i

Epidemiologic and Other Analyses of 

Avian Influenza Affected Poultry Flocks

September 2020 Report
ii

Suggested bibliographic citation for this report: 

USDA-APHIS (2020). Epidemiologic and other analyses of avian influenza affected poultry flocks: July 

21, 2020 Report. United States Department of Agriculture: Animal and Plant Health Inspection 

Service: Veterinary Services: Center for Epidemiology and Animal Health. Fort Collins, CO. pgs. 56

For more information, contact: 

Amy Delgado, DVM PhD

Associate Director, Monitoring and Surveillance

USDA:APHIS:Veterinary Services

Center for Epidemiology and Animal Health

2150 Centre Avenue, Bldg. B-2E7, Fort Collins, CO, 80526-8117

Phone (970) 494-7256 | Mobile (970) 237-0857 | Fax (970) 494-7228

Email: Amy.H.Delgado@aphis.usda.gov

Web: http://www.aphis.usda.gov/animal-health/ceah
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ii

Table of Contents

Executive Summary..................................................................................................................................5

Introduction .............................................................................................................................................7

A. Description of Outbreak ................................................................................................................................7

Detection of H7N3 LPAI ...............................................................................................................................7

Detection of Highly Pathogenic Avian Influenza in South Carolina .............................................................8

Epidemiologic Study to Investigate the H7N3 LPAI and HPAI Viruses in Commercial Poultry in 

North Carolina and South Carolina.................................................................................................11

A. Case Control Study.......................................................................................................................................11

Methods.....................................................................................................................................................11

Results........................................................................................................................................................12

Discussion ..................................................................................................................................................14

B. Contact network among case premises.....................................................................................................14

Methods and Results.................................................................................................................................14

Estimating the Time of H7N3 LPAI/HPAI Introduction into Commercial Turkey Flocks using 

Diagnostic Test Results and Production Data .................................................................................17

A. Summary.......................................................................................................................................................17

B. Methods .......................................................................................................................................................18

C. Results...........................................................................................................................................................19

Estimating the time of LPAI introduction for Anson 1...............................................................................21

Estimating the time of LPAI introduction for Union 6, Union 7, and Union 8 ...........................................22

Estimating the time of HPAI and LPAI introduction for Chesterfield 2......................................................24

Estimating the time of LPAI introduction for Union 2 and Union 3...........................................................25

Data Limitations/Sources of Uncertainty...................................................................................................28

Discussion ..................................................................................................................................................29

Phylogenetic Analysis and Diagnostics...................................................................................................30

A. North American H7N3 HPAI and LPAI from poultry (AM H7N3 2020).....................................................30

Comparison to Other Viruses/Lineages.....................................................................................................31

B. Public Health Aspects ..................................................................................................................................32

C. Diagnostics and Characterization for Influenza A Viruses ........................................................................32

General Information ..................................................................................................................................32

Using Geospatial Methods To Measure the Relative Risk of Environmental Persistence of 

Avian Influenza Virus in North Carolina and South Carolina...........................................................34

A. Summary.......................................................................................................................................................34

B. Model Inputs and Development .................................................................................................................34

C. Model Execution ..........................................................................................................................................35

D. Results and Discussion.................................................................................................................................37

References..............................................................................................................................................43

Acknowledgements................................................................................................................................47

Appendix A: Table of Variables Included in the Case-Control Questionnaire .........................................48

Appendix B: Data Factors and Processing for Geospatial Analysis Predicting Avian Influenza 

Virus Persistence in the Environment.............................................................................................53
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A. Data Preparation..........................................................................................................................................53

Predictive Factors and Data Sources .........................................................................................................53

List of Figures and Tables

Figure 1. Counties with confirmed findings of H7N3 LPAI or HPAI in March and April 2020. ..............................10

Figure 2. Categories of connections between LPAI/HPAI infected premises (grey dots) in North 

Carolina and South Carolina that were identified in epidemiologic surveys and/or interviews 

with premises owners.........................................................................................................................................16

Figure 3. Variation in daily mortality fraction among 4 barns of Union 7. The data points with 

mortality above 3 per 1000 birds are marked in red. ......................................................................................20

Figure 4.Variation in daily mortality fraction among 4 barns of Union 2. The data points with 

mortality above 3 per 1000 birds are marked in red. ......................................................................................20

