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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Chronic and persistent viral hemorrhagic septicemia virus infections in Pacific herring.

Chronic viral hemorrhagic septicemia virus (VHSV) infections were established in a laboratory stock of Pacific herring Clupea pallasii held in a large-volume tank supplied with pathogen-free seawater at temperatures ranging from 6.8 to 11.6 degrees C. The infections were characterized by viral persistence for extended periods and near-background levels of host mortality. Infectious virus was recovered from mortalities occurring up to 167 d post-exposure and was detected in normal-appearing herring for as long as 224 d following initial challenge. Geometric mean viral titers were generally as high as or higher in brain tissues than in pools of kidney and spleen tissues, with overall prevalence of infection being higher in the brain. Upon re-exposure to VHSV in a standard laboratory challenge, negligible mortality occurred among groups of herring that were either chronically infected or fully recovered, indicating that survival from chronic manifestations conferred protection against future disease. However, some survivors of chronic VHS infections were capable of replicating virus upon re-exposure. Demonstration of a chronic manifestation of VHSV infection among Pacific herring maintained at ambient seawater temperatures provides insights into the mechanisms by which the virus is maintained among populations of endemic hosts.

Sequence analysis of the internal transcribed spacer (ITS) region reveals a novel clade of Ichthyophonus sp. from rainbow trout.

The mesomycetozoean parasite Ichthyophonus hoferi is most commonly associated with marine fish hosts but also occurs in some components of the freshwater rainbow trout Oncorhynchus mykiss aquaculture industry in Idaho, USA. It is not certain how the parasite was introduced into rainbow trout culture, but it might have been associated with the historical practice of feeding raw, ground common carp Cyprinus carpio that were caught by commercial fisherman. Here, we report a major genetic division between west coast freshwater and marine isolates of Ichthyophonus hoferi. Sequence differences were not detected in 2 regions of the highly conserved small subunit (18S) rDNA gene; however, nucleotide variation was seen in internal transcribed spacer loci (ITS1 and ITS2), both within and among the isolates. Intra-isolate variation ranged from 2.4 to 7.6 nucleotides over a region consisting of approximately 740 bp. Majority consensus sequences from marine/anadromous hosts differed in only 0 to 3 nucleotides (99.6 to 100% nucleotide identity), while those derived from freshwater rainbow trout had no nucleotide substitutions relative to each other. However, the consensus sequences between isolates from freshwater rainbow trout and those from marine/anadromous hosts differed in 13 to 16 nucleotides (97.8 to 98.2% nucleotide identity).

Effects of temperature on disease progression and swimming stamina in Ichthyophonus-infected rainbow trout, Oncorhynchus mykiss (Walbaum).

Rainbow trout, Oncorhynchus mykiss, were infected with Ichthyophonus sp. and held at 10 degrees C, 15 degrees C and 20 degrees C for 28 days to monitor mortality and disease progression. Infected fish demonstrated more rapid onset of disease, higher parasite load, more severe host tissue reaction and reduced mean-day-to-death at higher temperature. In a second experiment, Ichthyophonus-infected fish were reared at 15 degrees C for 16 weeks then subjected to forced swimming at 10 degrees C, 15 degrees C and 20 degrees C. Stamina improved significantly with increased temperature in uninfected fish; however, this was not observed for infected fish. The difference in performance between infected and uninfected fish became significant at 15 degrees C (P = 0.02) and highly significant at 20 degrees C (P = 0.005). These results have implications for changes in the ecology of fish diseases in the face of global warming and demonstrate the effects of higher temperature on the progression and severity of ichthyophoniasis as well as on swimming stamina, a critical fitness trait of salmonids. This study helps explain field observations showing the recent emergence of clinical ichthyophoniasis in Yukon River Chinook salmon later in their spawning migration when water temperatures were high, as well as the apparent failure of a substantial percentage of infected fish to successfully reach their natal spawning areas.

Differential survival of Ichthyophonus isolates indicates parasite adaptation to its host environment.

In vitro viability of Ichthyophonus spp. spores in seawater and freshwater corresponded with the water type of the host from which the spores were isolated. Among Ichthyophonus spp. spores from both marine and freshwater fish hosts (Pacific herring, Clupea pallasii, and rainbow trout, Oncorhynchus mykiss, respectively), viability was significantly greater (P < 0.05) after incubation in seawater than in freshwater at all time points from 1 to 60 min after immersion; however, magnitude of the spore tolerances to water type differed with host origin. Ichthyophonus sp. adaptation to its host environment was indicated by greater seawater tolerance of spores from the marine host and greater freshwater tolerance of spores from the freshwater host. Prolonged aqueous survival of Ichthyophonus spp. spores in the absence of a host provides insight into routes of transmission, particularly among planktivorous fishes, and should be considered when designing strategies to dispose of infected fish carcasses and tissues.

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.

1,25-Dihydroxycholecalciferol (1,25-D3) inhibits the growth of squamous cell carcinoma and down-modulates p21(Waf1/Cip1) in vitro and in vivo.

1,25-Dihydroxycholecalciferol (1,25-D3) has significant antitumor effects in the murine squamous cell carcinoma (SCC) tumor model in vitro and in vivo. We investigated the basis for this antiproliferative activity and found that, in vitro, 1,25-D3 administration is associated with altered expression of cell cycle regulatory proteins, treatment results in retinoblastoma dephosphorylation, decreased expression of p21(Waf1/Cip1) (p21) mRNA and protein, and increased expression of p27Kip1 (p27) mRNA and protein. Dexamethasone, which acts synergistically with 1,25-D3 to inhibit SCC proliferation, enhanced 1,25-D3-induced down-modulation of p21 without affecting the ability of 1,25-D3 to increase p27 expression. 1,25-D3 did not induce cleavage of poly(ADP-ribose) polymerase. These in vitro data suggest that 1,25-D3 exerts antitumor activity in SCC by perturbing cell cycle progression rather than by inducing apoptosis. In vivo, a 1,25-D3 treatment regimen that results in a decrease in SCC tumor volume is associated with a statistically significant decrease in intratumoral p21 expression. p21 expression is not changed in tumors isolated from control animals or animals treated with a nontherapeutic dose of 1,25-D3. Intratumoral p27 levels were not modulated by 1,25-D3 treatment. Thus, both in vitro and in vivo, 1,25-D3-mediated growth inhibition is associated with p21 down-modulation.