Effects of temperature on viral load, inclusion body formation, and host response in Pacific Herring with viral erythrocytic necrosis (VEN).

OBJECTIVE: The primary objective of this study was to determine the effects of temperature on viral erythrocytic necrosis (VEN) progression under controlled conditions. Secondarily, this study was intended to evaluate the combined effects of temperature and VEN on the Pacific Herring Clupea palasii transcriptome. METHODS: The effects of temperature on VEN progression were assessed by waterborne exposure of laboratory-reared, specific-pathogen-free Pacific Herring to tissues homogenates containing erythrocytic necrosis virus (ENV) at 6.9, 9.0, or 13.5°C. RESULT: Exposure of Pacific Herring to ENV resulted in the establishment of infections characterized by high infection prevalence (89%; 40/45) and mean viral loads (5.5 log10 [gene copies/µg genomic DNA]) in kidney tissues at 44 days postexposure. Mean viral loads were significantly higher in fish from the ambient (mean = 9.0°C) and warm (mean = 13.5°C) treatments (6.1-6.2 log10 [gene copies/total genomic DNA]) than in fish from the cool (mean = 6.9°C) treatment (4.3 log10 [gene copies/µg genomic DNA]). Similarly, the peak proportion of diseased fish was directly related to temperature, with cytoplasmic inclusion bodies detected in 21% of fish from the cool treatment, 52% of fish from the ambient treatment, and 60% of fish from the warm treatment. The mean VEN load in each fish (enumerated as the percentage of erythrocytes with cytoplasmic inclusions) at 44 days postexposure increased with temperature from 15% in the cool treatment to 36% in the ambient treatment and 32% in the warm treatment. Transcriptional analysis indicated that the number of differentially expressed genes among ENV-exposed Pacific Herring increased with temperature, time postexposure, and viral load. Correlation network analysis of transcriptomic data showed robust activation of interferon and viral immune responses in the hepatic tissue of infected individuals independent of other experimental variables. CONCLUSION: Results from this controlled laboratory study, combined with previous observations of natural epizootics in wild populations, support the conclusion that temperature is an important disease cofactor for VEN in Pacific Herring.

Noninvasive Monitoring of Radiation-Induced Salivary Gland Vascular Injury.

Xerostomia is a common side effect of radiation therapy (RT) in patients with head and neck cancer. However, limited information is available on the temporal dynamics of parenchymal and vascular changes in salivary glands following RT. To address this gap in knowledge, we conducted experimental studies in mice employing ultrasound (US) with coregistered photoacoustic imaging (PAI) to noninvasively assess the early and late changes in salivary gland size, structure, vascularity, and oxygenation dynamics following RT. Multiparametric US-PAI of salivary glands was performed in immune-deficient and immune-competent mice before and after RT along with correlative sialometry and ex vivo histologic-immunohistochemical validation. US revealed reduction in gland volume and an early increase in vascular resistance postradiation. This was accompanied by a reduction in glandular oxygen consumption on PAI. Imaging data correlated strongly with salivary secretion and histologic evidence of acinar damage. The magnitude and kinetics of radiation response were impacted by host immune status, with immunodeficient mice showing early and more pronounced vascular injury and DNA damage response compared to immunocompetent animals. Our findings demonstrate the ability of noninvasive US-PAI to monitor dynamic changes in salivary gland hemodynamics following radiation and highlight the impact of the host immune status on salivary gland radiation injury.

Long-term shedding from fully convalesced individuals indicates that Pacific herring are a reservoir for viral hemorrhagic septicemia virus.

Processes that allow viral hemorrhagic septicemia (VHS) virus to persist in the marine environment remain enigmatic, owing largely to the presence of covert and cryptic infections in marine fishes during typical sub-epizootic periods. As such, marine host reservoirs for VHS virus have not been fully demonstrated, nor have the mechanism(s) by which infected hosts contribute to virus perpetuation and transmission. Here, we demonstrate that after surviving VHS, convalesced Pacific herring continue to shed virus at a low rate for extended periods. Further, exposure of previously naïve conspecific sentinels to this shed virus can result in infections for at least 6 mo after cessation of overt disease. This transmission mechanism was not necessarily dependent on the magnitude of the disease outbreak, as prolonged transmission occurred from 2 groups of donor herring that experienced cumulative mortalities of 4 and 29%. The results further suggest that the virus persists in association with the gills of fully recovered individuals, and long-term viral shedding or shedding relapses are related to cooler or decreasing water temperatures. These results provide support for a new VHS virus perpetuation paradigm in the marine environment, whereby the virus can be maintained in convalesced survivors and trafficked from these carriers to sympatric susceptible individuals.

