Evaluating observer bias and seasonal detection rates in amphibian pathogen eDNA collections by citizen scientists.

We trained volunteers from conservation organizations to collect environmental DNA (eDNA) from 21 ponds with amphibian communities that had a history of Batrachochytrium dendrobatidis (Bd) and ranavirus (Rv) infections. Volunteers were given sampling kits to filter pond water and preserve eDNA on filter paper, as were the principal investigators (PIs), who made independent collections within 48 h of volunteer collections. Using multi-scale occupancy modeling, we found no evidence to suggest the observer who collected the water sample (volunteer or PI) influenced either the probability of capturing eDNA on a filter or the probability of detecting extracted eDNA in a quantitative PCR (qPCR) reaction. The cumulative detection probability of Bd eDNA at a pond decreased from May through July 2017 because there was a decrease in the probability of detecting eDNA in qPCR reactions. In contrast, cumulative detection probability increased from May to July for Rv due to a higher probability of capturing eDNA on filters later in the year. Our models estimate that both pathogens could be detected with 95% confidence in as few as 5 water samples taken in June or July tested with either 4 or 3 qPCR reactions, respectively. Our eDNA protocols appeared to detect pathogens with 95% confidence using considerably fewer samples than protocols which typically recommend sampling ≥30 individual animals. In addition, eDNA sampling could reduce some biosecurity concerns, jurisdictional and institutional permitting, and stress to biota at ponds.

Development of a loop-mediated isothermal amplification assay for the detection and quantification of epizootic epitheliotropic disease virus (salmonid herpesvirus-3).

Epizootic Epitheliotropic Disease Virus (EEDV; Salmonid Herpesvirus-3) causes a serious disease hatchery-reared lake trout (Salvelinus namaycush), threatening restoration efforts of this species in North America. The current inability to replicate EEDV in vitro necessitates the search for a reproducible, sensitive, and specific assay that allows for its detection and quantitation in a time- and cost-effective manner. Herein, we describe a loop-mediated isothermal amplification (LAMP) assay that was developed for the quantitative detection of EEDV in infected fish tissues. The newly developed LAMP reaction was optimized in the presence of calcein, and the best results were produced using 2 mM MgCl2, 1.8 mM dNTPs and at an incubation temperature of 67.1 °C. This method was highly specific to EEDV, as it showed no cross-reactivity with several fish viruses, including Salmonid Herpesvirus-1, -2, -4, and -5, Infectious Pancreatic Necrosis Virus, Spring Viremia of Carp Virus, Infectious Hematopoietic Necrosis Virus, Golden Shiner Reovirus, Fathead Minnow Nidovirus, and Viral Hemorrhagic Septicemia Virus. The analytical sensitivity of the EEDV-LAMP method was estimated to be as low as 16 copies of plasmid per reaction. When infected fish tissue was used, a positive reaction could be obtained when an infected gill tissue sample that contained 430 viral copies/µg was diluted up to five orders of magnitude. The sensitivity and specificity of the newly developed LAMP assay compared to the SYBR Green qPCR assay were 84.3% and 93.3%, respectively. The quantitative LAMP for EEDV had a correlation coefficient (R2 = 0.980), and did not differ significantly from the SYBR Green quantitative PCR assay (p > 0.05). Given its cost- and time-effectiveness, this quantitative LAMP assay is suitable for screening lake trout populations and for the initial diagnosis of clinical cases.

Seasonal infection rates of Batrachochytrium dendrobatidis in populations of northern green frog Lithobates clamitans melanota tadpoles.

