Homo- and Heterosubtypic Low Pathogenic Avian Influenza Exposure on H5N1 Highly Pathogenic Avian Influenza Virus Infection in Wood Ducks (Aix sponsa)

Wild birds in the Orders Anseriformes and Charadriiformes are the natural reservoirs for avian influenza (AI) viruses. Although they are often infected with multiple AI viruses, the significance and extent of acquired immunity in these populations is not understood. Pre-existing immunity to AI virus has been shown to modulate the outcome of a highly pathogenic avian influenza (HPAI) virus infection in multiple domestic avian species, but few studies have addressed this effect in wild birds. In this study, the effect of pre-exposure to homosubtypic (homologous hemagglutinin) and heterosubtypic (heterologous hemagglutinin) low pathogenic avian influenza (LPAI) viruses on the outcome of a H5N1 HPAI virus infection in wood ducks (Aix sponsa) was evaluated. Pre-exposure of wood ducks to different LPAI viruses did not prevent infection with H5N1 HPAI virus, but did increase survival associated with H5N1 HPAI virus infection. The magnitude of this effect on the outcome of the H5N1 HPAI virus infection varied between different LPAI viruses, and was associated both with efficiency of LPAI viral replication in wood ducks and the development of a detectable humoral immune response. These observations suggest that in naturally occurring outbreaks of H5N1 HPAI, birds with pre-existing immunity to homologous hemagglutinin or neuraminidase subtypes of AI virus may either survive H5N1 HPAI virus infection or live longer than naïve birds and, consequently, could pose a greater risk for contributing to viral transmission and dissemination. The mechanisms responsible for this protection and/or the duration of this immunity remain unknown. The results of this study are important for surveillance efforts and help clarify epidemiological data from outbreaks of H5N1 HPAI virus in wild bird populations

Low Pathogenic Avian Influenza Isolates from Wild Birds Replicate and Transmit via Contact in Ferrets without Prior Adaptation

Direct transmission of avian influenza viruses to mammals has become an increasingly investigated topic during the past decade; however, isolates that have been primarily investigated are typically ones originating from human or poultry outbreaks. Currently there is minimal comparative information on the behavior of the innumerable viruses that exist in the natural wild bird host. We have previously demonstrated the capacity of numerous North American avian influenza viruses isolated from wild birds to infect and induce lesions in the respiratory tract of mice. In this study, two isolates from shorebirds that were previously examined in mice (H1N9 and H6N1 subtypes) are further examined through experimental inoculations in the ferret with analysis of viral shedding, histopathology, and antigen localization via immunohistochemistry to elucidate pathogenicity and transmission of these viruses. Using sequence analysis and glycan binding analysis, we show that these avian viruses have the typical avian influenza binding pattern, with affinity for cell glycoproteins/glycolipids having terminal sialic acid (SA) residues with α 2,3 linkage [Neu5Ac(α2,3)Gal]. Despite the lack of α2,6 linked SA binding, these AIVs productively infected both the upper and lower respiratory tract of ferrets, resulting in nasal viral shedding and pulmonary lesions with minimal morbidity. Moreover, we show that one of the viruses is able to transmit to ferrets via direct contact, despite its binding affinity for α 2,3 linked SA residues. These results demonstrate that avian influenza viruses, which are endemic in aquatic birds, can potentially infect humans and other mammals without adaptation. Finally this work highlights the need for additional study of the wild bird subset of influenza viruses in regard to surveillance, transmission, and potential for reassortment, as they have zoonotic potential

Role of Apoptosis in Modulating Effects of 2-Aminoanthracene in Pancreatic Tissue of Sprague Dawley Rat Dams

Modulation of the toxic effects of 2-aminoanthracene (2AA) on pancreatic tissue by apoptosis will be investigated. 2AA, also called anthramine, is a polycyclic aromatic hydrocarbon that is used in the manufacturing of chemicals, dyes, and inks, and it is also found in tobacco smoke and cooked foods. It is known to cause gene dysregulation, particularly of several genes of the pancreas that mediate protein and lipid metabolism (Gato and Means, 2011). To help determine the role of apoptosis in modulating the effects of 2AA, pancreatic tissue of Sprague Dawley rat dams exposed to various concentrations of 2AA for during gestation through postpartum will be analyzed for apoptotic activity using TUNEL apoptosis assay. This will be followed by total RNA extraction. The activity of Casp3, which plays a central role in the execution phase of apoptosis, will also be analyzed via assay, and relative gene expression of specific apoptotic genes will be quantified using qRT-PCR to test for significant differences in gene expression

Role of Oxidative Stress in the Pancreas of Pups Exposed to 2-Aminoanthracene in Utero

