# An Early Detection System for Highly Pathogenic H5N1 

# Avian Influenza in Wild Migratory Birds

# U.S. Interagency Strategic Plan

## Introduction

Avian influenza (AI) is a type A influenza virus that is naturally found
in certain species of waterfowl and shorebirds. However, the occurrence
of highly pathogenic avian influenza (HPAI) subtype highly pathogenic
H5N1 avian influenza has raised concern regarding the potential impact
on wild birds, domestic poultry, and human health should it be
introduced into the United States (U.S.). Numerous potential routes for
introduction of the virus into the U.S. exist including illegal movement
of domestic or wild birds, contaminated products, via an infected
traveler, as a bioterrorism event, and the migration of infected wild
birds. This plan focuses primarily on the detection of a potential
introduction of highly pathogenic H5N1 avian influenza virus by
migratory birds.

Avian influenza viruses are classified on the basis of two proteins,
hemagglutinin (H) and neuraminidase (N), found on the surface of the
virus. Specific viral subtypes have one of 16 different H proteins and
one of 9 different N proteins, resulting in 144 possible combinations or
subtypes based on this classification scheme. Within each subtype, there
are numerous combinations of genetic sequences that determine the
pathogenicity of the subtype to an infected host.

Wild birds, in particular certain species of waterfowl and shorebirds,
are considered to be the natural reservoirs for all 144 subtypes. These
subtypes are adapted to survive in these wild species and usually cause
little or no disease. However, gradual genetic drift (i.e., mutation)
can occur and a particular subtype can become adapted to infect other
species of wild birds and domestic birds. Although this slight genetic
change in the virus allows it to infect new species, it usually does not
cause disease in the new host. The virus can also change if a host is
simultaneously infected with another type A influenza virus. In such
situations, mixing of the genetic material from the two virus strains
(genetic shift) can occur, resulting in the formation of a new strain.
The combination of gradual drifts and rapid shifts results in the
production of a strain that now causes morbidity and mortality in
susceptible hosts. If the morbidity and mortality is significant, the
virus is classified as a highly pathogenic avian influenza (HPAI) virus.

During 1995-96, it is thought that antigenic drift occurred in an AI
virus of wild birds, allowing the virus to infect chickens in China.
This was followed by reassortment into the HPAI virus subtype highly
pathogenic H5N1 avian influenza. Since that time, this highly pathogenic
H5N1 has been circulating in Asian poultry and domestic fowl resulting
in significant mortality to these species. Highly pathogenic H5N1 avian
influenza likely underwent further antigenic drift and shift allowing
infection in additional species of birds, mammals, and humans. More
recently, this virus moved back into wild birds resulting in significant
mortality of species such as bar-headed geese, brown-headed gulls,
black-headed gulls, ruddy shelducks, and great cormorants in China
during April 2005.

Although the spread of H5N1 in Asia has been primarily due to movement
of domestic birds, the movement of this virus into wild birds raised the
possibility that these species may also spread the virus. This was
thought to be the case in August 2005, when bar-headed geese and whooper
swans died on Erkhel Lake, Mongolia, in an area not known to have
domestic poultry or fowl nearby.

Given the adaptation of highly pathogenic H5N1 avian influenza to wild
birds, increasing concern has developed over the potential for migrating
species to introduce the virus into new regions of the world such as
North America. Therefore, at the request of the Homeland Security
Council's Policy Coordinating Committee for Pandemic Influenza
Preparedness, the U.S. Departments of Agriculture (USDA) and Interior
(DOI) were asked to develop a coordinated National Strategic Plan for
early detection of HPAI introduction into North America by wild birds.
Dr. Tom DeLiberto (USDA-APHIS Wildlife Services) and Rick Kearney (USGS
Biological Resources Division) convened an interagency Working Group,
which consists of representatives from USDA, DOI, U.S. Department of
Health and Human Services (HHS), the International Association of Fish
and Wildlife Agencies IAFWA), and the state of Alaska (Attachment 1).

On 10 August 2005, the Working Group met by teleconference to initiate
development of a "Plan For the Detection of HPAI Virus in Migratory
Birds in the United States". After some discussion among the
participants it was decided that while the immediate concern was the
introduction of highly pathogenic H5N1 avian influenza virus via
migratory birds into Alaska and the Pacific Flyway (including Hawaii and
other Pacific Islands), the group would also begin to address detection
of the virus in all the North American flyways.

## Goal of the Strategic Plan

The goal of this plan is to describe the essential components of a
unified national system for the early detection of HPAI, specifically
highly pathogenic H5N1 avian influenza, in migratory birds. While the
immediate concern is a potential introduction of highly pathogenic H5N1
avian influenza into the U.S., the development of a system that is
capable of detecting the introduction of all HPAI viruses through
migratory birds would significantly improve the biosecurity of the
Nation. This document provides guidance to Federal, State, university,
and non-governmental organizations for conducting HPAI monitoring and
surveillance of migratory birds in the U.S. It is expected that this
document will be used by agencies and organizations to develop regional
and/or state-specific implementation plans for HPAI surveillance.

Data collected in accordance with the guidelines presented in this
document will be assimilated into a National database for use by all
agencies, organizations, and policy makers. Furthermore, although the
original charge of the Working Group was to monitor migratory birds as a
potential route of entry into the U.S., the standardized methodologies
and procedures identified in this document are applicable to other wild
birds as well. Agencies and organizations conducting monitoring and
surveillance in non-migratory birds are encouraged to follow these
guidelines so that their data can be incorporated into and tracked via
the National Early Detection System.

This system for highly pathogenic H5N1 avian influenza detection will
provide early warning for potentially catastrophic mortality events in
North American wild birds and poultry, and minimize the potential for
human exposures. Agencies and organizations are encouraged to
participate in this system by following the guidelines presented in this
document when conducting AI sampling in wild birds.

While this plan focuses on detection of highly pathogenic H5N1 avian
influenza virus, the Working Group fully supports efforts to
characterize all AI viruses in wild birds. Such information is critical
to our understanding of the ecology of AI viruses and their transmission
among wildlife, livestock, and humans. Birds will be sampled in
conjunction with existing studies when possible, and additional bird
captures will be initiated as necessary to provide a broad species and
geographic surveillance effort.

## A National Early Detection System for Highly pathogenic H5N1 avian influenza in Migratory Birds

The ability to efficiently control the spread of a highly infectious,
exotic disease such as highly pathogenic H5N1 avian influenza, is
dependent upon the capacity to rapidly detect the pathogen if
introduced. For this reason, a National Early Detection System for
Highly pathogenic H5N1 avian influenza in Wild Migratory Birds is not
only prudent, it is necessary. Effective implementation of this National
Detection System will require decentralized planning and execution at
regional and state levels, combined with centralized coordination to
ensure national level analysis of surveillance data for risk assessment.
It also must involve a partnership between public and private interests
and include efforts by Federal, State, and local governments as well as
nongovernmental organizations, universities, and other interest groups.
Lastly, it requires flexibility and commitment by all groups for
successful implementation.

### [Decentralized Planning and Execution]{.underline}

Wild migratory birds, by their very nature, are not subject to disease
containment controls as are domestic birds and people. While their
movements are generally uncontrollable, these movements are largely
predictable on both a daily and seasonal basis. Local movements within
or between breeding, feeding, and roosting areas are frequently well
known by State and local wildlife management authorities and others
familiar with local bird populations. Long range movements associated
with seasonal migration are also well known for many species, especially
those waterfowl and shorebird species of particular interest in highly
pathogenic H5N1 avian influenza detection and surveillance.

Coordinating groups such as the four Flyway Councils already exist to
deal with issues related to migratory bird management on a broad
geographic scale. These Councils include representation from each of the
States in their respective bird flyways as well as the U.S. Fish and
Wildlife Service. Therefore, the planning and execution of local and
regional highly pathogenic H5N1 avian influenza early detection efforts
will best be accomplished by the States in collaboration with Federal
agencies.

### [Centralized Coordination]{.underline}

States and flyways are exposed to varying degrees of threat from highly
pathogenic H5N1 avian influenza. Each has unique circumstances that will
shape the direction and intensity of its early detection efforts.
Consequently, gaps among regional programs may emerge over time.
Centralized coordination will evaluate the effectiveness of state and
regional efforts, allowing for prioritization of available federal
resources.

Integration of this National Early Detection System with similar
influenza surveillance systems in other species (e.g., domestic, feral,
zoo) as well as humans will also require centralized coordination.
Surveillance data from all of these systems will be incorporated into
national risk assessments, and preparedness and response planning
efforts.

## Geographic Prioritization of Sampling Efforts

This Strategic Plan targets bird species in North America that have the
highest risk of being exposed to or infected with the highly pathogenic
H5N1 avian influenza subtype because of their migratory movement
patterns. Currently, these include birds that migrate directly between
Asia and North America, birds that may be in contact with species from
areas in Asia with reported outbreaks, or birds that are known to be
reservoirs of AI. However, should highly pathogenic H5N1 avian influenza
virus be detected in domestic birds in the U.S., sampling of wild birds
within the affected flyway may become a high priority as well.

In general, bird flyways represent migration corridors within
continental landmasses. However, Alaska and areas in Eastern Siberia
represent a unique situation where major flyway systems cross
continental boundaries (Attachment 2, fig. 2-1). Two major Asian flyways
(the East Asian-Australasian and East Asian) include both Southeast Asia
and the Arctic regions of Siberia, the Russian Far East, and Alaska. The
East Asian-Australasian Flyway, defined primarily in the context of
shorebird use, extends across 20 countries from the Siberian and Alaskan
Arctic through North and Southeast Asia including U.S. trust territories
in the Pacific to Australia and New Zealand.

Similarly, in North America, the Pacific Flyway extends from Arctic
Canada, Alaska, and Eastern Siberia through coastal and western regions
of Canada, the United States and Mexico, and on to Central and South
America (Attachment 2, Fig. 2-2). Many migratory species that nest in
Arctic Siberia, Alaska, and Canada follow the Pacific Flyway to
wintering areas. Although not considered a major pathway, birds from
both Eastern Siberia and Alaska intermingle in both the Pacific and
Central Flyways. The overlap at the northern ends of these flyways and
in Hawaii and Oceania establishes a path for potential disease
transmission across continents and for mixing, re-assortment, and
exchange of genetic material among strains from Eurasia and North
America.

If highly pathogenic H5N1 avian influenza virus spreads to North America
via migratory birds, the above analysis of the major flyways suggests
that the virus would most likely arrive first in Alaska. Such a scenario
is reasonable, as the contribution of Eurasian AI viruses to the genetic
composition of viruses in North American migratory birds has already
been demonstrated. Given the current knowledge on highly pathogenic H5N1
avian influenza distribution, the Working Group developed a prioritized
sampling approach based on Alaska and the major North American flyways.

This approach prioritized the following regions in decreasing order of
importance:

> 1\. Alaska, the Pacific Flyway, and Oceania
>
> 2\. Central Flyway
>
> 3 Mississippi Flyway
>
> 4\. Atlantic Flyway

Agencies participating in the development of this plan are committed to
efforts that ensure adequate sampling based on the above prioritization.
However experiences with previous introductions of exotic diseases into
North America (e.g., West Nile Virus) have demonstrated that detection
and surveillance systems must be adaptable to changes in pathogens and
risk factors associated with their potential introduction. If changes in
the relative risks of highly pathogenic H5N1 avian influenza
introduction into the US result in regional reprioritization, agencies
must be prepared to redistribute resources accordingly.

## Sampling Strategies

This strategic plan recommends decentralized planning and execution of
highly pathogenic H5N1 avian influenza early detection efforts. To
provide a uniform structure for the development of local plans, it
recommends the consideration of five strategies for collecting
monitoring and surveillance data on highly pathogenic H5N1 avian
influenza virus in wild birds. Agencies and organizations are encouraged
to use one or more of these strategies when designing AI surveys in wild
birds. These strategies are:

### [Investigation of Morbidity/Mortality Events (Attachment 3):]{.underline} 

Over 40 species of wild birds have been shown to be susceptible to
infection with highly pathogenic H5N1 avian influenza virus. While not
all species infected necessarily exhibit disease, the current strain(s)
of H5N1 circulating in Asia have been shown to cause morbidity and
mortality in a wide variety of these species. The systematic
investigation of morbidity and mortality events in wild birds to
determine if highly pathogenic H5N1 avian influenza is playing a role in
causing illness and death offers the highest and earliest probability of
detecting the virus if it is introduced by migratory birds into the
United States. State natural resource agencies and Federal refuges and
parks, primarily within the DOI's U.S. Fish and Wildlife Service
National Wildlife Refuge System and the National Park Service, are the
principal authorities in a position to detect and respond to mortality
events involving wild birds. Morbidity and mortality events involving
wildlife are often detected by, or reported to, these agencies and
entities. This strategy capitalizes on an existing morbidity/mortality
program being conducted by DOI and its partners.

### [Surveillance in Live Wild Birds (Attachment 4):]{.underline} 

This strategy incorporates sampling of live-captured, apparently healthy
wild birds to detect the presence of highly pathogenic H5N1 avian
influenza virus. This effort will select bird species in North America
that represent the highest risk of being exposed to, or infected with,
Highly pathogenic H5N1 avian influenza virus because of their migratory
movement patterns, which include birds that migrate directly between
Asia and North America, or birds that may be in contact with species
from areas in Asia with reported outbreaks. Should highly pathogenic
H5N1 avian influenza virus be detected in domestic birds in the U.S.,
sampling of wild birds in the flyway in the affected area may become a
high priority as well. Data collected by organizations currently
conducting research and monitoring for avian influenza in Alaska will be
incorporated with additional bird captures as necessary to provide a
broad species and geographic surveillance effort. This strategy
capitalizes on research activities currently being conducted by DOI,
USDA and their partners.

### [Surveillance in Hunter-killed Birds (Attachment 5)]{.underline}

Check stations for waterfowl hunting are operated by the US Fish and
Wildlife Service and state natural resource agencies. Hunter check
stations provide an opportunity to collect additional samples to
determine the presence of HPAI and other subtypes of avian influenza
viruses and supplement data collected during surveillance of live wild
birds. As with surveillance of live wild birds, sampling of
hunter-killed birds will focus on hunted species that are most likely to
be exposed to HPAI in Asia; have relatively direct migratory pathways
from those areas to the U.S. via Alaska or directly to the Pacific
Coast; mix in Alaska staging areas with species that could bring the
virus from Asia; or should HPAI be detected in domestic birds in the
U.S., may mix with wild birds in the flyway of the affected area.
Collection of samples from these species will occur at hunter check
stations in the lower 48 states during hunting seasons in areas where
these birds stage during migration or over-wintering.

### [Sentinel Species (Attachment 6):]{.underline} 

Waterfowl, exhibition gamefowl, and poultry flocks reared on backyard
premises have been used as sentinels for active surveillance for avian
diseases of interest to the commercial poultry industry and regulatory
agencies. Currently in Alaska, the State veterinarian uses targeted
surveillance of domestic flocks at concentration points due to remote
location of villages and lack of resources; enthusiasts travel to
poultry exhibitions with birds from distant locations; and. surveillance
effectively covers a large geographic area. Enhancement of this approach
would be valuable. However, placement of sentinel ducks in strategic
locations may also prove useful. Placement of sentinel ducks has been
used successfully for surveillance of diseases of importance to the
poultry industry, including influenza A. Also, sentinel ducks in wild
pelagic bird colonies improved virus detection rates fivefold,
suggesting that this approach is advantageous in ecological studies.

### [Environmental Sampling (Attachment 7):]{.underline} 

Avian influenza viruses are generally released by waterfowl through the
intestinal tract and viable virus can be detected in both feces and the
water in which the birds swim, defecate and feed. This is the principal
means of virus spread to new avian hosts and potentially to poultry, and
other susceptible livestock. Analysis of both water and fecal material
from waterfowl habitat can provide evidence of AI virus circulating in
wild bird populations, the specific AI subtypes, levels of
pathogenicity, and possible risks to poultry and susceptible livestock.
Monitoring of water and/or fecal samples gathered from waterfowl habitat
is a reasonably cost effective, technologically achievable means to
assess risks to poultry.

## Sample Collection

Samples collected for AI surveillance may include carcasses, tracheal
and cloacal swabs, feces, and environmental samples (e.g., water). Prior
to initiating a surveillance activity, it is important to identify the
laboratory in which the samples will be submitted. Sample handling and
transportation procedures may differ among laboratories. It is
recommended that samples collected for inclusion into the National Early
Detection System be submitted to a laboratory that uses standardized
procedures identified in the Laboratory Diagnosis section of this
document or by using the attached detailed descriptions of sampling
methodologies.

If birds are found morbid or dead, it is important to use proper
personal protection techniques
(<http://www.nwhc.usgs.gov/publications/wildlife_health_bulletins/WHB_05_03.jsp>,
<http://www.cdc.gov/flu/avian/professional/protect-guid.htm>) and to
submit the entire carcass to a veterinary diagnostic laboratory for
necropsy (Attachment 8). Field biologists should contact the specific
laboratory that they will be working with well in advance of any
specimen collection and shipping to receive specific instructions for
specimen submissions to that laboratory. Laboratories should always be
notified ahead of time when a shipment is being made to their facility.

When collecting samples from live or hunter-killed birds, tracheal and
cloacal swabs are preferred. Most AI strains tend to replicate more
efficiently in the intestinal tract than in the respiratory tract of
natural host species (i.e., waterfowl and shorebirds). Consequently,
cloacal swabs are generally preferred. However, recent isolations of
highly pathogenic H5N1 avian influenza virus in wild birds have
documented higher levels of virus in tracheal samples. Therefore, it is
recommended that both samples be collected from birds when possible.
While the collection of cloacal swabs is a relatively easy procedure,
obtaining proper tracheal swabs can be problematic and requires
personnel trained in the sampling technique. Examples of
tracheal/cloacal swab collection protocols can be found in Attachment 9.
Tracheal and cloacal swabs should be placed in separate tubes, and swabs
should not be pooled across individuals.

Monitoring of water and/or fecal samples gathered from waterfowl habitat
is a reasonably cost effective, technologically achievable means to
detect the presence of HPAI and alert decision makers to the risks to
poultry in the Western Hemisphere from new, potentially highly
pathogenic subtypes of AI (Attachment 7). A surveillance system based on
water sampling is not ready to implement at the present. However, the
validation of this method could come on-line in a short period of time
and would represent considerable cost savings without loss of
sensitivity. Fecal sampling is an established technique and is ready for
use in surveillance with the establishment of sampling guidelines. Both
approaches yield advantages where individual bird sampling is too costly
or logistically impractical. Either approach could yield a spatial and
habitat risk assessment for site contamination with highly pathogenic
H5N1 avian influenza virus. The main considerations are where and when
to get the samples, ensuring proper storage and transport, and the
capacities and capabilities of the laboratories doing the analyses.
Real-time reporting and the infrastructure to support such reporting is
a serious constraint on any surveillance system. The ability to
integrate, analyze, and responsibly disseminate these data is critical
and needs to be addressed.

## Sample Size Determination

Prior to initiating a surveillance program, it is important to determine
the sample size necessary to make statistically valid inferences
concerning the presence of highly pathogenic H5N1 avian influenza virus
in a sample population. In the context of this plan, the population of
interest is not defined because this definition will vary by geographic
location, time of year, species of interest, and sampling method
employed. For example, sampling a breeding population versus a wintering
population, for a single species, may result in very different
interpretations of the geographic distribution of the population of
interest. If water samples are being collected, then the population may
consist of several water bodies. Therefore, it is crucial that prior to
collections beginning, statistically valid sample size estimations be
incorporated into regional and state implementation plans.

## Laboratory Diagnostics

All samples collected for inclusion in the National Early Detection
System should be analyzed in accordance with the standard procedures
included in this document. A list of laboratories certified to conduct
testing for highly pathogenic H5N1 avian influenza virus is included in
Attachment 11. Samples will be analyzed as soon as possible after
collection. Tracheal/cloacal swabs and fecal samples will be analyzed by
real-time reverse transcriptase-polymerase chain reaction (RT-PCR) using
the matrix gene RT-PCR assay (Attachment 12). The matrix gene RT-PCR
assay is capable of detecting all 16 hemagglutinin and nine
neuraminidase subtypes. Matrix gene RT-PCR-positive samples would
indicate the presence of avian influenza and they should be further
characterized by the H5- and H7-specific RT-PCR assays of Spackman et
al. (2002) as modified in Attachment 11. The H5 RT-PCR test is known to
detect the current Highly pathogenic H5N1 avian influenza viruses.

Positive H5 and H7 RT-PCR tests would indicate the presence of AI
viruses with the potential of causing pathology in domestic poultry.
Therefore, all samples positive for H5 and H7 by RT-PCR will be
submitted for virus isolation for verification. Samples positive for
live virus in virus isolation and positive for H5 or H7 by RT-PCR will
be submitted to the USDA APHIS National Veterinary Services Laboratory
(NVSL) for confirmation. The NVSL is capable of performing the
intracranial chicken pathogenicity index (ICPI) test on the resultant
virus to determine directly the pathogenicity of the virus in chickens.
Identification of a highly pathogenic H5 or H7 virus is a reportable
disease and immediate notification to the agency submitting the sample,
the state veterinarian, the area veterinarian in charge (AVIC), the
state public health official and the CDC/USDA Select Agent program.
Samples will be immediately secured as required by the Select Agent
Programs.

All positive H5 and H7 samples will also be sent to the USDA Agriculture
Research Service Southeastern Poultry Research Laboratory in Athens, GA,
for complete molecular sequencing. This will provide for complete typing
of the virus and allow for phylogenetic analysis.

## Data Management

Real-time reporting and the infrastructure to support such reporting is
a serious constraint on any surveillance system. The ability to
integrate, analyze, and responsibly disseminate these data is critical.
In addition, the data collected for this National Surveillance System
will consist of samples submitted by many agencies and organizations.
This will require a system to manage the input of animal and sample
collection data through multiple routes, the ability to easily match,
compare, and transfer laboratory data about these samples, and provide a
platform in which all data is secure, accessible, and able to be mapped
and used for spatial modeling.

The National Biological Information Infrastructure Wildlife Disease
Information Node (WDIN) managed by the U.S. Geological Survey's National
Wildlife Health Center has created a prototype web-enabled HPAI data
management system, which will serve as a template for data collected
from live and hunter-killed wild birds. (See
http://wildlifedisease.nbii.gov/ai). The WDIN has developed comparable
systems for the management of data from multiagency wildlife disease
surveillance efforts such as Chronic Wasting Disease, and for USDA APHIS
Wildlife Services Plague and Tularemia. General aspects of the proposed
WDIN Interagency HPAI Data Management System are described in Appendix
13.

Sentinel bird data will be incorporated into a web-enabled, national
data management system for backyard and small-flock poultry developed by
the USDA APHIS Veterinary Services' Application Information Management
Team at the Centers for Epidemiology and Animal Health. This database
system was developed to reduce the number of data-collection problems
experienced by field personnel and to generally improve the quality and
efficiency of data collection. The initial testing of the design
occurred in October 2005, with actual deployment scheduled for December
2005. If the project is successful in the pilot state of California, the
system will likely be expanded for national implementation in 2006. This
system will allow all necessary data collected in the field to be shared
among all approved organizations without the need for manual data entry,
and will provide greater chain-of-custody assurance from a legal and
diagnostic perspective. Field personnel will be equipped with computer
hardware and software which will facilitate the rapid and accurate
collection of samples and data. These devices will share the collected
information as needed with the diagnostic lab (National Veterinary
Services Laboratories), and will send the data to primary information
systems within USDA. To assist in data entry and to further improve data
quality, bar-coding will be implemented as key identifiers for samples
collected and for cases submitted.

## Recommendations

Given the current state of knowledge of the epidemiology of highly
pathogenic H5N1 avian influenza virus it is recommended that a
coordinated interagency/organization early detection system be
implemented in the U.S. An analysis of risk factors, including current
worldwide distribution of the virus and the migratory patterns of wild
birds, indicated that this system should primarily focus sampling
efforts in Alaska, Oceania, and the Pacific and Central flyways.
However, if adequate resources become available, the system should be
expanded to include surveillance of migratory birds in the Mississippi
and Atlantic Flyways as well.

State and Federal agencies should immediately begin developing
implementation plans based on the guidance provided in this Strategic
Plan. Development of these plans should be conducted with the
participation of all relevant management agencies and organizations such
that sampling designs are produced that allow for statistically sound
inference of the presence or absence of highly pathogenic H5N1 avian
influenza virus in wild birds. Furthermore, it is recommended that such
coordination be conducted through the Flyway Councils, so that
regionally based sampling designs can be implemented. Each Flyway
(Atlantic, Mississippi, Central, and Pacific) has a Flyway Council, a
formal organization generally composed of one member from each State and
Province in that Flyway. The Flyway Councils, established through the
International Association of Fish and Wildlife Agencies (IAFWA), also
assist in researching and providing management techniques for Federal,
State, and Provincial Governments, as well as private groups and the
public.

State and Federal agencies also should develop communication plans in
the event that a HPAI is detected in wild birds. For example, highly
pathogenic H5N1 avian influenza virus is a reportable disease that
requires notification of the State Veterinarian, and the Area
Veterinarian in Charge (AVIC). Highly pathogenic H5N1 avian influenza is
also a CDC/USDA Select Agent, requiring notification of the CDC/USDA
Select Agent Programs and adherence to Select Agent guidelines is
required.

Finally, it is recommended that a Steering Committee, consisting of one
representative each from USGS, FWS, USDA APHIS, IAFWA, HHS, the National
Flyway Council, and the State of Alaska be formed to coordinate wild
bird AI surveillance in the United States. Specific roles of this
Committee should include:

-   Facilitate communication between state and federal agencies, and
    organizations involved in AI surveillance for wild birds.

-   Coordinate implementation and data analysis of AI surveillance
    programs nationally.

-   Provide periodic summaries of AI surveillance for wild birds in the
    United States.

-   Provide periodic recommendations for AI surveillance in wild birds
    based on previous sampling efforts and changes in virus
    epidemiology.

-   Facilitate communication and coordination among state and federal
    agencies for contingency planning and other preparations for the
    appearance of highly pathogenic H5N1 avian influenza virus in wild
    birds in North America.

Sampling strategies to detect highly pathogenic H5N1 avian influenza
virus in wild bird populations will change depending upon the risk
assessment and management goals and prevailing status of the pathogen in
North America. For early detection of highly pathogenic H5N1 avian
influenza virus, efforts should focus on likely cross-over routes of
birds from Asia to North America (e.g., Alaska). Efforts should focus on
areas of high aggregations of waterfowl intersecting with logistical
sampling support such as the National Wildlife Refuge System and state
waterfowl management areas.

If highly pathogenic H5N1 avian influenza virus gains a foothold in
North America, the surveillance network should be placed along known
waterfowl movement paths from the point of origin (i.e., point of
detection). These paths can be inferred from known migration routes of
specific species. However, practically, and given the patterns emerging
in Eurasia, if highly pathogenic H5N1 avian influenza virus becomes
established in North America the likelihood of rapid and diffusive
spread across the continent is high. At this point local waterfowl and
environmental sampling should target areas of strategic value, e.g.,
human population centers and areas of high density of poultry
production. In the former case, such areas would be represented by urban
zoo-parks and lakes. These areas would represent the highest level of
risk of human contact with contaminated water and/or waterfowl. In the
latter case, ponds, lakes and waterfowl management areas around high
density poultry production areas would provide the best ability to
assess risk of transmission to humans and poultry. Surveillance efforts
patterned on these areas are most amenable to local and state efforts
for first detection and subsequent risk assessment if H5N1 subtype(s)
achieve enzootic status in North America.

