2010). Briefly, virus grown on EPC monolayers with complete cytopathic effect (CPE) were clarified by centrifugation and then applied to glycerol cushions (Granzow et al 2001, Betts et al 2003.…”

“…Animal experimentation was performed according to the Animal Care Guidelines of the University of Guelph. IHC was performed as described (Al-Hussinee et al 2010). Briefly, formalin-fixed tissues were deparaffinized, sectioned at 5 µm, and mounted on Snowcoat X-tra glass slides (Surgipath) to air dry.…”

“…To prepare the rabbit antibody, 2 rabbits were injected with purified virus as described (Al-Hussinee et al 2010). Pre-immune serum prepared in a similar manner was also available from both rabbits before immunization.…”