Morphological and molecular data establishes Clinostomum dolichorchum n. sp. (Digenea: Clinostomidae) in the great blue heron Ardea herodias L. and American bullfrog Rana catesbeiana Shaw.

Clinostomum is a cosmopolitan genus of trematodes that infect piscivorous birds, freshwater molluscs, freshwater fish and amphibians. Herein, a novel species of Clinostomum is described based on morphological and molecular data from an adult in the oral cavity of the great blue heron Ardea herodias and metacercariae collected from the gills and skin of American bullfrog tadpoles Rana catesbeiana. The novel species shares similar qualitative and quantitative morphological features with a congener, Clinostomum marginatum, which has overlap in host and geographic distribution. The most notable morphological difference when compared to C. marginatum is the greater posterior testis length of the novel species. Molecular data resolved similarities with morphological comparisons to nominal species and supports the establishment of a novel species. Molecular data include partial small ribosomal subunit (18S rRNA gene), ribosomal internal transcribed spacer regions (ITS1, 5.8S rRNA gene, and ITS2), partial large ribosomal subunit (28S rRNA gene), cytochrome c oxidase subunit 1 gene (cox1), and nicotinamide adenine dinucleotide dehydrogenase subunit 1 gene (nad1) sequences. Phylogenetic analyses place the novel species in a sister clade to C. marginatum. Morphological and molecular data, combined with phylogenetic analyses support the establishment of Clinostomum dolichorchum n. sp.

Characterization of an alloherpesvirus from wild lake sturgeon Acipenser fulvescens in Wisconsin (USA).

In the spring of 2017, 2 adult lake sturgeon (LS) Acipenser fulvescens captured from the Wolf River, Wisconsin (USA), presented with multiple cutaneous plaques that, upon microscopic examination, indicated proliferative epidermitis. Ultrastructural examination of affected keratinocytes revealed particles in the nucleus having a morphology typical of herpesviruses. A degenerate PCR assay targeting the DNA polymerase catalytic subunit (pol) gene of large double-stranded DNA viruses generated amplicons of the anticipated size from skin samples, and sequences of amplicons confirmed the presence of a novel alloherpesvirus (lake sturgeon herpesvirus, LSHV) related to acipenserid herpesvirus 1 (AciHV1). The complete genome (202660 bp) of this virus was sequenced using a MiSeq System, and phylogenetic analyses substantiated the close relationship to AciHV1. A PCR assay targeting the LSHV DNA packaging terminase subunit 1 (ter1) gene demonstrated the presence of the virus in 39/42 skin lesion samples collected from wild LS captured in 2017-2019 and 2021 in 4/4 rivers in Wisconsin. Future efforts to isolate LSHV in cell culture would facilitate challenge studies to determine the disease potential of the virus.

Genetic characterization of esocid herpesvirus 1 (EsHV1).

Blue spot disease, believed to be caused by esocid herpesvirus 1 (EsHV1), has been observed in wild northern pike Esox lucius in a number of cold-water locations, including the northern USA, Canada, and Ireland. In the spring of 2014, a northern pike was caught in Wisconsin displaying the characteristic bluish-white circular plaques on the dorsum and fins. Microscopic examination of hematoxylin and eosin-stained sections of the proliferative cutaneous lesions revealed a focally extensive abundance of panepidermal, megalocytic keratinocytes with karyomegaly. Enlarged nuclei stained basophilic, and an abundance of coarse eosinophilic granules were observed in the expanded cytoplasm. Transmission electron microscopy revealed aggregates of enveloped virus particles with electron-dense, hexagonal nucleocapsids surrounded by a uniformly staining ellipsoidal tegument layer within cytoplasmic vacuoles of megalocytic epidermal cells. More than 7000 bp of the EsHV1 genome were sequenced from infected skin tissues. Phylogenetic and phenetic analyses, based on the partial DNA-dependent DNA polymerase and terminase gene sequences, revealed EsHV1 forms a novel branch within the family Alloherpesviridae as the sister group to the clade that includes members of the genera Ictalurivirus and Salmonivirus. The gross, microscopic, and ultrastructural lesions reported in our study were identical to previous reports of blue spot disease in northern pike; however, here we provide the first molecular evidence supporting EsHV1 as a new species in the family Alloherpesviridae.

Temporal Variation in Viral Hemorrhagic Septicemia Virus Antibodies in Freshwater Drum (Aplodinotus grunniens) Indicates Cyclic Transmission in Lake Winnebago, Wisconsin.

