Assessment of a Serologic Diagnostic Test and Kinetics of Antibody Development in Northern Pike Experimentally Infected with Viral Hemorrhagic Septicemia Virus

Abstract

Viral hemorrhagic septicemia virus (VHSV) is an ongoing cause of disease and mortality in freshwater fishes across the Great Lakes region of the Midwestern United States. Antibody detection assays such as enzyme‐linked immunosorbent assay (ELISA) are nonlethal serological methods that can have significantly shorter turnaround times than the current validated viral detection diagnostic methodology for VHSV: cell culture with confirmation by polymerase chain reaction (PCR). This study evaluated an ELISA that detects nonneutralizing antinucleocapsid antibodies to VHSV in Northern Pike Esox lucius. Juvenile Northern Pike were experimentally infected with VHSV by intraperitoneal injection. The infected fish were monitored for 12 weeks for signs of disease, and weekly serum samples were obtained. An analysis of the survival data showed that mortality occurred significantly more quickly in inoculated fish than in control fish. Fish that were infected by injection showed a significant increase in antibody response by 2 weeks postinfection. However, variation in the rate and pattern of antibody response among the infected fish was high at any given point. The optimum window for detecting antibodies in Northern Pike is 2–12 weeks postinfection, which generally follows the median time to appearance of clinical signs (21 d postinfection). The receiver‐operating characteristic curve analysis showed the ELISA to have a sensitivity of 80.5% and a specificity of 63.2% in Northern Pike, but these values can be adjusted by choosing different percent inhibition cutoffs, which may facilitate the use of the test for specific management goals. The results of this study offer insights into the disease progression and immune kinetics of VHSV, including interindividual variation, which will aid in the management of this economically important virus.

Widespread Seropositivity to Viral Hemorrhagic Septicemia Virus (VHSV) in Four Species of Inland Sport Fishes in Wisconsin

“… f Percent positive using published threshold criteria of ≥25%I for Brown Trout (Wilson‐Rothering et al 2014) and ≥58.2%I for Northern Pike (Thiel et al 2020). …”

“…Although %I calculations reduce the effects of non‐specific binding on our results, there is still a risk of false positives. For this reason, highly conservative thresholds were adopted to maximize specificity (2 SDs above the mean), and alternative positive thresholds were also considered for Brown Trout and Northern Pike based on published data for these species (Wilson‐Rothering et al 2014; Thiel et al 2020; the published threshold for Northern Pike was altered to increase specificity for surveillance purposes—see Methods for details). The thresholds chosen (Table 1) may change as new data are collected, but the use of such baselines for management decisions is feasible.…”

“…Wilson‐Rothering et al (2014) developed an ELISA that detects nonneutralizing antinucleocapsid antibodies to VHSV across fish species by using nonlethal blood samples. The original publication showed that the test performed well in Brown Trout Salmo trutta , Yellow Perch Perca flavescens , Grass Carp Ctenopharyngodon idella , Pacific Herring, Muskellunge, and Freshwater Drum (sensitivity = 96.4%; specificity = 88.2%), and we (Thiel et al 2020) recently demonstrated that the test performed adequately in Northern Pike (sensitivity = 80.6%; specificity = 63.2%). However, this test has yet to be used for broad surveillance of wild fish populations.…”

“…The ELISA method developed by Wilson‐Rothering et al (2014, 2015), with minor alterations by Thiel et al (2020), provided the basis for this serological assessment. This blocking ELISA uses a monoclonal antibody (Aquatic Diagnostics, Stirling, Scotland; conjugated by American Qualex, San Clemente, California) against the nucleocapsid protein of the virus (Olesen et al 1991; Wilson‐Rothering et al 2014).…”

“…For Brown Trout, an alternative threshold of ≥25%I was used (Wilson‐Rothering et al 2014). For Northern Pike, an alternative threshold of ≥58.2%I was used (Thiel et al 2020; note that the published threshold of ≥41.3%I in experimentally infected Northern Pike was altered to improve results for surveillance purposes, which increased specificity to 95.4% and therefore decreased sensitivity of the assay to 34.5%).…”