Figure 5. Observed egg production and model fitted egg production for Barn 2, Anson 1 .................................21

Figure 6.Observed daily mortality and model fitted daily mortality for Barn 2, Anson 1.....................................22

Figure 7. The likelihood of observing individual and combined test results assuming different times 

of LPAI virus introduction for Union 6 under the slow rate of spread scenario.............................................22

Figure 8. The likelihood of observing individual and combined test results assuming different times 

of LPAI virus introduction for Union 7. ..............................................................................................................23

Figure 9. The likelihood of observing individual and combined test results assuming different times 

of LPAI virus introduction for barn 1, Union 8 ..................................................................................................24

Figure 10. Observed daily mortality and model fitted daily mortality for barn 3, Chesterfield 2........................25

Figure 11.The likelihood of observing individual and combined test results assuming different 

times of LPAI virus introduction for barn 2, Chesterfield 2 under the slow rate of spread 

scenario................................................................................................................................................................25

Figure 12. Observed daily mortality and model fitted daily mortality for Union 2, barn 5. .................................27

Figure 13. Observed daily mortality and model fitted daily mortality for Union 2, barn 6. .................................27

Figure 14. Observed daily mortality and model fitted daily mortality for Union 3, barn 4. .................................28

Figure 15. Observed daily mortality and model fitted daily mortality for Union 3, barn 3. .................................28

Figure 16. Phylogenetic analysis using a maximum-likelihood tree data suggests that the LPAI 

introduction was of wild bird origin, and that the H7N3 is distinct from other recent H7 viruses 

from poultry, including 2016 IN H7N8, 2017 TN H7N9, and Mexico H7N3 HPAI...........................................31

Figure 17. Percent of geographic area in each risk category by week (1 km resolution) in North 

Carolina, South Carolina, and both North and South Carolina (29 January – 30 June 2020)........................38

Figure 18. Percent of geographic area in each risk category by week (1 km resolution) in HPAI and 

LPAI affected counties (29 January 29 – 30 June 2020)...................................................................................39

Figure 19. Weekly risk (1 km resolution) of environmental persistence of AIV in counties affected 

by AI in spring 2020 in North Carolina and South Carolina (29 January – 30 June 2020)..............................40

Figure 20. Relative risk (1 km resolution) of environmental persistence of AIV in an undisclosed 

area within an AI-affected county. ....................................................................................................................41

Figure 21. Commercial poultry facilities in relation with relative risk for AIV environmental 

persistence for the week of 29 February - 3 March 2020................................................................................42
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Table 1. Location, production type, and confirmation date of flocks infected by HPAI/LPAI H7N3......................9

Table 2. Significant results from univariate logistic regression by characteristics of H7N3 LPAI/HPAI 

infected premises and control premises. ..........................................................................................................13

Table 3. Significant results from multivariate analysis including adjusted odds ratios (OR) for 

significant risk factors. ........................................................................................................................................14

Table 4.The most likely time of virus introduction with 95% CI and, when applicable, the most 

likely contact rate with the 95% CI for barns infected during the 2020 LPAI/HPAI H7N3 

outbreak. .............................................................................................................................................................18

Table 5. The most likely time of virus introduction with the 95% C.I. and the most likely contact 

rate with the 95% C.I. for barns infected on Union 2 and Union 3 during the 2020 LPAI/HPAI 

H7N3 outbreak. ...................................................................................................................................................26

Table 6. Environmental factors selected as model inputs, data sources, trends relative to AIV 

survival, and rationale for model inclusion. ......................................................................................................35

Table 7. Environmental factors, spatial layer values, and weights used to develop the final 

predictive model for the persistence of avian influenza virus in the environment. ......................................36

Table 8. Raw model output values (0-14) and reclassification into OIE risk categories. ......................................37

Table 9. Variables evaluated in the case-control study. All variables included in the table were not 

statistically significant at the p<0.1 level by univariate analysis .....................................................................48

Table 10. Common and scientific names of dabbling duck species that were included in the wild 

bird model. ..........................................................................................................................................................54
5

EXECUTIVE SUMMARY

An outbreak of low pathogenic avian influenza (LPAI) was detected in March 2020, in the States of North 

Carolina and South Carolina. A commercial breeder turkey operation in Anson County, North Carolina noted a 

slight drop in egg production. Samples were collected on 9 March and tested in accordance with National 