A geographic hot spot of Ichthyophonus infection in the southern Salish Sea, USA.

The prevalence of Ichthyophonus infection in Pacific herring Clupea pallasii was spatially heterogeneous in the southern Salish Sea, Washington State, USA. Over the course of 13 mo, 2232 Pacific herring were sampled from 38 midwater trawls throughout the region. Fork length was positively correlated with Ichthyophonus infection at all sites. After controlling for the positive relationship between host size and Ichthyophonus infection, the probability of infection was approximately 6-fold higher in North Hood Canal than in Puget Sound and the northern Straits (12 vs. 2% predicted probability for a 100 mm fish and 30 vs. 7% predicted probability for a 180 mm fish). Temporal changes in Ichthyophonus infection probability were explained by seasonal differences in fish length, owing to Pacific herring life history and movement patterns. Reasons for the spatial heterogeneity remain uncertain but may be associated with density-dependent factors inherent to the boom-bust cycles that commonly occur in clupeid populations.

Intra-Annual Changes in Waterborne Nanophyetus salmincola.

An analysis of daily water samples collected from an index site on Big Soos Creek, Washington indicated intra-annual differences in the concentrations of waterborne Nanophyetus salmincola. Waterborne concentrations, quantified as gene copies/L, peaked during the fall (October-November 2016), decreased to very low concentrations over the winter (January-March 2017), and then increased in the spring and throughout the summer. High waterborne concentrations of N. salmincola DNA (2 × 106 gene copies/L) corresponded with live N. salmincola cercariae (mean = 3 cercariae/L) that were detected in companion water samples. Spikes in waterborne N. salmincola concentrations in October and November typically coincided with increases in streamflow; this combination resulted in elevated infection pressures during high water events in the fall. The peak in waterborne N. salmincola concentrations corresponded with an accompanying peak in tissue parasite density (metacercariae/posterior kidney) in Coho Salmon Oncorhynchus kisutch that were reared in the untreated water.

Susceptibility of Nanophyetus salmincola Cercariae to Formalin, Hydrogen Peroxide, and Seawater.

The ability of formalin, PEROX-AID (hydrogen peroxide), and seawater to kill waterborne Nanophyetus salmincola cercariae was evaluated in vitro. Newly emerged cercariae survived for extended periods in freshwater, with 53-73% survival occurring in negative control groups after 24 h. Exposure to dilutions of formalin reduced this survival time, with 0% of cercariae surviving after 30 min in 450 µL/L, 40 min in 225 µL/L, and 300 min in 113 µL/L. Exposure to PEROX-AID (hydrogen peroxide) for 1 h resulted in reduced cercarial survival (16.4%) only at the highest concentration (100 µL/L), compared with 100% survival in the untreated controls and all lesser concentrations. Exposure to dilutions of seawater resulted in reduced cercarial survival only at high salinities (15.2-30.3‰), where 10-min exposures resulted in 0-20% survival. These results provide insights into options for prophylactic water treatment at salmonid enhancement facilities that experience high mortalities due to infections with Nanophyetus salmincola. Further, the intolerance of live cercariae to high salinities indicates that exposure to fish occurs primarily in the freshwater portions of watersheds.

Impact of Age on Disease Progression and Microenvironment in Oral Cancer.