Few studies have documented seasonal variation of Batrachochytrium dendrobatidis (Bd) infection rates in larval amphibians. We identified 4 natural populations of northern green frogs Lithobates clamitans melanota in Pennsylvania (USA) that contained Bd-infected tadpoles during post-wintering collections in May and June, after hibernating tadpoles had overwintered in wetlands. However, we failed to detect infected tadpoles at those wetlands when pre-wintering collections were made in late July through early September. We observed 2 cohorts of tadpoles that appeared to lack Bd-infected individuals in pre-wintering collections, yet contained Bd-infected individuals the following spring. We also observed 4 cohorts of pre-wintering tadpoles that were Bd-free, even though post-wintering tadpoles collected earlier in the year were infected with Bd. Our results suggest that tadpoles either reduce Bd infections during the summer months, and/or infections proliferate sometime prior to (or shortly after) tadpoles emerge from hibernation. It is unlikely that pre-wintering tadpoles were too small to detect Bd zoospores because (1) there was no correlation between Bd zoospore levels and tadpole size or stage, and (2) size was not a significant predictor of infection status. These results suggest that, while sampling larvae can be an effective means of collecting large sample sizes, investigators in our Mid-Atlantic region should conduct sampling by early summer to maximize the chances of detecting Bd. Further research is warranted to determine whether wetland topography and warm, shallow microhabitats within wetlands contribute to a population's ability to drastically reduce Bd prevalence prior to overwintering at ponds.

Initial Detection and Molecular Characterization of Namaycush Herpesvirus (Salmonid Herpesvirus 5) in Lake Trout.

A novel herpesvirus was found by molecular methods in samples of Lake Trout Salvelinus namaycush from Lake Erie, Pennsylvania, and Lake Ontario, Keuka Lake, and Lake Otsego, New York. Based on PCR amplification and partial sequencing of polymerase, terminase, and glycoprotein genes, a number of isolates were identified as a novel virus, which we have named Namaycush herpesvirus (NamHV) salmonid herpesvirus 5 (SalHV5). Phylogenetic analyses of three NamHV genes indicated strong clustering with other members of the genus Salmonivirus, placing these isolates into family Alloherpesviridae. The NamHV isolates were identical in the three partially sequenced genes; however, they varied from other salmonid herpesviruses in nucleotide sequence identity. In all three of the genes sequenced, NamHV shared the highest sequence identity with Atlantic Salmon papillomatosis virus (ASPV; SalHV4) isolated from Atlantic Salmon Salmo salar in northern Europe, including northwestern Russia. These results lead one to believe that NamHV and ASPV have a common ancestor that may have made a relatively recent host jump from Atlantic Salmon to Lake Trout or vice versa. Partial nucleotide sequence comparisons between NamHV and ASPV for the polymerase and glycoprotein genes differ by >5% and >10%, respectively. Additional nucleotide sequence comparisons between NamHV and epizootic epitheliotropic disease virus (EEDV/SalHV3) in the terminase, glycoprotein, and polymerase genes differ by >5%, >20%, and >10%, respectively. Thus, NamHV and EEDV may be occupying discrete ecological niches in Lake Trout. Even though NamHV shared the highest genetic identity with ASPV, each of these viruses has a separate host species, which also implies speciation. Additionally, NamHV has been detected over the last 4 years in four separate water bodies across two states, which suggests that NamHV is a distinct, naturally replicating lineage. This, in combination with a divergence in nucleotide sequence from EEDV, indicates that NamHV is a new species in the genus Salmonivirus. Received April 20, 2015; accepted October 11, 2015.

A Quantitative Polymerase Chain Reaction Assay for the Detection and Quantification of Epizootic Epitheliotropic Disease Virus (EEDV; Salmonid Herpesvirus 3).

