Detection of Viral Hemorrhagic Septicemia Virus (VHSV) from Diporeia spp. (Pontoporeiidae, Amphipoda) in the Laurentian Great Lakes, USA
© Faisal and Winters; licensee BioMed Central Ltd. 2011
Received: 4 September 2010
Accepted: 6 January 2011
Published: 6 January 2011
The mode of viral hemorrhagic septicemia virus (VHSV) transmission in the Great Lakes basin is largely unknown. In order to assess the potential role of macroinvertebrates in VHSV transmission, Diporeia spp., a group of amphipods that are preyed upon by a number of susceptible Great Lakes fishes, were collected from seven locations in four of the Great Lakes and analyzed for the presence of VHSV. It was demonstrated that VHSV is present in some Diporeia spp. samples collected from lakes Ontario, Huron, and Michigan, but not from Lake Superior. Phylogenetic comparison of partial nucleoprotein (N) gene sequences (737 base pairs) of the five isolates to sequences of 13 other VHSV strains showed the clustering of Diporeia spp. isolates with the VHSV genotype IVb. This study reports the first incidence of a fish-pathogenic rhabdovirus being isolated from Diporeia, or any other crustacean and underscores the role macroinvertebrates may play in VHSV ecology.
The viral hemorrhagic septicemia virus (VHSV), genotype IVb, is a recent invader to the Laurentian Great Lakes basin and has been associated with mortalities in a number of resident freshwater fish species , , , . While laboratory studies demonstrated that the virus can be transmitted to naïve fish by both immersion and injection , , , the mode of VHSV transmission in the Great Lakes basin is largely unknown. In a previous study, it was concluded the pisocolid intermittent leech Myzobdella lugubris harbors VHSV . Whether other macroinvertebrates can act as a vector or reservoir for VHSV remains to be elucidated.
In the Great Lakes foodweb, amphipods of the genera Diporeia, Gammarus, and Hyalella occupy a central position as they transform energy from lower to higher trophic levels . Unfortunately, Diporeia spp. have experienced a sharp decline sharp decline in abundance over the last two decades ; the cause(s) of which puzzle scientists. To tackle this enigma, a study was designed that involved comprehensive parasitological and microbiological analysis of Diporeia spp. collected from lakes Ontario, Huron, Michigan and Superior [Faisal and Winters: Pathogens impacting Diporeia spp. in the Great Lakes, submitted].
Since there is currently no amphipod cell line that could be used to aid in the isolation of amphipod-pathogenic viruses, virus isolation was performed according to the standard protocols detailed in the American Fisheries Society Blue Book  and the Office International des Epizooties , using the Epithelioma papulosum cyprinii (EPC) cell line . Inoculated 96-well plates containing EPC cells grown with MEM (5% fetal bovine serum) were incubated at 15°C for 21 days, and were observed for the formation of cytopathic effects (CPE). Second and third blind passages were performed and assessed for the presence of CPE. All cell culture positive samples of Diporeia homogenates (ON41, ON55-M, HU54-M, MI18-M, and MI27-M) caused CPE on EPC in the form of focal areas of rounded, refractile cells which progressed to full lysis of the cell monolayer.
Locations in the Laurentian Great Lakes from which Diporeia spp. were collected for this study (CPE = formation of cytopathic effect; RT-PCR = results for amplification of the viral hemorrhagic septicemia virus nucleoprotein gene).
Our findings provided evidence that VHSV can exist within Diporeia spp. Whether VHSV propagates in the cells of these amphipods or just existed in their viscera or gills is currently unknown and deserves further investigation. Indeed, the presence of VHSV in Diporeia spp. in three of the four Great Lakes sampled is surprising since these amphipods were collected from depths that ranged from 74-190 meters where none of the susceptible fish are known to reside. Diporeia spp. feed on detritus and planktonic organisms and is known to scavenge for food items in lower depths. Such a feeding habit has the potential to transfer VHSV from the benthos to the pelagic zone through their excreta or by being preyed upon by susceptible fish; lake whitefish for example. Moreover, based exclusively on data generated in this study, one cannot rule out that VHSV is a pathogen of Diporeia spp. or that Diporeia spp. can be a reservoir for VHSV in the Great Lakes. Regardless of these currently unanswered questions, this study reports the first incidence of a fish-pathogenic rhabdovirus being isolated from Diporeia, or other crustacean. This finding underscores the dire need to better understand the role of macroinvertebrates in disease ecology.
The authors thank the crew of the R/V Lake Guardian for their assistance with sample collection. In order to conduct the study, we are indebted to the generous funding provided by the United States Environmental Protection Agency - Great Lakes National Protection Office (Grant #: GL00E36101) and the United States Department of Agriculture - Animal and Plant Health Inspection Service (Grant#: 10-9100-1293-GR).
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