# ATTACHMENT 2

## Migratory Bird Flyways in Asia and North America

Figure 2-1: Asian Migratory Bird Flyways

![](media/image1.png){width="4.75in" height="3.4944444444444445in"}

![](media/image2.wmf){width="4.926377952755906in" height="3.55in"}

# ATTACHMENT 3

# Investigation of Morbidity and Mortality Events in Wild Birds

## Overview 

The systematic investigation of morbidity and mortality events in wild
birds to determine if the highly pathogenic H5N1 avian influenza subtype
of avian influenza (AI) is playing a role in causing illness and death
offers the highest and earliest probability of detecting the virus if it
is introduced by migratory birds into the United States. There is
increasing evidence that highly pathogenic H5N1 avian influenza virus is
capable of killing wild birds which is not the usual characteristic of
AI viruses. As such, the documentation of the movement of the virus
through Asia and into Europe has been discovered in part, through the
investigation of mortality events of wild migratory birds. Benefits
gained from conducting disease investigations of wildlife mortality
events are not unique to AI. Many other important diseases have been
discovered and described after initial detection through the wildlife
disease investigation process (e.g. West Nile Virus). The investigation
of wildlife diseases operates with consistent procedures while
maintaining enough flexibility to accommodate the unique characteristics
of specific disease agents involved. The initial detection of a
mortality event is strongly dependent upon well-trained and observant
field personnel. These people in turn communicate with an experienced
staff of disease investigation specialists that obtain the maximum
amount of information surrounding the event. Depending upon the
significance and intensity of the mortality event, these highly trained
investigators may visit the site of the mortality event to conduct field
investigations so as to obtain further information first hand. In
addition to determining a cause of death, disease investigation
specialists provide useful management recommendations to potentially
reduce further morbidity and mortality.

In the event that highly pathogenic H5N1 avian influenza is detected in
wild birds, it will be important to investigate the proximity of
domestic poultry and swine operations in order to initiate activities to
minimize contact between the wild birds and these other animals.
Morbidity and mortality of wild birds is most likely to occur in areas
where migratory birds infected with highly pathogenic H5N1 avian
influenza mingle with other wild bird species, particularly in wetland
habitats. Likewise, early outbreaks of highly pathogenic H5N1 avian
influenza would most likely occur in Alaska and along the Pacific Flyway
of the United States and Canada, where migratory birds from Asia stage
in the summer and early fall and subsequently migrate within North
America. However, given that migrants also move from Alaska to other
parts of North America, surveillance strategies should include other
flyways as well. In this surveillance plan, participating state, Federal
and tribal agencies, and cooperators will conduct targeted surveillance
for highly pathogenic avian influenza (HPAI) both in response to disease
outbreaks in wild birds focusing intensively in Alaska, the Pacific
Flyway, and Oceania, and in response to mortality events in
high-priority (i.e., most likely) species throughout the United States.

# Methodology

The key to success of this surveillance strategy involves: 1) early
detection of morbidity and mortality, 2) rapid reporting and submission
of appropriate biological specimens to qualified diagnostic facilities,
3) immediate assessment of the field event (descriptive epidemiology),
4) rapid, accurate, and consistent diagnosis and confirmation, 5)
immediate reporting of diagnostic results once confirmed, and 6)
pre-planned contingency and response training for the occurrence of
HPAI.

Specific steps necessary to orchestrate the early detection of highly
pathogenic H5N1 avian influenza include:

1)  State, Federal, and tribal resource personnel will be instructed to
    increase vigilance and to establish routine and systematic
    monitoring of wild bird populations for morbidity and mortality.
    Standard guidelines will be prepared with the assistance of the U.S.
    Geological Survey (USGS) - National Wildlife Health Center (NWHC)
    and the U.S. Department of Agriculture (USDA) - Wildlife Services
    (WS) to increase uniformity of effort. The most intensive monitoring
    will occur in Alaska and selected areas of high risk in the Pacific
    Flyway and Oceania where migratory birds from affected countries are
    likely to interact with North American bird species.

2)  A uniform protocol for reporting mortality events and instructions
    for the safe handling and shipment of specimens to identified
    diagnostic facilities will be developed. Field and response
    personnel will be trained. Reporting of mortality events will be
    through appropriate channels within each state, Federal, or tribal
    entity to the NWHC, where a centralized database (WDIN) will be
    maintained, made available to contributors, and summarized in
    modified form for public dissemination.

3)  Field personnel or teams designated by respective land management
    agencies will respond to mortality events by conducting field
    investigations to determine onset, course, duration, distribution,
    species, and other environmental conditions associated with
    mortality events. The NWHC and USDA-WS will assist in developing
    guidelines and training. In certain circumstances, NWHC and USDA-WS
    personnel will conduct field investigations or assist other
    agencies.

4)  Representative and suitable carcasses and other biological samples
    and specimens will be submitted to one or more identified diagnostic
    facilities capable of conducting immediate necropsy and laboratory
    analyses. Guidelines will be developed to assure that the
    appropriate number and types of samples are collected to ensure that
    there is a statistically-based confidence in the sample size
    analyzed in response to a mortality event. Necropsies, histology,
    and laboratory investigations will be utilized to substantiate a
    diagnosis of highly pathogenic H5N1 avian influenza virus. Virus
    isolation, hemagglutination inhibition tests, and molecular testing
    specifically for H5N1 will be performed to detect the presence of
    the virus in specimens.

5)  Reporting of results to submitters will be done as early as
    possible, including preliminary results that may refute or support
    the presence of HPAI. Highly pathogenic H5N1 avian influenza virus
    is a CDC/USDA Select Agent, thus the CDC/USDA Select Agent Programs
    will be notified immediately upon identification of the virus and
    all Select Agent guidelines will be followed as required. Final
    results of HPAI tests will be reported immediately to the submitter.
    As highly pathogenic H5N1 avian influenza is a reportable disease,
    the State Veterinarian, and the Area Veterinarian in Charge (AVIC)
    will be informed simultaneously of the discovery. A final report
    will also be provided to the WDIN. Public release of information
    will occur only after these final results are thus reported.

6)  Wildlife disease contingency plans will be established at an
    appropriate landscape scale to enable rapid deployment of personnel
    and resources to take action. Disease contingency plans can be
    developed for general response to a mortality event, with special
    reference and consideration for highly pathogenic H5N1 avian
    influenza virus. The NWHC and USDA-WS will assist in providing
    guidelines and training in the establishment of contingency plans.

To increase early detection and response capabilities to the extent
needed to protect the United States from highly pathogenic H5N1 avian
influenza virus, enhancements to current activities will need to
include:

> \- Personnel and resources in the field to intensively monitor for
> mortality events,

\- Systematic methods to detect mortality early in the field,

\- Resources to fully investigate all such events, and

\- Surge capacity at wildlife disease diagnostic facilities

Wildlife professionals employed by state natural resource agencies and
by the U.S. Department of Interior (DOI) Fish and Wildlife Service and
National Park Service are the principal authorities positioned to detect
and respond to morbidity and mortality events involving wild birds. The
DOI Bureau of Land Management, Tribal Nations, and several other state,
Federal, and local agencies (including the U.S. Department of Defense)
also have authority over lands that they administer and manage.
Morbidity and mortality events involving wildlife are often detected by,
or reported to these agencies and entities.

Investigations into the causes of wildlife mortality events are
dependent on the perceived significance of the event and on the
knowledge or availability of disease diagnostic facilities capable of
providing assistance. The USGS - NWHC, located in Madison, Wisconsin, is
a full-service wildlife diagnostic and research laboratory that assists
Federal, state, and tribal agencies in responding to wildlife disease
outbreaks. Together with its Honolulu Field Station, which serves Hawaii
and Pacific Trust Territories, the NWHC is the principal facility relied
upon by the DOI, as well as by most states, to investigate and diagnose
wildlife diseases, including those of migratory birds. Numerous state
natural resource agencies in the Pacific Flyway also have established
wildlife disease laboratories and programs with staff that respond to
wildlife disease outbreaks in their respective states. USDA, state and
university diagnostic laboratories, and regional entities such as the
Southeastern Cooperative Wildlife Disease Study are also involved in
wildlife disease investigations. The NWHC maintains an extensive
database on wildlife mortality events across the United States and
Canada to which Federal, state, provincial, and tribal agencies
contribute. The NWHC also supports and houses the Wildlife Disease
Information Node (WDIN), a part of the USGS - National Biological
Information Infrastructure (NBII).

# Discussion

The primary strength of the strategy of targeted investigations of avian
mortality events is based upon the observation that highly pathogenic
H5N1 avian influenza kills some species of wild birds. As such, a wild
bird die-off serves as a "trigger event" that immediately focuses the
investigation to a given area and species. Further, because the current
form of the virus circulating in Asia will be new to North America, the
hypothesis is that highly pathogenic H5N1 avian influenza will be
detected if it is the cause of an observed mortality event. Therefore
concentrating on recovering carcasses and samples from wild bird
die-offs affords a timely opportunity to detect HPAI. Conversely, live
bird surveillance provides the opportunity to detect birds that may shed
the virus without ill-effects and offers the possibility of early
detection of the arrival and especially, the spread of highly pathogenic
H5N1 avian influenza. Due to the size of the country and the number of
species of wild birds involved, careful consideration will be needed to
identify relevant species and sampling locations for live bird
surveillance. Hunter-harvested birds will provide an opportunity to
augment live bird surveillance by providing large numbers of birds using
a reduced level of field resources. However, as a limited number of
species are targeted for hunting, sound scientific judgment should be
exercised in choosing species and locations for analysis. All of the
strategies described above will require considerably more resources in
personnel to be effective, and the greater number of submissions (surge
capacity) will require laboratories to be prepared in advance.

# Recommendations

Because the primary goal of the process outlined in this plan is the
earliest possible detection of highly pathogenic H5N1 avian influenza in
wild birds, all of the strategies described are important, but not all
strategies are practical to conduct in all areas of the country. The
live wild bird surveillance strategy would be a most effective tool to
determine the pattern of virus spread subsequent to a die-off attributed
to highly pathogenic H5N1 avian influenza. Wild animal die-offs are
important to investigate for multiple reasons, however it should be
noted that highly pathogenic H5N1 avian influenza will not be the cause
of most of the mortality events investigated through a targeted
surveillance strategy. Rather, other bacterial and viral diseases that
are either zoonotic or important to agriculture may be detected through
these surveillance programs. Mortality event investigation provides the
opportunity to obtain the greatest amount of information about health
and disease in wild birds without an a priori bias. Supplemental
wildlife disease information will be prioritized and gathered as funding
and personnel allow. Surveillance for Highly pathogenic H5N1 avian
influenza will remain the top priority.

# Appendix

*Field Personal Safety:*

In an area where highly pathogenic H5N1 avian influenza has not been
detected, field personnel should follow the recommendations provided in
the NWHC Guidelines for Handling Birds
([http://www.nwhc.usgs.gov/publications/wildlife_health_bulletins/WHB_05_03.jsp](http://www.nwhc.usgs.gov/research/WHB/WHB_05_03.html)).
Personal protective equipment (PPE) should include boots, coveralls, and
gloves. In addition, the use of goggles and N95 masks are recommended.

In areas where highly pathogenic H5N1 avian influenza has been detected,
especially during a mass mortality event, field personnel should follow
the latest guidelines of the CDC
(<http://www.cdc.gov/flu/avian/professional/protect-guid.htm>). PPE
should include complete coveralls, gloves, and boot covers that are
either disposable or that can be disinfected. Goggles, N95 masks (NIOSH
respirator preferred) as well as a health monitoring plan are required.

# Attachment 4

# Surveillance for Highly pathogenic H5N1 avian influenza virus in Live Wild Birds

## Overview 

This surveillance strategy incorporates sampling of live-captured,
apparently healthy migratory birds to detect the presence of highly
pathogenic avian influenza (HPAI) H5N1 virus. Virus isolation from
tracheal and cloacal samples is a common method for detecting avian
influenza (AI) viruses and has been used before in various geographic
regions, including Alaska (Ito et al., 1995; Hanson et al., 2003;
Slemons et al., 2003; Krauss et al., 2004). This effort focuses on bird
species in North America that represent the highest risk of being
exposed to or infected with highly pathogenic H5N1 avian influenza virus
because of their migratory movement patterns, which include birds that
migrate directly between Asia and North America, or birds that may be in
contact with species from areas in Asia with reported outbreaks.

In general, bird flyways represent migration corridors within
continental land masses. However, Alaska and corresponding areas in the
Russian Far East represent a unique case where major flyway systems
cross continental boundaries. Two major Asian flyways (the East
Asian-Australasian and East Asian) include both Southeast Asia, Oceania,
and the arctic regions of Siberia, the Russian Far East, and Alaska. The
East Asian-Australasian Flyway, defined primarily in the context of
shorebird use, extends from the Siberian and Alaskan arctic through
North and Southeast Asia including U.S. trust territories in the Pacific
to Australia and New Zealand, covering 20 countries.

Similarly, in North America, the Pacific Flyway extends from Arctic
Canada, Alaska, and Eastern Siberia through coastal and western regions
of Canada, the United States and Mexico, and on to Central and South
America. Many migratory species that nest in Arctic Siberia, Alaska, and
Canada follow the Pacific Flyway to wintering areas. Although not
considered a major pathway, birds from both Eastern Siberia and Alaska
intermingle in both the Pacific and Central Flyways. The overlap at the
northern ends of these flyways and in Hawaii and Oceania establishes a
path for potential disease transmission across continents and for
mixing, re-assortment, and exchange of genetic material among strains
from Eurasia and North America.

There is concern about the spread of HPAI westward from Asia to Europe.
However, there is comparatively little movement of wild birds between
Europe and North America. Consequently, if highly pathogenic H5N1 avian
influenza virus arrives in the U.S. or a U.S. territory in migratory
birds, it would most likely arrive first in Alaska or one of the Pacific
Islands. Such an event is not unreasonable, as the contribution of
Eurasian AI viruses to the genetic composition of viruses in North
American wild birds has already been shown.

## Methodology

### [Identification of Priority Species]{.underline} 

Birds should be sampled in conjunction with existing studies when
possible, and additional bird captures should be initiated as necessary
to provide a broad species and geographic surveillance effort. Initial
efforts should focus on one or more species in each of the following
three groups that could potentially bring highly pathogenic H5N1 avian
influenza virus to the US Pacific Islands and trust territories and/or
Alaska and, subsequently, southward through the Pacific and potentially
the other North American flyways:

> 1\. Species that travel directly to Alaska or Oceania from Southeast
> Asia or Australasia. Some of these birds winter in Southeast Asia
> while others migrate along coastal Southeast Asia to and from
> wintering areas in Australasia. Based on what is known about the
> geographic distribution of the current highly pathogenic H5N1 avian
> influenza virus outbreaks, this is the group most likely to bring the
> virus to Alaska or the U.S. Islands and trust territories in Oceania.
> Before any species in this group can be a source of infection for
> birds in other areas of North America, inter-specific transmission of
> the virus to temperate migrants must occur in Alaska. Examples include
> the bar-tailed godwit (Fig. 4-1), dunlin, and red knot.
>
> 2\. Species that breed in Alaska, with some fraction of the population
> known to winter in Asia. Although the portion of the population that
> winters in Asia may be small, some of these species are highly
> gregarious at other times of the year, particularly during molting,
> staging, and on their primary wintering grounds. Because the primary
> wintering grounds of several of these species are in the North
> American Pacific Flyway, carriers arriving in Alaska from Asia could
> potentially transmit the virus to a large portion of the North
> American population. This scenario for highly gregarious species
> requires only intraspecific transmission in Alaska. The course of
> events for less gregarious species and those that tend to winter in
> more northerly latitudes is more likely to require interspecies
> transmission. Examples include the black brant, northern pintail (Fig.
> 4-1), long-tailed duck (Fig. 4-2), yellow-billed loon, and
> red-breasted merganser.
>
> 3\. Species that intermingle across Siberia, the Russian Far East, and
> Alaska. This group has become more important with the confirmation of
> HPAI in poultry near Novosibirsk in Siberia. However, unless highly
> pathogenic H5N1 avian influenza virus spreads further north and east
> in this region, the most likely way for this group to become infected
> would be contact with species that winter in southern Asia and breed
> in northern Asia. Under such circumstances, inter-specific
> transmission would be required on both sides of the Bering Strait
> before the virus could be carried from Alaska to temperate regions of
> North America. Examples include the Steller's eider, spectacled eider,
> emperor goose (Fig. 4-3), sharp-tailed sandpiper, sandhill crane.

Figure 4-1. Migratory routes of two species that illustrate movements of
birds between Asia and North America

![](media/image3.wmf){width="5.749305555555556in"
height="4.350694444444445in"}

Figure 4-2. Migratory routes of long-tailed ducks between Alaska and
Asia

![](media/image4.wmf){width="5.625in" height="4.257638888888889in"}

Figure 4-3. Migratory pathways of emperor geese between Alaska and
Russia.

![](media/image5.wmf){width="6.25in" height="4.25in"}The strategy for
selection of species to be sampled should initially focus on migrants
that have the greatest likelihood of making contact with wild migratory
birds, domestic flocks, and geographic areas in Asia where highly
pathogenic H5N1 avian influenza virus has been documented. Members of
three taxonomic groups of waterbirds---loons, waterfowl, and
shorebirds---offer the most immediate potential for meeting the
selection criteria and being carriers of the virus (see Tables 4-1to
4-3).

Table 4-1. Loon species as potential carriers of the highly pathogenic
H5N1 avian influenza subtype to North America.

  ---------------- ------------ -------------- ------------ ------------------ -------------
                   **Asian                                                     
                   Contact**                                                   

  **Species**      **Direct**   **Indirect**   **Timing**   **Specifics^1^**   **Point of
                                                                               Contact**

  Yellow-billed    Yes          Yes            Oct-Apr      Birds breeding on  Coastal
  Loon                                                      Alaskan north      Japan, Korea,
                                                            slope winter off   China
                                                            of Japan, Korea,   
                                                            China.             

  Red-throated     Yes          Yes            Oct-Apr      Birds breeding on  Coastal
  Loon                                                      Alaskan north      Chukotka and
                                                            slope winter off   Kamchatka
                                                            of Chukotka and    
                                                            Kamchatka.         

  Pacific/Arctic                                            Little data is     
  Loon                                                      available but      
                                                            probably follow    
                                                            similar patterns   
                                                            to yellow-billed   
                                                            and red-throated   
                                                            loons.             

  ^1^ For the most                                                             
  part, loon                                                                   
  species mix with                                                             
  other waterbirds                                                             
  along migration                                                              
  routes and at                                                                
  staging and                                                                  
  wintering areas.                                                             
  ---------------- ------------ -------------- ------------ ------------------ -------------

Table 4-2. Waterfowl species as potential carriers of the Highly
pathogenic H5N1 avian influenza subtype to North America.

  ----------------- ------------ -------------- ------------- ------------------------------------ ---------------
                    **Asian                                                                        
                    Contact**                                                                      

  **Species**       **Direct**   **Indirect**   **Timing**    **Specifics^1^**                     **Point of
                                                                                                   Contact**

  **Tundra Swan**   Yes          Yes            Jun-Sep       Portion of breeding range in         Chukotka
                                                              Chukotka. From there birds migrate   
                                                              to staging and wintering areas       
                                                              throughout Pacific Flyway.           

  **Whooper Swan**  Yes          Yes            Nov - Apr     Eastern Asian population breeds in   E. Russia and
                                                              E. Russia and NW China. Small        NW China;
                                                              numbers wintering in Aleutian and    Aleutian and
                                                              Pribilof islands.                    Pribilof
                                                                                                   islands

  **Emperor Geese** Yes          Yes            Year round    Portion of breeding range in         Chukotka and W.
                                                              Chukotka and some AK breeders molt   Alaska
                                                              in Chukotka. From there birds        
                                                              migrate to staging and wintering     
                                                              areas in the W. AK and Aleutian      
                                                              Islands. Small segment of population 
                                                              stage and winter in Commander        
                                                              Islands and Kamchatka.               

  **Black Brant**   Yes          Yes            Jun-Sep       Portion of breeding range in         Chukotka, and
                                                              Chukotka, some AK breeders molt on   N. and W.
                                                              Wrangel Island (Russia), and         Alaska, W.
                                                              Siberian breeders molt on Alaskan    coast of N.A.
                                                              North Slope (Teshekpuk). Most of     
                                                              population migrates to staging areas 
                                                              in W.AK and winters along Pacific    
                                                              coast to Mexico. Birds breeding west 
                                                              of Kolyma (Russia) winter in Korea,  
                                                              China, and Japan (mixing in fall and 
                                                              to some degree in spring).           

  **Aleutian Canada Yes          Yes            Jun-Sep       Portion of population breeds on      Commander
  Geese**                                                     Commander Island (Russia), then      Island
                                                              migrate through Alaska into Oregon   
                                                              and California.                      

  **Snow Geese**    Yes          Yes            Jun-Sep       Portion of population breeds on      Wrangel Island
                                                              Wrangel Island (Russia), then        
                                                              migrates through Alaska into Pacific 
                                                              Flyway states (mixing with other     
                                                              waterbird species).                  

  **Eurasian        Yes          No                           Regular vagrant along west coast of  W. coast of
  Wigeon**                                                    North America, especially Aleutian   North America,
                                                              Islands.                             especially
                                                                                                   Aleutian Is.

  **Northern        Yes          Yes            May-Sep       Some Siberian breeders winter in W.  Siberia
  Pintail**                                                   US (California). Also birds banded   
                                                              in North America have been recovered 
                                                              over large areas of E. Siberia and   
                                                              Kamchatka.                           

  **Baikal Teal**   Yes          No                           Occasional vagrant to North America  Aleutians &
                                                              chiefly in Aleutians & extreme       extreme Western
                                                              Western Alaska.                      Alaska

  **Common          Yes          No                           Vagrant in W. Alaska (Aleutian and   W. Alaska
  Pochard**                                                   Pribilof Islands).                   (Aleutian and
                                                                                                   Pribilof
                                                                                                   Islands)

  **Steller's       Yes          Yes            Nov-Sep       Most of the population breeds E.     Alaska
  Eiders**                                                    Siberian arctic, these birds molt on Peninsula, and
                                                              Alaska Peninsula (Izembek and Nelson Aleutian
                                                              Lagoons). Birds winter in largest    Islands; E.
                                                              numbers in Commander and Kuril       Siberian
                                                              Islands (Russia) and in smaller      arctic,
                                                              numbers in Northern Japan, along     Commander and
                                                              Alaska Peninsula, and Aleutian       Kuril Islands
                                                              Islands.                             (Russia),
                                                                                                   Northern Japan

                    **Asian                                                                        
                    Contact**                                                                      

  **Species**       **Direct**   **Indirect**   **Timing**    **Species**                          **Point of
                                                                                                   Contact**

  **Common Eiders** Yes          Yes            Year round    Portion of AK and Canada breeders    Chukotka and
                                                              molting and wintering in Chukotka.   Aleutian Is.
                                                              Portion of E. Siberian breeding      
                                                              population winters in Bering Sea     
                                                              (Aleutian Islands) mixing with AK    
                                                              and Canada breeders.                 

  **King Eiders**   Yes          Yes            Year round    Portion of AK and Canada breeding    Kamchatka and
                                                              population molt and winter in        Kuril islands
                                                              Kamchatka and Kuril islands          (Russia) and E.
                                                              (Russia). Portion of E. Siberian     Siberian arctic
                                                              breeding population winters in       
                                                              Bering Sea (Aleutian Islands) mixing 
                                                              with AK and Canada breeders.         

  **Spectacled      No           Yes            Nov-Apr       E. Siberian arctic and Alaska        Bering Sea
  Eiders**                                                    breeders mix during winter in Bering 
                                                              Sea.                                 

  **Long-tailed     **Yes**      **Yes**        **Oct-Apr**   **Yukon Kuskokwim Delta breeders and **Chukotka and
  Ducks**                                                     North Slope molters molt and winter  Kamchatka**
                                                              along Chukotka, Kamchatka, south     
                                                              along the Russian coast, and Kuril   
                                                              and Sakhalin islands.**              

  **Tufted Duck**   Yes          No                           Regular vagrant along west coast of  W. coast of
                                                              North America.                       North America

                                                                                                   

  **Other                                                                                          
  Possibilities**                                                                                  

  **Species**                                                 **Specifics**                        

  **Greater                                                   Circumpolar distribution.            
  White-fronted                                                                                    
  Geese**                                                                                          

  **Green-winged                                              Breed throughout middle latitude     
  Teal**                                                      Northern Hemisphere.                 

  **Mallard**                                                 Holarctic distribution.              

  **Northern                                                  Holarctic distribution.              
  Shoveler**                                                                                       

  **Gadwall**                                                 Breed on Alaskan peninsula,          
                                                              Kamchatka, China, Russia.            

  **Greater Scaup**                                           Holarctic distribution. AK breeders  
                                                              winter on Atlantic coast             

  **Harlequin                                                 Pacific population breeds from E.    
  Ducks**                                                     Siberia through Alaska to W. Canada. 

  **Black Scoters**                                           Pacific population breeds in Siberia 
                                                              and Kamchatka, into western Alaska   
                                                              and sparsely across Canada.          

  **Common                                                    Circumpolar distribution.            
  Goldeneye**                                                                                      

  **Red-breasted                                              Holarctic distribution.              
  Merganser**                                                                                      

  ^1^ For the most                                                                                 
  part, all the                                                                                    
  waterfowl species                                                                                
  mix with other                                                                                   
  waterbirds along                                                                                 
  migration routes                                                                                 
  and at staging                                                                                   
  and wintering                                                                                    
  areas.                                                                                           
  ----------------- ------------ -------------- ------------- ------------------------------------ ---------------

Table 4-3. Shorebird species as potential carriers of the Highly
pathogenic H5N1 avian influenza subtype to North America.

  ------------------- ------------ -------------- ------------ --------------------------------- ----------------
                      **Asian                                                                    
                      Contact**                                                                  

  **Species**         **Direct**   **Indirect**   **Timing**   **Specifics**                     **Point of
                                                                                                 Contact**

  **Pacific           Yes          Yes            Jun-Sep      Pop. nesting in Siberia/Chukotka  Russian Far East
  Golden-Plover**                                              returns to North America; during  and w. Alaska;
                                                               passage through AK mixes with     Oceania, main
                                                               local nesting Pacific             Hawaiian
                                                               Golden-Plovers that winter in     Islands,
                                                               Hawaii and central Oceania.       Northwestern
                                                               Breeding birds from Siberia       Hawaiian
                                                               thought to also migrate overland  Islands,
                                                               to Southeast Asia and Oceania.    Marshall
                                                               Birds wintering in the Marshall   Islands, Guam
                                                               and Mariana Islands migrate       and the Northern
                                                               through Southeast Asia whereas    Marianas
                                                               birds wintering in the Hawaiian   
                                                               Islands thought to migrate        
                                                               through Alaska.                   

  **Black- bellied    Yes          Yes            Jun-Sep      Pop. nesting in Siberia/Chukotka  Russian Far East
  Plover**                                                     returns to North America, mixing  and w. Alaska
                                                               with birds in w. Alaska before    
                                                               both migrate to nonbreeding areas 
                                                               in North and Central America.     

  **Semipalmated      Yes          Yes            Jun-Sep      North American breeding and North Chukotka, w.
  Plover**                                                     and South American nonbreeding    Alaska
                                                               species with small breeding pop.  
                                                               recently established in Chukotka. 
                                                               Chukotka pop. mixes with other    
                                                               species from the East Asian       
                                                               flyway (EAF) before returning to  
                                                               the Americas.                     

  **Bar-tailed        Yes          Yes            Apr-Nov      About 90,000 birds migrate along  Birds spending
  Godwit**                                                     the coast of E. Asia en-route to  nonbreeding
                                                               breeding grounds in n. and w.     season in
                                                               Alaska. Coastal w. Alaska         Australia with
                                                               principal autumn staging area     potential to
                                                               where birds mix with 15-20 spp.   also mix with
                                                               of shorebirds and equal number of other pop. of
                                                               waterfowl spp. that migrate to    godwits (*L. l.
                                                               the Americas.                     menzberi*) that
                                                                                                 are restricted
                                                                                                 to coastal E.
                                                                                                 Asia

  **Marbled Godwit**  No           Yes            Aug-Oct      Mixes with Bar-tailed Godwits at  AK Peninsula
                                                               staging sites on AK Pen. Migrates 
                                                               to Pacific NW.                    