Poultry Improvement Program (NPIP) requirements. Two additional meat-type turkey flocks in Union County, 

North Carolina submitted pre-slaughter surveillance samples on 10 March. All three flocks were confirmed 

with H7N3 LPAI of North American wild bird lineage on 12 March. A total of 12 operations were confirmed 

with H7N3 LPAI, and one operation in South Carolina was confirmed with H7N3 highly pathogenic avian 

influenza (HPAI) over the course of this outbreak. Depopulation and disposal were completed for poultry

immediately on each farm with the final infected premises being completed by 15 May. A total of 337,362 

turkeys were depopulated in response to this outbreak.

Following initial response activities, a series of epidemiologic, genetic and environmental investigations were 

initiated to better understand virus introduction and transmission. These investigations were a collaboration 

between the poultry industry, North Carolina Department of Agriculture and Consumer Services (NCDA&CS), 

State officials from Clemson University’s Livestock Poultry Health (CULPH), and the USDA-APHIS-VS. 

Analysis of epidemiologic surveys conducted at case and control premises identified a higher number of 

turkey housing structures and having a common gathering place for workers on the premises as risk factors 

for avian influenza in this commercial turkey population. However, the results of the case control study should 

be interpreted with caution since both cases and controls were associated with a single integrator. Results 

may not be generalizable to the entire poultry industry in the Carolinas. There were extensive overlapping 

networks of connections identified among the infected premises; however, the level of information provided 

about network relationships was not sufficient to evaluate the risk from particular movements or connections.

Using diagnostic testing, mortality, and egg production data, a range of dates of possible virus introduction 

were estimated for six of the infected premises. The earliest most likely date of introduction among the six 

premises was 6 February 2020. The latest most likely date of introduction, to the premises that was infected 

with HPAI in one barn and LPAI in four other barns, was 28 March 2020. However, no birds were sent to 

slaughter from these flocks prior to testing and the subsequent detection of avian influenza. Though NPIP 

recommended biosecurity measures were in place, the overlap of ranges of detection dates among the 

premises support the possibility of lateral spread among the infected premises, including the HPAI infected 

premises.

Phylogenetic analysis of viruses from these premises support a single introduction of North American wild bird 

lineage H7N3 LPAI into turkey farms in North Carolina followed by lateral spread to other farms, with a single 

mutation event resulting in HPAI in a single barn on a commercial turkey operation in South Carolina. Analysis 

suggests the precursor virus likely emerged in wild waterfowl from the Mississippi flyway. The 2020 H7N3 

virus is distinguishable from other recent H7 poultry detections (2016 H7N8, 2017 H7N9, 2018 H7N1, and 

2018-2019 H7N3) across the entire genome. Although no recent single wild bird origin precursor with all 8 

gene segments was identified, highly similar progenitor genes were identified from wild bird surveillance 
6

efforts, particularly from the Mississippi flyway. Analysis also suggests that this H7 HA clade may represent a 

repetitive threat to poultry.

Introduction of avian influenza virus (AIV) into domestic poultry can be initiated by exposures to infectious 

wild birds or to virus surviving in the environment. A temporal spatial analysis of environmental factors known 

to influence AIV environmental persistence in North Carolina and South Carolina was used to create risk maps 

of AIV persistence from January 2020 through June 2020. The analysis identified an increasing level of weekly 

relative environmental risk from the beginning of the study period through the week ending with April 7th. 

From the week beginning April 8th through the end of the study period, weekly relative risk remained similar. 

Small areas with high and very high risk persisted in all weeks of the study period. 

The epidemiologic investigation focused on factors and management practices which have been implicated as 

risk factors for infection in previous avian influenza outbreaks. Phylogenetic analysis supports a single 

introduction followed by subsequent lateral spread. The affected premises were part of a highly connected 

network and transmission through the movement of fomites such as people, vehicles, and equipment 

between farms, were likely; however data collected on potential epidemiologic links during the window of 

introduction did not reveal a clear mechanism of spread. Additionally, breaches in biosecurity, for example, 

contact with wild birds, may have had a role in introduction and transmission. These results can be used to 

improve surveillance activities and to inform biosecurity practices and emergency preparedness efforts within 

the Carolinas.
7

INTRODUCTION

In response to the H7N3 LPAI/HPAI outbreak in commercial turkey operations in Anson and Union counties in 

North Carolina and Chesterfield county in South Carolina, the North Carolina Department of Agriculture and 

Consumer Services (NCDA&CS), State officials from Clemson University’s Livestock Poultry Health (CULPH), 

and the USDA-APHIS-VS initiated epidemiologic and genetic investigations to understand factors associated 

with introduction and transmission of avian influenza virus into and between poultry flocks.