Epizootic epitheliotropic disease virus (EEDV; salmonid herpesvirus [SalHV3]; family Alloherpesviridae) causes a systemic disease of juvenile and yearling Lake Trout Salvelinus namaycush. No cell lines are currently available for the culture and propagation of EEDV, so primary diagnosis is limited to PCR and electron microscopy. To better understand the pervasiveness of EEDV (carrier or latent state of infection) in domesticated and wild Lake Trout populations, we developed a sensitive TaqMan quantitative PCR (qPCR) assay to detect the presence of the EEDV terminase gene in Lake Trout tissues. This assay was able to detect a linear standard curve over nine logs of plasmid dilution and was sensitive enough to detect single-digit copies of EEDV. The efficiency of the PCR assay was 99.4 ± 0.06% (mean ± SD), with a 95% confidence limit of 0.0296 (R(2) = 0.994). Methods were successfully applied to collect preliminary data from a number of species and water bodies in the states of Pennsylvania, New York, and Vermont, indicating that EEDV is more common in wild fish than previously known. In addition, through the development of this qPCR assay, we detected EEDV in a new salmonid species, the Cisco Coregonus artedi. The qPCR assay was unexpectedly able to detect two additional herpesviruses, the Atlantic Salmon papillomatosis virus (ASPV; SalHV4) and the Namaycush herpesvirus (NamHV; SalHV5), which both share high sequence identity with the EEDV terminase gene. With these unexpected findings, we subsequently designed three primer sets to confirm initial TaqMan qPCR assay positives and to differentiate among EEDV, ASPV, and NamHV by detecting the glycoprotein genes via SYBR Green qPCR. Received April 20, 2015; accepted November 10, 2015.

Isolation and molecular characterization of a novel infectious pancreatic necrosis virus strain in returning Atlantic salmon Salmo salar from the Connecticut River, USA.

After 22 years of negative viral screening results, the viral pathogen infectious pancreatic necrosis virus (IPNV) was isolated from the ovarian fluid of two pooled samples of returning Connecticut River Atlantic salmon Salmo salar during the 2007 spawning season at Richard Cronin National Salmon Station (RCNSS), Hadley, Massachusetts. Cytopathic effect was observed in Chinook salmon embryo (CHSE-214) cells, and IPNV was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR). Sequence analysis conducted by the U.S. Geological Survey's Western Fisheries Research Center determined that the isolate closely resembled the Canada_3 strain, falling into Genogroup 4 rather than Genogroup 1, which is more common in the United States. This allowed us to speculate that the Atlantic salmon were not infected during their freshwater life stage in the Connecticut River watershed but somewhere on their migratory route or feeding grounds in the Northwest Atlantic. On November 20, 2007, the Connecticut River Atlantic Salmon Commission voted to depopulate the infected stock at RCNSS and the entire suspect egg lots held at White River National Fish Hatchery, Vermont. Approximately one and a half months later, the 121 Connecticut River Atlantic salmon were euthanized and sampled for a follow-up investigation to determine the prevalence of infection. Only one kidney-spleen homogenate (male) was confirmed IPNV positive via cell culture and RT-PCR. A total of 2,983 base pairs from segment A of the RNA genome were sequenced from this fish and determined to be from a new strain (Connecticut-1) of IPNV that closely resembles Canada_2 and Canada_3 in Genogroup 4. The new strain is genetically identical to one of the first ovarian fluid isolates over a shared 130-nucleotide region, possibly indicating original transmission from a single source. The absence of IPNV from the Connecticut River's subsequent four returning Atlantic salmon year-classes may indicate that the aggressive corrective action was prudent.

Description of two new gill myxozoans from smallmouth (Micropterus dolomieu) and largemouth (Micropterus salmoides) bass.