  **Whimbrel**        No           Yes            Jul-Aug      Contact occurs with Bar-tailed    AK Peninsula
                                                               Godwits and plovers               
                                                               (Black-bellied and Pacific        
                                                               Golden) in w. AK & on AK Pen.     
                                                               Estuaries.                        

  **Bristle-thighed   Yes          Yes            May-Sep      Possible direct contact on        Seward Pen.,
  Curlew**                                                     nonbreeding grounds in Oceania;   Andreafsky
                                                               indirect through contact with     Wilderness,
                                                               Bar-tailed Godwits on w. AK       Yukon Delta NW
                                                               breeding and YKD staging grounds  Hawaiian Is. and
                                                                                                 the Marshall Is.

  **Greater           No           Yes            Aug-Sep      Mixes with Bar-tailed Godwits at  W. Alaska
  Yellowlegs**                                                 staging sites on YKD & AK Pen.    

  **Wandering         Yes          Likely         May-Sep      Birds breeding in AK migrate to   Chukotka, W.
  Tattler**                                                    Hawaii and likely elsewhere in    Alaska
                                                               Oceania. Bird in Australia during 
                                                               nonbreeding season may be from    
                                                               Alaska and/or part of breeding    
                                                               range in Chukotka.                

  **Ruddy Turnstone** Possibly     Likely         May-Sep      Not known if birds nesting on     W. Alaska;
                                                               Chukotka come to AK postbreeding. Oceania, NW
                                                               However, birds nesting in AK      Hawaiian
                                                               known to migrate to sites in EAF  Islands,
                                                               & Oceania. Birds from Eastern     Marshall Island,
                                                               Siberia and Western Alaska also   Guam and Mariana
                                                               migrate along the East Asian      Islands
                                                               coast, and a portion winter in    
                                                               the Mariana and Marshall Islands. 

                      **Asian                                                                    
                      Contact**                                                                  

  **Species**         **Direct**   **Indirect**   **Timing**   **Specifics**                     **Point of
                                                                                                 Contact**

  **Black Turnstone** No           Yes            Jul-Sep      On YKD mingles with species       W. Alaska
                                                               (Bar-tailed Godwit, Sharp-tailed  
                                                               Sandpiper, and *C. a. arcticola*  
                                                               Dunlin) that pass along EAF       
                                                               during migration.                 

  **Long-billed       Yes          Yes            Jun-Oct      Birds nesting in Chukotka/Siberia Siberia, W.
  Dowitcher**                                                  return to AK and then migrate to  Alaska
                                                               nonbreeding areas in temp. NA.    
                                                               When in Russia there exists       
                                                               potential to have contact with    
                                                               numerous species that migrate     
                                                               along the EAF.                    

  **Surfbird**        No           Yes            Jul-Sep      At AK Peninsula estuaries mixes   SW Alaska
                                                               with flocks of Bar-tailed Godwits 
                                                               that migrate along the EAF.       

  **Red Knot**        Yes          Yes            May-Oct      Subspecies *C. c. roselaari*      Wrangel Is., W.
                                                               breeds in w. and n. Alaska and on Alaska
                                                               Wrangel l. and spends nonbreeding 
                                                               season along Pacific coast of N.  
                                                               & C. America. Birds staging on    
                                                               YKD in spring possibly mix with   
                                                               *C. c. rogersi* pop. that reaches 
                                                               Alaska via Australia & EAF. If no 
                                                               mixing with *C. c. rogersi*, then 
                                                               birds on Wrangel I. are in direct 
                                                               contact with other spp. of waders 
                                                               and waterfowl from EAF.           

  **Sanderling**      ?            Likely         Sep-Oct      Birds in autumn in W. Alaska      W. Alaska
                                                               likely from Asian nesting areas   
                                                               where direct contact likely.      
                                                               Nonbreeding areas of birds in w.  
                                                               AK in autumn unknown.             

  **Semipalmated      No           Yes            Jun-Aug      Widespread nesting species in n.  N. and W. Alaska
  Sandpiper**                                                  Alaska where in direct contact    
                                                               with Dunlin (*C. a. arcticola*)   
                                                               that migrate through and winter   
                                                               in EAF.                           

  **Western           Possibly     Yes            Jun-Sep      Breeds in Chukotka with birds     N. and W. Alaska
  Sandpiper**                                                  returning to nonbreeding areas in 
                                                               NA; also has contact with *C. a.  
                                                               arcticola* Dunlin in N. AK.       

  **Red-necked        Yes          Yes            Jun-Sep      Old World species that            N. and W. Alaska
  Stint**                                                      occasionally nest in w. Alaska    
                                                               with Western Sandpipers.          

  **Pectoral          Yes          Yes            Jun-Aug      Birds nesting in Siberia/Russian  W. & N Alaska
  Sandpiper**                                                  Far East return to nonbreeding    
                                                               areas in SA via passage through   
                                                               N. America. In Siberia the        
                                                               species is in direct contact with 
                                                               numerous spp. of waders and       
                                                               waterfowl from the EAF.           

  **Sharp-tailed      Yes          Yes            Aug-Oct      Possibly the entire annual cohort Mostly W. Alaska
  Sandpiper**                                                  of juveniles comes to w. AK from  
                                                               Siberian nesting grounds where    
                                                               they have had contact with        
                                                               several pop. of birds that have   
                                                               migrated along the EAF.           

  **Buff-breasted     Yes          Yes            Jun-Aug      Breeds in Canada, AK but pop.     Wrangel Is., W.
  Sandpiper**                                                  also nests on Wrangel Is. and     Alaska
                                                               migrates through AK & N.A. to     
                                                               reach nonbreeding areas in S.A.   

                      **Asian                                                                    
                      contact**                                                                  

  **Species**         **Direct**   **Indirect**   **Timing**   **Specifics**                     **Point of
                                                                                                 Contact**

  **Rock Sandpiper**  Yes          Yes            Jun-Oct      *C. p. tschuktschorum* subspecies W. & SW Alaska
                                                               has portion of breeding range in  
                                                               Chukotka. Birds from there        
                                                               migrate to nonbreeding areas in   
                                                               the Pacific NW via w. AK staging  
                                                               sites where they mix with Dunlin  
                                                               (C*. a. pacifica)* & Rock         
                                                               Sandpipers (C*. p. ptilocnemis*). 

  **Dunlin**          Yes          Yes            May-Oct      *C. a. arcticola* nests in n. AK  N. & W. Alaska
                                                               and migrates to nonbreeding areas 
                                                               in central EAF (Japan, Korea,     
                                                               Taiwan). While in AK it has       
                                                               contact with numerous spp. of     
                                                               waders and waterfowl that migrate 
                                                               to N, C. & S. America.            

  **Buff-breasted     Yes          Yes            Jun-Aug      Breeds in Canada, AK but pop.     Wrangel Is., W.
  Sandpiper**                                                  also nests on Wrangel Is. and     Alaska
                                                               migrates through AK & N.A. to     
                                                               reach nonbreeding areas in S.A.   
  ------------------- ------------ -------------- ------------ --------------------------------- ----------------

To further focus sampling, five criteria were employed to rank these
migratory waterbirds and other migrants that are potential carriers of
highly pathogenic H5N1 avian influenza virus (see Tables 4-4 to 4-6
below). These ranking criteria include 1) proportion of the population
occurring in Asia, 2) contact with a known area of highly pathogenic
H5N1 avian influenza virus, 3) habitats used in Asia, 4) population size
in Alaska, and 5) likelihood of obtaining a representative sample of
sufficient size. Table 4-7 is a summary of primary and secondary species
that should be considered as sampling targets for highly pathogenic H5N1
avian influenza virus in the four major flyways (see also Attachment 5).

Table 4-4. Ranking matrix for populations of waterfowl and cranes to be
sampled for HPAI during the 2006 field season in Alaska.

  --------------- ------------------- ------------- ---------------- --------------- ---------------- -----------
  **Taxon**       **Total or partial  **Contact     **Habitat used   **Pop. in       **Can samples be **Score**
                  contact with Asia** with known    in Asia**        Alaska**        obtained?**      
                                      \"hot                                                           
                                      spot\"**                                                        

                                                                                                      

  **Steller\'s    **4**               **1**         **4**            **3**           **3**            **15**
  Eider**                                                                                             

                  Most (\>90%) of the No known use  Uses estuarine   Winter pop      Relatively easy  
                  Pacific-wintering   of            and freshwater   approx 80,000   to trap during   
                  population          AI-infected   habitats         Breeding        fall molting     
                  (250,000) breeds in areas                          population      period           
                  northeastern Asia                                  \<1,000                          

  **Northern      **2**               **2**         **4**            **4**           **3**            **15**
  Pintail**                                                                                           

                  Unknown number of   Asian summer  Freshwater       Summer          Easy to capture  
                  Siberian-breeding   range         marshes,         population      in Alaska in     
                  birds migrate       overlaps with ephemeral        approximately 1 autumn           
                  through Alaska to   known         wetlands         million                          
                  winter in North     AI-infected                                                     
                  America             areas                                                           

  **Taxon**       **Total or partial  **Contact     **Habitat used   **Pop. in       **Can samples be **Score**
                  contact with Asia** with known    in Asia**        Alaska**        obtained?**      
                                      \"hot                                                           
                                      spot\"**                                                        

  **Lesser Snow   **5**               **1**         **4**            **3**           **2**            **14**
  Goose**                                                                                             

                  The Wrangel Island  No known use  Freshwater       Entire breeding Could be         
                  colony of 110,000   of            marshes,         population of   difficult to     
                  breeding birds is   AI-infected   ephemeral        110,000         obtain target    
                  managed as a        areas         wetlands         breeding birds  number depending 
                  discreet population                                plus young of   on timing and    
                                                                     the year        route of         
                                                                     migrate through migration.       
                                                                     Alaska en-route                  
                                                                     to the west                      
                                                                     coast states                     

  **Emperor       **2**               **1**         **4**            **3**           **3**            **13**
  Goose**                                                                                             

                  Approximately       No known use  Breeds moist     approximately   Relatively easy  
                  20,000 birds molt   of            tundra meadows   90% of the      to trap during   
                  in Chukotka,        AI-infected   and near         population      summer and fall  
                  several thousand    areas         wetlands         winters in      molting period   
                  breed in the Anadyr                                Alaska and                       
                  lowlands                                           approximately                    
                                                                     60% summers in                   
                                                                     Alaska                           

  **Black Brant** **1**               **1**         **4**            **3**           **3**            **12**

                  Several thousand    No known use  Breeds in moist  Near entire     Samples could be 
                  birds nest in the   of            sedge coastal    Pacific         obtained easily  
                  Anadyr lowlands and AI-infected   tundra areas     population of   from fall birds  
                  on Wrangel Island   areas                          130,000 birds                    
                                                                     stage at                         
                                                                     Izembek Lag                      
                                                                     prior to fall                    
                                                                     migration to                     
                                                                     winter from                      
                                                                     B.C. to Mexico                   

  **Spectacled    **4**               **1**         **4**            **2**           **1**            **12**
  Eider**                                                                                             

                  Over 90% of the     No known use  Breeds moist     Approximately   Could be         
                  world population    of            tundra meadows   9,000 birds     difficult to     
                  (approx 300,000)    AI-infected   and near         breed on the    obtain target    
                  nests in Arctic     areas         wetlands         Arctic Slope,   number           
                  Russia                                             and 8,000 on                     
                                                                     the Yukon-                       
                                                                     Kuskokwim Delta                  

  **Aleutian      **1**               **1**         **4**            **3**           **2**            **11**
  Cackling                                                                                            
  Goose**                                                                                             

                  Small numbers breed No known use  Breeds on        Approximately   Could be         
                  on Commander        of            Aleutian Islands 70,000 birds in difficult to     
                  Islands and winter  AI-infected   in wet, grassy   fall population obtain target    
                  in Asia             areas         freshwater                       number           
                                                    meadows                                           

  **Long-tailed   **2**               **1**         **2**            **3**           **3**            **11**
  Duck**                                                                                              

                  Approx 250,000      No known use  Nests coastal    Approx 80,000   Samples could be 
                  breed in            of            tundra;          summer in       obtained easily  
                  northeastern        AI-infected   postbreeding use western Alaska, from fall birds  
                  Russia, unknown     areas         estuarine areas  600,000 in                       
                  numbers cross to                                   northern Alaska                  
                  North America                                      and western                      
                                                                     Canada                           

  **Taxon**       **Total or partial  **Contact     **Habitat used   **Pop. in       **Can samples be **Score**
                  contact with Asia** with known    in Asia**        Alaska**        obtained?**      
                                      \"hot                                                           
                                      spot\"**                                                        

  **Tundra Swan** **1**               **1**         **4**            **3**           **2**            **11**

                  Unknown numbers     No known use  Nests coastal    Approximately   Could be         
                  breed in eastern    of            tundra;          150,000 summer  difficult to     
                  Chukotka; may be    AI-infected   migration and    in Alaska       obtain target    
                  associated with     areas         non-breeding in                  number           
                  Pacific Flyway                    coastal habitats                                  

  **Common        **2**               **1**         **2**            **3**           **2**            **10**
  Eider**                                                                                             

                  Approx 30,000 breed No known use  Breeds in wet or Alaska          Could be         
                  in northeastern     of            moist tundra     population      difficult to     
                  Russia              AI-infected   meadows near     believed to be  obtain target    
                                      areas         wetlands or on   25,000 western  number in most   
                                                    barrier islands  Alaska plus     locations        
                                                                     120,000 in                       
                                                                     northern Alaska                  
                                                                     plus western                     
                                                                     Canada                           

  **King Eider**  **2**               **1**         **2**            **3**           **2**            **10**

                  Approx 150,000      No known use  Breeds in moist  Approx 360,000  Could be         
                  breed in            of            and upland       breed in        difficult to     
                  northeastern Russia AI-infected   tundra           northern Alaska obtain target    
                                      areas                          and western     number in most   
                                                                     Canada          locations        

  **Lesser        **2**               **1**         **3.5**          **3**           **2**            **11.5**
  Sandhill                                                                                            
  Crane**                                                                                             

                  unknown numbers of  No known use  Breeds in wet or Alaska          Could be         
                  mid-continent       of            moist tundra     population      difficult to     
                  population breed in AI-infected   meadows near     believed to be  obtain target    
                  Siberia             areas         wetlands or on   in the low tens number           
                                                    barrier islands, of thousands                     
                                                    often feeds in                                    
                                                    agricultural                                      
                                                    areas where                                       
                                                    available                                         

  Ranking                                                                                             
  criteria:                                                                                           

                  1\. Proportion of   2\. Contact   3\. Habitats     4\. Population  5\. Can we       
                  the population      with a known  used in context  size in Alaska  obtain a         
                  occurring in Asia.  \'hotspot\'   of likelihood of during 2006.    representative   
                  Score as 1-5 where  or source.    exposure         Score to the    sample of        
                  5=100%              Score as 1=no 1=Offshore       closest number  sufficient size  
                                      contact,      marine,          1=1,000,        (n=200)? Score   
                                      2=contact     2=Estuary,       2=10,000,       1=no, 2=maybe,   
                                                    3=Terrestrial,   3=100,000,      3=yes.           
                                                    4=Freshwater.    4=1,000,000                      
  --------------- ------------------- ------------- ---------------- --------------- ---------------- -----------

Table 4-5. Ranking matrix for populations of shorebirds to be sampled
for avian influenza during the 2006 field season in Alaska.

