Vertebrates mount a strong innate immune response against viruses, largely by activating the interferon system. Double-stranded RNA (dsRNA), a common intermediate formed during the life cycle of many viruses, is a potent trigger of this response. In contrast, no general inducible antiviral defense mechanism has been reported in any invertebrate. Here we show that dsRNA induces antiviral protection in the marine crustacean Litopenaeus vannamei. When treated with dsRNA, shrimp showed increased resistance to infection by two unrelated viruses, white spot syndrome virus and Taura syndrome virus. Induction of this antiviral state is independent of the sequence of the dsRNA used and therefore distinct from the sequence-specific dsRNAmediated genetic interference phenomenon. This demonstrates for the first time that an invertebrate immune system, like its vertebrate counterparts, can recognize dsRNA as a virus-associated molecular pattern, resulting in the activation of an innate antiviral response.

ABSTRACT:In vivo bioassay is the predominant method for evaluating the infectivity of materials potentially harboring viable shrimp pathogens and determining the relative susceptibility of shrimp species to viral infections. A controlled bioassay system for white spot syndrome virus (WSSV) and Taura syndrome virus (TSV) was developed utilizing 260 ml tissue culture flasks modified with an air exchange vent. Individual shrimp (1.00 ± 0.25 g) were placed in separate flasks containing artificial seawater (100 to 150 ml) and held in an incubator at 27°C. After a 48 h acclimation period, shrimp were either injected intramuscularly with viral inoculum or exposed to virus-laden water. Water was exchanged and shrimp were fed a commercial food pellet daily except 24 h post-infection (p.i.). Bioassays were performed with serial dilutions of stock viral preparations and shrimp mortality was recorded for 7 d p.i. Mortality rates of test animals permitted the estimation of the lethal infective doses, LD 50 and LD 90 . The LD 50 of the TSV injection preparation was estimated at viral dilutions of 1:7.692 × 10 7 (Trial 1) and 1:6.667 × 10 7 (Trial 2). The LD 50 s of 2 different WSSV injection preparations were estimated at 1:4.444 × 10 6 and 1:4.505 × 10 6 . The LD 50 for the TSV waterborne challenge was 1:9916 (Trial 1) and 1:15 710 (Trial 2) at 20°C and 1:1272 at 27°C. A second waterborne TSV inoculum challenge at 27°C produced an LD 50 of 1:2857. WSSV doses used in the waterborne challenge only reached 39% mortality, which did not allow for the estimation of effective lethal doses. Bioassay by injection proved to be a more reliable method of estimating viral infectivity compared to waterborne method. The dose-response curves developed can serve as a basis for controlled comparisons of relative levels of viral infectivity of specific tissue preparations and for controlled comparisons of relative susceptibility of shrimp species or stocks to viral pathogens. KEY WORDS: TSV · WSSV · Bioassay · Shrimp · Litopenaeus vannameiResale or republication not permitted without written consent of the publisher

The two major pathways for the metabolism of estradiol-17beta (E2) are the 2- and 16-hydroxylase pathways. Research has suggested that the increased production of the estrogenically active 16-hydroxy products such as estriol (E3) may be involved in increased susceptibility to breast cancer. 4-Nonylphenol (4-NP) is an environmental estrogen that also can activate the pregnane-X receptor (PXR) and induce P-450 enzymes responsible for the production of E3. It is hypothesized that 4-NP may act in part as an environmental estrogen by increasing E3 production. Based on its affinity for the estrogen receptor (ER) alone, 4-NP may be more potent than predicted at increasing mammary cancer incidence in the MMTVneu mouse. Female mice were treated per os for 7 days at 0, 25, 50 or 75 mg kg(-1) day(-1) 4-NP to investigate the effects of 4-NP on hepatic estrogen metabolism after an acute treatment. 4-Nonylphenol increased the hepatic formation of E3 in a dose-dependent manner. However, serum E3 concentrations were only increased at 25 mg kg(-1) day(-1) presumably due to direct inhibition of E3 formation by 4-NP. MMTVneu mice were then treated for 32 weeks at 0, 30 or 45 mg kg(-1) day(-1) 4-NP to determine its effects on mammary cancer formation and estrogen metabolism. 4-Nonylphenol increased mammary cancer formation in the MMTVneu mice at 45 mg kg(-1) day(-1) but not at 30 mg kg(-1) day(-1). Mice treated with an equipotent dose of E2, 10 microg kg(-1) day(-1), based on the relative binding affinities of nonylphenol and estradiol for ER alpha, did not develop mammary cancer. This suggests that nonylphenol is more potent than predicted based on its affinity for the estrogen receptor. However, no changes in serum E3 concentrations or hepatic E3 production were measured after the chronic treatment. Changes in E3 formation were correlated with increased CYP2B levels after the 7 day 4-NP treatment, and repression of CYP2B and CYP3A after 32 weeks of 4-NP treatment. Microarray analysis and Q-PCR of liver mRNA from the mice treated for 32 weeks demonstrated a decrease in RXR alpha, the heterodimeric partner of the PXR, which may in part explain the repressed transcription of the P450s measured. In conclusion, 4-NP treatment for 32 weeks increased mammary cancer formation at a dose of 45 mg kg(-1) day(-1). However, chronic treatment with 4-NP did not increase hepatic E3 formation or serum E3 concentrations. The transient induction by 4-NP of hepatic E3 formation and serum concentrations is most likely not involved in the increased incidence of mammary cancer in MMTVneu mice since E3 serum concentrations were only increased at 25 mg kg(-1) day(-1), a dose that was not sufficient to induce mammary tumor formation. Nevertheless, the induced hepatic E3 production in the acute exposures to 4-NP was indicative of an increase in mammary cancer incidence after the chronic exposure.

Avian vacuolar myelinopathy (AVM) is a neurologic disease primarily affecting bald eagles (Haliaeetus leucocephalus) and American coots (Fulica americana). The disease was first characterized in bald eagles in Arkansas in 1994 and then in American coots in 1996. To date, AVM has been confirmed in six additional avian species. Attempts to identify the etiology of AVM have been unsuccessful to date. The objective of this study was to evaluate dermal and oral routes of exposure of birds to hydrilla (Hydrilla verticillata) and associated materials to evaluate their ability to induce AVM. Mallards (Anas platyrhynchos) were used in all trials; bobwhite quail (Colinus virginianus) also were used in one fresh hydrilla material exposure trial. Five trials were conducted, including two fresh hydrilla material exposure trials, two cyanobacteria exposure trials, and a frozen hydrilla material exposure trial. The cyanobacteria exposure trials and frozen hydrilla material trial involved gavaging mallards with either Pseudanabaena catenata (live culture), Hapalosiphon fontinalis, or frozen hydrilla material with both cyanobacteria species present. With the exception of one fresh hydrilla exposure trial, results were negative or inconclusive. In the 2002 hydrilla material exposure trial, six of nine treated ducks had histologic lesions of AVM. This established the first cause-effect link between aquatic vegetation and AVM and provided evidence supporting an aquatic source for the causal agent.

Our previous studies have demonstrated that transforming growth factor e (TGFe) acts as a mitogen for epithelial and fibroblastic cells in both monolayer and soft agar. We have also identified TGFe in both normal and neoplastic tissues of mostly epithelial origin, and in body fluids. In this study we report on the purification of TGFe to homogeneity from bovine kidney using a multistep purification protocol which utilizes high performance electrophoresis chromatography in the final step. Amino acid analysis of TGFe revealed high content of proline, aspartate and glutamate. Examination of partial amino acid sequence indicated no similarity to other, already characterized, growth factors.