The goal of this experiment is to understand the role oxidative stress plays in the pancreas of pups exposed 2-aminoanthracene (2AA) in utero. Environmental exposure to 2AA may increase the risk of developing diseases, such as diabetes. Oxidative stress has been linked with beta-cell dysfunction of the pancreas. Oxidative stress refers to the imbalance between production of reactive oxygen species (ROS) and antioxidant defenses. To examine oxidative stress response in pups exposed to 2AA in utero, specific gene expression of Duox1, Gpx1, Ncf2, Prdx1, Prkcg, Ptgs2 and Sod1 in the pancreas were quantified by qRT-PCR. Duox1, Gpx1, Ncf2, Pdx1, Prkcg and Ptgs2 were not expressed in the pancreas of pups. No significant expression differences in these genes were noted. PRDX6 was dose-dependently up-regulated in the pancreas of pups from dams that ingested 2AA during gestation. Sod1 was significantly up-regulated in pups exposed to 2AA in utero. Additional feeding study involving moderately high fat is being implemented after three months post-wean. Oxidative stress immunohistochemistry staining will also be performed. Results will demonstrate the role of oxidative stress in understanding vulnerability to diabetes in pups exposed to arylamine in utero

Role of Oxidative Stress in the Pancreas of Pups Exposed to 2-Aminoanthracene In Utero

The goal of this experiment is to understand the role oxidative stress plays in the pancreas of pups exposed 2-aminoanthracene (2AA) in utero. Environmental exposure to 2AA may increase the risk of developing diseases, such as diabetes. Oxidative stress has been linked with beta-cell dysfunction of the pancreas. Oxidative stress refers to the imbalance between production of reactive oxygen species (ROS) and antioxidant defenses. To examine oxidative stress response in pups exposed to 2AA in utero, specific gene expression of Duox1, Gpx1, Ncf2, Prdx1, Prkcg, Ptgs2 and Sod1 in the pancreas were quantified by qRT-PCR. Duox1, Gpx1, Ncf2, Pdx1, Prkcg and Ptgs2 were not expressed in the pancreas of pups. No significant expression differences in these genes were noted. PRDX6 was dose-dependently up-regulated in the pancreas of pups from dams that ingested 2AA during gestation. Sod1 was significantly up-regulated in pups exposed to 2AA in utero. Additional feeding study involving moderately high fat is being implemented after three months post-wean. Oxidative stress immunohistochemistry staining will also be performed. Results will demonstrate the role of oxidative stress in understanding vulnerability to diabetes in pups exposed to arylamine in utero

Investigating Pancreatic Response of Pups Exposed to 2-Aminoanthracene in Utero

Environmental exposures to toxic chemicals increase the risk of developing diseases such as type 2 diabetes. 2-Aminoanthracene (2AA) is an aromatic amine commonly found in the environment including road tars, dyes, and cigarette smoke. Previous studies have found a link between 2AA and insulin-dependent gene expression changes. The goal of the present investigation is to examine the expression of pancreatic genes associated with metabolic syndrome in response to 2AA in utero. Timed Sprague Dawley pregnant rats were fed 0 mg/kg (control), 50 mg/kg (low dose), and 100 mg/kg (high dose) concentrations of 2AA diet. After sacrifice, a histological examination of the pancreas was performed to determine the microscopic anatomy of pancreatic cells followed by specific insulin staining and β-cell mass analysis. Quantification of insulin-dependent transcripts via qRT-PCR show that FTO, GCK, IGF2BP2, PPARG1 genes were not expressed. Diabetic related genes such as GLUT2 and INS1 were up-regulated in pancreas of pups whose mothers consumed various amounts of 2AA. Insulin IHC staining indicated that pancreatic islet of the low dose progeny had the highest total percentage of pancreatic tissue, followed by the control offspring and then the high dose exposed rats. This experiment is ongoing to examine how these parameters change with moderately high fat diet three months postwean. Quantification of the expression of genes associated with the regulation of insulin and obesity might suggest a link between diabetes and exposure to environmental chemicals such as an aromatic amine

Investigating the Toxic Effects of 2-Aminoanthracene Ingestion in Pregnant Sprague Dawley Dams