+-----------+-------------+--------+--------+---+---------+---------+---+
| **Taxon** | **Total or  | **C    | **H    | * |         | **Can   | * |
|           | partial     | ontact | abitat | * |         | samples | * |
|           | contact     | with   | used   | P |         | be      | S |
|           | with Asia** | known  | in     | o |         | obt     | c |
|           |             | \"hot  | Asia** | p |         | ained** | o |
|           |             | sp     |        | . |         |         | r |
|           |             | ot\"** |        | i |         |         | e |
|           |             |        |        | n |         |         | * |
|           |             |        |        | A |         |         | * |
|           |             |        |        | l |         |         |   |
|           |             |        |        | a |         |         |   |
|           |             |        |        | s |         |         |   |
|           |             |        |        | k |         |         |   |
|           |             |        |        | a |         |         |   |
|           |             |        |        | * |         |         |   |
|           |             |        |        | * |         |         |   |
+-----------+-------------+--------+--------+---+---------+---------+---+
|           |             |        |        |   |         |         |   |
+-----------+-------------+--------+--------+---+---------+---------+---+
| *         | **5**       | **2**  | **3**  | * |         | **3**   | * |
| *Dunlin** |             |        |        | * |         |         | * |
|           |             |        |        | 4 |         |         | 1 |
| **(*C. a. |             |        |        | * |         |         | 7 |
| arct      |             |        |        | * |         |         | * |
| icola*)** |             |        |        |   |         |         | * |
+-----------+-------------+--------+--------+---+---------+---------+---+
|           | Entire pop. | W      | Est    |   | Est. at | Rela    |   |
|           | winters     | inters | uarine |   | 650,000 | tively. |   |
|           | from Taiwan | thro   | and    |   |         | easy to |   |
|           | north to    | ughout | fres   |   |         | trap on |   |
|           | Yellow Sea  | areas  | hwater |   |         | nest    |   |
|           | and n.      | where  | hab    |   |         | and     |   |
|           | Japan       | H5N1   | itats; |   |         | during  |   |
|           |             | iden   | also   |   |         | post-b  |   |
|           |             | tified | eph    |   |         | reeding |   |
|           |             |        | emeral |   |         | when in |   |
|           |             |        | inland |   |         | flocks  |   |
|           |             |        | lakes  |   |         |         |   |
|           |             |        | where  |   |         |         |   |
|           |             |        | do     |   |         |         |   |
|           |             |        | mestic |   |         |         |   |
|           |             |        | wat    |   |         |         |   |
|           |             |        | erfowl |   |         |         |   |
|           |             |        | raised |   |         |         |   |
+-----------+-------------+--------+--------+---+---------+---------+---+
| **Sha     | **5**       | **1**  | *      |   | **2**   | **3**   | * |
| rp-tailed |             |        | *3.5** |   |         |         | * |
| Sa        |             |        |        |   |         |         | 1 |
| ndpiper** |             |        |        |   |         |         | 4 |
|           |             |        |        |   |         |         | . |
|           |             |        |        |   |         |         | 5 |
|           |             |        |        |   |         |         | * |
|           |             |        |        |   |         |         | * |
+-----------+-------------+--------+--------+---+---------+---------+---+
|           | Breeding    | Mig    | Fres   |   | Between | Easy to |   |
|           | restricted  | rating | hwater |   | 10,000  | capture |   |
|           | to n.       | adults | ma     |   | and     | in      |   |
|           | central     | pass   | rshes, |   | 40,000  | Alaska  |   |
|           | Siberia     | t      | br     |   | de      | in      |   |
|           | with annual | hrough | ackish |   | pending | autumn  |   |
|           | cohort of   | known  | wet    |   | on      |         |   |
|           | immatures   | \"hot  | lands, |   | annual  |         |   |
|           | coming to   | s      | salt   |   | pro     |         |   |
|           | Alaska;     | pots\" | ponds, |   | duction |         |   |
|           | adults move | in     | sewage |   |         |         |   |
|           | through EAA | c      | farms, |   |         |         |   |
|           | flyway      | entral | eph    |   |         |         |   |
|           |             | E.     | emeral |   |         |         |   |
|           |             | Asia.  | we     |   |         |         |   |
|           |             | S      | tlands |   |         |         |   |
|           |             | pecies |        |   |         |         |   |
|           |             | of     |        |   |         |         |   |
|           |             | c      |        |   |         |         |   |
|           |             | oncern |        |   |         |         |   |
|           |             | if     |        |   |         |         |   |
|           |             | adults |        |   |         |         |   |
|           |             | can    |        |   |         |         |   |
|           |             | pass   |        |   |         |         |   |
|           |             | virus  |        |   |         |         |   |
|           |             | to     |        |   |         |         |   |
|           |             | off    |        |   |         |         |   |
|           |             | spring |        |   |         |         |   |
|           |             | on     |        |   |         |         |   |
|           |             | br     |        |   |         |         |   |
|           |             | eeding |        |   |         |         |   |
|           |             | g      |        |   |         |         |   |
|           |             | rounds |        |   |         |         |   |
+-----------+-------------+--------+--------+---+---------+---------+---+
| **B       | **5**       | **2**  | **2**  |   | **3**   | **2**   | * |
| ar-tailed |             |        |        |   |         |         | * |
| Godwit**  |             |        |        |   |         |         | 1 |
|           |             |        |        |   |         |         | 4 |
| **(*L. l. |             |        |        |   |         |         | * |
| b         |             |        |        |   |         |         | * |
| aueri*)** |             |        |        |   |         |         |   |
+-----------+-------------+--------+--------+---+---------+---------+---+
|           | Entire pop. | On     | Est    |   | Est. at | Could   |   |
|           | nests w.    | mig    | uarine |   | 1       | be      |   |
|           | and n.      | ration |        |   | 20,000, | di      |   |
|           | Alaska &    | stops  |        |   | but     | fficult |   |
|           | stages      | in     |        |   | 2005    | to      |   |
|           | central E.  | c      |        |   | census  | obtain  |   |
|           | Asia        | entral |        |   | efforts | target  |   |
|           | (Yellow     | E.     |        |   | ac      | number  |   |
|           | Sea, Korea, | Asia   |        |   | counted |         |   |
|           | Japan) in   | (      |        |   | for     |         |   |
|           | spring;     | Yellow |        |   | \       |         |   |
|           | southward   | Sea,   |        |   | <50,000 |         |   |
|           | migration   | Japan, |        |   |         |         |   |
|           | direct      | Korea) |        |   |         |         |   |
|           | across      |        |        |   |         |         |   |
|           | Pacific     |        |        |   |         |         |   |
+-----------+-------------+--------+--------+---+---------+---------+---+
| **Ruddy   | **3**       | **2**  | *      |   | **2.5** | **3**   | * |
| Tu        |             |        | *2.5** |   |         |         | * |
| rnstone** |             |        |        |   |         |         | 1 |
|           |             |        |        |   |         |         | 3 |
| **(*A. i. |             |        |        |   |         |         | * |
| inte      |             |        |        |   |         |         | * |
| rpres*)** |             |        |        |   |         |         |   |
+-----------+-------------+--------+--------+---+---------+---------+---+
|           | Portion of  | On     | Breeds |   | \>35%   | Unless  |   |
|           | W. Alaska   | mig    | upland |   | of      | post-b  |   |
|           | nesting     | ration | t      |   | North   | reeding |   |
|           | pop.        | stops  | undra; |   | A       | concent |   |
|           | migrates to | in     | mig    |   | merican | rations |   |
|           | SE and E    | c      | ration |   | pop. (= | found   |   |
|           | Asia; pop.  | entral | and    |   | \       | (e.g.,  |   |
|           | nesting     | E.     | non-br |   | ~20,000 | P       |   |
|           | Chukotka    | Asia   | eeding |   | birds)  | ribilof |   |
|           | moves to W. | (      | c      |   | in      | Is.)    |   |
|           | Alaska in   | Yellow | oastal |   | Alaska, | could   |   |
|           | fall before | Sea,   | (rocky |   | plus    | be      |   |
|           | returning   | Japan, | inter  |   | histo   | di      |   |
|           | to E and SE | Korea) | tidal, |   | rically | fficult |   |
|           | Asia.       |        | sand   |   | large   | to meet |   |
|           | Eastern     |        | be     |   | numbers | target  |   |
|           | Siberia and |        | aches, |   | visit   | sample. |   |
|           | West Alaska |        | &      |   | (\      | 200     |   |
|           | breeding    |        | mud    |   | >20,000 | tur     |   |
|           | birds also  |        | flats) |   | on      | nstones |   |
|           | migrate     |        |        |   | P       | can be  |   |
|           | down the    |        |        |   | ribilof | c       |   |
|           | East Asian  |        |        |   | Is.)    | aptured |   |
|           | coast, with |        |        |   | from    | in      |   |
|           | some birds  |        |        |   | C       | either  |   |
|           | wintering   |        |        |   | hukotka | the     |   |
|           | in the      |        |        |   |         | M       |   |
|           | Mariana and |        |        |   |         | arshall |   |
|           | Marshall    |        |        |   |         | or      |   |
|           | Islands     |        |        |   |         | Mariana |   |
|           |             |        |        |   |         | Islands |   |
|           |             |        |        |   |         | or both |   |
+-----------+-------------+--------+--------+---+---------+---------+---+
| **Taxon** | **Total or  | **C    | **H    | * |         | **Can   | * |
|           | partial     | ontact | abitat | * |         | samples | * |
|           | contact     | with   | used   | P |         | be      | S |
|           | with Asia** | known  | in     | o |         | obta    | c |
|           |             | \"hot  | Asia** | p |         | ined?** | o |
|           |             | sp     |        | . |         |         | r |
|           |             | ot\"** |        | i |         |         | e |
|           |             |        |        | n |         |         | * |
|           |             |        |        | A |         |         | * |
|           |             |        |        | l |         |         |   |
|           |             |        |        | a |         |         |   |
|           |             |        |        | s |         |         |   |
|           |             |        |        | k |         |         |   |
|           |             |        |        | a |         |         |   |
|           |             |        |        | * |         |         |   |
|           |             |        |        | * |         |         |   |
+-----------+-------------+--------+--------+---+---------+---------+---+
| *         | **3**       | **1**  | **4**  | * |         | **2**   | * |
| *Pectoral |             |        |        | * |         |         | * |
| Sa        |             |        |        | 3 |         |         | 1 |
| ndpiper** |             |        |        | * |         |         | 3 |
|           |             |        |        | * |         |         | * |
|           |             |        |        |   |         |         | * |
+-----------+-------------+--------+--------+---+---------+---------+---+
|           | Greater     | To     | Breeds | 2 |         | Could   |   |
|           | than 50% of | date   | m      | 0 |         | be      |   |
|           | pop. nests  | no     | arshy/ | 0 |         | di      |   |
|           | in Russia   | known  | grassy | , |         | fficult |   |
|           | west to     | use of | t      | 0 |         | to      |   |
|           | Eastern     | \"hot  | undra; | 0 |         | obtain  |   |
|           | Taimyr      | s      | p      | 0 |         | target  |   |
|           | Peninsular. | pots\" | ost-br | - |         | number  |   |
|           |             |        | eeding | 3 |         |         |   |
|           |             |        | uses   | 0 |         |         |   |
|           |             |        | br     | 0 |         |         |   |
|           |             |        | ackish | , |         |         |   |
|           |             |        | ponds  | 0 |         |         |   |
|           |             |        | fres   | 0 |         |         |   |
|           |             |        | hwater | 0 |         |         |   |
|           |             |        | m      |   |         |         |   |
|           |             |        | arshes |   |         |         |   |
+-----------+-------------+--------+--------+---+---------+---------+---+
| **Red     | **4**       | **2**  | **2**  | * |         | **2**   | * |
| Knot**    |             |        |        | * |         |         | * |
|           |             |        |        | 2 |         |         | 1 |
| **(*C. c. |             |        |        | . |         |         | 2 |
| rogersi & |             |        |        | 5 |         |         | . |
| rose      |             |        |        | * |         |         | 5 |
| laari*)** |             |        |        | * |         |         | * |
|           |             |        |        |   |         |         | * |
+-----------+-------------+--------+--------+---+---------+---------+---+
|           | *C.c.       | On     | Est    | * |         | Could   |   |
|           | roselaari*  | mig    | uarine | C |         | be      |   |
|           | pop. nests  | ration |        | . |         | di      |   |
|           | Wrangel I.  | *C. c. |        | c |         | fficult |   |
|           | and w.      | ro     |        | . |         | to      |   |
|           | Alaska and  | gersi* |        | r |         | obtain  |   |
|           | winters     | passes |        | o |         | target  |   |
|           | Pacific     | t      |        | s |         | number  |   |
|           | coast of    | hrough |        | e |         |         |   |
|           | the         | areas  |        | l |         |         |   |
|           | Americas.   | where  |        | a |         |         |   |
|           | *C. c.      | H5N1   |        | a |         |         |   |
|           | rogersi*    | iden   |        | r |         |         |   |
|           | nests       | tified |        | i |         |         |   |
|           | C           |        |        | * |         |         |   |
|           | hukotka/New |        |        | \ |         |         |   |
|           | Siberian    |        |        | < |         |         |   |
|           | Isl. &      |        |        | 5 |         |         |   |
|           | winters     |        |        | 0 |         |         |   |
|           | Aust./New   |        |        | , |         |         |   |
|           | Zealand,    |        |        | 0 |         |         |   |
|           | passing     |        |        | 0 |         |         |   |
|           | through c.  |        |        | 0 |         |         |   |
|           | E. Asia     |        |        | ; |         |         |   |
|           |             |        |        | C |         |         |   |
|           |             |        |        | . |         |         |   |
|           |             |        |        | c |         |         |   |
|           |             |        |        | . |         |         |   |
|           |             |        |        | * |         |         |   |
|           |             |        |        | r |         |         |   |
|           |             |        |        | o |         |         |   |
|           |             |        |        | g |         |         |   |
|           |             |        |        | e |         |         |   |
|           |             |        |        | r |         |         |   |
|           |             |        |        | s |         |         |   |
|           |             |        |        | i |         |         |   |
|           |             |        |        | * |         |         |   |
|           |             |        |        | 2 |         |         |   |
|           |             |        |        | 2 |         |         |   |
|           |             |        |        | 0 |         |         |   |
|           |             |        |        | , |         |         |   |
|           |             |        |        | 0 |         |         |   |
|           |             |        |        | 0 |         |         |   |
|           |             |        |        | 0 |         |         |   |
|           |             |        |        | . |         |         |   |
|           |             |        |        | * |         |         |   |
|           |             |        |        | C |         |         |   |
|           |             |        |        | . |         |         |   |
|           |             |        |        | c |         |         |   |
|           |             |        |        | . |         |         |   |
|           |             |        |        | r |         |         |   |
|           |             |        |        | o |         |         |   |
|           |             |        |        | g |         |         |   |
|           |             |        |        | e |         |         |   |
|           |             |        |        | r |         |         |   |
|           |             |        |        | s |         |         |   |
|           |             |        |        | i |         |         |   |
|           |             |        |        | * |         |         |   |
|           |             |        |        | t |         |         |   |
|           |             |        |        | h |         |         |   |
|           |             |        |        | o |         |         |   |
|           |             |        |        | u |         |         |   |
|           |             |        |        | g |         |         |   |
|           |             |        |        | h |         |         |   |
|           |             |        |        | t |         |         |   |
|           |             |        |        | t |         |         |   |
|           |             |        |        | o |         |         |   |
|           |             |        |        | s |         |         |   |
|           |             |        |        | t |         |         |   |
|           |             |        |        | o |         |         |   |
|           |             |        |        | p |         |         |   |
|           |             |        |        | i |         |         |   |
|           |             |        |        | n |         |         |   |
|           |             |        |        | A |         |         |   |
|           |             |        |        | l |         |         |   |
|           |             |        |        | a |         |         |   |
|           |             |        |        | s |         |         |   |
|           |             |        |        | k |         |         |   |
|           |             |        |        | a |         |         |   |
|           |             |        |        | i |         |         |   |
|           |             |        |        | n |         |         |   |
|           |             |        |        | s |         |         |   |
|           |             |        |        | p |         |         |   |
|           |             |        |        | r |         |         |   |
|           |             |        |        | i |         |         |   |
|           |             |        |        | n |         |         |   |
|           |             |        |        | g |         |         |   |
|           |             |        |        | b |         |         |   |
|           |             |        |        | u |         |         |   |
|           |             |        |        | t |         |         |   |
|           |             |        |        | n |         |         |   |
|           |             |        |        | u |         |         |   |
|           |             |        |        | m |         |         |   |
|           |             |        |        | b |         |         |   |
|           |             |        |        | e |         |         |   |
|           |             |        |        | r |         |         |   |
|           |             |        |        | s |         |         |   |
|           |             |        |        | u |         |         |   |
|           |             |        |        | n |         |         |   |
|           |             |        |        | k |         |         |   |
|           |             |        |        | n |         |         |   |
|           |             |        |        | o |         |         |   |
|           |             |        |        | w |         |         |   |
|           |             |        |        | n |         |         |   |
|           |             |        |        | ( |         |         |   |
|           |             |        |        | p |         |         |   |
|           |             |        |        | o |         |         |   |
|           |             |        |        | s |         |         |   |
|           |             |        |        | s |         |         |   |
|           |             |        |        | i |         |         |   |
|           |             |        |        | b |         |         |   |
|           |             |        |        | l |         |         |   |
|           |             |        |        | y |         |         |   |
|           |             |        |        | s |         |         |   |
|           |             |        |        | e |         |         |   |
|           |             |        |        | v |         |         |   |
|           |             |        |        | e |         |         |   |
|           |             |        |        | r |         |         |   |
|           |             |        |        | a |         |         |   |
|           |             |        |        | l |         |         |   |
|           |             |        |        | 1 |         |         |   |
|           |             |        |        | 0 |         |         |   |
|           |             |        |        | , |         |         |   |
|           |             |        |        | 0 |         |         |   |
|           |             |        |        | 0 |         |         |   |
|           |             |        |        | 0 |         |         |   |
|           |             |        |        | s |         |         |   |
|           |             |        |        | ) |         |         |   |
+-----------+-------------+--------+--------+---+---------+---------+---+
| **Lo      | **3**       | **1**  | **3**  | * |         | **2**   | * |
| ng-billed |             |        |        | * |         |         | * |
| Do        |             |        |        | 3 |         |         | 1 |
| witcher** |             |        |        | * |         |         | 2 |
|           |             |        |        | * |         |         | * |
|           |             |        |        |   |         |         | * |
+-----------+-------------+--------+--------+---+---------+---------+---+
|           | \>30% of    | To     | Breeds | N |         | Could   |   |
|           | pop. breeds | date   | c      | o |         | be      |   |
|           | in Russia   | no     | oastal | r |         | di      |   |
|           | where range | known  | lo     | t |         | fficult |   |
|           | expanding   | use of | wlands | h |         | to      |   |
|           | w. to       | \"hot  | in     | A |         | obtain  |   |
|           | Taimyr      | s      | wet,   | m |         | target  |   |
|           | Pen.; \>95% | pots\" | grassy | e |         | number  |   |
|           | of entire   |        | fres   | r |         |         |   |
|           | pop.        |        | hwater | i |         |         |   |
|           | winters in  |        | me     | c |         |         |   |
|           | North and   |        | adows; | a |         |         |   |
|           | Central     |        | uses   | n |         |         |   |
|           | America.    |        | est    | p |         |         |   |
|           | Unknown     |        | uarine | o |         |         |   |
|           | numbers     |        | and    | p |         |         |   |
|           | winter in   |        | m      | . |         |         |   |
|           | Asia        |        | anaged | = |         |         |   |
|           | (Japan)     |        | we     | 4 |         |         |   |
|           |             |        | tlands | 5 |         |         |   |
|           |             |        | during | 0 |         |         |   |
|           |             |        | mig    | , |         |         |   |
|           |             |        | ration | 0 |         |         |   |
|           |             |        | &      | 0 |         |         |   |
|           |             |        | winter | 0 |         |         |   |
|           |             |        |        | ( |         |         |   |
|           |             |        |        | \ |         |         |   |
|           |             |        |        | > |         |         |   |
|           |             |        |        | 9 |         |         |   |
|           |             |        |        | 0 |         |         |   |
|           |             |        |        | % |         |         |   |
|           |             |        |        | o |         |         |   |
|           |             |        |        | f |         |         |   |
|           |             |        |        | t |         |         |   |
|           |             |        |        | h |         |         |   |
|           |             |        |        | i |         |         |   |
|           |             |        |        | s |         |         |   |
|           |             |        |        | i |         |         |   |
|           |             |        |        | n |         |         |   |
|           |             |        |        | A |         |         |   |
|           |             |        |        | l |         |         |   |
|           |             |        |        | a |         |         |   |
|           |             |        |        | s |         |         |   |
|           |             |        |        | k |         |         |   |
|           |             |        |        | a |         |         |   |
|           |             |        |        | d |         |         |   |
|           |             |        |        | u |         |         |   |
|           |             |        |        | r |         |         |   |
|           |             |        |        | i |         |         |   |
|           |             |        |        | n |         |         |   |
|           |             |        |        | g |         |         |   |
|           |             |        |        | m |         |         |   |
|           |             |        |        | i |         |         |   |
|           |             |        |        | g |         |         |   |
|           |             |        |        | r |         |         |   |
|           |             |        |        | a |         |         |   |
|           |             |        |        | t |         |         |   |
|           |             |        |        | i |         |         |   |
|           |             |        |        | o |         |         |   |
|           |             |        |        | n |         |         |   |
|           |             |        |        | ) |         |         |   |
+-----------+-------------+--------+--------+---+---------+---------+---+
| **Rock    | **3**       | **1**  | *      | * |         | **3**   | * |
| Sandpiper |             |        | *2.5** | * |         |         | * |
| (*C. p.   |             |        |        | 2 |         |         | 1 |
| tschuktsc |             |        |        | * |         |         | 1 |
| horum*)** |             |        |        | * |         |         | . |
|           |             |        |        |   |         |         | 5 |
|           |             |        |        |   |         |         | * |
|           |             |        |        |   |         |         | * |
+-----------+-------------+--------+--------+---+---------+---------+---+
|           | \~ 20-30%   | To     | Nests  | T |         | Easy to |   |
|           | of pop.     | date   | upland | o |         | trap on |   |
|           | nests in    | no     | t      | t |         | nest    |   |
|           | Chukotka    | known  | undra; | a |         | and     |   |
|           |             | use of | p      | l |         | during  |   |
|           |             | \"hot  | ost-br | p |         | post-b  |   |
|           |             | s      | eeding | o |         | reeding |   |
|           |             | pots\" | use    | p |         | f       |   |
|           |             |        | est    | . |         | locking |   |
|           |             |        | uarine | 5 |         |         |   |
|           |             |        | areas  | 0 |         |         |   |
|           |             |        |        | , |         |         |   |
|           |             |        |        | 0 |         |         |   |
|           |             |        |        | 0 |         |         |   |
|           |             |        |        | 0 |         |         |   |
|           |             |        |        | . |         |         |   |
|           |             |        |        | \ |         |         |   |
|           |             |        |        | ~ |         |         |   |
|           |             |        |        | 2 |         |         |   |
|           |             |        |        | 0 |         |         |   |
|           |             |        |        | K |         |         |   |
|           |             |        |        | n |         |         |   |
|           |             |        |        | e |         |         |   |
|           |             |        |        | s |         |         |   |
|           |             |        |        | t |         |         |   |
|           |             |        |        | C |         |         |   |
|           |             |        |        | h |         |         |   |
|           |             |        |        | u |         |         |   |
|           |             |        |        | k |         |         |   |
|           |             |        |        | o |         |         |   |
|           |             |        |        | t |         |         |   |
|           |             |        |        | k |         |         |   |
|           |             |        |        | a |         |         |   |
|           |             |        |        | b |         |         |   |
|           |             |        |        | u |         |         |   |
|           |             |        |        | t |         |         |   |
|           |             |        |        | a |         |         |   |
|           |             |        |        | l |         |         |   |
|           |             |        |        | l |         |         |   |
|           |             |        |        | r |         |         |   |
|           |             |        |        | e |         |         |   |
|           |             |        |        | t |         |         |   |
|           |             |        |        | u |         |         |   |
|           |             |        |        | r |         |         |   |
|           |             |        |        | n |         |         |   |
|           |             |        |        | t |         |         |   |
|           |             |        |        | o |         |         |   |
|           |             |        |        | A |         |         |   |
|           |             |        |        | K |         |         |   |
|           |             |        |        | e |         |         |   |
|           |             |        |        | n |         |         |   |
|           |             |        |        | r |         |         |   |
|           |             |        |        | o |         |         |   |
|           |             |        |        | u |         |         |   |
|           |             |        |        | t |         |         |   |
|           |             |        |        | e |         |         |   |
|           |             |        |        | t |         |         |   |
|           |             |        |        | o |         |         |   |
|           |             |        |        | n |         |         |   |
|           |             |        |        | o |         |         |   |
|           |             |        |        | n |         |         |   |
|           |             |        |        | - |         |         |   |
|           |             |        |        | b |         |         |   |
|           |             |        |        | r |         |         |   |
|           |             |        |        | e |         |         |   |
|           |             |        |        | e |         |         |   |
|           |             |        |        | d |         |         |   |
|           |             |        |        | i |         |         |   |
|           |             |        |        | n |         |         |   |
|           |             |        |        | g |         |         |   |
|           |             |        |        | a |         |         |   |
|           |             |        |        | r |         |         |   |
|           |             |        |        | e |         |         |   |
|           |             |        |        | a |         |         |   |
|           |             |        |        | s |         |         |   |
|           |             |        |        | i |         |         |   |
|           |             |        |        | n |         |         |   |
|           |             |        |        | P |         |         |   |
|           |             |        |        | a |         |         |   |
|           |             |        |        | c |         |         |   |
|           |             |        |        | i |         |         |   |
|           |             |        |        | f |         |         |   |
|           |             |        |        | i |         |         |   |
|           |             |        |        | c |         |         |   |
|           |             |        |        | N |         |         |   |
|           |             |        |        | W |         |         |   |
+-----------+-------------+--------+--------+---+---------+---------+---+
| **Taxon** | **Total or  | **C    | **H    | * |         | **Can   | * |
|           | partial     | ontact | abitat | * |         | samples | * |
|           | contact     | with   | used   | P |         | be      | S |
|           | with Asia** | known  | in     | o |         | obta    | c |
|           |             | \"hot  | Asia** | p |         | ined?** | o |
|           |             | sp     |        | . |         |         | r |
|           |             | ot\"** |        | i |         |         | e |
|           |             |        |        | n |         |         | * |
|           |             |        |        | A |         |         | * |
|           |             |        |        | l |         |         |   |
|           |             |        |        | a |         |         |   |
|           |             |        |        | s |         |         |   |
|           |             |        |        | k |         |         |   |
|           |             |        |        | a |         |         |   |
|           |             |        |        | * |         |         |   |
|           |             |        |        | * |         |         |   |
+-----------+-------------+--------+--------+---+---------+---------+---+
| **Pacific | **3**       | **2**  | *      | * |         | **2**   | * |
| Golden    |             |        | *2.5** | * |         |         | * |
| -Plover** |             |        |        | 2 |         |         | 1 |
|           |             |        |        | * |         |         | 1 |
|           |             |        |        | * |         |         | . |
|           |             |        |        |   |         |         | 5 |
|           |             |        |        |   |         |         | * |
|           |             |        |        |   |         |         | * |
+-----------+-------------+--------+--------+---+---------+---------+---+
|           | > Nesting   | Likely | Nests  | 1 |         | Could   |   |
|           | > occurs w. | in c.  | upland | 6 |         | be      |   |
|           | > & sw      | East   | t      | , |         | di      |   |
|           | > Alaska    | Asia   | undra; | 0 |         | fficult |   |
|           | > and over  |        | mig    | 0 |         | to      |   |
|           | > large     |        | ration | 0 |         | obtain  |   |
|           | > portion   |        | and    |   |         | target  |   |
|           | > of n.     |        | nonbr  |   |         | number  |   |
|           | > Siberia   |        | eeding |   |         | in      |   |
|           | > and       |        | in     |   |         | Alaska; |   |
|           | > Chukotka. |        | c      |   |         | h       |   |
|           | >           |        | oastal |   |         | owever, |   |
|           | Interchange |        | ha     |   |         | 200     |   |
|           | > known     |        | bitats |   |         | plovers |   |
|           | > between   |        |        |   |         | can be  |   |
|           | > Asia and  |        |        |   |         | c       |   |
|           | > Alaska    |        |        |   |         | aptured |   |
|           | > but not   |        |        |   |         | in      |   |
|           | >           |        |        |   |         | either  |   |
|           | quantified. |        |        |   |         | the     |   |
|           | > Ala       |        |        |   |         | M       |   |
|           | ska-nesting |        |        |   |         | arshall |   |
|           | > birds     |        |        |   |         | or      |   |
|           | > disperse  |        |        |   |         | Mariana |   |
|           | > to        |        |        |   |         | Islands |   |
|           | > Oceania   |        |        |   |         | or both |   |
|           | > and       |        |        |   |         |         |   |
|           | > Pacific   |        |        |   |         |         |   |
|           | > coast of  |        |        |   |         |         |   |
|           | > N & C     |        |        |   |         |         |   |
|           | > America.  |        |        |   |         |         |   |
|           | > Birds     |        |        |   |         |         |   |
|           | > wintering |        |        |   |         |         |   |
|           | > in the    |        |        |   |         |         |   |
|           | > Marshall  |        |        |   |         |         |   |
|           | > or        |        |        |   |         |         |   |
|           | > Mariana   |        |        |   |         |         |   |
|           | > Islands   |        |        |   |         |         |   |
|           | > are       |        |        |   |         |         |   |
|           | > believed  |        |        |   |         |         |   |
|           | > to be     |        |        |   |         |         |   |
|           | > birds     |        |        |   |         |         |   |
|           | > that have |        |        |   |         |         |   |
|           | > migrated  |        |        |   |         |         |   |
|           | > overland  |        |        |   |         |         |   |
|           | > from      |        |        |   |         |         |   |
|           | > Siberia   |        |        |   |         |         |   |
|           | > to        |        |        |   |         |         |   |
|           | > Southeast |        |        |   |         |         |   |
|           | > Asia and  |        |        |   |         |         |   |
|           | > Oceania.  |        |        |   |         |         |   |
+-----------+-------------+--------+--------+---+---------+---------+---+
| **Buff    | **2**       | **1**  | **3**  | * |         | **2**   | * |
| -breasted |             |        |        | * |         |         | * |
| Sa        |             |        |        | 2 |         |         | 1 |
| ndpiper** |             |        |        | * |         |         | 0 |
|           |             |        |        | * |         |         | * |
|           |             |        |        |   |         |         | * |
+-----------+-------------+--------+--------+---+---------+---------+---+
|           | Small       | To     | Va     | 3 |         | Could   |   |
|           | portion of  | date   | riable | , |         | be      |   |
|           | pop. nests  | no     | but    | 0 |         | di      |   |
|           | Wrangel I.  | known  | gen    | 0 |         | fficult |   |
|           | & Chukotka  | use of | erally | 0 |         | to      |   |
|           | then        | \"hot  | dry    | , |         | obtain  |   |
|           | returns to  | s      | upland | i |         | target  |   |
|           | n           | pots\" | tundra | n |         | number  |   |
|           | on-breeding |        |        | c |         |         |   |
|           | area in     |        |        | l |         |         |   |
|           | southern S. |        |        | u |         |         |   |
|           | America     |        |        | d |         |         |   |
|           |             |        |        | i |         |         |   |
|           |             |        |        | n |         |         |   |
|           |             |        |        | g |         |         |   |
|           |             |        |        | \ |         |         |   |
|           |             |        |        | ~ |         |         |   |
|           |             |        |        | 1 |         |         |   |
|           |             |        |        | 0 |         |         |   |
|           |             |        |        | 0 |         |         |   |
|           |             |        |        | 0 |         |         |   |
|           |             |        |        | b |         |         |   |
|           |             |        |        | i |         |         |   |
|           |             |        |        | r |         |         |   |
|           |             |        |        | d |         |         |   |
|           |             |        |        | s |         |         |   |
|           |             |        |        | f |         |         |   |
|           |             |        |        | r |         |         |   |
|           |             |        |        | o |         |         |   |
|           |             |        |        | m |         |         |   |
|           |             |        |        | C |         |         |   |
|           |             |        |        | h |         |         |   |
|           |             |        |        | u |         |         |   |
|           |             |        |        | k |         |         |   |
|           |             |        |        | o |         |         |   |
|           |             |        |        | t |         |         |   |
|           |             |        |        | k |         |         |   |
|           |             |        |        | a |         |         |   |
|           |             |        |        | / |         |         |   |
|           |             |        |        | W |         |         |   |
|           |             |        |        | r |         |         |   |
|           |             |        |        | a |         |         |   |
|           |             |        |        | n |         |         |   |
|           |             |        |        | g |         |         |   |
|           |             |        |        | e |         |         |   |
|           |             |        |        | l |         |         |   |
|           |             |        |        | I |         |         |   |
|           |             |        |        | . |         |         |   |
|           |             |        |        | s |         |         |   |
|           |             |        |        | t |         |         |   |
|           |             |        |        | o |         |         |   |
|           |             |        |        | p |         |         |   |
|           |             |        |        | p |         |         |   |
|           |             |        |        | i |         |         |   |
|           |             |        |        | n |         |         |   |
|           |             |        |        | g |         |         |   |
|           |             |        |        | o |         |         |   |
|           |             |        |        | n |         |         |   |
|           |             |        |        | s |         |         |   |
|           |             |        |        | o |         |         |   |
|           |             |        |        | u |         |         |   |
|           |             |        |        | t |         |         |   |
|           |             |        |        | h |         |         |   |
|           |             |        |        | w |         |         |   |
|           |             |        |        | a |         |         |   |
|           |             |        |        | r |         |         |   |
|           |             |        |        | d |         |         |   |
|           |             |        |        | m |         |         |   |
|           |             |        |        | i |         |         |   |
|           |             |        |        | g |         |         |   |
|           |             |        |        | r |         |         |   |
|           |             |        |        | a |         |         |   |
|           |             |        |        | t |         |         |   |
|           |             |        |        | i |         |         |   |
|           |             |        |        | o |         |         |   |
|           |             |        |        | n |         |         |   |
+-----------+-------------+--------+--------+---+---------+---------+---+
| Ranking   |             |        |        |   |         |         |   |
| criteria: |             |        |        |   |         |         |   |
+-----------+-------------+--------+--------+---+---------+---------+---+
| 1\.       |             |        |        |   |         |         |   |
| P         |             |        |        |   |         |         |   |
| roportion |             |        |        |   |         |         |   |
| of the    |             |        |        |   |         |         |   |
| p         |             |        |        |   |         |         |   |
| opulation |             |        |        |   |         |         |   |
| occurring |             |        |        |   |         |         |   |
| in Asia.  |             |        |        |   |         |         |   |
| Score as  |             |        |        |   |         |         |   |
| 1-5 where |             |        |        |   |         |         |   |
| 5=100%    |             |        |        |   |         |         |   |
+-----------+-------------+--------+--------+---+---------+---------+---+
| 2\.       |             |        |        |   |         |         |   |
| Contact   |             |        |        |   |         |         |   |
| with a    |             |        |        |   |         |         |   |
| known     |             |        |        |   |         |         |   |
| \'        |             |        |        |   |         |         |   |
| hotspot\' |             |        |        |   |         |         |   |
| or        |             |        |        |   |         |         |   |
| source.   |             |        |        |   |         |         |   |
| Score as  |             |        |        |   |         |         |   |
| 1=no      |             |        |        |   |         |         |   |
| contact,  |             |        |        |   |         |         |   |
| 2=contact |             |        |        |   |         |         |   |
+-----------+-------------+--------+--------+---+---------+---------+---+
| 3\.       |             |        |        |   |         |         |   |
| Habitats  |             |        |        |   |         |         |   |
| used in   |             |        |        |   |         |         |   |
| context   |             |        |        |   |         |         |   |
| of        |             |        |        |   |         |         |   |
| l         |             |        |        |   |         |         |   |
| ikelihood |             |        |        |   |         |         |   |
| of        |             |        |        |   |         |         |   |
| exposure  |             |        |        |   |         |         |   |
| 1         |             |        |        |   |         |         |   |
| =Offshore |             |        |        |   |         |         |   |
| marine,   |             |        |        |   |         |         |   |
| 2         |             |        |        |   |         |         |   |
| =Estuary, |             |        |        |   |         |         |   |
| 3=Ter     |             |        |        |   |         |         |   |
| restrial, |             |        |        |   |         |         |   |
| 4=Fr      |             |        |        |   |         |         |   |
| eshwater. |             |        |        |   |         |         |   |
+-----------+-------------+--------+--------+---+---------+---------+---+
| 4\.       |             |        |        |   |         |         |   |
| P         |             |        |        |   |         |         |   |
| opulation |             |        |        |   |         |         |   |
| size in   |             |        |        |   |         |         |   |
| Alaska    |             |        |        |   |         |         |   |
| during    |             |        |        |   |         |         |   |
| 2006.     |             |        |        |   |         |         |   |
| Score to  |             |        |        |   |         |         |   |
| the       |             |        |        |   |         |         |   |
| closest   |             |        |        |   |         |         |   |
| number    |             |        |        |   |         |         |   |
| 1=1,000,  |             |        |        |   |         |         |   |
| 2=10,000, |             |        |        |   |         |         |   |
| 3         |             |        |        |   |         |         |   |
| =100,000, |             |        |        |   |         |         |   |
| 4=        |             |        |        |   |         |         |   |
| 1,000,000 |             |        |        |   |         |         |   |
+-----------+-------------+--------+--------+---+---------+---------+---+
| 5\. Can   |             |        |        |   |         |         |   |
| we obtain |             |        |        |   |         |         |   |
| a         |             |        |        |   |         |         |   |
| repre     |             |        |        |   |         |         |   |
| sentative |             |        |        |   |         |         |   |
| sample of |             |        |        |   |         |         |   |
| s         |             |        |        |   |         |         |   |
| ufficient |             |        |        |   |         |         |   |
| size      |             |        |        |   |         |         |   |
| (n=200)?  |             |        |        |   |         |         |   |
| Score     |             |        |        |   |         |         |   |
| 1=no,     |             |        |        |   |         |         |   |
| 2=maybe,  |             |        |        |   |         |         |   |
| 3=yes.    |             |        |        |   |         |         |   |
+-----------+-------------+--------+--------+---+---------+---------+---+

Table 4-6. Ranking matrix for populations of passerines and a larid to
be sampled for HPAI during the 2006 field season in Alaska.

  -------------------- -------------- ------------- --------------------- --------------- --------------- -----------
  **Taxon**            **Proportion   **Contact     **Habitat used in     **Population.   **Can samples   **Score**
                       of population  with known    Asia**                in Alaska**     be obtained**   
                       in Alaska**    \"hot                                                               
                                      spot\"**                                                            

                                                                                                          

  **Arctic Warbler     **5**          **2**         **3**                 **4**           **3**           **17**
  (*Phylloscopus                                                                                          
  borealis                                                                                                
  kennicotti*)**                                                                                          

                       Endemic        Winters in    Terrestrial. Wooded   Est. at         Many locations  
                       subspecies to  Myanmar,      habitats, cultivated  2,700,000       where the most  
                       Alaska         Thailand, se. areas, grasslands,                    abundant        
                                      China,        gardens, and                          breeding bird.  
                                      Taiwan,       mangroves                             Easy to capture 
                                      Philippines                                         during breed    
                                      south to                                            and migration   
                                      Andaman Is.,                                                        
                                      Malay                                                               
                                      Peninsula,                                                          
                                      and Indonesia                                                       
                                      east to                                                             
                                      Moluccas                                                            

  **Eastern Yellow     **5**          **2**         **3.5**               **4**           **3**           **17.5**
  Wagtail (*Motacilla                                                                                     
  tschutschensis*)**                                                                                      

                       Endemic        Taiwan,       Terrestrial. Open     Est. 1,400,000  Easy to capture 
                       species to     Indonesia,    areas with water,                     and areas with  
                       Alaska         Sunda Isles,  sugarcane fields,                     known           
                                      and Moluccas  rice fields, sparse                   concentration   
                                                    grasslands, cassava                   of breeding     
                                                    plots; usually in                     birds already   
                                                    association with wild                 identified      
                                                    and domestic grazing                                  
                                                    mammals                                               

  **Gray-cheeked       **3**          **2**         **3**                 **4**           **3**           **15**
  Thrush (*Catharus                                                                                       
  minimus*)**                                                                                             

                       48% of global  Breeds in E.  Terrestrial. Shrubs   Est. 5,000,000  Most abundant   
                       population in  Siberia       often in riparian                     bird in many    
                       Alaska                       habitats                              locations.      
                                                                                          Already         
                                                                                          captured at     
                                                                                          many banding    
                                                                                          sites.          