These investigations included:

• A field-based study of infected farms using data collected through site visits and interviews with farm 

personnel

• A case-control study of infected and non-infected premises

• Analysis of barn-level mortality, egg production, and diagnostic test data from multiple infected farms to 

estimate the dates of virus introduction into this population

• Virus phylogenetic analysis

• Geospatial estimation of the relative risks of environmental persistence of avian influenza viruses in the 

environment in North Carolina and South Carolina.

This report includes the results from these investigations, to provide producers, industry, and other 

stakeholders with epidemiologic data from this outbreak. This is a final report of findings resulting from these 

investigations.

A. Description of Outbreak 

Detection of H7N3 LPAI

On 9 March 2020, a commercial breeder turkey operation in Anson County, North Carolina (Error! Reference 

source not found.), submitted samples for testing as a part of the National Poultry Improvement Program 

(NPIP) requirements after the flock manager noted a slight drop in egg production. On 10 March 2020, two 

commercial meat-type turkey flocks in Union County, North Carolina submitted samples to the Rollins Animal 

Diagnostic Laboratory for testing as part of routine pre-slaughter surveillance. When the samples from all 

three of these premises were determined by Rollins Animal Disease Diagnostic Laboratory to be non-negative 

for avian influenza on 11 March 2020, oropharyngeal (OP) swab samples were collected and tested H7 

positive at the Rollins Animal Disease Diagnostic Laboratory. Samples were received at the National Veterinary 

Services Laboratories (NVSL) on 12 March 2020, and H7 LPAI was confirmed the same day for all three flocks.

NVSL confirmed North American wild bird lineage H7N3 LPAI on all farms based upon partial 

Hemagglutinin/Neuraminidase (HA/NA) sequencing. 

On 12 March, a North Carolina incident command team began surveillance for commercial flocks in control 

zones established around all three positive premises. Surveillance was immediately initiated for commercial 

poultry premises located within 10km zones around all infected farms and for epidemiologically linked 

premises. Between 12 March 2020 and 2 April 2020, a total of twelve commercial turkey flocks, two breeder 
8

and ten meat-type flocks, were confirmed by NVSL as infected in Anson and Union counties in North Carolina 

and Chesterfield county South Carolina (Table 1). The owners of confirmed flocks reported no respiratory 

disease or increased mortality. The only clinical sign reported was a decrease in egg production in the breeder

flock confirmed on 12 March. 

Following initial detection, NCDA&CS and CULPH, with assistance from USDA-APHIS-VS, immediately began 

two rounds of active surveillance testing on commercial poultry premises located within 10km zones around 

all infected farms and for epidemiologically linked premises. In North Carolina, 1,645 Influenza-A tests were 

conducted on an estimated 18,095 birds tested as a part of the surveillance efforts. In South Carolina, 89 

Influenza-A tests were conducted on an estimated 890 birds as a part of the surveillance efforts. A total of 190

facilities were tested in the 10km zones around the infected farms in North Carolina. A total of 16 facilities 

were tested in the 10km zones around the infected farms in South Carolina. The lead State agencies and 

Veterinary Services agreed that poultry on the infected premises would be depopulated in accordance with 

State Response and Containment Plans and USDA-APHIS-VS guidance documents. Depopulation was 

completed immediately on each farm with the final LPAI infected flock being completed by 2 April 2020. 

Testing in surveillance zones was completed by 15 April 2020. 

Detection of Highly Pathogenic Avian Influenza in South Carolina

On 6 April 2020, CULPH was contacted after an increase in mortality and respiratory signs (snicking) was noted 

in one of five barns at a commercial meat-type turkey flock located in Chesterfield County, South Carolina. 

