Development of duplex qPCR targeting Carnobacterium maltaromaticum and Vagococcus salmoninarum.

In November 2018, Vagococcus salmoninarum was identified as the causative agent of a chronic coldwater streptococcosis epizootic in broodstock brook trout (Salvelinus fontinalis) at the Iron River National Fish Hatchery in Wisconsin, USA. By February 2019, the epizootic spread to adjacent raceways containing broodstock lake trout (Salvelinus namaycush), whereby fish were found to be coinfected with Carnobacterium maltaromaticum and V. salmoninarum. To differentiate these two pathogens and determine the primary cause of the lake trout morbidity, a quantitative real-time PCR (qPCR) was developed targeting the C. maltaromaticum phenylalanyl-tRNA synthase alpha subunit (pheS) gene. The qPCR was combined with a V. salmoninarum qPCR, creating a duplex qPCR assay that simultaneously quantitates C. maltaromaticum and V. salmoninarum concentrations in individual lake trout tissues, and screens presumptive isolates from hatchery inspections and wild fish from national fish hatchery source waters throughout the Great Lakes basin. Vagococcus salmoninarum and C. maltaromaticum were co-detected in broodstock brook trout from two tribal hatcheries and C. maltaromaticum was present in wild fish in source waters of several national fish hatcheries. This study provides a powerful new tool to differentiate and diagnose two emerging Gram-positive bacterial pathogens.

Isolations of the Spring Viremia of Carp Virus in the Upper Mississippi River (USA), Including a New Host, the Quillback.

In July of 2018 and 2019, wild fish health surveys were conducted along the Wisconsin and Minnesota portions of the upper Mississippi River. Spring viremia of carp virus (SVCV) was isolated from Common Carp Cyprinus carpio as well as a newly identified host species, the Quillback Carpiodes cyprinus. Sanger sequencing of the gene encoding for the G protein revealed a high similarity of the Quillback isolate to various SVCV isolates identified from Common Carp that were collected during earlier wild fish health surveys and mortality events in the USA. Despite annual monitoring, this virus has been infrequently identified. The speculative role of native fish and invertebrates in allowing the virus to persist for long periods without detection is discussed.

Vagococcus salmoninarum II-qPCR, tropism and egg-associated transmission.

Vagococcus salmoninarum was identified as the causative agent of a chronic epizootic in broodstock "coaster" brook trout (Salvelinus fontinalis) at the Iron River National Fish Hatchery. The epizootic spanned more than a year, was unresponsive to multiple florfenicol treatments, and resulted in >50% mortality of the affected fish. The decision was made to cull the remaining fish during spawning, which presented an opportunity to more thoroughly examine V. salmoninarum sampling methods, organ tropism and vertical transmission. A newly developed qPCR targeting the pheS gene was used in concert with bacterial culture to show that V. salmoninarum indeed disproportionately affects females and has a tropism for female reproductive tissues. The study demonstrates that some female reproductive tissues (e.g. ovarian fluid, unfertilized eggs) are also an effective option for non-lethal detection. Despite the widespread presence of V. salmoninarum in ovarian fluid and on egg surfaces, we found no evidence of intra-ova transmission.

Vagococcus salmoninarum I-A chronic coldwater streptococcosis in broodstock brook trout (Salvelinus fontinalis) in Wisconsin, USA.

In 2018, Vagococcus salmoninarum was isolated from two lots of broodstock "coaster" brook trout (Salvelinus fontinalis) containing ~1,500 fish at the Iron River National Fish Hatchery, at which time it was identified as the causative agent of a chronic coldwater streptococcosis epizootic. Clinical signs included exophthalmia, lethargy, erratic swimming and loss of equilibrium. Female fish experienced disproportionately higher morbidity and mortality than male co-inhabitants, and routinely retained eggs following spawning. The most consistent gross clinical sign was heart pallor and turbid pericardial effusion. An attempted treatment using florfenicol was ineffective at halting the epizootic, which spanned more than a year and resulted in >50% mortality before remaining fish were culled. As there is no previous documentation of V. salmoninarum at this hatchery or in this species, it is still unclear what circumstances led to this epizootic. The inability to treat this chronic disease led to the loss of valuable broodstock, hampering ongoing fishery conservation efforts in the Great Lakes Basin.

Genomic characterization of a novel calicivirus, FHMCV-2012, from baitfish in the USA.