Despite the recognized link between aging and cancer, most preclinical studies in experimental tumor models are conducted with 6- to 8-wk-old rodents. The goal of the present study was to examine the impact of age on tumor incidence, growth, and microenvironmental characteristics in mouse models of head and neck squamous cell carcinoma (HNSCC). Experimental studies were conducted with the 4-nitroquinoline-oxide (4NQO) oral carcinogenesis model and orthotopic FaDu HNSCC xenografts, established in young (7 to 12 wk of age) and old (65 to 70 wk of age) female C57BL/6 mice ( n = 44; 4NQO model) and severe combined immunodeficient mice ( n = 13; HNSCC xenografts). Noninvasive whole body magnetic resonance imaging revealed increased subcutaneous and visceral fat in aging animals of both strains. On histologic examination, a higher incidence ( P < 0.001) of severe dysplasia/invasive squamous cell carcinoma was observed in old mice (92%) as compared with young mice (69%). Old C57BL/6 mice exposed to 4NQO exhibited increased incidence of oral and extraoral (peritoneal masses) neoplasms (42%) versus their young counterparts ( P < 0.05). The incidence of extraoral neoplasms was significantly lower (16%) in the younger cohort. Interestingly, no difference in growth rate and oxygen saturation was observed between orthotopic FaDu xenografts established in old and young severe combined immunodeficient mice. Our observations suggest that host age may have an impact on the growth kinetics and progression of HNSCC in the immunocompetent 4NQO model. Further investigation into the impact of aging on tumor response to preventive and therapeutic intervention is warranted.

Infection by Nanophyetus salmincola and Toxic Contaminant Exposure in Out-migrating Steelhead from Puget Sound, Washington: Implications for Early Marine Survival.

Out-migrating steelhead Oncorhynchus mykiss from four Puget Sound rivers and associated marine basins of Puget Sound in Washington State were examined for the parasite, Nanophyetus salmincola in 2014 to determine whether recent trends in reduced marine survival are associated with the presence of this pathogen. A subset of steelhead from three of these river-marine basin combinations was analyzed for the presence of persistent organic pollutants (POPs) to assess whether exposure to these contaminants is a contributing factor to their reduced marine survival. The prevalence and parasite load of N. salmincola were significantly higher in fish from central and southern Puget Sound than in fish from river systems in northern Puget Sound. The proportion of steelhead samples with concentrations of POPs higher than adverse effects thresholds (AETs) or concentrations known to cause adverse effects was also greater in fish from the central and southern regions of Puget Sound than in those from the northern region. Polybrominated diphenyl ether concentrations associated with increased disease susceptibility were observed in 10% and 40% of the steelhead sampled from central and southern Puget Sound regions, respectively, but in none of the fish sampled from the northern region. The AET for polychlorinated biphenyls was exceeded in steelhead collected from marine habitats: 25% of the samples from the marine basins in the central and southern regions of Puget Sound and 17% of samples from northern Puget Sound region. Both N. salmincola and POP levels suggest there are adverse health effects on out-migrating steelhead from one southern and one central Puget Sound river that have lower early marine survival than those from a river system in northern Puget Sound.

The parasite Ichthyophonus sp. in Pacific herring from the coastal NE Pacific.

The protistan parasite Ichthyophonus occurred in populations of Pacific herring Clupea pallasii Valenciennes throughout coastal areas of the NE Pacific, ranging from Puget Sound, WA north to the Gulf of Alaska, AK. Infection prevalence in local Pacific herring stocks varied seasonally and annually, and a general pattern of increasing prevalence with host size and/or age persisted throughout the NE Pacific. An exception to this zoographic pattern occurred among a group of juvenile, age 1+ year Pacific herring from Cordova Harbor, AK in June 2010, which demonstrated an unusually high infection prevalence of 35%. Reasons for this anomaly were hypothesized to involve anthropogenic influences that resulted in locally elevated infection pressures. Interannual declines in infection prevalence from some populations (e.g. Lower Cook Inlet, AK; from 20-32% in 2007 to 0-3% during 2009-13) or from the largest size cohorts of other populations (e.g. Sitka Sound, AK; from 62.5% in 2007 to 19.6% in 2013) were likely a reflection of selective mortality among the infected cohorts. All available information for Ichthyophonus in the NE Pacific, including broad geographic range, low host specificity and presence in archived Pacific herring tissue samples dating to the 1980s, indicate a long-standing host-pathogen relationship.