  **Glaucous Gull      **2**          **2**         **3**                 **2**           **3**           **12**
  (*Larus                                                                                                 
  hyperboreus)***                                                                                         

                       40,000 (100%)  Contact with  Terrestrial/coastal   Approx 40,000   Samples easily  
                       of US breeding humans and                                          obtainable      
                       population in  garbage dumps                                                       
                       AK                                                                                 
  -------------------- -------------- ------------- --------------------- --------------- --------------- -----------

Table 4-7. Suggested migratory bird species for highly pathogenic H5N1
avian influenza surveillance in the four North American flyways.

  ------------------------------------------- ---------------------------
  **[Pacific Flyway]{.underline}**            

  **Taxon**                                   **Ranking**

  Tundra Swan (Western Population)            Primary

  Lesser Snow Goose (Wrangel Island           Primary
  Population)                                 

  Northern Pintail                            Primary

  Long-billed Dowitcher                       Primary

  Red Knot (small numbers)                    Primary

  Pacific Golden Plover (small numbers)       Primary

  Ruddy Turnstone (very small numbers)        Primary

  Black Brant (Pacific Population)            Secondary

  Cackling Goose                              Secondary

  Pacific Greater White-fronted Goose         Secondary

  Mallard                                     Secondary

  American Wigeon                             Secondary

  American Green-winged Teal                  Secondary

  Northern Shoveler                           Secondary

                                              

  **[Central Flyway]{.underline}**            

  **Taxon**                                   **Ranking**

  Lesser Sandhill Crane (Mid-continent)       Primary

  Tundra Swan (Eastern Population)            Primary

  Northern Pintail (low percentage from       Primary
  Alaska)                                     

  Pectoral Sandpiper                          Primary

  Buff-breasted Sandpiper                     Primary

  Long-billed Dowitcher                       Primary

  Greater White-fronted Goose (Mid-continent) Secondary

  Lesser Snow Goose (Western Central Flyway)  Secondary

  Mallard                                     Secondary

  American Wigeon                             Secondary

  American Green-winged Teal                  Secondary

  Northern Shoveler                           Secondary
  ------------------------------------------- ---------------------------

  ------------------------------------------- ---------------------------
  **[Mississippi Flyway]{.underline}**        

  **Taxon**                                   **Ranking**

  Pectoral Sandpiper                          Primary

  Dunlin                                      Primary

  Long-billed Dowitcher                       Primary

  Greater White-fronted Goose                 Secondary

  Northern Pintail                            Secondary

  Mallard                                     Secondary

  American Wigeon                             Secondary

  American Green-winged Teal                  Secondary

  Northern Shoveler                           Secondary

  Lesser Scaup                                Secondary

  Greater Yellow-legs                         Secondary

  Lesser Yellow-legs                          Secondary

  Ruddy Turnstone                             Secondary

  Gray-cheeked Thrush                         Secondary

                                              

  **[Atlantic Flyway]{.underline}**           

  **Taxon**                                   **Ranking**

  Tundra Swan (Eastern Population)            Primary

  Greater Scaup                               Primary

  Horned Grebe (possibly Europe/Greenland     Primary
  breeders)                                   

  Lesser Scaup                                Secondary

  Canvasback                                  Secondary

  Long-tailed Duck (unknown east-west         Secondary
  interchange)                                

  Western Sandpiper                           Secondary

  Least Sandpiper (do not breed in Asia)      Secondary

  Greater Yellow-legs (do not breed in Asia)  Secondary

  Black-bellied Plover                        Secondary
  ------------------------------------------- ---------------------------

## Sample Size

When sampling for highly pathogenic H5N1 avian influenza virus it is
critical that an appropriate sample size for each species or species
group in each designated sample population is obtained. Equation 1
provides a method for calculating the recommended sample size:

n = log (1-c) / log(1-p) (eq. 1)

where n is the sample size, c is the desired level of confidence, and p
is the prevalence of positive samples in the population. An adequate
sample size should allow for \>95% confidence that AI is detected at
[\<]{.underline} 1.5% prevalence. These criteria result in an estimated
sample size of 200:

n = log (1-.95) / log (1-0.015) = 200

Thus, a minimum of 200 samples should be collected from the population
of interest based on an assumed prevalence of 1.5% of highly pathogenic
H5N1 avian influenza. We caution that this calculation is very sensitive
to the assumed prevalence, which we can not know a priori. For example,
if prevalence of the disease at the time of sampling is 0.1% (i.e., 1 in
1000 birds is infected) the necessary sample size is 3000. As prevalence
decreases the likelihood of detecting the disease in an individual bird
also decreases due to the low probability of detection and practical
limitations on laboratory processing capability. We also caution that
this formula is weakened here because it is based on assumptions that
may not apply to H5N1 virus in wild birds, namely that the agent is
homogeneously distributed within a host population that also is
homogeneously distributed.

## Sample Collection

Tracheal and cloacal swabs should be collected from individuals of each
species at each location using the procedures identified in Attachment
9.

## Discussion 

Wild birds, particularly waterfowl and other waterbirds, are natural
hosts of avian influenza viruses and are believed to play an important
role in the epizootiology of these viruses. All hemagglutinin and
neuraminidase subtypes have been found in waterfowl and shorebirds
(Webster et al., 1992; Krauss et al., 2004; Widjaja et al., 2004). This
proposed sampling effort provides the best opportunity for detection of
highly pathogenic H5N1 avian influenza virus in live migratory birds
that may bring the disease from Asia to Alaska, the Pacific Islands and
the west coast of the U.S. The primary advantage to this approach is
that species will be sampled that travel directly to Alaska or the US
Pacific Islands and trust territories from Southeast Asia or
Australasia, have some fraction of the population known to winter in
Asia or Pacific trust territories, or intermingle with other species
across Siberia, the Russian Far East, and Alaska. The primary
disadvantage is the logistical considerations in live capture of the
birds in remote areas.

## Recommendations 

Sampling live birds will allow us to determine if they are currently
infected with highly pathogenic H5N1 avian influenza virus or other AI
viruses. When collecting samples from live birds, tracheal and cloacal
swabs are preferred. Most AI strains tend to replicate more efficiently
in the intestinal tract than in the respiratory tract of natural host
species (i.e., waterfowl and shorebirds). Consequently, cloacal swabs
are generally preferred. However, recent isolations of highly pathogenic
H5N1 avian influenza virus in wild birds have documented higher levels
of virus in tracheal samples. Therefore, it is recommended that both
samples be collected from birds when possible. While, the collection of
cloacal swabs is a relatively easy procedure, obtaining proper tracheal
swabs can be problematic and requires personnel trained in the sampling
technique. Examples of tracheal/cloacal swab collection protocols can be
found in Attachment 9. Tracheal and cloacal swabs should be placed in
separate tubes, and swabs should not be pooled across individuals.

Specific implementation plans should be developed for each state/flyway.
It is strongly advised that agencies and organizations coordinate their
sampling efforts to assure that adequate sample sizes are obtained from
each species within each state/flyway. Coordination can be achieved
through the existing migratory bird flyway councils.

## Literature Cited

Hanson B.A., D.E. Stallknecht, D.E. Swayne, L.A. Lewis, and D.A. Senne.
2003. Avian influenza viruses in Minnesota ducks during 1998-2000. Avian
Diseases 47:867-871.

Krauss, S., D. Walker, S.P., Pryor, L. Niles, L. Chenghong, V.S.
Hinshaw, and R.G. Webster. 2004. Influenza A viruses of migrating wild
aquatic birds in North America. Vector-Bourne and Zoonotic Diseases
4:177-189.

Ito, T., K. Okazaki, Y. Kawaoka, A. Takada, R.G. Webster, and H. Kida.
1995. Perpetuation of influenza A viruses in Alaskan Waterfowl
reservoirs. Archives of Virology 140:1163-1172.

Slemons R.D. et al. 2003. Type A influenza virus surveillance in
free-flying, nonmigratory ducks residing on the eastern shore of
Maryland. Avian Diseases 47:1107-1110.

Swayne, D.E. and D.A. Halvorson. Influenza. 2003. Pp 135-160 *in*
Diseases of Poultry, 11^th^ ed. Saif, Y.M., H.J. Barnes, J.R. Glisson,
A.M. Fady, L.R. McDougald, and D.E. Swayne (eds.). Iowa State University
Press, Ames, IA.

Widjaja, L., S.L. Krauss, R.J. webby, T. Xie, and R.G. Webster. 2004.
Matrix gene of influenza A viruses isolated from wild aquatic birds:
ecology and emergence of influenza A viruses. Journal of Virology
78:8771-8779.

Webster, R.G., W.J. Bean, O.T. Gorman, T.M. Chambers, and Y. Kawaoka.
1992. Evolution and ecology of influenza A viruses. Microbiological
Reviews 56:152-179.

# ATTACHMENT 5

# Surveillance for Highly pathogenic H5N1 avian influenza in Hunter-killed Birds

## Overview

Check stations for waterfowl hunting are operated by some state natural
resource agencies and National Wildlife Refuges to collect information
on local waterfowl harvest. Hunter check stations provide an opportunity
to collect additional samples to monitor for the presence of Highly
pathogenic H5N1 avian influenza virus and other avian influenza (AI)
virus subtypes. This sampling would supplement live bird surveillance
(Attachment 4) by increasing the number of selected species, geographic
locations, and time periods represented in surveillance efforts.
Previous and current studies that have sampled hunter-killed waterfowl
to detect AI include an ongoing 20-year study in Ohio by Dr. Richard
Slemons of Ohio State University, current work in North Carolina by Dr.
David Stallknecht of the Southeastern Cooperative Wildlife Disease Study
(the North Carolina Wildlife Resources Commission has been collecting
samples from hunter-killed ducks and tundra swans for Dr. Stallknecht),
and sampling in New Mexico, Texas, and Maryland by state wildlife
agencies and/or university researchers. A number of the state natural
resource agencies and other researchers are developing plans to sample
hunter-killed birds for AI in 2005 and/or 2006.

This conceptual surveillance strategy includes (1) sampling birds killed
in fall by sport hunters and in summer by subsistence hunters in Alaska:
(2) sampling birds killed in fall in the lower 48 states. As with
surveillance of live-captured birds, sampling hunter-killed birds in
Alaska will focus on hunted species that are most likely to be exposed
to HPAI in Asia, and that have relatively direct migratory pathways from
those areas to Alaska (primary species). Additional samples collected on
the wintering grounds in the lower 48 states will include both primary
species and species that mix with the primary species in Alaska staging
areas (secondary species). Currently, the probability of highly
pathogenic H5N1 avian influenza virus transmission from primary species
to secondary species is poorly understood, but AI viruses are known to
remain viable for months in cold freshwater. From a surveillance
standpoint, if secondary transmission proves to be potent and extensive,
a very large number of species could be involved. However, this
conceptual program is focused on early detection, with adaptation to
more intensive efforts as needed. Thus, it is recommended that sampling
efforts involving hunter-killed birds in the lower 48 states should
concentrate on the species/populations and wintering areas in which the
presence of highly pathogenic H5N1 avian influenza virus is most likely
to be detected. Some research indicates that susceptibility may vary
among game birds. The complete design, implementation and development of
an operational plan and the funding necessary for this strategy requires
closer coordination with states through the Flyway Council system.

A similar approach to investigate the possible movement of Highly
pathogenic H5N1 avian influenza virus from Europe could be developed.
There are few data on migratory bird movement rates between North
America and Europe, but the band recovery data that are available
suggest very low exchange rates. Presently we only have data on movement
from North America to Europe, but if we make the tenuous assumption that
movement rates are similar in both directions, then only 3 species
emerge as likely primary species: Eurasian wigeon, northern pintail, and
green-winged teal. Of American wigeon banded in the Atlantic Flyway,
only 6 of 2,211 recoveries (0.27%) were from Europe. The rates for
northern pintail and green-winged teal are 0.06% (3 of 5,341 recoveries)
and 0.04% (5 of 12,274 recoveries), respectively. If immigration rates
are similar to emigration rates, and if those rates are indicative of
the proportion of eastern North American birds that are immigrants from
Europe, the chance of sampling even one hunter-killed immigrant is very
low. Some common eiders (Keith McAloney, Canadian Wildlife Service,
personal communication) and North Atlantic Population Canada geese (Fox
et al. 1996) move back and forth between northeastern North America and
Greenland, where they could interact with birds from mainland Europe.
Likewise, the high-arctic Atlantic brant that breed in North America and
winter in Ireland also come into contact with European birds. Thus,
these are additional species that should be considered when implementing
surveillance for highly pathogenic H5N1 avian influenza virus in the
Atlantic flyway.

## Methodology 

### [Alaska]{.underline} 

The surveillance of live-captured birds strategy (Attachment 4) has
provided the biological basis for identifying the primary species
recommended for sampling in Alaska. Below is a list of these target
species and sampling locations recommended for surveillance of
hunter-killed birds.

> *Samples from fall hunters:*

-   Northern pintail: Mendenhall Refuge, Minto Flats, and Cook Inlet

-   Lesser sandhill crane: Delta Junction

-   Black brant: Cold Bay

-   Common eider, king eider, long-tailed duck: Kodiak National Wildlife
    Refuge, and perhaps cooperating hunting guides along the coast of
    Alaska.

> *Samples from summer hunters:*

-   Seward Peninsula: lesser sandhill crane, bar-tailed godwit,
    long-billed dowitcher

-   Yukon-Kuskokwim Delta: black brant, emperor goose, common eider,
    king eider, northern pintail, long-tailed duck

-   Barrow: common eider, king eider, black brant, long-tailed duck,
    glaucous gull

-   St. Lawrence Island: emperor goose, black brant, common eider, king
    eider

### [North American Flyways]{.underline}

There are at least 25 states in which either the state wildlife agencies
or USFWS routinely check hunter harvested birds: 9 in the Atlantic
Flyway, 7 in the Mississippi Flyway, 4 in the Central Flyway, and 5 in
the Pacific Flyway. Those 25 states and most other states have already
expressed some willingness to collect samples from hunter-killed birds.
States are encouraged to develop specific implementation plans in
consultation with their respective flyway council using the guidance
provided in this strategic plan.

Four primary target species/populations have been identified for highly
pathogenic H5N1 avian influenza virus sampling in the lower 48 states:
northern pintail, Pacific black brant, Wrangel Island snow geese, and
lesser sandhill crane (mid-continent population). Also, several
secondary species that mix with the primary species in Alaska (and thus
have an increased risk of exposure to highly pathogenic H5N1 avian
influenza virus) and later winter in the 4 flyways have been identified
(see below). Given the ephemeral nature of birds at specific sites along
their migration routes, sampling efforts for both primary and secondary
species should be concentrated on the wintering grounds.

The target species/populations and general sampling locations presented
in this document are based on band recovery and in some cases radio
telemetry data for birds banded in Alaska. Specific sampling sites
(i.e., check stations or other areas where hunter-harvested birds could
be sampled) will be determined by the individual states and National
Wildlife Refuges that elect to participate in the sampling.

> *Pacific Flyway*:

-   Northern pintail (primary species): Central Valley of California

-   Wrangel Island snow geese (primary): Skagit-Fraser Rivers Delta,
    Washington and British Columbia

-   Black brant (primary): Humboldt Bay, California and San Quintin Bay,
    Mexico if permits to collect samples and ship them to the U.S. can
    be obtained

-   American wigeon combined (secondary): Central Valley of California

-   American green-winged teal (secondary): Central Valley of California

-   Northern shoveler (secondary): Central Valley of California

-   Cackling goose (secondary): Northwest Oregon/Southwest Washington
    permit goose areas

-   Pacific greater white-fronted geese (secondary): Central Valley of
    California

-   Tundra Swan (secondary): Montana and Utah

-   Mallard (secondary): western Washington

> *Central Flyway*:

-   Lesser sandhill crane (primary): New Mexico, west Texas, Nebraska

-   Northern pintail (primary, but few from Alaska): Gulf Coast, Texas

-   Lesser snow goose (secondary): North Dakota, Nebraska

-   Mid-continent greater white-fronted geese (secondary): Texas

-   American wigeon (secondary): Gulf Coast, Texas

-   American green-winged teal (secondary): Gulf Coast, Texas

-   Northern shoveler (secondary): Gulf Coast, Texas

-   Tundra Swan (secondary): North Dakota, South Dakota

-   Mallard (secondary): Oklahoma, Texas, Nebraska

> *Mississippi Flyway*:

-   Northern pintail (primary, but few from Alaska): Gulf Coast,
    Louisiana

-   Mid-continent greater white-fronted geese (secondary): Louisiana

-   American wigeon (secondary): Gulf Coast, Louisiana

-   American green-winged teal (secondary): Gulf Coast, Louisiana

-   Northern shoveler (secondary): Gulf Coast, Louisiana

-   Mallard (secondary): Arkansas, Mississippi, Louisiana

-   Lesser scaup (secondary): Gulf Coast of Louisiana, Mississippi,
    Alabama

> *Atlantic Flyway*

-   Tundra swan (secondary): North Carolina, Virginia

-   Greater scaup (secondary): East Coast from Massachusetts to Virginia

-   Lesser scaup (secondary): Florida, East Coast from Chesapeake Bay
    south

-   Canvasback (secondary): Chesapeake Bay

-   Long-tailed duck (secondary): East Coast from Massachusetts to
    Virginia

Duck breeding population estimates can provide some indication of the
relative likelihood (among species) that a given hunter-killed bird came
from Alaska. That is, samples from species that have a larger proportion
of their breeding population in Alaska are more likely to contain birds
from Alaska than samples from species with a lower proportion of their
breeding population occurring in Alaska. Based on that premise, we
ranked the secondary species of ducks listed above according to sampling
priority. In 2005, 39% of the American wigeon breeding population
occurred in Alaska, making that the highest priority species according
to this criterion. The others are ranked as follows: 2. northern pintail
(35% of the breeding population occurred in Alaska), 3. American
green-winged teal (33%), 4. greater and lesser scaup combined (28%), 5.
northern shoveler (18%), 6. canvasback (18%), and 7. mallard (10%).

As in Alaska, a sample size of 200 birds from each sample population is
needed to detect highly pathogenic H5N1 avian influenza virus prevalence
of 1.5% or greater with 95% power. Therefore, it is recommended that a
minimum sample size goal of 200 birds per species per wintering area.
Tracheal and/or cloacal samples should be collected in accordance with
protocols identified in this document (Attachment 8).

## Discussion

Sampling of hunter-killed birds would supplement targeted surveillance
in live wild birds (Attachment 4) and other strategies identified in
this strategic plan. The advantage to this approach is that it is
cost-effective because for most of the species that are classified as
game birds, existing infrastructure (e.g., check stations) is in place
in most wintering areas and sufficient numbers of birds are expected to
be encountered. The disadvantages to this approach are: 1) most of the
sampling in the lower 48 states will be of secondary species, thus the
likelihood of sampling birds that have come into contact with infected
primary species birds is small, especially in the Atlantic, Mississippi,
and Central Flyways; and 2) numerous sampling sites throughout the U.S.
will require sufficient training of sampling personnel to ensure samples
are properly acquired, preserved, and shipped. There are advantages and
disadvantages in terms of public perceptions of sampling
hunter-harvested birds. Public perceptions could be positive if
user-groups will appreciate that samples are being taken. Conversely, if
hunters do not have accurate information about highly pathogenic H5N1
avian influenza virus, they could become unnecessarily alarmed about
exposure (especially if agency samplers are wearing protective gear).
For spring and summer subsistence users, providing access to birds that
were historically taken illegally may make sampling difficult and basic
information on highly pathogenic H5N1 avian influenza virus and the
sampling program may be harder to deliver.

## Recommendation

Sampling hunter-killed birds would supplement other approaches in a
cost-effective manner and may allow us to determine if certain species
of birds (e.g., migratory game birds) are currently infected with highly
pathogenic H5N1 avian influenza subtype or other AI viruses. This
expanded effort to identify highly pathogenic H5N1 avian influenza virus
should be considered a supplemental part of any surveillance system.
Specific implementation plans and budgets should be developed in concert
with affected state agencies and the flyway councils.

Unlike other surveillance approaches, the use of hunter-killed birds has
high public profile. Therefore, the implementation of this action should
be discussed with agencies and organizations that have experience
regarding the public relations aspects of researching and assessing
zoonoses (e.g., chronic wasting disease and West Nile virus) to ensure
that we develop an appropriate and consistent message to hunters.

## References

Fox, A.D., Christian Glahder, Carl R. Mitchell, David A. Stroud, Hugh
Boyd, and John Frikke. 1996. North American Canada geese (*Branta
canadensis*) in west Greenland. Auk 113(1): 231-233.

# ATTACHMENT 6

# Sentinel Animal Methods

## Overview

This methods section reviews two sentinel animal methods that have been
used in avian disease surveillance programs and that may be used for the
early detection of avian influenza (AI) virus infection along migratory
flyways in the U.S.

### [Non-commercial Backyard Poultry Flocks]{.underline}

Backyard poultry are defined as domesticated fowl, including chickens,
turkeys, waterfowl, and game birds (except doves and pigeons) maintained
for hobby or noncommercial egg and meat production (NAHRS FAQ 2005).
Backyard poultry are typically allowed to forage freely or may be
confined in partially enclosed fenced areas. The evaluation of poultry
flocks reared on backyard premises for diseases of interest to the
commercial poultry industry has been used as a surveillance method to
estimate seroprevalence of selected disease agents as part of health
surveys in backyard flocks adjacent to commercial operations. (McBride;
Hird; Carpenter; Snipes; Danaye-Elmi, and Utterback 1991;Johnson; Colby;
Tablante; Hegngi; Salem; Gedamu, and Pope C. 2004)

In 2005, State animal health officials in Alaska sampled birds at fairs
and exhibitions (concentration points). Most exhibitors were 4H or hobby
farmers. Fairs and exhibitions are social events and are attended by
large numbers of game bird fanciers from remote regions of Alaska. State
animal health officials offered testing to exhibitors at three
agricultural fairs with the goal of testing every entry to the fair (600
samples representing 100 flocks). This was a voluntary sampling program,
but nearly 100% of owners of exhibition game agreed to test in 2005.

-   150 birds were sampled at the Fairbanks fair. Tanana Fair entries
    represent flocks from a 40 mile radius around the city of Fairbanks
    (Healy, Tanana, and the North Pole).

-   100 birds were sampled at the Kenai fair. Kenai Fair entries include
    flocks from Homer to Anchorage

-   300 samples were collected at the Alaskan State Fair in Palmer.
    Palmer Fair entries include Anchorage, Matanuska Valley (includes
    flocks as far north as Fairbanks)

In Alaska, poultry chicks are either purchased through mail order or
from a few local breeders and may be reared in suburban areas or in
remote villages throughout the State. Most backyard birds are reared for
egg production and slaughtered for meat prior to the winter season,
although there are a growing number of hobbyists that raise show birds.
Birds are often reared on open range or in outside enclosures and
sometimes have an opportunity to intermingle with wild waterfowl.
Limited resources prohibited widespread backyard bird surveillance
testing over the large expanse of the state. Backyard flock surveillance
is presently passive and owners request testing after morbidity or
mortality events occur in their flock or after noting dead waterfowl or
sick waterfowl on their premises. There is currently no census available
to estimate the number of backyard flocks in Alaska.

Cloacal swab samples are placed in ethanol and evaluated at the
University of Alaska, at Fairbanks by RT-PCR. If surveillance screening
samples are positive by PCR, the premise is placed under quarantine and
additional cloacal samples taken during the epidemiological
investigation are placed in viral transport media and submitted for
virus isolation to NVSL in Ames, Iowa.

In 2006, the Alaska Department of Environmental Conservation, Office of
the State Veterinarian will sample backyard flocks, near summer water
systems where wild and domestic waterfowl congregate and collect
environmental samples (bird droppings, water samples) in six general
areas:

-   Southeast- 2 cities (Juneau, Ketchikan)

-   Southcentral- 4 cities (Homer, Soldotna, Anchorage, Matanuska Valley

-   Interior- 3 cities (Fairbanks, Healy, Talkeetna)

-   Southwest- 2 cities (Bethel, King Salmon)

-   Northwest- 2 cities (Nome, Kotezebue)

-   Aleutians/Bering Sea- 4 cities (Kodiak, Dutch Harbor, Cold Bay,
    Pribilof Islands)

The areas listed in bold have the highest priority and cover a majority
of the population where domestic poultry is kept. The other 3 areas have
substantial populations of wild birds but few domestic backyard flocks.
A sample size of 11 is needed to detect avian influenza at a prevalence
rate of 25% at a 95% confidence interval in flocks ranging from 10 to
10,000 or more birds.

The Office of the State Veterinarian will sample poultry exhibited at
the six agricultural fairs (concentration points):

-   Deltana Fair Date to be announced

-   Haines Fair 7/26-30/2006

-   Tanana Valley Fair 8/4-12/2006

-   Kenney Lake Fair Date to be announced (2^nd^ Week August 2006)

-   Kenai Peninsula Fair (Ninilchik) 8/18-20/2006

-   Alaska State Fair (Palmer) 8/24 - 9/4/2006

The fair boards have expressed interest in promoting this sampling
effort and it is expected that all entries will be tested. The state has
just finished construction of a new diagnostic laboratory, Alaska
Environmental Health Laboratory in Anchorage, and will develop the
capacity to run the diagnostic tests using RT-PCR. If surveillance
screening samples are positive by PCR, the premise will be placed under
quarantine and additional cloacal samples taken during the
epidemiological investigation are placed in viral transport media and
submitted for virus isolation to NVSL in Ames, Iowa.

This approach to sampling non-commercial poultry flocks may be adapted
in other areas of the U.S. where there is widespread non-commercial
poultry production.

### [Sentinel Duck Flocks]{.underline}

The second method described is the placement of sentinel duck flocks in
wetland environments where they are potentially exposed to and infected
with disease agents as they commingle with wild birds. The placement of
sentinel flocks of domestic ducks has been used to recover AI and detect
influenza epizootics in pelagic bird colonies, and yielded much higher
isolation rates compared to isolations from wild birds (Turek;
Gresikova, and Tumova 1984;Sinnecker; Sinnecker; Zilske, and Koehler
1982;Sinnecker; Sinnecker, and Zilske 1982). Sentinel ducks have been
used to determine the presence of AI and timing of infection associated
with the arrival of wild migratory waterfowl in wetland habitats
adjacent to market turkey production flocks (Halvorson; Karunakaran;
Senne; Kelleher; Bailey; Abraham; Hinshaw, and Newman 1983;Halvorson;
Kelleher, and Senne 1985;Kelleher; Halvorson; Newman, and Senne 1985).

In North America, AI isolations from waterfowl have been reported from
approximately 30 locations over the past 35 years (Hanson; Stallknecht;
Swayne; Lewis, and Senne 2003). Many of these sites are located along
each of the four migratory waterfowl flyways (Pacific, Central,
Mississippi, and Atlantic) in the continental U.S. Bodies of water with
large concentrations of migratory waterfowl and shorebirds might also
serve as sentinel sites.

Ideally, surveillance activities should occur at sites at a time when
migratory birds are actively nesting and at locations where they marshal
and intermingle with other migratory birds transiting the area prior to
winter migration. The onset of avian influenza infection in sentinel
ducks has been shown to occur in late July and early August in summer
breeding areas (infection of range reared turkey flocks was shown to
occur about 6 to 8 weeks later) (Halvorson et al. 1985). Avian influenza
virus prevalence estimates from published waterfowl surveys indicate
that virus can first be detected in naïve juvenile birds in summer
breeding areas in July or August (prevalence ranged from 11% to 61% in
published surveys) as juveniles emerge from hiding and intermingle with
other broods and a subsequent high rate of re-infection as birds marshal
for winter migration in October (Hanson et al. 2003; Hinshaw et al.
1985). Avian influenza virus prevalence generally decreases during late
fall and winter and may reach a level of 1% or less in over-wintering
areas.(Stallknecht ;Webster; Bean; Gorman; Chambers, and Kawaoka 1992)
However, virus was isolated from 11% of teals and from 15% of northern
pintails in one recent survey of wintering ducks in Texas, suggesting
that the avian influenza season may not be a fall season event (Hanson
2003). As a result of early migration, blue winged teal are thought to
serve as an immunologically naïve host in wintering areas.

