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.

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.

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.

Folate supplementation in methotrexate-treated rheumatoid arthritis patients.

Two hundred rheumatoid arthritis (RA) patients taking low dose methotrexate (MTX) were evaluated for adverse effects. During a mean follow up of 41.5 months, the mean cell volume (MCV) was elevated at some time during the course of treatment in 42 patients. The MCV was normal in the remaining 158 patients. One hundred ninety-eight patients were treated simultaneously with oral folic acid. With the exception of heartburn, which was seen more often in the high MCV group, there was no difference in the frequency of adverse effects attributable to MTX between groups. Severity of side effects and the frequency of MTX dose reduction and MTX discontinuation due to toxicity were also similar between groups. This analysis suggests that elevation of MCV in RA patients treated simultaneously with MTX and folate does not predict MTX toxicity. The authors also discuss the mechanism of action of MTX with regard to folate metabolism.

Outcome of first-trimester exposure to low-dose methotrexate in eight patients with rheumatic disease.

PURPOSE: Methotrexate (MTX), when used to treat malignancy or psoriasis, has been implicated in anecdotal reports as a teratogen or abortifacient in the first trimester of pregnancy. We are unaware of any previous reports that describe the course of gestation and the effect on subsequent offspring in patients treated with low-dose oral MTX for rheumatoid arthritis, and therefore present our experience. PATIENTS AND METHODS: We report on eight women experiencing 10 pregnancies. Mean number of weeks of gestation while taking MTX was 7.5 (range 2 to 20 weeks). Outcome of pregnancies included five full-term babies (FTB), three spontaneous abortions (SAB), and two elective abortions. RESULTS: There were no significant differences in either the FTB or SAB group when considering risk factors including smoking, alcohol, concomitant medications, and age. One of three in the SAB group had recurrent abortions prior to MTX therapy. All five of the FTB group had uncomplicated pregnancies and deliveries. All offspring were of normal height and weight at birth with no physical abnormalities. All children reached growth, development, and intellectual stages normally, and their present mean age is 11.5 years. No observed learning disabilities or medical abnormalities have occurred in any of these children. CONCLUSION: In this uncontrolled study we failed to demonstrate tertogenicity of MTX. However, the possibility of abortion due to MTX use remains.

Differential diagnosis of rheumatoid arthritis.

The differential diagnosis of subacute or chronic polyarthritic diseases, including rheumatoid arthritis, is based on recognizing a pattern of changes, with special emphasis placed on certain key features that possess higher specificity. The clinical history is by far the most important diagnostic tool and involves clear assessment of the distribution of joint involvement, whether pain is articular or extra-articular, whether a syndrome follows trauma or infection, and the duration of the process. The distribution of involved joints is a major contributor to differential diagnosis but can be misleading unless a skilled physical examination confirms objective synovitis. If present, nodules, which represent localization of the disease process, also offer a powerful tool in differential diagnosis. Rheumatoid nodules occur in about 20 percent of patients with well-developed rheumatoid arthritis. The effects of inflammation may be clinically detectable, but laboratory tests--e.g., the erythrocyte sedimentation rate and quantitative C-reactive protein--provide the most reliable evidence of inflammation. Synovial fluid analysis may reveal inflammatory changes and other abnormalities permitting diagnosis. Rheumatoid factor is present in about 80 percent of patients with rheumatoid arthritis but is not specific for this diagnosis.

Antimalarial drugs for rheumatoid arthritis.

Chloroquine and hydroxychloroquine effectively suppress rheumatoid arthritis with a superior benefit to risk ratio. Controlled studies demonstrate moderate efficacy in about 70 percent of patients. High-grade suppression is seen in 15 percent and partial suppression in 55 percent. The dropout rate for poor efficacy is 30 percent and for side effects 3 to 7 percent. Most studies show antimalarials to be almost equivalent to chrysotherapy in potency. Antimalarials are indicated for active rheumatoid arthritis not optimally controlled with nonsteroidal anti-inflammatory drugs and for all cases of progressive disease. Therapy is continued indefinitely. Safe use of these drugs depends on daily dosage. With the single exception of late stage retinopathy, other adverse effects are fully reversible. Strict adherence to three tested safety rules virtually eliminates retinopathy and prevents loss of vision: (1) limit the daily dosage: chloroquine 3.5 to 4.0 mg/kg per day or hydroxychloroquine 6.0 to 6.5 mg/kg per day based on lean body weight; (2) subject the patient to an annual ocular examination to age 65, twice annually thereafter; (3) adjust treatment for pharmacokinetic variables. The lower risk and nearly comparable efficacy make antimalarials first choice among remittive drugs.

Dose refinements in long-term therapy of rheumatoid arthritis with antimalarials.

No eye disease was detected in over 900 rheumatoid arthritis patients treated with less than 4.0 mg/kg per day of chloroquine or less than 6.5 mg/kg per day of hydroxychloroquine for a mean of about seven years. I therefore consider these dosage rates safe, since they are below the threshold of retinal toxicity. This is based on more than 6,000 patient-years of drug exposure. That dosage threshold for retinopathy appears to be 5.1 mg/kg per day for chloroquine and 7.8 mg/kg per day for hydroxychloroquine according to my studies with these compounds. The daily dosage rate, rather than total drug accumulation, seems to determine the development of eye disease. To prevent overdosage, dosing should be calculated not on the actual weight of the patient but on ideal (lean) body weight. Furthermore, the patient's renal and liver function should also be taken into account to avoid overdosage. Since exposure to light amplifies the risk of retinopathy in patients treated with antimalarials, dark sunglasses are recommended for patients spending much time in sunlight.

Pharmacologic actions of 4-aminoquinoline compounds.

The pharmacokinetics, physiologic effects, and the metabolization of chloroquine and hydroxychloroquine are all similar. Their concentrations in plasma and tissue are directly related to daily dosing. The highest concentrations are found in melanin-containing tissues, particularly the choroid and ciliary body of the eye. The pharmacologic effects of 4-aminoquinoline compounds are reviewed in detail. It is likely that the rheumatologic effectiveness of these agents is primarily related to lysosomal actions. The drug-induced lysosomal abnormalities include diminished vesicle fusion, diminished exocytosis, and reversible "lysosomal storage disease." It is likely that the retinal toxicity of these drugs is one manifestation of the altered lysosomal physiology involving the retinal pigmented epithelium. Tissue of retinal pigmented epithelium is similar to that of the bone-marrow-derived macrophage. Depression of extra-oculogram is an early sign of excessive dosage and can be used to measure potential toxicity during therapy with 4-aminoquinolines. Dosages ranging from 3.5 to 4.0 mg/kg per day for chloroquine and 6.0 to 6.5 mg/kg per day for hydroxychloroquine are clinically safe.