Persistence of external signs in Pacific herring Clupea pallasii Valenciennes with ichthyophoniasis.

The progression of external signs of Ichthyophonus infection in Pacific herring Clupea pallasii Valenciennes was highly variable and asynchronous after intraperitoneal injection with pure parasite preparations; however, external signs generally persisted through the end of the study (429 days post-exposure). Observed signs included papules, erosions and ulcers. The prevalence of external signs plateaued 35 days post-exposure and persisted in 73-79% of exposed individuals through the end of the first experiment (147 days post-exposure). Among a second group of infected herring, external signs completely resolved in only 10% of the fish after 429 days. The onset of mortality preceded the appearance of external signs. Histological examination of infected skin and skeletal muscle tissues indicated an apparent affinity of the parasite for host red muscle. Host responses consisted primarily of granulomatous inflammation, fibrosis and necrosis in the skeletal muscle and other tissues. The persistence and asynchrony of external signs and host response indicated that they were neither a precursor to host mortality nor did they provide reliable metrics for hindcasting on the date of exposure. However, the long-term persistence of clinical signs in Pacific herring may be useful in ascertaining the population-level impacts of ichthyophoniasis in regularly observed populations.

Infecting Pacific Herring with Ichthyophonus sp. in the Laboratory.

The protistan parasite Ichthyophonus sp. occurs in coastal populations of Pacific Herring Clupea pallasii throughout the northeast Pacific region, but the route(s) by which these planktivorous fish become infected is unknown. Several methods for establishing Ichthyophonus infections in laboratory challenges were examined. Infections were most effectively established after intraperitoneal (IP) injections with suspended parasite isolates from culture or after repeated feedings with infected fish tissues. Among groups that were offered the infected tissues, infection prevalence was greater after multiple feedings (65%) than after a single feeding (5%). Additionally, among groups that were exposed to parasite suspensions prepared from culture isolates, infection prevalence was greater after exposure by IP injection (74%) than after exposure via gastric intubation (12%); the flushing of parasite suspensions over the gills did not lead to infections in any of the experimental fish. Although the consumption of infected fish tissues is unlikely to be the primary route of Ichthyophonus sp. transmission in wild populations of Pacific Herring, this route may contribute to abnormally high infection prevalence in areas where juveniles have access to infected offal.

A multicenter phase II study of cetuximab in combination with chest radiotherapy and consolidation chemotherapy in patients with stage III non-small cell lung cancer.

BACKGROUND: Cetuximab has demonstrated improved efficacy in combination with chemotherapy and radiotherapy. We evaluated the integration of cetuximab in the combined modality treatment of stage III non-small cell lung cancer (NSCLC). METHODS: Patients with surgically unresectable stage IIIA or IIIB NSCLC were treated with chest radiotherapy, 73.5 Gy (with lung and tissue heterogeneity corrections) in 35 fractions/7 weeks, once daily (63 Gy without heterogeneity corrections). Cetuximab was given weekly during radiotherapy and continued during consolidation therapy with carboplatin and paclitaxel up to a maximum of 26 weekly doses. The primary endpoint was overall survival. Baseline tumor tissue was analyzed for EGFR by fluorescence in situ hybridization (FISH). RESULTS: Forty patients were enrolled in this phase II study. The median overall survival was 19.4 months and the median progression-free survival 9.3 months. The best overall response rate in 31 evaluable patients was 67%. No grade 3 or 4 esophagitis was observed. Three patients experienced grade 3 rash; 16 patients (69%) developed grade 3/4 neutropenia during consolidation therapy. One patient died of pneumonitis, possibly related to cetuximab. EGFR gene copy number on baseline tumor tissues, analyzed by FISH, was not predictive of efficacy outcomes. CONCLUSIONS: The addition of cetuximab to chest radiotherapy and consolidation chemotherapy was tolerated well and had modest efficacy in stage III NSCLC. Taken together with the lower incidence of esophagitis, our results support evaluation of targeted agents instead of chemotherapy with concurrent radiotherapy in this setting.