This flock had an epidemiologic link to another LPAI-infected flock in Chesterfield County but had tested 

negative in three previous rounds of testing, with most recent test negative samples collected on 31 March 

2020. On 6 April 2020, the company flock supervisor collected and submitted samples to CULPH for PCR and 

ELISA testing. CULPH Diagnostic lab reported PCR presumptive positive results for H7 avian influenza for this 

flock on 7 April 2020. On 8 April 2020, the results were confirmed at the NVSL as H7N3 HPAI from the house 

that had turkeys showing clinical signs. On 9 April 2020, H7 LPAI was confirmed in 3 houses that had turkeys 

without clinical signs based on partial sequence results. The flock of 33,000 commercial meat-type turkeys 

was depopulated on 8 April 2020 by State personnel.

Following the confirmation of the H7N3 HPAI, four rounds of testing occurred on all commercial flocks located 

in a 10km radius control area around the infected premises. Ten of the thirteen confirmed LPAI/HPAI positive 

facilities were part of a single integrator. As a result, multiple rounds of targeted integrator-based network 

surveillance were performed on premises located outside of the control area. Testing was completed by 29 

April 2020 with all results negative. Routine surveillance for NPIP avian influenza programs is ongoing 

statewide in both North Carolina and South Carolina.
9

Table 1. Location, production type, and confirmation date of flocks infected by HPAI/LPAI H7N3.

State County Production Type Confirmation Date

North Carolina Anson Commercial Breeder Turkey 12 March 2020

North Carolina Union Commercial Meat Turkey 12 March 2020

North Carolina Union Commercial Meat Turkey 12 March 2020

North Carolina Union Commercial Meat Turkey 17 March 2020

North Carolina Anson Commercial Meat Turkey 17 March 2020

South Carolina Chesterfield Commercial Meat Turkey 17 March 2020

North Carolina Union Commercial Meat Turkey 17 March 2020

North Carolina Union Commercial Breeder Turkey 17 March 2020

North Carolina Union Commercial Meat Turkey 17 March 2020

North Carolina Union Commercial Meat Turkey 18 March 2020

North Carolina Union Commercial Meat Turkey 21 March 2020

North Carolina Union Commercial Meat Turkey 2 April 2020

South Carolina Chesterfield Commercial Meat Turkey 8 April 2020
10

Figure 1. Counties with confirmed findings of H7N3 LPAI or HPAI in March and April 2020.
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EPIDEMIOLOGIC STUDY TO INVESTIGATE THE H7N3 LPAI AND HPAI VIRUSES IN 

COMMERCIAL POULTRY IN NORTH CAROLINA AND SOUTH CAROLINA

A. Case Control Study

In collaboration with the State Animal Health Officials, USDA-APHIS-VS conducted a case-control study of the 

H7N3 infected commercial turkey farms and some non-infected commercial turkey farms located in North 

Carolina and South Carolina. A questionnaire was administered to individual(s) on each farm most familiar 

with the farm’s management and operations. Questions focused on the time period beginning three weeks 

before initial detection of avian influenza in the population up to the date the survey was administered. The 

purpose of the study was to generate hypotheses for potential risk factors for LPAI/HPAI infection based on a 

statistical comparison of farm characteristics and management practices of case (infected) farms and control 

(non-infected) farms.

Methods

An online questionnaire was developed using the ArcGIS Survey 123 platform. The questionnaire was adapted 

from similar documents previously developed for a case-control study conducted during the 2014-2015 HPAI 

outbreak in the Midwest, and from a case-series questionnaire developed specifically for turkey operations 

during the 2019 LPAI outbreak in Minnesota (Garber et al., 2016, USDA-APHIS, 2019). The questionnaire was 

adapted to meet the requirements of the online survey platform. Transmission of virus in previous outbreaks 

of avian influenza in the United States has been attributed to the movement of live birds, transportation of 

manure, equipment sharing, and contaminated feed trucks, vehicles, water, and people (McQuiston et al., 

2005, Halvorson, 2009, Garber et al., 2016). The online questionnaire focused on these categories of risk 

factors and consisted of approximately 100 questions (yes/no or multiple-choice answer format).

Participating turkey operations were recruited with the help of NCDA&CS and CULPH. Five commercial turkey 

companies operating in North Carolina and South Carolina were invited to participate, including the three that 

were affected by the outbreak. Due to challenges caused by the COVID-19 pandemic, the two unaffected 

companies were unable to participate. The three remaining companies provided contact information for 45 

operations, including the 13 LPAI/HPAI infected premises. Invitation letters were sent to the potential 

participants, which included: a description of the study, the questionnaire URL, and a unique PIN to access the 

online survey.

