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Ohio Sea Grant College Program
and Stone Laboratory

Ohio Sea Grant and Stone Laboratory

Environmental tolerance of type A influenza virus isolated from wild waterfowl in Ohio

Project Number: R/ES-009, Progress Report

Start Date: 2/1/2011

Completion Date: 1/31/2013

Revision Date: 5/14/2012

Principal Investigator(s)1.Richard D. Slemons, College of Food, Agricultural, and Environmental Sciences The Ohio State University
2.Robert J. Gates, College of Food, Agricultural, and Environmental Sciences The Ohio State University


  1. Determine the environmental tolerance of four waterfowl-origin type A influenza isolates in Lake Erie marsh substrates.
  2. Compare the observed environmental tolerance to the yearly frequency of each strain recovered from Lake Erie marshes since 1986.


The marshes adjoining the southwestern basin of Lake Erie comprise the largest area of coastal wetlands in the Great Lakes ecosystem (Bookhout 1989), attracting hundreds of thousands of waterfowl, shorebirds, and other marsh birds during autumn migration (Bookhout 1989, Baranowski 2007). An important link between migratory flyways, these marshes have been designated a regionally significant migration stopover by the Western Hemisphere Shorebird Reserve Network (WHSRN). By connecting the eastern U.S. coast with the Mississippi, Central, and Pacific Flyways, this vital staging area for migratory waterfowl can potentially expedite the spread of avian-origin type-A influenza viruses (AIVs) across the continent (Olsen et al. 2006).

Arriving in a region via infected migratory birds, individual AIVs may persist for varying lengths of time in some environments with more tolerant viruses surviving longer and more able infect naive birds as population density increases and birds are stressed. The ability of AIVs to persist under natural environmental conditions, defined as environmental tolerance (ET) (Schwarten et al. 2009) has been inadequately examined; however, Stallknecht et al. (1990) and Schwarten et al. (2009) have shown that AIVs can persist for varying periods under different chemical conditions and simulated summer environmental conditions, respectively. Preliminary results by Schwarten et al. (2009) from one study site, with one individual type A influenza virus strain, show slower virus degradation than expected, over one month at variable temperature across a range of environmental substrates. These results indicate that heavily used waterfowl habitats may serve as a potential environmental reservoir for AIVs, spreading infections via indirect transmission to birds from a source of viruses persisting for longer periods of time in nature.

It is hypothesizes that commonly recovered strains of AIVs have a higher ET and persist in the environment for sufficient periods of time to infect susceptible birds after originally infected birds have left the area and sporadically isolated strains have a lower ET and do not persist in the environment, requiring a constant influx of new susceptible birds into the environment to maintain the presence of the virus. Therefore, we propose to test the ET of commonly and sporadically isolated strains of AIV that have been monitored in waterfowl from the coastal marshes of the southwestern Lake Erie basin over the past 20 years (Slemons, OSU Type A Influenza Repository). Furthermore, we hypothesize that more stable, anoxic water conditions raise virus persistence because oxidizing agents that inactivate AIVs by lipid peroxidation are absent (Murray et al 2008). Also, the more permanent wetlands retain higher levels of organic matter, which may stabilize AIVs that adhere to it, resulting in increased virus persistence.

The research proposes addresses goals and focus areas in the National Sea grant College Program 2009- 2013 Strategic Plan. We will develop microcosms that simulate hydrologically stable, non-vegetated wetland systems (i.e. estuarine or littoral wetlands) and more hydrologically variable wetland that are managed to produce waterfowl habitat (i.e. managed marshes). By characterizing the physical and chemical parameters of these systems we will develop environmental models to compare virus ET under different environmental conditions. This will advance understanding of the ecology of AIVs in coastal marshes and estuaries of Lake Erie and create a system where manipulations of the physical and chemical environment to reduce persistence of AIV can be tested. The research will be conducted by a Veterinary Medicine professional student in the College of Veterinary Medicine and an undergraduate Honor's student in the School of Environment and Natural Resources. In addition to developing a novel scientific approach to better understand ecological processes that affect persistence of type A influenza viruses and the introduction and/or spread of a potentially economically dangerous virus (see Cross-cutting Goal 1, Strategy 1), we will explore new avenues of research and create new educational opportunities for professional and undergraduate students (see Cross-cutting Goal 2, Strategy 2).

The water bird resource supports both consumptive and non-consumptive uses that contribute to the culture and economy of the Lake Erie region. This resource and attendant cultural and economic benefits will be threatened if a highly pathogenic AIV is introduced into Lake Erie marshes by migrating marsh birds, particularly during hunting seasons and popular bird-watching seasons. Understanding how environmental conditions can suppress or enhance virus ET and spread of AIVs will contribute to three of four focus areas of the 2009-2013 Plan including: using ecosystem-based processes to maintain healthy coastal ecosystems, ensure a safe and sustainable "seafood" supply and improve the hazard resilience (e.g. to disease outbreaks) of coastal wildlife and human communities. Our results will also influence policy decisions and emergency management plans dealing with a potential introduction and/or outbreak of highly pathogenic AIV infections in migrating birds along Ohio's coastal marshlands.