Experimental infection studies demonstrating Atlantic salmon as a host and reservoir of viral hemorrhagic septicemia virus type IVa with insights into pathology and host immunity.

In British Columbia, Canada (BC), aquaculture of finfish in ocean netpens has the potential for pathogen transmission between wild and farmed species due to the sharing of an aquatic environment. Viral hemorrhagic septicemia virus (VHSV) is enzootic in BC and causes serious disease in wild Pacific herring, Clupea pallasii, which often enter and remain in Atlantic salmon, Salmo salar, netpens. Isolation of VHSV from farmed Atlantic salmon has been previously documented, but the effects on the health of farmed salmon and the wild fish sharing the environment are unknown. To determine their susceptibility, Atlantic salmon were exposed to a pool of 9 isolates of VHSV obtained from farmed Atlantic salmon in BC by IP-injection or by waterborne exposure and cohabitation with diseased Pacific herring. Disease intensity was quantified by recording mortality, clinical signs, histopathological changes, cellular sites of viral replication, expression of interferon-related genes, and viral tissue titers. Disease ensued in Atlantic salmon after both VHSV exposure methods. Fish demonstrated gross disease signs including darkening of the dorsal skin, bilateral exophthalmia, light cutaneous hemorrhage, and lethargy. The virus replicated within endothelial cells causing endothelial cell necrosis and extensive hemorrhage in anterior kidney. Infected fish demonstrated a type I interferon response as seen by up-regulation of genes for IFNα, Mx, and ISG15. In a separate trial infected salmon transmitted the virus to sympatric Pacific herring. The results demonstrate that farmed Atlantic salmon can develop clinical VHS and virus can persist in the tissues for at least 10 weeks. Avoiding VHS epizootics in Atlantic salmon farms would limit the potential of VHS in farmed Atlantic salmon, the possibility for further host adaptation in this species, and virus spillback to sympatric wild fishes.

Viral tropism and pathology associated with viral hemorrhagic septicemia in larval and juvenile Pacific herring.

Viral hemorrhagic septicemia virus (VHSV) genotype IVa causes mass mortality in wild Pacific herring, a species of economic value, in the Northeast Pacific Ocean. Young of the year herring are particularly susceptible and can be carriers of the virus. To understand its pathogenesis, tissue and cellular tropisms of VHSV in larval and juvenile Pacific herring were investigated with immunohistochemistry, transmission electron microscopy, and viral tissue titer. In larval herring, early viral tropism for epithelial tissues (6d post-exposure) was indicated by foci of epidermal thickening that contained heavy concentrations of virus. This was followed by a cellular tropism for fibroblasts within the fin bases and the dermis, but expanded to cells of the kidney, liver, pancreas, gastrointestinal tract and meninges in the brain. Among wild juvenile herring that underwent a VHS epizootic in the laboratory, the disease was characterized by acute and chronic phases of death. Fish that died during the acute phase had systemic infections in tissues including the submucosa of the gastrointestinal tract, spleen, kidney, liver, and meninges. The disease then transitioned into a chronic phase that was characterized by the appearance of neurological signs including erratic and corkscrew swimming and darkening of the dorsal skin. During the chronic phase viral persistence occurred in nervous tissues including meninges and brain parenchymal cells and in one case in peripheral nerves, while virus was mostly cleared from the other tissues. The results demonstrate the varying VHSV tropisms dependent on the timing of infection and the importance of neural tissues for the persistence and perpetuation of chronic infections in Pacific herring.

Susceptibility of Pacific herring to viral hemorrhagic septicemia is influenced by diet.

Groups of specific-pathogen-free Pacific herring Clupea pallasii were highly susceptible to infection by viral hemorrhagic septicemia virus (VHSV); however, the level of mortality was influenced by diet during the 40-71 d before, during, and after the first exposure to the virus. Cumulative mortality was highest among the herring maintained on an experimental soy-based pellet, intermediate among those maintained on a commercially available fish-meal-based pellet, and lowest among those maintained on a second commercially available fish-meal-based pellet containing beta-glucans. Additionally, the herring maintained on the experimental soy-based feed demonstrated less growth than those on the commercially available feeds. The results indicate the importance of standardizing diet during empirical determinations of disease susceptibility and provide insights into the risk factors affecting VHS susceptibility in wild populations.