Two previously undescribed species of myxozoan parasites were observed in the gills of bass inhabiting the Potomac and James River basins. They are described using morphological characteristics and small-subunit (SSU) rDNA gene sequences. Both were taxonomically identified as new species of Myxobolus; Myxobolus branchiarum n. sp. was found exclusively in smallmouth bass, and Myxobolus micropterii n. sp. was found in largemouth and smallmouth bass. Small, spherical, white plasmodia of M. branchiarum from smallmouth bass were observed grossly in the gills; these plasmodia had an average length of 320.3 µm and width of 246.1 µm. The development of the plasmodia is intralamellar in the secondary lamellae of the gills. Mature spores were pyriform in shape with a length of 12.8 ± 1.4 (8.1-15.1) µm and width of 6.9 ± 1.1 (4.0-9.0) µm. Analysis of SSU rDNA identified M. branchiarum in a sister-group to 3 species of Henneguya , although morphologically caudal appendages were absent. Myxobolus micropterii observed in the gills of largemouth and smallmouth bass had larger, ovoid, cream-colored plasmodia with an average length of 568.1 µm and width of 148.1 µm. The cysts developed at the distal end of the gill filament within the primary lamellae. The mature spores were ovoid in shape with a length of 10.8 ± 0.7 (9.2-12.2) µm and width of 10.6 ± 0.6 (9.0-11.8) µm. SSU rDNA analysis placed M. micropterii in a sister group with Henneguya lobosa and Myxobolus oliveirai . The highest prevalence of M. branchiarum was observed in the gills of bass collected from the Cowpasture River (50.9%). Prevalence was 44.6% in bass from the Potomac River and only 4.3% in bass collected from the Shenandoah River. A seasonal study of M. branchiarum , which included both infected and uninfected smallmouth bass, determined that a significantly higher intensity was observed in the spring than in the summer (P < 0.001) or fall (P  =  0.004). In an analysis excluding uninfected bass, a higher intensity was observed in the spring than in the summer (P  =  0.001) or fall (P  =  0.008). Prevalence and seasonal differences were not determined for M. micropterii .

Origin and evolution of TNF and TNF receptor superfamilies.

The tumor necrosis factor superfamily (TNFSF) and the TNF receptor superfamily (TNFRSF) have an ancient evolutionary origin that can be traced back to single copy genes within Arthropods. In humans, 18 TNFSF and 29 TNFRSF genes have been identified. Evolutionary models account for the increase in gene number primarily through multiple whole genome duplication events as well as by lineage and/or species-specific tandem duplication and translocation. The identification and functional analyses of teleost ligands and receptors provide insight into the critical transition between invertebrates and higher vertebrates. Bioinformatic analyses of fish genomes and EST datasets identify 14 distinct ligand groups, some of which are novel to teleosts, while to date, only limited numbers of receptors have been characterized in fish. The most studied ligand is TNF of which teleost species possess between 1 and 3 copies as well as a receptor similar to TNFR1. Functional studies using zebrafish indicate a conserved role of this ligand-receptor system in the regulation of cell survival and resistance to infectious disease. The increasing interest and use of TNFSF and TNFRSF modulators in human and animal medicine underscores the need to understand the evolutionary origins as well as conserved and novel functions of these biologically important molecules.

Preliminary amphibian health survey in the Delaware Water Gap National Recreation Area.

To detect aquatic animal diseases of national concern, 111 individual amphibians, including wood frogs Rana sylvatica (28), spring peepers Pseudacris crucifer (35), red-spotted newts Notophthalmus viridescens (41), and gray tree frogs Hyla versicolor (7), were sampled at seven different sites in the Delaware Water Gap National Recreation Area (DGNRA), Pennsylvania, from June 14 to July 19, 2007. These samples were screened for Batrachochytrium dendrobatidis and viral pathogens at the U.S. Fish and Wildlife Service's Fish Health Center in Lamar, Pennsylvania. Cell culture revealed cytopathic effect (CPE) in two cell lines (epithelioma papillosum cyprini and fathead minnow) inoculated with liver, kidney, and spleen samples from one sample pool of Notophthalmus viridescens (4 individuals). Polymerase chain reaction was conducted on cell culture supernatant exhibiting CPE. Sequencing revealed the resulting product to be identical to frog virus 3, a ranavirus in the family Iridoviridae. Upon gross examination, two Notophthalmus viridescens were found to exhibit dermal swelling and lethargy. Histological examination of these lesions revealed involvement by an Ichthyophonus sp. In summary, two pathogens of concern were found in amphibians in the DGNRA: a ranavirus with a major capsid protein sequence identical to that of frog virus 3 and a mesomycetozoan, Ichthyophonus sp. Although no epizootic die-offs were observed during this health survey, the results warrant further research into the distribution of these pathogens throughout the DGNRA because they have the potential to cause mass mortalities in amphibians.