Most virus isolations have occurred in mallards and other species of
dabbling ducks, but less commonly in wood ducks and similar species
(Stallknecht ). Mallards are commonly associated with habitats located
near man, livestock, and poultry and would be more likely to interact
with backyard poultry flocks compared with other waterfowl species
(Stallknecht and Shane 1988). Although H5, H7, and H9 subtypes have been
poorly represented in most waterfowl surveys (H3, H4, or H6 subtypes
have been isolated most frequently), pintails and mallards have been
shown to be significant reservoirs in one recent survey where H5, H7,
and H9 virus subtypes were isolated 21.5% of the time in Minnesota
(Hanson and others 2003). The prevalence of AI isolated from blue winged
teal on wintering grounds in February in Texas was found to be 22% in
2001 and 15% in 2002 (Hanson 2003). Migration of blue winged teal occurs
in late summer and early fall (typically September), prior to the
highest period of AI prevalence. Early migration of this species is
thought to play a role in maintenance of AI infection on wintering
grounds by providing a susceptible population with little or no prior
exposure or immunity. However, blue winged teal are less likely to
interact with man or livestock, so sites where blue winged teal
congregate may not serve as the best sites for surveillance using
backyard flocks of domestic waterfowl.

The role of shorebirds in avian influenza ecology should be considered
separately from that of migratory waterfowl. The highest prevalence of
avian influenza virus in shorebirds has been shown to occur in May and
in September, which coincides with the times of peak shorebird migration
in the northeastern U.S. (Kawaoka; Chambers; Sladen, and Webster 1988).
Shorebirds migrating through the Delaware Bay have been shown to have
the highest prevalence of AI virus compared with other shorebird
populations surveyed at four other locations along the Atlantic flyway
(Hanson 2003). Although most isolates reported from shorebirds in this
survey were H10 and H12 (H9 and H13 in previous studies), H5 and H7
subtypes were isolated from a small percentage of shorebirds. During
May, virus was isolated mostly from ruddy turnstones (9.1%).

The approach to the design of a targeted surveillance method for the
detection of avian influenza using either of these two sentinel animal
methods should incorporate what is presently known about the ecology and
natural history of avian influenza infection in wild waterfowl reservoir
species. Sentinel animals are most likely to become infected with AI if
exposed to reservoirs in nature during periods of highest viral
shedding. As described above, prevalence of infection as measured by
virus isolations in published waterfowl surveys has been shown to vary
temporally by location, age, season, and species. A targeted approach to
sentinel animal surveillance should be designed to:

-   Target specific locations where AI has been isolated from wild
    waterfowl historically;

-   Target locations where known primary reservoir species (mallards,
    blue winged teal, ruddy turnstones) congregate for breeding
    (resulting in higher concentrations of juveniles susceptible to
    infection) or wintering (higher concentrations of species with
    little or no previous exposure) resulting in a higher prevalence of
    infection;

-   Be timed to coincide with periods (seasons) of highest prevalence in
    the reservoir species, in particular migratory species that
    originate from an area having high incidence of AI (Southeast Asia).

## Methods

### [Backyard Poultry Surveillance Method]{.underline}

> *Flock Selection*

-   Targeted flocks should consist of free range domestic waterfowl or
    poultry flocks located near marshlands or wetlands.

-   Marshlands should contain high density populations of waterfowl or
    shorebirds.

-   Flocks should have an opportunity to directly intermingle with
    waterfowl (especially mallards) at or near the common watershed via
    open range or open enclosure or by sharing a common source of water.

-   Chose sites adjacent to wetlands where AI virus has been isolated
    historically.

> *Timing of Surveillance*

-   Surveillance should begin in late July and continue through October
    at sites near northern breeding areas.

-   Although, prevalence rates in wild waterfowl were shown to decrease
    significantly in wintering areas in Louisiana (1%), prevalence in
    blue winged teal in wintering areas in Texas during February of
    \>10% indicates that some wintering sites may be useful for sentinel
    surveillance.

-   The seasonal peak of AI prevalence in shorebirds occurs in May
    rather than late summer, so surveillance of backyard flocks in the
    Delmarva (Delaware Bay) area should be planned for May to coincide
    with the time of highest prevalence.

*Sample Size Estimates*

-   The average size of backyard poultry flocks in the U.S. is 35 birds
    (varies from 28 to 49 birds per flock by region). A prevalence
    estimate for avian influenza of 25% is assumed (NAHMS Poultry \'04
    Part I 2004).

-   A sample size of 11 is needed to detect avian influenza at a
    prevalence rate of 25% at a 95% confidence interval in flocks
    ranging from 10 to 10,000 or more birds.(Cannon and Roe 1982)

-   Cloacal and tracheal swab samples would be submitted to the
    appropriate diagnostic labs for RT-PCR testing and to a reference
    lab for virus isolation.

### [Sentinel Duck Method]{.underline}

> *Flock Preparation and Placement*

-   Construct pens or plan for open fenced enclosures that will hold 10
    to 20 ducks and allow contact with released "messenger" ducks and
    wild ducks. Pens should allow exposure to water contaminated with
    wild duck feces.

-   Deploy pens to selected wetlands (or construct fenced enclosures).

-   Arrange to provide basic husbandry.

-   Rear one day old ducks in isolation facilities for 6 to 7 weeks.

-   Establish AI free status by cloacal swabbing and serologic testing.

-   Release 10 to 20 isolation reared "messenger" pinioned mallard or
    white Peking ducks on selected body of water.

-   Place 10 to 20 ducks in pens on selected body of water to
    intermingle with "messenger" ducks and wild ducks.

-   Periodically bleed ducks to determine serologic status and replace
    H5 seropositive ducks with immunologically naïve ducks.

> *Timing of Surveillance*

-   Placement of sentinel duck flocks should coincide with backyard
    flock surveillance seasonally.

> *Sampling*

-   Retrieve fecal samples via cloacal swabs from 10 to 20 penned ducks
    to detect virus weekly and periodically trap messenger ducks for
    cloacal sampling.

## Data Collection

For backyard flocks, a database similar to the one used for Exotic
Newcastle Disease (END) surveillance would provide the ability to trace
positive samples back to their flock of origin (Accession number;
sending facility premises ID; submitter name, address, and contact
information; location of animals including premises ID, latitude, and
longitude; owner name; flock information including size, number
affected, number dead; purpose of submission and relevant clinical
information).

Data needed to create predictive geospatial models to evaluate spatial
and temporal risk for sentinel duck flocks include: (1) lat/long (in
unprojected decimal degrees with a WGS-84 or NAD-83 datum) of the
sentinel cage\'s location; (2) front gate coordinates for the premises;
(3) name, address, county, zip code, contact information for the land
owner/manager, and occupations of all residents; (4) age, sex, and breed
of birds; (5) number of sentinel birds and each bird must have a unique
ID (e.g., numbered aluminum leg or wing bands work well); (6)
environmental description of area where cage containing sentinel birds
is placed; (7) AI virus test status (birds are bled periodically to
evaluate immune status and need for replacement); (8) presence and
approximate distance to other birds and mammals; (9) exposure to wild
birds and free ranging domestic birds; (10) estimated density of birds
and mammals on premises and in the vicinity of the sentinels; (11)
exposure of sentinels to human contact other than the avian
phlebotomist; and (12) an environmental assessment of the vicinity
(e.g., within 100 meters, within 500 meters, and within 1000 meters).
These data should be captured on a site survey form. However, a separate
form should be used to record: date and time blood samples were
collected, the birds\' ID number, and the vial number for the blood
specimen. With this basic information, other data sources can be used to
evaluate proximity to wetlands, bird roosts, position with in normal
flyways, terrain features, and more. Access to extensive datasets (e.g.
the National Wetlands Inventory and the National Landcover Dataset) and
hydrologic models could be used to identify wetlands.

## Discussion

### [Major advantages of the use of sentinel animals to detect AI:]{.underline}

-   Backyard bird surveillance programs are already in existence in most
    states.

-   State animal health officials are familiar with a targeted
    surveillance approach (i.e. surveillance of backyard flocks within a
    designated radius adjacent to commercial poultry operations).

-   The placement of sentinel ducks has been used successfully to
    isolate AI from wild waterfowl in previous published surveys.

-   Mortality in backyard poultry from H5N1 has occurred in other
    countries.

-   Could be done in conjunction with other surveillance methods at the
    same location for comparison.

### [Major disadvantages of the use of sentinel animals:]{.underline} 

-   Locating suitable surveillance sites will require field surveillance
    or input from wildlife biologists.

-   Expense of rearing AI free birds.

-   Pen construction and husbandry costs.

-   Sentinel flocks are subject to predation.

## Recommendations

In order to implement an efficient active sentinel animal surveillance
system, sentinel flock locations should be purposefully chosen.
Appropriately allocating limited resources to achieve targeted sampling
and reduce costs is an important objective of animal disease
surveillance programs (McCluskey 2003). Knowledge of disease
distribution allows us to focus surveillance activities. In this case,
we can use our knowledge of the most likely entry points for H5N1
through migratory waterfowl to locate sentinel animal flocks. In order
to target areas for sentinel surveillance with a higher probability of
disease, flyway information should be plotted over waterfowl management
areas in order to select sites most likely to have migratory birds from
areas where commingling with Eurasian species is most likely to occur.
Specific locations in areas where migratory birds from possible northern
exposure sites are most likely to be in highest concentration have been
identified in other methods sections of this plan. National information
on the health and management practices of backyard and small production
flocks adjacent to commercial poultry operations in 18 states is
available. All of this information should be combined with information
on the geographic distribution of poultry producers including sizes and
densities of operations in order to produce a risk map. Local animal
health officials could then locate sentinel backyard flocks adjacent to
waterfowl management areas in poultry dense regions where there is the
highest probability of disease transmission. The health status of
sentinel backyard flocks could be evaluated on a recurring basis
(quarterly, or more often during seasons of the year that pose the
highest probability of disease transmission due to higher prevalences)
for an active disease surveillance program.

## References

NAHMS Poultry \'04 Part I; 2004.

NAHRS FAQ \[Web Page\]. Accessed 2005 Nov 14. Available at:
http://www.aphis.usda.gov/vs/ceah/ncahs/nahrs/NAHRS_faq.htm.\
Notes: The National Animal Health Reporting System (NAHRS) is a
cooperative effort between the American Association of Veterinary
Laboratory Diagnosticians (AAVLD), the U.S. Animal Health Association
(USAHA) and USDA\'s Animal and Plant Health Inspection Service (APHIS).

Alfonso, C. P.; Cowen, B. S., and van Campen, H. Influenza A viruses
isolated from waterfowl in two wildlife management areas of
Pennsylvania. J Wildl Dis. 1995 Apr; 31(2):179-85.

Cannon, R. M. and Roe, R. T. Livestock Disease Surveys A Field Manual
for Veterinarians Department of Primary Industry Bureau of Rural
Science. Canberra: Australian Government Publishing Service; 1982.

Halvorson, D.; Karunakaran, D.; Senne, D.; Kelleher, C.; Bailey, C.;
Abraham, A.; Hinshaw, V., and Newman, J. Epizootiology of avian
influenza\--simultaneous monitoring of sentinel ducks and turkeys in
Minnesota. Avian Dis. 1983 Jan-1983 Mar 31; 27(1):77-85.

Halvorson, D. A.; Kelleher, C. J., and Senne, D. A. Epizootiology of
avian influenza: effect of season on incidence in sentinel ducks and
domestic turkeys in Minnesota. Appl Environ Microbiol. 1985 Apr;
49(4):914-9.

Hanson, B. A. Temporal, spatial and species patterns of avian influenza
viruses among wild birds. Athens, Georgia: University of Georgia; 2003.

Hanson, B. A.; Stallknecht, D. E.; Swayne, D. E.; Lewis, L. A., and
Senne, D. A. Avian influenza viruses in Minnesota ducks during
1998-2000. Avian Dis. 2003; 47(3 Suppl):867-71.

Hinshaw, V. S.; Nettles, V. F.; Schorr, L. F.; Wood, J. M., and Webster,
R. G. Influenza virus surveillance in waterfowl in Pennsylvania after
the H5N2 avian outbreak. Avian Dis. 1986 Jan-1986 Mar 31; 30(1):207-12.

Hinshaw, V. S.; Wood, J. M.; Webster, R. G.; Deibel, R., and Turner, B.
Circulation of influenza viruses and paramyxoviruses in waterfowl
originating from two different areas of North America. Bull World Health
Organ. 1985; 63(4):711-9.

Johnson, Y. J.; Colby, M. M.; Tablante, N. L.; Hegngi, F. N.; Salem, M.;
Gedamu, N., and Pope C. Application of commercial and backyard poultry
geographic information system databases for the identification of risk
factors for clinical infectious laryngotracheitis in a cluster of cases
on the delmarva peninsula. International Journal of Poultry Science.
2004; 3(3):201-205.

Kawaoka, Y.; Chambers, T. M.; Sladen, W. L., and Webster, R. G. Is the
gene pool of influenza viruses in shorebirds and gulls different from
that in wild ducks? Virology. 1988 Mar; 163(1):247-50.

Kelleher, C. J.; Halvorson, D. A.; Newman, J. A., and Senne, D. A.
Isolation of avian paramyxoviruses from sentinel ducks and turkeys in
Minnesota. Avian Dis. 1985 Apr-1985 Jun 30; 29(2):400-7.

McBride, M. D.; Hird, D. W.; Carpenter, T. E.; Snipes, K. P.;
Danaye-Elmi, C., and Utterback, W. W. Health survey of backyard poultry
and other avian species located within one mile of commercial California
meat-turkey flocks. Avian Dis. 1991 Apr-1991 Jun 30; 35(2):403-7.

McCluskey, Brian J. Epidemiology of Vesicular Stomatitis Viruses in the
Southwestern United States: Colorado State University; c2003.

Sinnecker, H.; Sinnecker, R., and Zilske, E. Detection of influenza A
viruses by sentinel domestic ducks in an ecological survey. Acta Virol.
1982 Jan; 26(1-2):102-4.

Sinnecker, H.; Sinnecker, R.; Zilske, E., and Koehler, D. Detection of
influenza A viruses and influenza epidemics in wild pelagic birds by
sentinels and population studies. Zentralbl Bakteriol Mikrobiol Hyg
\[A\]. 1982 Dec; 253(3):297-304.

Slemons, R. D.; Hansen, W. R.; Converse, K. A., and Senne, D. A. Type A
influenza virus surveillance in free-flying, nonmigratory ducks residing
on the eastern shore of Maryland. Avian Dis. 2003; 47(3 Suppl):1107-10.

Slemons, R. D.; Johnson, D. C.; Osborn, J. S., and Hayes, F. Type-A
influenza viruses isolated from wild free-flying ducks in California.
Avian Dis. 1974 Jan-1974 Mar 31; 18(1):119-24.

Slemons, R. D.; Shieldcastle, M. C.; Heyman, L. D.; Bednarik, K. E., and
Senne, D. A. Type A influenza viruses in waterfowl in Ohio and
implications for domestic turkeys. Avian Dis. 1991 Jan-1991 Mar 31;
35(1):165-73.

Stallknecht, D. E. Ecology and epidemiology of avian influenza viruses
in wild bird populations: waterfowl, shorebirds, pelicans, cormorants,
etc. Proceedings of the Fourth International Symposium on Avian
Influenza; Athens, GA. U.S. Animal Health Association.

Stallknecht, D. E. and Shane, S. M. Host range of avian influenza virus
in free-living birds. Vet Res Commun. 1988; 12(2-3):125-41.

Stallknecht, D. E.; Shane, S. M.; Zwank, P. J.; Senne, D. A., and
Kearney, M. T. Avian influenza viruses from migratory and resident ducks
of coastal Louisiana. Avian Dis. 1990 Apr-1990 Jun 30; 34(2):398-405.

Turek, R.; Gresikova, M., and Tumova, B. Isolation of influenza A virus
and paramyxoviruses from sentinel domestic ducks. Acta Virol. 1984 Mar;
28(2):156-8.

Webster, R. G.; Bean, W. J.; Gorman, O. T.; Chambers, T. M., and
Kawaoka, Y. Evolution and ecology of influenza A viruses. Microbiol Rev.
1992 Mar; 56(1):152-79.

Webster, R. G.; Morita, M.; Pridgen, C., and Tumova, B. Ortho- and
paramyxoviruses from migrating feral ducks: characterization of a new
group of influenza A viruses. J Gen Virol. 1976 Aug; 32(2):217-25.

# ATTACHMENT 7

# 

# Environmental Sampling (Feces/Water)

## Introduction

Avian Influenza viruses (AI) are released by waterfowl through the
intestinal tract and viable virus can be detected in both feces and the
water in which the birds swim, defecate and feed. This is the principle
means of virus spread to new avian hosts and potentially to poultry and
other susceptible livestock. Analysis of both water and fecal material
from waterfowl habitat can provide evidence of AI virus circulating in
wild bird populations, the specific AI subtypes, levels of
pathogenicity, and possible risks to livestock.

### [Technical Aspects of Sampling Water]{.underline}

AI is relatively stable in water, especially at colder temperatures. The
longevity of viable virus (weeks to months) allows for an integration of
activity on a site basis. However, in the absence of established serial
sampling, pinpointing the time at which a site becomes contaminated
would be difficult. The advantages of including waterfowl sites as a
point of sampling lie in the ease of collecting samples and the
potential to sample the potential contaminating influence many birds at
once. This method would provide a cost-effective, geographically
explicit methodology. Moreover, given the ease of sampling, more sites
could be sampled, providing for a higher resolution surveillance
network.

Technical aspects to monitor water samples for AI involve collecting
specified volumes of water (usually 50-500ml), transporting the samples
on ice or frozen, concentrating the virus present either by filtration
or precipitation/centrifugation, and inoculating the virus onto chicken
eggs or cell culture for virus growth. The virus replicates in the
cultures and is characterized by serological or molecular methods to
determine specific subtypes. Alternative methods of analysis involve
extracting viral genetic material from the sample with detection using
molecular techniques such as reverse transcription-polymerase chain
reaction (RT-PCR) and subsequent sequencing to determine subtype.
Refinement of these methods still needs to be done. However, these
techniques have the advantage of rapid results. All of the procedures
for monitoring AI in water samples are generally established, and with
proper expertise and equipment can be easily adapted to most laboratory
settings.

### [Technical Aspects of Sampling Feces]{.underline}

Fecal sampling is used extensively in monitoring studies for AI in wild
bird populations. The principal advantages of this method are that the
costs and effort of capturing birds are avoided and large sample numbers
can be quickly and easily obtained. It also is a good method to
determine the presence or absence of virus in bird populations present
at a specific location. The disadvantage is that species identification
is not always possible, determination of prevalence is complicated by
the possibility of repeated sampling of individuals, and the sensitivity
of the method is lower than for oral swabs. However, the increased
sampling effort occurring because of reduced sampling costs are
anticipated to offset any short comings owing to decreased sensitivity.
Information from the field could be used to generate an environmental
risk map related to specific areas (habitats) associated with potential
AIV transmission.

Infectivity of the virus is maintained up to 4 days in wet feces at
25^o^C. Best analytical results come from fresh fecal samples that are
either processed quickly or frozen until processing. Thus this method of
sampling, while providing good information, is best applied while birds
are present at a location such that the samples are as fresh as
possible. By restricting fecal collection to fresh samples, it allows
for population census data to be collected, and by inference estimates
of the species sources of the contamination. Species and individual
identification through genetic typing of feces would allow estimates of
prevalence.

Accredited laboratories have the capacity and infrastructure to analyze
a limited number of samples for AI. The anticipated sampling effort for
this surveillance study will require an investment in equipment and
staff to provide results in a timely fashion. Equipment needs include
real-time PCR thermalcyclers, RNA extraction capabilities, DNA
sequencing capabilities, tissue culture and egg culture facilities,
ultracold freezers, centrifuges and vacuum pumps.

## Methodology

Sampling for highly pathogenic avian influenza (HPAI), such as highly
pathogenic H5N1 avian influenza, from environmental deposition of virus
by waterfowl should be accomplished by collecting and analyzing feces
(Attachment 10) and water from areas of known use by high risk species
(e.g., transcontinental migrants). The general challenges faced
include; 1) Determining locations used by high risk species, 2)
Refinement of existing methods for detecting the virus in water and
fecal samples and developing the analytical infrastructure and capacity,
3) Design of a sampling system using composite samples for analysis.

Table 7-1. Qualitative comparison of environmental sampling methods.

  ----------------------------------- -----------------------------------
  **Fecal Sampling**                  

  **Pros**                            **Cons**

  Technically easy sample             Viable virus restricted to fresh
  acquisition. Sampling represents    samples (1-4 days)
  non technical approach and would    
  not require extensive training or   
  experience by field personnel.      

  Generate large sample numbers       Large sample numbers can swamp lab
  quickly.                            systems (applies to all methods)

  Does not require handling or        
  capturing animals                   

  Low cost, well established          
  technique amenable for high         
  through-put screening (modified     
  APHIS RT-PCR method). Sample        
  analysis is transferable across     
  labs.                               

  Capable of identifying HPAI         Identity of species and individuals
  contaminated                        unlikely, estimates of prevalence
  sites/locations/regions. Prevalence not possible. Species
  would be estimated on a site basis. identification possible through
  Information from the field could be molecular fingerprinting, but at
  used to generate an environmental   additional cost.
  risk map related to specific areas  
  (habitats) associated with          
  potential AIV transmission.         

  BSL-2 laboratory conditions         Requires Biosafety level 3
  sufficient for initial diagnostic   capabilities for virus isolation
  screening.                          

  **[Summary]{.underline}**: An       
  approach based on fecal sampling    
  could be immediately implemented    
  and may represent the only          
  reasonable approach in areas where  
  bird capture is not practical.      

  **Water Sampling**                  

  **Pros**                            **Cons**

  Low cost                            Biosafety level 3 capabilities for
                                      virus isolation

  Effectively sample all or most      Analyses potentially complicated if
  birds present on the body of water  multiple strains of AI present in
                                      water samples.

  Samples easily, quickly obtained    Large volumes of water needed to
                                      concentrate virus for analysis,
                                      transportation and logistical issue

  Virus stable, especially at         Longevity complicates
  moderate pH and low temperature     interpretation on initial timing of
                                      contamination.

  Does not require handling or        Identity of species and individuals
  capturing animals                   not possible/difficult. Prevalence
                                      calculation restricted to a site
                                      basis system.

  Generate large sample numbers       Large sample numbers can swamp lab
                                      systems- need analysis
                                      infrastructure

  Can provide large scale spatial     May need to validate technique
  risk assessment of HPAI             
  contamination.                      
  ----------------------------------- -----------------------------------

Sampling strategies to detect highly pathogenic H5N1 avian influenza
virus in waterfowl populations will change depending upon the risk
assessment and management goals and prevailing status of the pathogen in
North America. For first detection of highly pathogenic H5N1 avian
influenza virus in migratory birds efforts should focus on likely
cross-over routes of birds from Asia to North America (e.g., Alaska and
North Slope). Efforts should focus on areas of high aggregations of
waterfowl intersecting with logistical sampling support, e.g., National
Wildlife Refuge (NWR) system and state waterfowl management areas. While
highly pathogenic H5N1 avian influenza virus may cross from Asia to
North America at any point the surveillance network needs to be
tactically practical without compromising its ability for detection.
Once highly pathogenic H5N1 avian influenza virus gains a foothold in
North America the surveillance network should be placed along known
waterfowl movement paths from the point of origin (i.e., point of
detection). These paths can be inferred from known migration routes
based on waterfowl telemetry data. However, practically, and given the
patterns emerging in Eurasia, once highly pathogenic H5N1 avian
influenza virus gains a foothold in North America the likelihood of
rapid and diffusive spread across the continent is high. At this point
local waterfowl and environmental sampling should target areas of
strategic value, e.g., human population centers and areas of high
density of poultry production. In the former case, such areas would be
represented by urban zoo-parks and lakes. These areas would represent
the highest level of risk of human contact with contaminated water
and/or waterfowl. In the latter case, ponds, lakes and waterfowl
management areas around high density poultry production areas would
provide the best ability to assess risk of transmission to humans and
poultry. Surveillance efforts patterned on these areas are best amenable
to local and state efforts for first detection and subsequent risk
assessment once the highly pathogenic H5N1 avian influenza virus
achieves enzootic status in North America.

There is an inherent conflict between the need for high resolution
surveillance, the number of samples generated, the time to analyze those
samples, and the cost of analysis. If the goal is first detection,
methods that integrate across many individuals and species at a
particular site without loss of sensitivity should be preferred.
Currently analysis of fresh fecal samples is the best method to achieve
these goals. For logistically practical and economic reasons sample
analysis should focus on composite samples on a per site basis; this
bulk sample minimizes effort in both data collection and analysis, while
greatly increasing the probability of detection. Given the expected
rarity of highly pathogenic H5N1 avian influenza virus in current
migratory bird populations, this approach will allow for a substantially
reduced number of samples to be analyzed. Table 7-2 provides a
hypothetical, but plausible, example of the expected number of tests per
composite fecal sample necessary to detect Highly pathogenic H5N1 avian
influenza virus. When prevalence is very low (e.g., 10^-7^) almost all
composites will test negative and on average only a single test will be
needed to determine the absence of highly pathogenic H5N1 avian
influenza virus in that composite sample.

The approximate sample sizes necessary for assuring a high probability
of detecting highly pathogenic H5N1 avian influenza virus depends on its
prevalence in the population, which is currently unknown. However, a
preliminary estimator is:

p\* = 1- (1-r/m)^1/n^ (eq. 1)

where p\* is the proportion of infected individual samples across all
composite samples, r is the number of composite samples that test
positive for the presence of highly pathogenic H5N1 avian influenza
virus, m is the total number of composite samples tested, and n is the
number of individual samples in each composite sample (e.g., fecal count
or volume). Rearranging eq. 1 provides an estimate of the number of
individual fecal samples needed to detect highly pathogenic H5N1 avian
influenza virus, for a given population level prevalence;

n = ln(1-r/m) / ln(1-p^\*^) (eq. 2)

Table 7-2. Expected number of tests needed for a single positive
reaction for each composite sample containing 100 individual fecal
samples, *n*, as a function of expected prevalence of HPAI, *p*.
Calculation is based on the binomial probability model describing the
average number of tests needed as (*n*+1) -- *n*(1-*p*)*^n^*

The results for various hypothetical values of r, m, n, and p^\*^ are
shown in Table 7-2. Thus, if highly pathogenic H5N1 avian influenza
virus prevalence is 10^-6^ and 10,000 independent fecal samples are
collected, analysis of 100 composite samples would result in detecting
the presence of highly pathogenic H5N1 avian influenza virus in one
composite. These two equations allow us to initially estimate the number
of fecal samples to be collected and to estimate prevalence of highly
pathogenic H5N1 avian influenza virus in the population.

Table 7-3. Number of individual fecal samples *n*, for a fixed
prevalence *p^\*^*, needed to detect the presence of HPAI in 1 out of
100 composite samples. Calculation is based on the probability model
given by eq. 2.

## Safety

Given the concern of introduction of highly pathogenic H5N1 avian
influenza virus into North America, and the potential for human
infection, significant precautions should be taken by workers conducting
the environmental sampling and those handling the samples in the
laboratory. In the lab, standard BSL-3 precautions are required for
virus isolation, and BSL-2 precautions for molecular diagnostics. In the
field, workers should wear disposable gloves and garments. Gloves should
be decontaminated with 70% ethanol frequently, or changed often as
necessary. Mucous membranes (eyes, nose, throat) should be protected
from splashes and aerosols. This may require covering with protective
equipment such as goggles and hepafiltered masks in some cases. Field
workers should avoid direct contact with animals after handling
environmental samples until decontamination procedures are completed
(e.g. changing garments and gloves). Untrained workers (such as the
general public) should be discouraged from collecting and submitting
environmental samples for testing.