Inability to demonstrate fish-to-fish transmission of Ichthyophonus from laboratory infected Pacific herring Clupea pallasii to naïve conspecifics.

The parasite Ichthyophonus is enzootic in many marine fish populations of the northern Atlantic and Pacific Oceans. Forage fishes are a likely source of infection for higher trophic level predators; however, the processes that maintain Ichthyophonus in forage fish populations (primarily clupeids) are not well understood. Lack of an identified intermediate host has led to the convenient hypothesis that the parasite can be maintained within populations of schooling fishes by waterborne fish-to-fish transmission. To test this hypothesis we established Ichthyophonus infections in Age-1 and young-of-the-year (YOY) Pacific herring Clupea pallasii (Valenciennes) via intraperitoneal (IP) injection and cohabitated these donors with naïve conspecifics (sentinels) in the laboratory. IP injections established infection in 75 to 84% of donor herring, and this exposure led to clinical disease and mortality in the YOY cohort. However, after cohabitation for 113 d no infections were detected in naïve sentinels. These data do not preclude the possibility of fish-to-fish transmission, but they do suggest that other transmission processes are necessary to maintain Ichthyophonus in wild Pacific herring populations.

Factors controlling the early stages of viral haemorrhagic septicaemia epizootics: low exposure levels, virus amplification and fish-to-fish transmission.

Viral haemorrhagic septicaemia virus, Genogroup IVa (VHSV), was highly infectious to Pacific herring, Clupea pallasii (Valenciennes), even at exposure doses occurring below the threshold of sensitivity for a standard viral plaque assay; however, further progression of the disease to a population-level epizootic required viral amplification and effective fish-to-fish transmission. Among groups of herring injected with VHSV, the prevalence of infection was dose-dependent, ranging from 100%, 75% and 38% after exposure to 19, 0.7 and 0.07 plaque-forming units (PFU)/fish, respectively. Among Pacific herring exposed to waterborne VHSV (140 PFU mL(-1) ), the prevalence of infection, geometric mean viral tissue titre and cumulative mortality were greater among cohabitated herring than among cohorts that were held in individual aquaria, where fish-to-fish transmission was prevented. Fish-to-fish transmission among cohabitated herring probably occurred via exposure to shed virus which peaked at 680 PFU mL(-1) ; shed virus was not detected in the tank water from any isolated individuals. The results provide insights into mechanisms that initiate epizootic cascades in populations of wild herring and have implications for the design of VHSV surveys in wild fish populations.

Viral replication in excised fin tissues (VREFT) corresponds with prior exposure of Pacific herring, Clupea pallasii (Valenciennes), to viral haemorrhagic septicaemia virus (VHSV).

Procedures for a viral replication in excised fin tissue (VREFT) assay were adapted to Pacific herring, Clupea pallasii, and optimized both to reduce processing time and to provide the greatest resolution between naïve herring and those previously exposed to viral haemorrhagic septicaemia virus (VHSV), Genogroup IVa. The optimized procedures included removal of the left pectoral fin from a euthanized fish, inoculation of the fin with >10(5) plaque-forming units (PFU) mL(-1) VHSV for 1 h, rinsing the fin in fresh medium six times to remove unadsorbed virions, incubation of the fin in fresh medium for 4 days and enumeration of the viral titre in a sample of the incubation medium by plaque assay. The optimized VREFT assay was effective at identifying the prior exposure history of laboratory-reared Pacific herring to VHSV. The geometric mean VREFT value was significantly greater (P < 0.01) among naïve herring (1.2 × 10(3) PFU mL(-1) ) than among groups that survived exposure to VHSV (1.0-2.9 × 10(2) PFU mL(-1) ); additionally, the proportion of cultures with no detectable virus was significantly greater (P = 0.0002) among fish that survived exposure to VHSV (39-47%) than among naïve fish (3.3%). The optimized VREFT assay demonstrates promise for identifying VHSV exposure history and forecasting disease potential in populations of wild Pacific herring.