Spleen size predicts resistance of rainbow trout to Flavobacterium psychrophilum challenge.

Selective breeding of animals for increased innate resistance offers an attractive strategy to control disease in agriculture. However, this approach is limited by an incomplete knowledge of the heritability, duration, and mechanism(s) of resistance, as well as the impact of selection on the immune response to unrelated pathogens. Herein, as part of a rainbow trout broodstock improvement program, we evaluated factors involved in resistance against a bacterial disease agent, Flavobacterium psychrophilum. In 2005, 71 full-sibling crosses, weighing an average of 2.4 g, were screened, and resistant and susceptible crosses were identified. Naive cohorts were evaluated at 10 and 800 g in size, and most maintained their original relative resistant or susceptible phenotypes, indicating that these traits were stable as size increased >300-fold. During the course of these studies, we observed that the normalized spleen weights of the resistant fish crosses were greater than those of the susceptible fish crosses. To test for direct association, we determined the spleen-somatic index of 103 fish crosses; created high, medium, and low spleen-index groups; and determined survival following challenge with F. psychrophilum or Yersinia ruckeri. Consistent with our previous observations, trout with larger spleen indices were significantly more resistant to F. psychrophilum challenge; however, this result was pathogen-specific, as there was no correlation of spleen size with survival following Y. ruckeri challenge. To our knowledge, this is the first report of a positive association between spleen size and disease resistance in a teleost fish. Further evaluation of spleen index as an indirect measure of disease resistance is warranted.

Early diversification of the TNF superfamily in teleosts: genomic characterization and expression analysis.

The TNF superfamily (TNFSF) of proteins are cytokines involved in diverse immunological and developmental pathways. Little is known about their evolution or expression in lower vertebrate species. Bioinformatic searches of Zebrafish, Tetraodon, and Fugu genome and other teleost expressed sequence tag databases identified 44 novel gene sequences containing a TNF homology domain. This work reveals the following: 1) teleosts possess orthologs of BAFF, APRIL, EDA, TWEAK, 4-1BBL, Fas ligand, LIGHT, CD40L, RANKL, and possibly TL1A; 2) the BAFF-APRIL subfamily is enriched by a third member, BALM, unique to fish; 3) orthologs of lymphotoxins alpha and beta were not clearly identified in teleosts and are substituted by a related ligand, TNF-New; 4) as many as four TRAIL-like genes are present in teleosts, as compared with only one in mammals; and 5) T cell activation ligands OX40L, CD27L, CD30L, and GITRL were not identified in any fish species. Finally, we characterize mRNA expression of TNFSF members CD40L, LIGHT, BALM, APRIL, Fas ligand, RANKL, TRAIL-like, and TNF-New in rainbow trout, Oncorhynchus mykiss, immune and nonimmune tissues. In conclusion, we identified a total of 14 distinct TNFSF members in fishes, indicating expansion of this superfamily before the divergence of bony fish and tetrapods, approximately 360-450 million years ago. Based on these findings, we extend a model of TNFSF evolution and the co-emergence of the vertebrate adaptive immune system.

Identification of novel rainbow trout (Onchorynchus mykiss) chemokines, CXCd1 and CXCd2: mRNA expression after Yersinia ruckeri vaccination and challenge.