## Summary

Monitoring of water and/or fecal samples gathered from waterfowl habitat
is a reasonably cost effective, technologically achievable means to
assess risks to poultry in the western hemisphere to new, potentially
highly pathogenic subtypes of AI. A surveillance system based on water
sampling is not ready to implement. However, the validation of this
method could come on-line in a short period of time and would represent
considerable cost savings without loss of sensitivity. Fecal sampling is
an established technique and is ready for use in surveillance with the
establishment of sampling guidelines. Both approaches yield advantages
where individual bird sampling is too costly or logistically
impractical. Either approach could yield a spatial and habitat risk
assessment for site contamination with highly pathogenic H5N1 avian
influenza virus. The main considerations are where and when to get the
samples, ensuring proper storage and transport, and the capacities and
capabilities of the laboratories doing the analyses. Real-time reporting
and the infrastructure to support such reporting is a serious constraint
on any surveillance system. The ability to integrate, analyze, and
responsibly disseminate these data is critical and needs to be
addressed.

# ATTACHMENT 8

# Instructions for Collection and Shipment of Avian Carcasses for Diagnostic Evaluation

The following are general guidelines for collecting and shipping
wildlife carcasses to veterinary diagnostic labs to insure adequate and
well preserved specimens. Field biologists should contact the specific
laboratory that they will be working with well in advance of any
specimen collection and shipping to receive specific instruction for
specimen submissions to that lab. Labs should always be notified ahead
of time when a shipment is being made to their facility. Once you have
determined what equipment and supplies will be needed for specimen
shipping, keep adequate numbers of shipping containers, frozen ice
packs, shipping labels and packing materials available at all times. If
you plan to collect animals while in the field, take along a cooler with
ice packs to chill the carcasses.

1\. More than one disease may be affecting the population
simultaneously. Different species may have varying susceptibility to
disease agents. Therefore, collect and ship specimens representative of
all species and geographic areas affected.

> Obtain good specimens for necropsy. Carcasses that are decomposed or
> scavenged are unacceptable. If the carcass has an odor, is soft and
> mushy, has skin discoloration, feathers or skin that easily rubs off,
> or has maggots present, it is too decomposed for testing.

2\. Collect animals under the assumption that an infectious disease or
toxic substance is involved and other animals or humans may be at risk.
Remember to protect yourself as some of these diseases and toxins are
hazardous to humans. Guidelines for personal protection against disease
exposure for individuals working with sick or dead wild animals can be
obtained from the USGS National Wildlife Health Center, the Centers for
Disease Control and Prevention, and OSHA websites.

> Always wear latex or nitrile gloves when picking up sick or dead
> animals. If you are dealing with a significant number of dead animals,
> or you suspect the presence of a zoonotic disease agent, additional
> protective equipment including coveralls, eye protection and N95
> respiratory protection should be used.
>
> Attach a leg tag to each animal with the following information in
> pencil/waterproof ink:

-   species![](media/image6.png){width="2.615972222222222in"
    height="1.1194444444444445in"}

-   date collected

-   location (state, county, location name, and latitude/longitude if
    available)

-   found dead or euthanized

-   collector (name/address/phone)

-   additional history or comments on back of tag

> Place each animal in a plastic bag, tie shut, then place inside a
> second bag and tie shut. This system of double bagging prevents
> cross-contamination of individual specimens and leaking shipping
> containers that can contaminate vehicle surfaces and handlers during
> transportation. Contact the diagnostic lab for guidance in assistance
> with collecting samples from animals that are too large to
> ship.![](media/image7.png){width="2.5631944444444446in"
> height="2.3361111111111112in"}
>
> 3\. Ship animals in a sturdy hard sided plastic cooler. These coolers
> can be disinfected and returned to you if a pre-paid shipping label or
> commercial shipping company account number is provided to the
> diagnostic lab. Be sure to provide a street address for return of the
> cooler.
>
> Line the shipping cooler with a large plastic bag and pack the
> individually bagged animal(s) in the cooler with enough blue ice to
> keep carcasses cold. Disperse blue ice packs among the carcasses so
> that all carcasses are kept chilled. If you are shipping blood tubes,
> culture tubes, or other specimen containers along with the carcasses,
> these specimens should be placed within a sturdy cardboard or plastic
> box or screw cap container with padding material to prevent breakage.
> That container should be place next to blue ice packs within the large
> cooler. Do not use bagged wet ice for shipments in order to avoid
> fluid leakage during shipment. Do not use dry ice unless instructed to
> do so by the diagnostic lab. Place crumpled newspaper or similar
> absorbent material in the cooler with the bagged carcasses to fill
> unused space, hold the ice in contact with carcasses, provide
> insulation, and absorb any liquids. Tape the cooler shut with sturdy
> strapping tape.![](media/image8.png){width="2.2215277777777778in"
> height="2.1083333333333334in"}![](media/image9.png){width="2.5569444444444445in"
> height="2.1486111111111112in"}
>
> Place a detailed history of the animal and circumstances associated
> with the mortality event in a paper envelope or a plastic sleeve and
> tape it to the outside of the cooler. A copy of this history should be
> faxed or e-mailed to the diagnostic lab at the time of shipment. A
> standard wildlife specimen history form can be found on the last page
> of these instructions.
>
> 4\. Prior to shipping contact the diagnostic lab to inform them of the
> type and number of specimens being shipped. Ship specimens for next
> day delivery (overnight service) from Monday through Wednesday to
> guarantee arrival at the diagnostic lab before the weekend. If
> specimens are fresh and need to be shipped on Thursday or Friday
> contact the diagnostic lab to make special arrangements for receipt of
> specimens.
>
> Freezing and thawing can make isolation of some pathogens difficult
> and damage tissues needed for microscopic examination. Diagnostic labs
> prefer unfrozen specimens if they can be sent within 24 -- 48 hours of
> collection or death. The diagnostic lab can provide guidance on when
> or if to freeze samples on a case-by-case basis. If you are in the
> field and cannot call or ship within 24-48 hours, freeze the
> animal(s).

5.  Prior to shipping contact the commercial shipping company to obtain
    guidelines for shipping diagnostic or biological specimens. Label
    coolers with clear, legible labels including the diagnostic lab
    name, street address, and telephone number. In addition to the
    mailing address, attach a label reading "**DIAGNOSTIC SPECIMENS
    --WILDLIFE"** to the side of the cooler. If dry ice was used in the
    shipment a standard dry ice warning label will be required. These
    can be obtained from the shipping company. [Please make note of the
    tracking number]{.underline} [in case packages are
    delayed.]{.underline}

**Wildlife Specimen History Form**

**Always contact the diagnostic lab before shipping specimens!**

Submitter's name: Affiliation:

Address: Telephone: E-mail:

Date collected: Collector\'s Name:

Method of collection: \[found dead, euthanized (describe method) etc.\]

Method of storage: \[chilled, frozen, fixed, etc.\]

Species Submitted:

Specific die-off location:

State: County: Latitude/longitude:

Environmental factors: (Record conditions such as storms, precipitation,
temperature changes, or other changes that may contribute to stress.)

Disease onset: (The best estimate of when the outbreak started.)

Species affected: (The diversity of species affected may provide clues
to the disease involved.)

Age/sex: (Any selective mortality related to age and sex.)

Morbidity/mortality: (Ratio of sick animals to dead animals.)

Known dead: (Actual carcass count)

Estimated dead: (Consider removal by scavengers or other means.)

Clinical signs: (Any unusual behavior and physical appearance.)

Population at risk: (Number of animals in the area that could be exposed
to the disease.)

Population movement: (Recent changes in the number of animals on the
area and their source or destination, if known.)

Problem area description: (Land use, habitat types, and other
distinctive features.)

Comments: (Additional information/observations that may be of value such
as past occurrences of disease in area.)

**PLEASE USE ADDITIONAL SHEETS IF NECESSARY.**

# ATTACHMENT 9

# Protocol for the Collection of Tracheal/Cloacal Swab Samples

1.  Contact Laboratory to determine specific protocol to use.
    Laboratories may request samples be placed in tubes containing Viral
    Transport Medium (VTM) or brain-heart infusion broth (BHI).

2.  Thaw appropriate number of pre-labeled tubes of Viral Transport
    Medium (VTM) or brain-heart infusion broth (BHI) at refrigerator
    temperature (4 °C) overnight and keep chilled with wet/blue ice
    packs in a cooler during the day of collection.

3.  Unwrap a Dacron swab from the stem-end of the packaging.

4.  Remove swab and insert the entire head of the swab into the trachea
    or cloaca. Use gentle pressure and in a circular motion, swab the
    inside circumference of the trachea/cloaca two or three times.

5.  ![](media/image10.jpeg){width="3.75in" height="2.8125in"}For Cloacal
    swabs, shake off large pieces of feces.

6.  Inserting the swab into the tube containing VTM or BHI broth. With
    the swab in the media, swirl the stem end of the swab between
    fingers vigorously. Lift the swab approximately ¼" from the bottom
    of the vial and bend the stem over the edge of the vial to break off
    the stem so that the swab remains in the vial and the cap can be
    screwed tight.

The entire swab end and a portion of the stem will be left in the tube.
If the stems are unable to be broken (some small swabs will have metal
stems) then they can be cut with scissors. Scissors should be wiped with
70% alcohol each time they are used to cut a stem.

7.  Record sample tube number on banding sheet or the Sample History
    Sheet along with date, species, age, sex, and location data (GIS
    coordinates if possible)..

8.  Replace tube into cooler for transport back to the base camp.
    Samples should be kept cold (\<4 °C, frozen if possible) and out of
    direct sunlight.

9.  At camp, transfer tubes into liquid nitrogen shippers or into a
    freezer as soon as possible. Note any exceptions to the collection
    or storage conditions in field sheets and note such information on
    the \"Sample History and Packing List Form\".

10. Place tubes into a hard plastic shipping container with enough
    frozen gel packs to keep samples cold for at least two days.

11. Notify laboratory that samples are being shipped, the method of
    shipment (FEDEX is preferred), and the expect date of arrival.
    Packages should only be shipped on Monday, Tuesday, or Wednesday.

**Sample History and Packing List**

**H5N1 Avian Influenza Project**

Submitter
\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Address
\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

Phone
\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

E mail
\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

<table>
<colgroup>
<col style="width: 8%" />
<col style="width: 8%" />
<col style="width: 8%" />
<col style="width: 8%" />
<col style="width: 8%" />
<col style="width: 8%" />
<col style="width: 8%" />
<col style="width: 8%" />
<col style="width: 8%" />
<col style="width: 8%" />
<col style="width: 8%" />
<col style="width: 8%" />
</colgroup>
<tbody>
<tr class="odd">
<td><blockquote>
<p>Comments or Sampling problems</p>
</blockquote></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td><blockquote>
<p>Location</p>
</blockquote></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td><blockquote>
<p>Band Number</p>
</blockquote></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td><blockquote>
<p>Sex</p>
</blockquote></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td><blockquote>
<p>Age</p>
</blockquote></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td><blockquote>
<p>Bird Species</p>
</blockquote></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td><blockquote>
<p>Sample ID</p>
</blockquote></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
</tbody>
</table>

# ATTACHMENT 10

# Fecal Sampling and Shipping Protocol

## Fecal Sampling

### [Purpose]{.underline}

The purpose of this standard operating procedure is to describe the
essential elements of proper handling and collection of field fecal
samples for surveillance of avian influenza.

### [Procedures]{.underline} 

-   Before collecting samples, personnel should don the appropriate
    personal protective equipment (PPE). These include latex or nitrile
    gloves and face shields, and if it is determined to be necessary,
    PAPRs.

-   Label plastic whirl pack bags with necessary information including
    date, location (GPS coordinate if possible), species (if possible),
    investigator and sample identification. Record all required
    information on data sheets.

-   Feces must be less than 24 hours old. Feces should appear moist.

-   For collection, turn a sterile Whirl-Pak inside out and pick up
    feces using the Whirl-Pak as a glove, then turn the bag right side
    in with the feces inside the closed bag. Release as much air from
    the inside of the bag.

-   Label the Whirl-Pak using an indelible ink marker. The sample should
    be labeled with the sample number, date, time, collector\'s name,
    location, and Quality Assurance number (Protocol Number). This
    latter information describes in detail the context, purpose, and
    other procedural and review information of the samples collected.

-   Place the Whirl-Paks with fecal samples into a large zip-lock bag,
    tape (e.g., duct tape or packaging tape) the opening and label the
    outer bulk bag with name, date, location, and Protocol number.

-   Place the bulk collection bag in a cooler with wet or blue ice to
    keep the specimen cool. This precaution is for maximizing the
    chances for subsequent viral isolation. Pack samples with enough ice
    or frozen gel packs to keep samples cold for at least two days.

-   Maintain the temperature of samples as constant as possible.

-   Change gloves if soiled or contaminated. When finished collecting,
    wash hands with suitable antibacterial agent.

-   Notify laboratory that samples are being shipped, the method of
    shipment (FEDEX is preferred), and the expect date of arrival.
    Packages should only be shipped on Monday, Tuesday, or Wednesday;
    this allows the laboratories time to process samples during a normal
    work-week, or allows for tracking if the shipment is delayed.

## Shipping

### [Purpose]{.underline}

The purpose of this standard operating procedure is to ensure diagnostic
specimens are shipped safely and in compliance with governing
regulations and requirements. Shippers of diagnostic specimens where a
relatively low probability exists that infectious substances are present
(diagnostic specimens being transported to undergo routine screening
tests or for the purpose of initial diagnosis may be considered to fall
under this category) must comply with the International Air
Transportation Association (IATA) Dangerous Goods Regulations. The
shipper must also ensure that shipments are prepared in such a manner
that they arrive at their destination in good condition and that they
present no hazard to persons or animals during shipment.

### [Procedures:]{.underline} 

-   Federal Express is the preferred carrier for the USDA/NWRC. Use next
    day service.

-   The inner packaging (appropriately labeled) must be comprised of a
    watertight primary receptacle, and must no exceed 500 ml total
    volume. Primary receptacles include those of glass, metal, or
    plastic (i.e., test tube, plastic jar, or taped zip-loc bag).
    Positive means of ensuring a leak-proof seal must be provided. Screw
    caps on primary receptacles must be reinforced with adhesive tape.

-   A secondary packaging (also water tight) must be used, but must not
    exceed 4 L total volume.

-   An absorbent material sufficient to absorb the entire contents of
    all primary receptacles must be placed between the primary
    receptacle and the secondary packaging.

-   The outer packaging (i.e., cardboard box or cooler) must be of
    adequate strength for its capacity, weight, and intended use
    (capable to withstand being dropped at least 1.2 meters, without
    leakage of the primary receptacle or significant damage to the outer
    packaging).

-   An itemized list of contents must be enclosed between the secondary
    packaging and the outer packaging. To protect against possible
    leakage, the list should be enclosed in a sealed plastic bag.

-   A Shippers Declaration for Dangerous Goods is not required. However,
    both the air waybill and the outer box must show the text
    \"***DIAGNOSTIC SPECIMEN PACKED IN COMPLIANCE WITH IATA PACKING
    INSTRUCTION 650\"***.

# ATTACHMENT 11

# Veterinary Laboratories Currently Certified to Conduct Highly pathogenic H5N1 avian influenza Virus Diagnostics

  ----------------------------------------------------------------------------------------------------------------------------------------------------------------
  **State**   **Laboratory   **Laboratory   **Telephone**   **Shipping     **Shipping   **City**       **Zip code** **Lab Director E-Mail**           **AI/END
              Director**     Name**                         address 1**    address 2**                                                                Contact**
  ----------- -------------- -------------- --------------- -------------- ------------ -------------- ------------ --------------------------------- ------------
  AL                         Charles S.     334-844-4987    1001 Wire Road              Auburn         36830                                          Dr. Fred
                             Roberts                                                                                                                  Hoerr
                             Veterinary                                                                                                               
                             Diagnostic Lab                                                                                                           

  AR          Dr. Konnie     Arkansas       501-907-2400    One Natural                 Little Rock    72205        kpluml@arlpc.org                  Dr. Paul
              Plumlee        Livestock &                    Resources Dr.                                                                             Norris
                             Poultry                                                                                                                  
                             Commission Lab                                                                                                           

  AZ          Dr. Greg       Arizona        520-621-2356    2831 N.                     Tucson         85705        gabrad@ag.arizona.edu             Dr. Greg
              Bradley        Veterinary                     Freeway                                                                                   Bradley
                             Diagnostic                                                                                                               
                             Laboratory                                                                                                               

  CA          Dr. Alex       California     530-752-8709    University of  W. Health    Davis          95616        aaardans@ucdavis.edu              Dr. Alex
              Ardans         Animal Health                  California,    Science                                                                    Ardans
                             & Food Safety                  School of Vet  Drive                                                                      
                             Lab                            Med                                                                                       

  CO          Dr. Barbra     Colorado State 970-297-1281    College of     300 West     Fort Collins   80523        bep@lamar.colostate.edu           Dr. Barbara
              Powers         University                     Vet. Med. &    Drake                                                                      Powers
                             Veterinary                     Biomedical                                                                                
                             Diag. Lab                      Sciences                                                                                  

  CT          Dr. Herbert    Department of  860-486-0837    University of  61 N.        Storrs         06269-3089   herbert.vandruiningen@uconn.edu   Dr. Sandra
              Van Kruiningen Pathobiology &                 Connecticut,   Eagleville                                                                 Bushmich
                             Veterinary                     Unit 3089      Rd.                                                                        
                             Science                                                                                                                  

  DE                         University of  302-856-1997    16684 County                Georgetown     19947                                          Dr. Mariano
                             Delaware                       Seat Hi-Way                                                                               Salem
                             Poultry                                                                                                                  
                             Laboratory                                                                                                               

  FL          Dr. Betty      Kissimmee      407-846-5200    Florida        2700 N. John Kissimmee      34745        miguelb@doacs.state.fl.us         Dr. Betty
              Miguel         Diagnostic                     Department of  Young                                                                      Miguel
                             Laboratory                     Agriculture    Parkway                                                                    

  GA                         Georgia        770-535-5996    4457 Oakwood                Oakwood        30566                                          Dr. James
                             Poultry                        Road                                                                                      Scroggs
                             Laboratory                                                                                                               

  GA          Dr. Doris      Athens         706-542-5568    University of  Building     Athens         30602        miller@vet.uga                    Dr. Doris
              Miller         Veterinary                     Georgia        1079                                                                       Miller
                             Diagnostic                     College of Vet                                                                            
                             Laboratory                     Med                                                                                       

  GA          Dr. Charles A. University of  229-386-3340    43 Brighton                 Tifton         31793-3000   cbaldwin@uga.edu                  Dr. Charles
              Baldwin        Georgia                        Road                                                                                      A. Baldwin
                             Veterinary                                                                                                               
                             Diag.                                                                                                                    
                             Laboratory                                                                                                               

  HI          Dr. David T.   State          808-453-5990    2725 Waimano                Pearl City     96782        david.horio@doh.hawaii.gov        Dr. David T.
              Horio          Laboratories                   Home Road                                                                                 Horio
                             Division                                                                                                                 

  IA          Dr. Bruce      Iowa State     515-294-1950    Veterinary     1600 S. 16th Ames           50011        bhjanke@iastate.edu               Dr.
              Janke          University                     Diagnostic     St.                                                                        Kyoung-Jin
                                                            Laboratory                                                                                Yoon

  IN          Dr. Leon       Purdue         765-494-7460    406 South                   West Lafayette 47907        thackerl@purdue.edu               Dr. Leon
              Thacker        University                     Lafayette                                                                                 Thacker
                             Animal Disease                                                                                                           
                             Diagnostic Lab                                                                                                           

  LA          Dr. H.W.       Louisiana      225-578-9777    Veterinary Med 1909 Skip    Baton Rouge    70803        hwt@vetmed.lsu.edu                Dr. Alma Roy
              Taylor         State                          Diag.          Bertman                                                                    
                             University                     Laboratory     Drive                                                                      

  MD          Dr. Daniel     Maryland Dept. 410-543-6610    27722                       Salsbury       21801                                          Dr. Daniel
              Bautista       of Ag & Animal                 Nanticoke Road                                                                            Bautista
                             Health                                                                                                                   
                             Laboratory                                                                                                               

  MI          Dr. Willie     Diagnostic     517-353-0635    Michigan State 4125         Lansing        48910        reed@dcpah.msu.edu                Dr. Willie
              Reed           Center of                      University     Beaumont Rd,                                                               Reed
                             Population and                                Ste 201H                                                                   
                             Animal Health                                                                                                            

  MN          Dr. James E.   Minnesota      612-625-8787    University of  1333 Gortner St. Paul       55108        colli002@unm.edu                  Dr. James E.
              Collins        Veterinary                     Minnesota, Vet Ave, 244 Vet                                                               Collins
                             Diagnostic                     Diag Lab       D L                                                                        
                             Laboratory                                                                                                               

  MO          Dr. Alex       University of  573-882-6811    Veterinary     1600 East    Columbia       65211        bermudeza@missouri.edu            Dr. Stanley
              Bermudez       Missouri                       Medical        Rollins                                                                    Casteel
                                                            Diagnostic                                                                                
                                                            Laboratory                                                                                

  MS          Dr. Lanny Pace Mississippi    601-354-6089    2531 North                  Jackson        39216        pace@cvm.msstate.edu              Dr. Lanny
                             Vet Research &                 West Street                                                                               Pace
                             Diagnostic                                                                                                               
                             Laboratory                                                                                                               

  NC          Dr. Gene       North Carolina 919-733-3986    Rollins Animal 2101 Blue    Raleigh        27607        gene.erickson@ncmail.net          Dr. Gene
              Erickson       Department of                  Disease        Ridge Rd.                                                                  Erickson
                             Agriculture                    Diagnostic Lab                                                                            

  NE          Dr. David      Veterinary     402-472-1434    University of  137 VDC UNL  Lincoln        68583-0907   dsteffen1@unl.edu                 Dr. David
              Steffen        Diagnostic                     Nebraska                                                                                  Steffen
                             Center                                                                                                                   

  NJ          Dr. Robert     New Jersey     609-984-2293    State          Rm 201 John  Trenton        08625        rjeisner1@comcast.net             Dr. Robert
              Eisner         Dept of Ag,                    Diagnostic     Fitch Plaza,                                                               Eisner
                             Division of                    Lab, H & A     P.O. Box 330                                                               
                             Animal Health                  Building                                                                                  

  NM          Dr. Flint      New Mexico     505-841-2576    Veterinary     700 Camino   Albuquerque    87106        ftaylor@nmda.nmsu.edu             Dr. David
              Taylor         Department of                  Diagnostic     de Salud, NE                                                               Mills
                             Agriculture                    Services                                                                                  

  NV          Dr. Anette     Nevada Animal  775-668-1182    Nevada         350 Capitol  Reno           89502-2923   arink@govmail.state.nv.us         Dr. Anette
              Rink           Disease                        Department of  Hill Ave.                                                                  Rink
                             Laboratory                     Agriculture                                                                               

  NY          Dr. Alfonso    Animal Health  607-253-4136    Cornell        S3 110       Ithaca         14853        at97@cornell.edu                  Dr. Sung Kim
              Torres         Diagnostic                     University,    Schurman                                                                   
                             Center                         College of     Hall, Upper                                                                
                                                            Vet. Med.      Tower Rd.                                                                  

  OH          Dr. Beverly    Ohio           614-728-6220    Animal Disease 8995 E. Main Reynoldsburg   43068        byrum@mail.agri.state.oh.us       Dr. Beverly
              Byrum          Department of                  Diagnostic     Street,                                                                    Byrum
                             Agriculture                    Laboratory     Building 6                                                                 

  OK          Dr. Bill J.    Oklahoma       405-744-6623    Oklahoma State Farm Road &  Stillwater     74078        billyjj@cvm.okstate.edu           Dr. W. C.
              Johnson        Animal Disease                 Univ., College Ridge Road                                                                 Edwards
                             Diagnostic                     of Vet. Med.                                                                              
                             Laboratory                                                                                                               

  OR          Dr. Jerry      Oregon State   541-737-3261    Oregon State   30th &       Corvallis      97331        jerry.heidel@oregonstate.edu      Dr. Jerry
              Heidel         Veterinary                     Univ., College Washington                                                                 Heidel
                             Diagnostic Lab                 of Vet. Med.                                                                              

  PA          Dr. Helen      Pennsylvania   717-787-8808    2305 N.                     Harrisburg     17110        hacland@state.pa.us               Dr.
              Acland         State Vet                      Cameron Street                                                                            Deepanker
                             Diagnostic                                                                                                               Tewari
                             Laboratory                                                                                                               

  PA                         University of  610-925-6210    Lab of Large   New Bolton   Kennett Square 19348-1692                                     Dr. Sherrill
                             Pennsylvania                   Animal         Center, 382                                                                Davison
                                                            Pathology &    West Street                                                                
                                                            Toxicology     Rd                                                                         

  SC          Dr. Pamela     Clemson        803-788-2260    500 Clemson                 Columbia       29229        pprnll@clemson.edu                Dr. Pamela
              Parnell        Veterinary                     Road                                                                                      Parnell
                             Diagnostic                                                                                                               
                             Center                                                                                                                   

  TX          Dr. Lelve      Texas Vet      979-845-9000    1 Sippel Road  Drawer 3040  College        77843        1-gayle@tvmdl.tamu.edu            Dr. Lelve
              Gayle          Medical                                                    Station                                                       Gayle
                             Diagnostic                                                                                                               
                             Laboratory                                                                                                               

  UT          Dr. Tom        Utah           435-797-1895    950 E. 1400                 Logan          84322-5700   tjbald@cc.usu.edu                 Dr. Tom
              Baldwin        Veterinary                     North                                                                                     Baldwin
                             Diagnostic                                                                                                               
                             Laboratory                                                                                                               

  VA          Dr. David W.   Virginia Dept  540-434-3897    116 Reservoir               Harrisonburg   22801                                          Dr. David
              Brown          of Agriculture                                                                                                           Brown
                             and Animal                                                                                                               
                             Health Lab                                                                                                               

  WA          Dr. Terry      Washington     509-335-9696    Bustad Hall    Room 155-N   Pullman        99164        tfm@vetmed.wsu.edu                Dr. Terry
              McElwain       Animal Disease                                                                                                           McElwain
                             Diagnostic                                                                                                               
                             Laboratory                                                                                                               

  WA                         Avian Health   253-445-4537    7613 Pioneer                Puyallup       98371-4919                                     Dr. A. S.
                             and Food                       Way E.                                                                                    Dhillon
                             Safety                                                                                                                   
                             Laboratory                                                                                                               

  WI          Dr. Leslie     USGS National  608-270-2400    6006 Schroeder              Madison        53711        ldierauf@usgs.gov                 Dr. Leslie
              Dierauf        Wildlife                       Road                                                                                      Dierauf
                             Health Center                                                                                                            

  WI          Dr. Kathy      Wisconsin      608-262-5432    Wisconsin      6101 Mineral Madison        53705        Kathy.Kurth@WVDL.wisc.ed          Dr. Kathy
              Kurth and Dr.  Veterinary                     Department of  Point Road                                                                 Kurth and
              Pete Vanderloo Diagnostic                     Agriculture                                                                               Dr. Pete
                             Laboratory                                                                                                               Vanderloo

  WV                         West Virginia  304-558-2214    1900                        Charleston     25305-0172                                     Dr. Jewell
                             Dept of                        Boulevard,                                                                                Plumley
                             Agriculture                    East                                                                                      
  ----------------------------------------------------------------------------------------------------------------------------------------------------------------

^a^ This list represents the National Animal Health Laboratory Network
(NAHLN) labs certified as of 3/14/06 to conduct avian influenza
screening. This list will be updated as new labs become certified. For
the latest list of certified laboratories, please contact
Thomas.J.Deliberto@aphis.usda.gov

# Attachment 12

# Real-Time Reverse Transcriptase-Polymerase Chain Reaction for the Detection of Type A Influenza and the Avian H5 and H7 Ha Subtypes

# In Tracheal and Cloacal Samples 

**Cepheid Smart Cycler Protocol**

**I.
MATERIALS\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\....74**

> **General recommendations regarding
> reagents\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...74**
>
> **General\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...74**
>
> **RNA
> extraction\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\.....74**
>
> **Real-time
> RT-PCR\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...74**
>
> **Primer and probe
> sequences\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\.....75**
>
> **Primer and probe handling and
> dilution...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\.....75**
>
> **Suppliers\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\.....76**

**II.
METHODS\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...77**

> **Note on sample types and RNA extraction
> methods\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...77**
>
> **RNA extraction with the Qiagen RNeasy kit: Centrifuge
> method\...\...\...\...\...\...\...\...\...\....77**
>
> **RNA extraction with the Qiagen RNeasy kit: Vacuum manifold
> method\...\...\...\...\...\....78**
>
> **RNA Extraction with Trizol
> reagent\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\....78**
>
> **RNA handling and
> storage\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...79**
>
> **Real-time RT-PCR for type A influenza and the avian H5 and H7 HA
> subtypes\...\....79**

**III. ANALYSIS OF
RESULTS\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\.....81**

> **Recommendations for evaluating
> fluorograms\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\.....81**

**IV.
APPENDIX\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\.....84**

> **Troubleshooting\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\.....84**
>
> **Cross-contamination prevention
> \...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\....84**

**Real-time PCR
basics\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\...\.....85**

Prepared by:\
Erica Spackman, Ph.D.\
Southeast Poultry Research Lab

U.S. Dept. of Agriculture, Agricultural Research Service\
934 College Station Rd.\
Athens, GA 30605\
706-546-3617\
[espackman@seprl.usda.gov\
]{.underline}Updated March 15, 2005\
**I. MATERIALS**

Mention of trade names or commercial products in this procedure is
solely for the purpose of providing specific information and does not
imply recommendation or endorsement by the U.S. Department of
Agriculture.