During regulatory sampling of fathead minnows (Pimephales promelas), a novel calicivirus was isolated from homogenates of kidney and spleen inoculated into bluegill fry (BF-2) cells. Infected cell cultures exhibiting cytopathic effects were screened by PCR-based methods for selected fish viral pathogens. Illumina HiSeq next generation sequencing of the total RNA revealed a novel calicivirus genome that showed limited protein sequence similarity to known homologs in a BLASTp search. The complete genome of this fathead minnow calicivirus (FHMCV) is 6564 nt long, encoding a polyprotein of 2114 aa in length. The complete polyprotein shared only 21% identity with Atlantic salmon calicivirus,followed by 11% to 14% identity with mammalian caliciviruses. A molecular detection assay (RT-PCR) was designed from this sequence for screening of field samples for FHMCV in the future. This virus likely represents a prototype species of a novel genus in the family Caliciviridae, tentatively named "Minovirus".

Novel reovirus associated with epidemic mortality in wild largemouth bass (Micropterus salmoides).

Reoviruses (family Reoviridae) infect vertebrate and invertebrate hosts with clinical effects ranging from inapparent to lethal. Here, we describe the discovery and characterization of Largemouth bass reovirus (LMBRV), found during investigation of a mortality event in wild largemouth bass (Micropterus salmoides) in 2015 in WI, USA. LMBRV has spherical virions of approximately 80 nm diameter containing 10 segments of linear dsRNA, aligning it with members of the genus Orthoreovirus, which infect mammals and birds, rather than members of the genus Aquareovirus, which contain 11 segments and infect teleost fishes. LMBRV is only between 24 % and 68 % similar at the amino acid level to its closest relative, Piscine reovirus (PRV), the putative cause of heart and skeletal muscle inflammation of farmed salmon. LMBRV expands the known diversity and host range of its lineage, which suggests that an undiscovered diversity of related pathogenic reoviruses may exist in wild fishes.

Development and evaluation of reverse transcription loop-mediated isothermal amplification assay for the detection of the fathead minnow nidovirus.

Fathead minnow nidovirus (FHMNV) is a serious baitfish-pathogenic virus in North America. Studies to trace the spread of the virus and determine its host range are hampered by the absence of reliable diagnostic assays. In this study, a one-step, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed that targets a region in the FHMNV spike protein gene. The assay was optimized, and the best results were obtained at 8 mM of Mg(2+) with an incubation time of 40 min at 63 °C in the presence of calcein. The analytical sensitivity of the RT-LAMP method was estimated to be as low as 5 viral copies and was 1000-fold more sensitive than the conventional reverse transcription polymerase chain reaction (RT-PCR) method. The diagnostic sensitivity and specificity of the developed RT-LAMP assay versus the RT-PCR assay was 100% and 95.7%, respectively. A quantitative RT-LAMP of FHMNV with a high correlation coefficient (r(2)=0.9926) was also developed and the result of quantitation of viral copies in tissue samples of infected fish showed that the viral loads of the infected fish tissue samples reached up to 4.7×10(10) copies per mg. It is anticipated that the developed RT-LAMP and quantitative RT-LAMP methods will be instrumental for diagnosis and surveillance of FHMNV.

Isolation and molecular characterization of a novel picornavirus from baitfish in the USA.

During both regulatory and routine surveillance sampling of baitfish from the states of Illinois, Minnesota, Montana, and Wisconsin, USA, isolates (n = 20) of a previously unknown picornavirus were obtained from kidney/spleen or entire viscera of fathead minnows (Pimephales promelas) and brassy minnows (Hybognathus hankinsoni). Following the appearance of a diffuse cytopathic effect, examination of cell culture supernatant by negative contrast electron microscopy revealed the presence of small, round virus particles (∼ 30-32 nm), with picornavirus-like morphology. Amplification and sequence analysis of viral RNA identified the agent as a novel member of the Picornaviridae family, tentatively named fathead minnow picornavirus (FHMPV). The full FHMPV genome consisted of 7834 nucleotides. Phylogenetic analysis based on 491 amino acid residues of the 3D gene showed 98.6% to 100% identity among the 20 isolates of FHMPV compared in this study while only 49.5% identity with its nearest neighbor, the bluegill picornavirus (BGPV) isolated from bluegill (Lepomis macrochirus). Based on complete polyprotein analysis, the FHMPV shared 58% (P1), 33% (P2) and 43% (P3) amino acid identities with BGPV and shared less than 40% amino acid identity with all other picornaviruses. Hence, we propose the creation of a new genus (Piscevirus) within the Picornaviridae family. The impact of FHMPV on the health of fish populations is unknown at present.