Chemokines play important roles in controlling leukocyte trafficking under normal and inflammatory conditions. Sixteen CXC chemokines have been identified in the human and mouse genomes, while considerably fewer teleost fish CXC chemokines have been reported. Here, we describe a novel clade of trout (Onchorynchus mykiss) CXC chemokines, designated Onmy CXCd, and we identify a novel gene, CXCd1, and a putative duplicate, CXCd2. The trout CXCd proteins contain 112 amino acids and the CXCd1 gene is comprised of four exons and three introns. Constitutive CXCd mRNA expression was detected in skin, gill, visceral fat, and posterior kidney tissues, while low transcript levels were present in the anterior kidney and spleen. Spleen CXCd transcript abundance increased 1 day after bath vaccination (fourfold) and subsided to basal levels by 7 days postvaccination. Challenge with viable Yersinia ruckeri induced expression of trout CXCd RNA up to ninefold in the spleen. The number of viable Y. ruckeri were significantly correlated with CXCd gene transcript abundance (P = 0.0051, Spearman correlation 0.497, n = 30 fish), and fish with the highest bacterial loads had the highest CXCd expression. In contrast, pro-inflammatory cytokine IL-1-beta2 mRNA levels were elevated in fish infected with low numbers of Y. ruckeri, while diminishing in heavily infected fish. CXCd mRNA expression was not increased in rainbow trout infected with infectious hematopoietic necrosis virus, suggesting that up-regulation may be pathogen-specific. Taken together, these results indicate that CXCd transcript elevation follows the pro-inflammatory cytokine response to Y. ruckeri and may be a relevant immunological marker of exposure.

Fate of intraperitoneally injected fluorescent microspheres in developing Ictalurus punctatus.

Fluorescent microspheres (FMS) were injected intraperitoneally into channel catfish fry at 2 days post hatch (dph), 1, 2, 3, 4 and 8 weeks post hatch (wph). The FMS were observed in the vasculature almost immediately after injection in all age groups except 2 dph. Fluorescent microspheres were observed within mononuclear phagocytes in the vasculature after 0.16 dph in all age groups. Fluorescent microspheres were first phagocytized in the coelomic cavity immediately after injection, while the majority of coelomic FMS were phagocytized between 0.16 and 1 dph for all ages. Enzyme cytochemical staining indicated that both polymorphonuclear (neutrophilic granulocytes) and mononuclear phagocytes had phagocytized FMS in the coelomic cavity and organs, with a predominance of FMS found in mononuclear phagocytic cells in all age groups across all sample periods. The predominant organs associated with the observed cellular responses were the posterior kidney, spleen, and anterior kidney. Splenic organization and melanomacrophage development and activity were more pronounced as the fish aged from 2 wph on. Particulate clearance rates were faster in the 2 dph and 1 wph fish than the older ages of fish. These results suggest that to facilitate particulate retention, channel catfish should be vaccinated at 4 wph or older.

Fate of fluorescent microspheres in developing Ictalurus punctatus following prolonged immersion.

Particulate antigen uptake by the mucosa of developing channel catfish was determined by immersing larvae and fry [2-day post-hatch (dph), 1-, 2-, 3-, 4-, and 8-week post-hatch (wph)] to two forms of fluorescent microspheres (FMS): blue FMS were carboxylated, and green FMS were coated via conjugation with a crude extract of Edwardsiella ictaluri outer membrane protein (OMP). Phagocytosis, destination, and clearance appeared similar for the two types of FMS used. In the older age classes, primary uptake was observed in epithelial cells of the torso, fins, nares and to a lesser extent the gills. Fluorescent microspheres were less frequently observed within mononuclear phagocytes in the epidermis, dermis and underlying connective tissue of the tissue mentioned above. Limited FMS trafficking was observed from 4- to 24-h post-immersion (hpi). Significantly higher numbers of FMS (blue and green)/mm(3) of tissue were observed in the posterior kidney of the 4- and 8-wph age classes and in the anterior kidney and spleen of the 8-wph age class when compared to younger age classes (p < 0.05). Significantly higher FMS (blue and green)/mm(3) of tissue were observed in the posterior kidney of 4- and 8-wph fish when compared to all other organs (p < 0.05). The present study indicates that FMS uptake increases with age in channel catfish. The younger age classes may possess an increased ability to exclude particulate antigen, or lack the specific mechanisms that needed to take up particulates in the form of FMS.