Polycyclic aromatic hydrocarbons (PAHs) refer to organic compounds that are byproducts of incomplete combustion of fossil fuels and wood. One specific polycyclic aromatic hydrocarbon, 2-aminoanthracene (2AA), is a member of a broader group of compounds known as anthracenes, which have been classified by the United States Agency for Toxic Substances and Disease Registry (ASTDR) as one of a group of PAHs of top concern based on their greater potential risk for exposure and greater harmful effects to humans, compared to other PAHs. Previous research has shown that 2AA affects genes involved in carbohydrate and lipid metabolism, inflammatory stress responses, and immune system responses, among other processes. The objective of the present study was to examine the toxicity of dietary ingestion of 2AA from gestation through the postnatal period. Pregnant dams (Day 1) were purchased from Taconic Hudson, NY, and assigned into dose regimens of 0 mg/kg- (control-C), 50 mg/kg- (low dose-LD) and 100 mg/kg-diet (high dose-HD) 2AA. Dams were fed 2AA contaminated diet during the period of gestation and postpartum. Insulin and H&E immunohistochemical staining were undertaken and indicated no significant changes between control and treated groups. However, percent pancreatic islets (islets within the pancreas) were larger in the exposed groups. The value was 1.5% in the control dams compared to 3.2% and 4.3% low dose and high dose groups respectively. Serum concentrations of albumin and lactate dehydrogenase (LDH) were increased in the exposed groups, with the HD group experiencing the greater increase. Analyses of Fabp4, Mgmt , Fas, Nhej1, Aldh1a1 and Ncam1 were conducted via real-time quantitative polymerase chain reaction (RT-pPCR), using β-Actin as the control gene. There was an up-regulation of the Mgmt and Nhej1 gene transcripts in the exposed groups, with the extent of upregulation being highest in the HD group. Taken together, a link between environmental exposure to 2AA and pancreatic effects appears to exist

Inflammatory Effect of 2-Aminoanthracene (2AA) on Adipose Tissue Gene Expression in Pregnant Sprague Dawley

The effect of 2-Aminoanthracene (2AA) on adipose tissue gene expression in pregnant Sprague Dawley rats was investigated. Adipocyte dysfunction may be a critical link between obesity and insulin resistance as a result of abnormal fat storage and mobilization. We have previously observed insulin-signaling related altered gene expression in animals exposed to 2AA. 2AA is an amino-substituted polycyclic aromatic hydrocarbon used in manufacturing dyes, chemical, inks, resins, and polyurethanes. 2AA is a known mutagen and carcinogen that occurs naturally and can be found in tobacco smoke and cooked foods. To examine insulin-dependent 2AA effects on the adipose tissue, nine timed pregnant dams were assigned into dose regimens of 0 mg/kg- (control-C), 50 mg/kg- (low dose-LD) and 100 mg/kg-diet (high dose-HD) 2AA. Dams were fed 2AA contaminated diet during the period of gestation and postpartum. Body weight gain during gestation and postnatal period indicated no significant differences in animals. Examination of the AT for microscopic changes suggests no alterations between control and low dose animals. However, AT of the high dose animals exhibited clusters of mononuclear cells and small numbers of eosinophils and mast cells. Inflammatory response was noted in dams fed 2AA. This is observed as phagocytic cells which are most likely macrophages as part of the inflammatory response. In addition, analysis of the mRNA expression of cytokines and adipokines demonstrate the importance of inflammation in ATs. For instance, TNFα, LEPTIN and IL-6 transcripts were relatively more expressed in the low dose animals than the high dose and control rats. It appears the effects of 2AA on pregnant dams were more pronounced in the low dose group than the high dose group. This means that rat offspring within this group might be more susceptible to diabetic-type conditions

Investigating Pancreatic Response of Pups Exposed to 2-Aminoanthracene In Utero

Environmental exposures to toxic chemicals increase the risk of developing diseases such as type 2 diabetes. 2-Aminoanthracene (2AA) is an aromatic amine commonly found in the environment including road tars, dyes, and cigarette smoke. Previous studies have found a link between 2AA and insulin-dependent gene expression changes. The goal of the present investigation is to examine the expression of pancreatic genes associated with metabolic syndrome in response to 2AA in utero. Timed Sprague Dawley pregnant rats were fed 0 mg/kg (control), 50 mg/kg (low dose), and 100 mg/kg (high dose) concentrations of 2AA diet. After sacrifice, a histological examination of the pancreas was performed to determine the microscopic anatomy of pancreatic cells followed by specific insulin staining and β-cell mass analysis. Quantification of insulin-dependent transcripts via qRT-PCR show that FTO, GCK, IGF2BP2, PPARG1 genes were not expressed. Diabetic related genes such as GLUT2 and INS1 were up-regulated in pancreas of pups whose mothers consumed various amounts of 2AA. Insulin IHC staining indicated that pancreatic islet of the low dose progeny had the highest total percentage of pancreatic tissue, followed by the control offspring and then the high dose exposed rats. This experiment is ongoing to examine how these parameters change with moderately high fat diet three months postwean. Quantification of the expression of genes associated with the regulation of insulin and obesity might suggest a link between diabetes and exposure to environmental chemicals such as an aromatic amine