Viral hemorrhagic septicemia virus (VHSV) is an emerging pathogen that causes mass mortality in multiple fish species. In 2007, the Great Lakes freshwater strain, type IVb, caused a large die-off of freshwater drum (Aplodinotus grunniens) in Lake Winnebago, Wisconsin, USA. To evaluate the persistence and transmission of VHSV, freshwater drum from Lake Winnebago were tested for antibodies to the virus using recently developed virus neutralization (VN) and enzyme-linked immunosorbent (ELISA) assays. Samples were also tested by real-time reverse transcription-PCR (rRT-PCR) to detect viral RNA. Of 548 serum samples tested, 44 (8.03%) were positive by VN (titers ranging from 1:16 to 1:1,024) and 45 (8.21%) were positive by ELISA, including 7 fish positive by both assays. Antibody prevalence increased with age and was higher in one northwestern area of Lake Winnebago than in other areas. Of 3,864 tissues sampled from 551 fish, 1 spleen and 1 kidney sample from a single adult female fish collected in the spring of 2012 tested positive for VHSV by rRT-PCR, and serum from the same fish tested positive by VN and ELISA. These results suggest that VHSV persists and viral transmission may be active in Lake Winnebago even in years following outbreaks and that wild fish may survive VHSV infection and maintain detectable antibody titers while harboring viral RNA. Influxes of immunologically naive juvenile fish through recruitment may reduce herd immunity, allow VHSV to persist, and drive superannual cycles of transmission that may sporadically manifest as fish kills.

Detection of viral hemorrhagic septicemia virus-IVb antibodies in sera of muskellunge Esox masquinongy using competitive ELISA.

A competitive enzyme-linked immunosorbent assay (cELISA) was developed for the detection of antibodies to viral hemorrhagic septicemia virus genotype IVb (VHSV-IVb) in fish sera. Assay conditions were standardized using known negative and positive muskellunge Esox masquinongy. A positive-negative threshold of 14.6% inhibition was established based on analysis of sera of 60 muskellunge with no previous exposure to VHSV-IVb. The cELISA was then used to investigate immune responses of wild muskellunge sampled from 5 water bodies in Michigan and Wisconsin, USA, between 2005 and 2012. Antibodies were detected in fish from Lake St. Clair, Michigan, and Lower Fox River/Green Bay, Wisconsin. Both water systems were considered enzootic for VHSV-IVb. Additionally, antibodies were detected in muskellunge from Thornapple Lake, a Michigan inland lake previously considered negative for VHSV-IVb based on virus isolation methods. Muskellunge populations from Lake Hudson, Michigan, and Butternut Lake, Wisconsin, lacked evidence of an immune response to VHSV-IVb. When results of the cELISA were compared to the 50% plaque neutralization test for several groups of fish, there was 78.4% agreement between the tests for antibody presence. The cELISA is a rapid and efficient test for the detection of binding antibodies to VHSV-IVb and will be a useful non-lethal tool for monitoring the spread of this serious pathogen.

Development and evaluation of a blocking enzyme-linked immunosorbent assay and virus neutralization assay to detect antibodies to viral hemorrhagic septicemia virus.

Viral hemorrhagic septicemia virus (VHSV) is a target of surveillance by many state and federal agencies in the United States. Currently, the detection of VHSV relies on virus isolation, which is lethal to fish and indicates only the current infection status. A serological method is required to ascertain prior exposure. Here, we report two serologic tests for VHSV that are nonlethal, rapid, and species independent, a virus neutralization (VN) assay and a blocking enzyme-linked immunosorbent assay (ELISA). The results show that the VN assay had a specificity of 100% and sensitivity of 42.9%; the anti-nucleocapsid-blocking ELISA detected nonneutralizing VHSV antibodies at a specificity of 88.2% and a sensitivity of 96.4%. The VN assay and ELISA are valuable tools for assessing exposure to VHSV.

Decreased mortality of Lake Michigan Chinook salmon after bacterial kidney disease challenge: evidence for pathogen-driven selection?

In the late 1960s, Chinook salmon Oncorhynchus tshawytscha from the Green River, Washington, were successfully introduced into Lake Michigan. During spring from 1988 to 1992, large fish die-offs affecting Chinook salmon occurred in the lake. Multiple ecological factors probably contributed to the severity of the fish kills, but the only disease agent found regularly was Renibacterium salmoninarum, the causative agent of bacterial kidney disease. In this study, survival after challenge by R. salmoninarum was compared between two Chinook salmon stocks: a Lake Michigan stock from Wisconsin (WI) and the progenitor stock from the Green River. We found that the WI stock had significantly greater survival than the Green River stock. Next, the WI and Green River stocks were exposed to the marine pathogen Listonella anguillarum (formerly Vibrio anguillarum), one of the causative agents of vibriosis; survival after this challenge was significantly poorer for the WI stock than for the Green River stock. A close genetic relationship between the Green River and WI stocks was confirmed by analyzing 13 microsatellite loci. These results collectively suggest that disease susceptibility of Lake Michigan Chinook salmon has diverged from that of the source population, possibly in response to pathogen-driven selection.