An established laboratory environmental model will be refined and used to simulate the conditions of different waterfowl habitats located along Ohio's Lake Erie coast. Using this model, the environmental tolerance (ET) will be determined for four strains of AIV isolated from ducks along the southern shore of Lake Erie in Ohio. Two of the AIV isolates we will be testing have been frequently isolated from the ducks year after year, while the other two AIV isolates being tested have been seldom isolated from ducks in Ohio. We will be examining the AIVs for a possible relationship between virus ET and frequency of virus isolation. By examining the ET of the four different strains of AIV, we will gain a better understanding of the environment's role in the natural history of these viruses.

Assessing the potential of an environmental reservoir for AIVs, we will base our study on late summer or autumn environmental conditions, when the waterfowl population is increasing in the region. Using the Winous Point Marsh Conservancy as our study site, we will record temperature, conductivity, pH, dissolved oxygen, and oxidation-reduction potential data and collect soil and water samples to develop a system of laboratory microcosms.

These laboratory microcosms will simulate the environment, allowing the study of the effects the environment upon AIV persistence. To develop these microcosms, we will first examine our physical and chemical parameter data recorded during the late summer/autumn. Using these field data and the soil and water samples collected from Winous Point, we will create two microcosms (soil and water). Three replicates of each microcosm will be spiked with known concentrations of 4 different AIV isolates and subjected to the maximum and minimum temperatures recorded at Winous Point. A negative control (no virus) will also be established,

The virus-spiked microcosms will be exposed to the determined late summer/autumn temperatures for 60-90 days, and sampled on a weekly basis, to allow us to reach endpoints for each. To evaluate the ET of the AIVs being used, we will determine the egg infective dose concentration (EID50/ml) of virus in the samples collected from the first and last sampling period, and then work backward from the last sample to determine if significant decreases are noted, providing us with quantitative data to detect declines in infectious virus concentrations, possibly allowing us to identify relationships between virus ET and individual microenvironments.


1Environmental Tolerance of Waterfowl-Origin Influenza A Virus Isolates in Lake Erie Marsh Substrates (2011)
Focus Area: Healthy Coastal Ecosystems

RELEVANCE- The marshes adjoining the southwestern basin of Lake Erie comprise the largest area of coastal wetlands in the Great Lakes Ecosystem which attract hundreds of thousands of waterfowl. These marshes connect the eastern U.S. coast Atlantic Flyway with the Mississippi, Central and Pacific Flyways. As a vital staging area for migratory waterfowl the potential exists for expediting the spread of avian-origin influenza A viruses across the continent.  At the local level this water bird resource supports both consumptive and non-consumptive recreational and commercial uses that contribute to the culture and economy of the Lake Erie region which would be threatened if a highly pathogenic avian influenza virus was introduced into the Lake Erie marshes by migrating marsh birds. Understanding how environmental conditions can suppress or enhance virus environmental tolerance will contribute to improving the hazard resilience if a highly pathogenic avian-origin influenza A virus is introduced into these coastal wildlife and human communities.   


RESPONSE-Water quality parameters and submerged organic surface layers were collected in numerous marshes in the Lake Erie coastal wetlands during the summer of 2010.  During 2010-2011 school- year the wetlands data were evaluated and served as the basis for developing a laboratory model to simulate the general conditions of the different waterfowl habitats. The first test-run of the selected model included six influenza virus isolates in two different wetlands substrates, water and the bottom organic material.  The results were unexpected and inconclusive.  The environmental model was reevaluated and the sampling protocol was modified.  The environmental tolerance of the six influenza A virus isolates will be retested this summer (2012) and six additional isolates will be tested during Fall 2012 bringing the total number of isolates tested to 12.       

RESULTS-As mentioned above the results from the first test-run were inconsistent.  It is unclear whether organic material was tying up viruses or the sampling protocol was not obtaining representative samples.  The virus isolation procedure and protocol for detecting viral nucleic acid both produced positives.   

RECAP- Water and sediment quality parameters were determined for coastal wetlands in the Great Lakes Ecosystem and a laboratory model simulating these parameters was tested using six influenza A virus isolates but, inconsistent (or unexpected) results required that the model and sampling protocols be modified for retesting the six influenza A virus isolates and six additional isolates during the balance of 2012.


For additional information contact: 


Richard Slemons, Department of Veterinary Preventive, The Ohio State University

Robert Gates, School of Natural Resources, The Ohio State University

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