**[General recommendations regarding reagents]{.underline}**

This assay was optimized using the using the Qiagen RNeasy kit and
Qiagen one-step RT-PCR kit, therefore these reagents are recommended for
uniformity, if available. Additionally, it is highly recommended that
the PCR primers and probe be highly purified (i.e. HPLC purified).

**[General]{.underline}**

Reagent grade H2O (nuclease free)\
Pipetors and tips for volumes between 1µl and 1ml\
1.5 ml microcentrifuge tubes Microcentrifuge TE buffer pH 8.3 (Promega
#V6231 or V6232) (optional)

**[RNA Extraction]{.underline}**

Qiagen RNeasy Mini Kit (Qiagen #74104 or #74106)\
100% Ethanol\
70% Ethanol (in nuclease free water)\
QiaVac 24 vacuum manifold (Optional)\
2-Mercaptoethanol (BME) (Sigma #M-6250)

Trizol LS reagent (Invitrogen #10296028, 200ml)\
Chloroform (Sigma #C-2432)\
Isopropanol (Sigma #I-9516)\
Glycogen 5mg/ml (Ambion #9510)

**[Real-time RT-PCR]{.underline}**

Qiagen one step RT-PCR Kit (Qiagen #210210 or #210212)\
Hydrolysis probes (IDT, Idaho Tech/Biochem or Qiagen-Operon) (Table 1)\
Primers (IDT, Idaho Tech/Biochem or Qiagen-Operon) (Table 1)\
25 mM MgCl2 (Promega #A3511 or #A3513)\
RNase Inhibitor (Promega #N2511 or \# N2515)\
Positive control RNA\
Nuclease free H2O\
25µl Smart Cycler tubes (Cepheid #900-0022 or 900-0003)

**Table 1.** Influenza real-time RT-PCR probe and primer sequences.
Protocols for H7 subtype Eurasian, H7 subtype South American strains,
the H6 subtype and the H9 subtype strains are available as supplementary
protocols.

  --------------------- ----------- ------------------------------------------
  **Specificity**                   **Sequence**

  **Type A              M+25 5'     5'-AGA TGA GTC TTC TAA CCG AGG TCG-3'
  influenza**-Matrix    Primer      
  gene                              

                        M+64 Probe  5'-FAM-TCA GGC CCC CTC AAA GCC GA-BHQ1-3'

                        M-124       5'-TGC AAA AAC ATC TTC AAG TCT CTG-3'
                        3'Primer    

                                    

  **H7Subtype**-North   H7+ 1244 5' 5'-ATT GGA CAC GAG ACG CAA TG-3'
  American strains      Primer      

                        H7+1281     5'-FAM-TAA TGC TGA GCT GTT GGT GGC-BHQ1-3'
                        Probe       

                        H7-1342     5'-TTC TGA GTC CGC AAG ATC TAT TG-3'
                        3'Primer    

                                    

  **H5 subtype**-Any    H5+1637     5'-FAM-TCA ACA GTG GCG AGT TCC CTA
  straina               Probe       GCA-BHQ1-3'

                        H5-1685     5'-AGA CCA GCT AYC ATG ATT gC-3'
                        3'Primer    

                                    

  **H5 subytpe**-North  H5+1456 NA  5'-ACG TAT GAC TAT CCA CAA TAC TCA-3'
  American strains      5' Primer   

  **H5                  H5+1456 EA  5'-ACG TAT GAC TAC CCG CAG TAT TCA-3'
  subtype**-Eurasian    5' Primer   
  strains                           
  --------------------- ----------- ------------------------------------------

a\. H5 strain specificity is determined by the 5' primer. Use only one
primer in the reaction.

Note on subtype determination: Due to the high level of sequence
variation within each HA subtype, a negative RRT-PCR result for a
specific subtype does not exclude the possibility that that subtype is
present.

**[Primer and probe handling and dilution]{.underline}**

Lyophilized primers and probes must be centrifuged briefly, to ensure
that the DNA pellet is at the bottom of the tube, before they are opened
and reconstituted. TE buffer should be used for the initial
reconstitution of lyophilized primers and probes (Idaho Tech sends TE
with probes and primers and it is commercially available). Concentrated
stock solutions should be stored at -20°C. Primer stock solutions should
be 200µM (200pmol/µl), probes should be 120µM (120pmol/µl). Quantitation
information will be supplied for each oligo (primers and probes are DNA
oligos) by the manufacturer.

An example of calculation for oligo reconstitution:

You have 17786 pmol of oligo (will be on oligo information sheet from
manufacturer).

Need 200pmol/µl for stock concedntration.

> Divide pmol of oligo by the pmol/µl needed or: [17786
> pmol]{.underline} = 88.9µl 200 pmol/µl

For 200pmol/µl resuspend the pellet in 89µl of TE or nuclease free H2O.
The calculation for the probe is the same, except divide the number of
probe pmol by 120pmol/µl. Mix gently by tapping the tube and allow the
oligo to resuspend for about 10 minutes before use.

Working stocks of primers should be 20pmol/ul (20µM) and working stocks
of probes should be 6µM. Dilute the primers 1:10 and dilute the probe
1:20 in nuclease free H2O (do not use TE buffer) for the working stocks.

Working stocks should be stored at 4°C. The probes are stable at this
concentration at 4° C for approximately 1 month. It may be useful to
make up several aliquots (5-6) of working stocks of primers and probes
which are a volume that can be used in about one month. Store the unused
aliquots at -20°C.

Note: the probes are light sensitive; store them in amber tubes if
available, and minimize their exposure to light.

Additional information on fluorescent probe handling and storage can
found at: www.idahotech.com, www.operon.com and www.idtdna.com.

**Suppliers**

Biosearch Technologies, Inc. Promega

81 Digital Drive 2800 Woods Hollow Rd

Novato, CA 94949-5750 Madison, WI 53711-5399

1.800.436.6631 1-800-356-9526

WWW.Biosearchtech.com www.promega.com

Idaho Technology/Biochem IDT

390 Wakara Way 1710 Commercial Park

Salt Lake City, UT 84108 Coralville, IA 52241

1-800-735-6544 1-800-328-2661

www.idahotech.com www.idtdna.com

Qiagen Inc./Operon

28159 Avenue Stanford

Valencia, CA 91355

1-800-426-8157 www.qiagen.com www.operon.com

**II. METHODS**

**NOTE ON SAMPLE TYPES AND RNA EXTRACTION METHODS**

The types of samples collected and the processing of those samples
varies by species. The optimal sample types and processing methods for
many species are given below.

**Table 2.** Sample types and optimal processing methods.

  ------------------ ----------------- ------------------ ---------------------
                     **Recommended**   **Processing**     

  **Species/ Type**  **Specimen**      **Method**         **Notes**

                                                          

  Gallinaceous       Tracheal swab     RNeasy RNA         Virus primarily
  Poultry                                                 replicates in

  (chickens,                           extraction, then   the respiratory tract
  turkeys,                             RRT-               (LPAI)

  quail)                               PCR                

  Waterfowl/ducks    Cloacal Swab      Trizol Reagent RNA Virus primarily
                                       extraction, then   replicates in the
                                       RRT­PCR             intestinal tract. RNA
                                                          extraction method
                                                          must be modified for
                                                          cloacal

                                                          samples

  Any species        Tissue samples    RNA extraction     For HPAI viruses high
                                       with               

                                       Trizol Reagent,    levels of virus may
                                       then               be in

                                       RRT-PCR            tissues.

  Environmental                        Virus isolation,   RRT-PCR can detect
                                       RRT-               

  samples            (Swab)            PCR not            inactivated virus

                                       recommended        
  ------------------ ----------------- ------------------ ---------------------

**REVISED RNA EXTRACTION PROTOCOL FOR TRACHEAL SWABS (7/03)**

**[RNA Extraction with Qiagen RNeasy Kit- Centrifuge
Method]{.underline}**

Notes:

• Adaptation of kit for fluid samples from manufacturer.

> • All kit supplied buffers and reagents should be prepared in
> accordance with the kit instructions.

• Use only RNA grade reagents and supplies

1.  Vortex the sample (cloacal or tracheal swabs in BHI or other media)
    for 3-5 seconds and withdraw 500µl and place in a 1.5 ml
    microcentrifuge tube.

2.  Add 500 µl of RLT buffer. Close the tube and vortex the sample for 5
    seconds.

3.  Add 500 µl of RNA grade 70% ethanol to the tube and mix. Centrifuge
    the sample for 5 minutes at \~5KXg to pellet any debris.

4.  Add 750µl of the supernatant from step 3 to the RNeasy column and
    centrifuge for 15 seconds at \~12 KXg, empty the flow through from
    the collection tube and repeat (all of the sample/RLT/70% ethanol
    mix should be applied to the column).

5.  Add 700µl RW1 buffer to the RNeasy column and centrifuge for 15
    seconds at \~12 KXg and place the column in a clean collection tube
    (the collection tube with RW1 flow through may be discarded and
    replaced with a fresh collection tube).

6.  Add 500µl RPE buffer to the RNeasy column and centrifuge for 15
    seconds at \~12 KXg, empty the flow through from the collection
    tube.

7.  Repeat step 6 for a total of 2 washes with RPE buffer.

8.  Centrifuge the empty RNeasy column an extra 2 minutes at \~14 KXg
    and discard the collection tube.

9.  Place the RNeasy column in an elution tube (or 1.5ml microfuge tube)
    and add 50 µl nuclease free H2O to the column. Incubate at room
    temperature for 1 minute. Elute RNA by centrifuging for 1 minute at
    \~14KXg. Discard RNeasy column.

**[RNA Extraction with Qiagen RNeasy Kit- QiaVac 24 Vacuum Manifold
Method]{.underline}**

Notes:

• Adaptation of the RNeasy kit for fluid samples from manufacturer.

> • All kit supplied buffers and reagents should be prepared in
> accordance with the kit instructions.

• RNeasy column lids should be open whenever vacuum is being applied.

• Use only RNA grade reagents and supplies

1.  Vortex the sample (cloacal or tracheal swabs in BHI or other media)
    for 3-5 seconds and withdraw 500 µl and place in a 1.5 ml
    microcentrifuge tube.

2.  Add 500 µl of RLT buffer. Close the tube and vortex the sample for 5
    seconds.

3.  Add 500 µl of RNA grade 70% ethanol to the tube and mix. Centrifuge
    the sample for 5 minutes at \~5KXg to pellet any debris.

4.  Place the appropriate number of RNeasy columns in the luer locks of
    the vacuum manifold, cover any empty positions with the luer caps
    supplied with the vacuum manifold.

5.  Apply vacuum and add the entire sample/RLT/ethanol mixture to an
    RNeasy column for each sample.

6.  Wash by applying 700µl RW1 buffer to each column.

7.  Wash again by applying 500µl RPE buffer to the column and repeat for
    a total of 2 washes with buffer RPE.

8.  Shut off the vacuum and place each RNeasy column in a 2ml collection
    tube. Centrifuge the column for 2 minutes at \~14 KXg and discard
    the collection tube.

9.  Place each column in an elution tube (or 1.5ml microfuge tube) and
    add 50 µl nuclease free H2O and incubate at room temperature 1
    minute. Elute RNA by centrifuging for 1 minute at \~14KXg.

10. Use 8µl per PCR reaction. Store at -70°C for long term storage.

**RNA EXTRACTION FROM CLOACAL SWABS OR TISSUE WITH TRIZOL REAGENT**

1\. Sample Prparation:

> a\. Cloacal Swabs: Vortex vigorously for 7-10 seconds. Centrifuge for
> 5 min. at 12,000Xg.Extract RNA from the supernatant.
>
> b\. Tissues: Make a 10% homogenate of tissue in PBS. Centrifuge for 10
> min. at 12,000Xg. Extract RNA from the supernatant.

2\. Add 250µl of the supernatant from the sample prepared as described
in step 1, to 750µl of Trizol LS reagent. Vortex. Pulse spin to remove
liquid from the tube lid.

3\. Add 200µl 100% chloroform to the sample/Trizol homogenate. Vortex
for 15 sec. Incubate at room temperature for 7 min.

4\. Centrifuge at 12,000 x g for 15 min at room temperature.

5\. Transfer 400-450µl of the upper aqueous layer to a separate
microcentrifuge tube marked with sample number. **Caution: The transfer
of organic phase material with the aqueous layer will inhibit the PCR
reaction.** Add 500µl of 100% isopropanol. Add carrier to the
isopropanol to aid precipitation i.e. glycogen: 1µl of 5mg/ml stock (may
be added prior to addition of the aqueous phase from the trizol). Invert
tube several times to mix. Incubate at room temperature for 10 min.

6\. Centrifuge at 10,000 x g for 10 min at 4 C.

7\. Decant liquid. Care should be taken to assure that the RNA pellet is
not disturbed. Add 1.0 ml of 70% or 80% ethanol. Mix gently.

8\. Centrifuge at 10,000 X g for 5 min at 4 C.

9\. Decant ethanol. Invert tube on a clean tissue wipe and allow to air
dry for 10 min. or until all visible signs of moisture are gone. It is
important not to let the RNA pellet over-dry, as this will decrease its
solubility.

10\. Hydrate pellet in 100µl of RNase free water and allow to sit at 4 C
for 1 hr to overnight.

**[RNA Handling and Storage]{.underline}**

The RNA sample may be stored at 4°C for \< 1 week, storage for longer
than one week should be at --70°C. Always wear gloves when handling RNA
and use only RNase or nuclease free materials and reagents with RNA.
Additional RNA handling and storage information can be found in:
Sambrook, J. and Russell, D. *Molecular Cloning, A Laboratory Manual*,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.

**[Procedure for Real-time RT-PCR for type A Influenza (MA gene), the
H5, H6, H7 and H9 HA subtypes]{.underline}**

This procedure was designed for the Cephied Smart-Cycler (Cepheid,
Sunnyvale, CA). A protocol for the Roche Light Cycler and Idaho Tech
R.A.P.I.D. is available. The original reference and validation study for
this assay is: Spackman, et. al.; Journal of Clinical Microbiology
40:3256-3260, 2002.

Information on setting-up and programming the Smart Cycler can be found
in the Smart Cycler user's manual. The conditions for the influenza
primers and probes on the Smart Cycler are shown in tables 2 and 3. The
RT step is the same for all primer and probe sets, these conditions are
specific for the Qiagen OneStep RT-PCR kit. Cycle times for the PCR
phase may vary among different real-time PCR instruments.

**Table 3.** RT step thermocycling for Qiagen one-step RT-PCR Kit.

  ------------------------- ----------------- ----------------- ---------
  RT Step                   1 cycle           30 min.           50° C

                                              15 min.           95° C
  ------------------------- ----------------- ----------------- ---------

**Table 4.** Thermocycling conditions for gene specific probe and primer
sets.

  -------------------- ------------ -------------- ----------- --- ----------
  **Probe/Primer set**              **Step**       **Time**        **Temp**

  **Type A influenza** 45 cycles    Denaturation   1 sec.          94° C

                                    Annealing^a^   20 sec.         60° C

                                                                   

  **H7 Subtype** North 40 cycles    Denaturation   1 sec.          94° C
  American                                                         

                                    Annealing^a^   20 sec.         58° C

                                                                   

  **H5 subtype** North 40 cycles    Denaturation   10 sec.         94° C
  American or                                                      
  Eurasian^b^                                                      

                                    Annealing^a^   20 sec.         57° C

                                    Extension      10 sec.         72° C
  -------------------- ------------ -------------- ----------- --- ----------

a\. Fluorescence is acquired at the annealing step.

b\. Use only one H5 subtype 5' primer for the H5 test.

The real-time RT-PCR reactions for type A influenza (M gene) and the H5
and H7 HA subtypes should be set­up with the following components and
volumes using the appropriate primer and probe set and cycling
conditions. Set-up the reactions with the tubes in the cooling block and
use aerosol resistant pipet-tips.

1.  Prepare the reaction mix (everything but the template) by pipetting:
    H2O, kit supplied 5X reaction buffer, kit supplied dNTP's and 25mM
    MgCl2 into a nuclease free microcentrifuge tube using the volumes
    per reaction for each reagent given in table 4. Next add the RNase
    inhibitor and enzyme. Add the probe last. Mix by vortexing for 3-5
    seconds and centrifuge briefly. Once the probe has been added
    minimize exposure of the reaction mix to light.

2.  Add the reaction mix (17µl) to the Smart Cycler tubes (add the mix
    to the bottom of the cup at the top of the reaction tube).

**Table 5.** Real-time RT-PCR reaction mix volumes and conditions for
type A influenza (M gene), H5 and H7 HA subtypes.

  -------------------- ------------------------- ------------------------
                       **Volume Per**            **Final**

                       **Reaction**              **Concentration**

  H2O                  6.95µl                    

  5X                   5                         1X

  25mM MgCl2           1.25                      3.75 mM

  Enzyme Mix           1                         

  Forward Primer       0.5                       10 pmol

  Reverse Primer       0.5                       10 pmol

  dNTP's               0.8                       320 µM ea. dNTP

  Probe                0.5                       0.12 µM

  Rnase Inhibitor      0.5                       13 units
  -------------------- ------------------------- ------------------------

> MM per rxn 17
>
> Template 8
>
> Total 25µl

3\. Add the template to the smart cycler tubes (8µl per reaction). Note:
The template for the positive controls is *in vitro* transcribed RNA
from the appropriate gene and the template for the negative controls is
H2O.

4\. Centrifuge the reaction tubes briefly in the Smart Cycler centrifuge
and run the real-time RT-PCR with the conditions described in tables 2
and 3 depending on the probe and primer set used. Note the RT step is
the same for all probe and primer sets (the RT step is specific for the
Qiagen one-step RT-PCR kit).

**III. ANALYSIS OF RESULTS**

Positive results on the Smart Cycler may be determined by the Smart
Cycler software (shown on the results table in the Smart Cycler
software) and are generally reliable; however results should be manually
confirmed by examination of the fluorogram.

On the Smart Cycler the default minimum increase in fluorescence for a
sample to be classified as positive by the software is 30 units. Because
this is an arbitrary threshold, any samples which have an increase in
fluorescence between 20 and 40 should be considered suspect and should
be re-tested with the type A influenza (M gne) assay and/or subtype
specific assays. In general, any questionable samples should be
re­tested. If results of the second test are unsatisfactory additional
sampling from the flock or premises should be considered if possible.

**[Recommendations for evaluating fluorograms]{.underline}**

Evaluation of the fluorogram with the following conditions may be
helpful in determining results manually:

>  All reactions with default settings.
>
>  Remove all reactions with greater than 100 units increase in
> fluoresce from the graph (this changes the scale, making it easier to
> identify weak negatives). (Figures 1a and 1b).
>
>  If there are samples which have a "V" shaped fluorescence trace
> incrementally lower the "background maximum cycles" (analysis settings
> table) to approximately 2 cycles below the cycle number where the base
> of the "V" is (Figures 2a and 2b).

**Figure 1a.** Example of a fluorogram from samples run on the Smart
Cycler. All samples shown. Background subtraction is on. All analysis
criteria are set to the default values. Note that scale is from 0 to
1000 fluorescence units (Y axis), making it difficult to evaluate weak
positive samples.

![](media/image11.jpeg){width="5.677777777777778in"
height="3.0340277777777778in"}

**Figure 1b.** Same fluorogram as figure 1a, however all samples which
increased greater than 100units in fluorescence were removed from the
graph. Note that the scale is from 0 to 120 fluorescence units (Y axis)
making it easier to recognize weak positives.

![](media/image12.jpeg){width="5.677777777777778in"
height="3.0034722222222223in"}

**Figure 2a.** Example of a "V" shaped fluorescence trace. The
background maximum cycle is set to the default of 40 (red circle). All
other analysis criteria are set to the default values. The negative
control is shown for

![](media/image13.jpeg){width="6.844444444444444in"
height="8.041666666666666in"}

**IV. APPENDIX**

**[Troubleshooting]{.underline}**

• Positive controls are negative:

• Control template has degraded.

• Probe may be old and the fluorescence may be dead.

• The enzymes may be inactivated.

> • Incorrect thermal-cycling program or fluorescence acquisition (wrong
> setting or wrong channel being viewed).

• Negative controls are positive:

• There may be cross contamination among the samples.

• There may be non-specific probe degradation, use fresh probe and
primers.

• Background level too high or too low (should be approximately 100-200
units):

• The probe concentration may be wrong.

• The probe may be degraded or too old if the background level is too
low.

• Warning message in sample status on results screen.

> • Probe concentration too high (may cause 'railing'; a sharp decrease
> in fluorescence after a steady increase).

**[Cross-contamination prevention]{.underline}**

Due to the high sensitivity of RT-PCR based assays cross-contamination
is an important issue. The following guidelines will help to prevent
contamination of PCR samples in the lab:

>  Use aerosol resistant pipet tips
>
>  Centrifuge all reagents prior to use, especially freeze-dried
> materials
>
>  Preparation of samples in a biosafety cabinet
>
>  Use of separate areas (separate biosafety cabinets for RNA
> extraction and RT-PCR reaction
>
> preparation).\
> Minimizing sample handling\
> Change gloves often

**[Real-time PCR Basics]{.underline}**

The general principle of real-time PCR is the same as standard PCR,
however the reaction product can be monitored in real-time with a
fluorogenic probe. There are several types of probes for real-time PCR:
hydrolysis probes, hybridization probes and molecular beacons. This
assay utilizes hydrolysis probes.

In the hydrolysis probe system, a DNA probe which binds the PCR product
and which has a fluorogenic reporter dye on one end and a quencher dye
on the other end, is added to the PCR reaction (figure 4). As the target
PCR product increases the probe binds the amplicons and reporter dye is
cleaved from the 5' end of the probe by *taq* polymerase (due to 5'
exonuclease activity). As the reporter is cleaved from more and more
probe molecules the fluorescence signal from the increases. The
fluorescence signal is monitored every cycle, revealing increases in the
PCR product as it occurs.

Additional information about Real-time PCR, primers and probes can be
found at www.operon.com and www.idtdna.com.

**a.** Reporter Quencher

![](media/image14.jpeg){width="2.8090277777777777in"
height="0.26666666666666666in"}

**b.**

> Reporter Quencher

![](media/image15.jpeg){width="2.6979166666666665in"
height="0.23541666666666666in"}

![](media/image16.jpeg){width="3.1708333333333334in"
height="0.7444444444444445in"}

**Figure 4.** Hydrolysis probe mechanism. **a.** The probe (\-\--) binds
the PCR product (-) during amplification. **b.** The polymerase (
![](media/image17.jpeg){width="0.2569444444444444in"
height="9.027777777777778e-2in"} ) runs into the probe during synthesis
of the PCR product. **c.** Taq polymerase cleaves the reporter dye from
the probe, increasing the detectable fluorescence of the reporter dye.

# ATTACHMENT 13

# Proposed Highly pathogenic H5N1 avian influenza Data Integration, Data Management and Spatial Modeling

## Data Standards

The Wildlife Disease Information Node (WDIN) system will accommodate a
common set of data standards as developed by the Interagency HPAI Early
Detection Working Group. It is expected that these standards will be
compliant with those under consideration for use in the National Animal
Health Laboratory Network (NAHLN). The needs for standards utilized in
other systems (e.g. USGS Bird Banding Laboratory) will also be
addressed. The WDIN will provide a data schema of required fields and
elements that will be used through the system, as defined by the Working
Group and collaborators in the data management field.

## Data Security

Security for the system will be achieved through various checkpoints
throughout the application. There will be several access roles each user
can be assigned or revoked ("data entry", "data edit", "data
verification", and "data administrator"). Users can be granted any
number of the roles available. Without sufficient access, the user will
be rejected from entering that portion of the system. Depending on
access, a user's roles may include: (1) Data entry; (2) Data edit; (3)
Data verification, and (4) Data administrator.

Data sharing will be achieved alongside the security measures. Each
user/institution will have the ability to grant or revoke access to
their data in agreed upon levels of access, and this access will be
determined by the WDIN in collaboration with the Working Group. WDIN is
envisioning low and high level access roles that can be granted to
partner institutions.

## Data System Environment

These are the existing components of the system proposed for HPAI data
management:

> • Java J2EE environment 1.4.2 (HTML, javascript, JSP) for the web
> application;
>
> • Microsoft SQL (database) to house the entered data;
>
> • Apache 2 (web server);
>
> • Tomcat 5; (application server);
>
> • ESRI ArcIMS 9 (web-enabled mapping);
>
> • ESRI ArcSDE (spatial data engine component on top of MS SQL).

To keep the system up-to-date and fully functional, additional
components, such as Rhapsody/Chameleon or another HL7 messaging software
system will be needed to accommodate the transfer and receipt of HL7
messages.

## Data Entry

Because of the multiple agencies and groups involved in sampling, there
may be different procedures for field data capture and diversity of
abilities and mechanisms for entering these data into an electronic
system. The data management platform must accommodate these differences,
and allow data entry to proceed in an efficient manner. WDIN is
exploring the following options for data entry. Some or all of these may
be implemented depending on user needs: (1) Direct web access; (2) File
transfer; (3) Optical Mark-Read data forms; or (4) Handheld/PDA. WDIN
will work with users whose preferences for data entry may change over
time.

## Data Access and Mapping 

Based on the security protocols described above, and within the access
guidelines determined by the Interagency Working Group, through the web
portal, partners will be able to view all data that has been entered in
a number of ways. Data can be browsed in entirety, or filtered by
various parameters (e.g. species, sex, location). Standardized reports
for individual partners, as well as grouped data will also be available.
If permission has been obtained, subsets of raw data could also be
downloaded.

Through the use of an interactive mapping tool (ArcIMS), maps will be
available both on-line and printable. These maps are created on demand
and can show whatever data fields the user desires, overlaid on a wide
range of backgrounds, such as roads, political boundaries, species
populations, topography, etc.

## Spatial Analysis and Modeling

Once surveillance data has been collected and mapped, they can be used
in spatial analysis both to assess the progress of the surveillance
effort, and if HPAI is detected, observe the course of the disease and
potentially model its spread, providing guidance for operational staff
undertaking control and eradication measures. As the proposed WDIN
Interagency Data Management System already contains a Geographic
Information System (GIS) component, this process can be easily
instituted. WDIN will work closely with the USDA APHIS Centers for
Epidemiology and Animal Health (CEAH) to integrate GIS surveillance,
mapping and modeling tools for application to HPAI analysis and
response.