Odds ratios, and p-values for each question were estimated by univariate logistic regression. To identify 

significant risk factors while controlling for possible confounding variables, a multivariable logistic regression 

analysis was performed using all variables that were statistically significant (p-values <0.1) in the univariate 

analysis. Backward, stepwise elimination was used to obtain the final model. The number (n) of cases and 

controls that responded to questions, odds ratios, and p-values for variables that were significant at the p<0.1 

level are provided in 
12

.

Results

A total of 36 participants (10 cases and 26 controls) completed the questionnaire. All completed 

questionnaires were submitted by operations belonging to one integrator company.

Premises characteristics 

All farms that responded were single-age, meat-turkey grower operations. Most farms (n=34, 

including all case farms) reported raising toms, while only 2 reported raising hens. Risk factors that 

were statistically significant at the p<0.1 level are summarized below and in 
13

. Additional tabulation of the variables that had p-values>0.1 are provided in Error! Reference source 

not found. in Appendix A.

Risk Factors – Univariate Analysis 

• Size

Infected (case) farms were larger than non-infected (control) farms. Although the odds of being 

infected was only slightly higher for operations with more birds, the odds ratio was statistically 

significant (OR=1.0001, p=0.038). Likewise, the odds of being infected was greater for farms with a 

higher number of poultry houses on site (OR=2.4, p=0.015).

• Waterfowl presence

The odds of being infected were higher for farms which reported having seen waterfowl on the farm 

(OR=10.7, p=0.025) and for those that had a pond on their property (OR 4.2, p=0.065).

• Potential for workers to act as fomites

The odds of being infected were also greater for farms that reported that workers mingled in a 

common gathering place on farm (OR = 6.8, p = 0.036) and that workers mingled off farm with 

workers from other poultry premises (OR = 10.7, p = 0.054). 

• Disposal of daily mortality

The odds of being infected were much greater for farms which reported on-farm disposal by 

incinerator (OR = 8, p = 0.019). Other methods of daily mortality disposal (burial, composting, 

rendering, landfill) were not statistically significant. The odds of being infected were also greater for 

farms that reported workers performed off-farm disposal versus the growers (OR=6.3, p=0.017).

• Cleanout

The odds of being infected were greater when cleanout was performed by the grower versus a 

contractor (OR=4.1, p=0.64).
14

Table 2. Significant1results from univariate logistic regression by characteristics of H7N3 LPAI/HPAI infected premises 

and control premises.

 

1 Univariate results should be interpreted with caution. For a tabulation of variables with p>0.1 see Appendix A, Table 9.

Characteristic Level

Case 

Farms

Control 

Farms

Odds 

Ratio

pvalue

Number of birds in 1000’s 

Median (range)

35.9

(20.7-75.2)

26.8

(14.0-50.0)

1.0 0.038

Number of houses 

Median (range)

5.5 (4-10) 4 (2-8) 2.4 0.015

Pond on the property Yes 5/10 5/26 4.2 0.065

Workers gather/mingle in 

centralized location on farm

Yes 8/10 10/26

6.8 0.036

Workers ever interact with 

workers from other poultry 

premises

Yes 3/10 1/26 10.7

0.054

Grower does cleanout vs 

Contractor

Grower 6/10 7/26

4.1 0.064

On farm disposal by incinerator Yes 4/10 2/26 8.0 0.019

Worker does off farm disposal 

vs Grower

Worker 6/10 5/26

6.3 0.017

Waterfowl seen on farm Yes 3/10 1/26 10.7 0.054
15

Risk Factors - Multivariate analysis 

Some of the risk factors described above are related, which can made univariate odds ratios misleading. A 

multivariate analysis was performed in order to provide adjusted odds ratios for risk factors, while considering 

interrelationships among these farm characteristics. Adjusting for all of the significant factors identified in the 

univariate analysis, a higher number of poultry houses and having workers gather and mingle in a common 

area remained significant risk factors for infection (

).

Table 3. Significant results from multivariate analysis including adjusted odds ratios (OR) for significant risk factors.

Discussion

These results suggest that some factors and management practices were shared across infected farms; 

however, the sample size of this study was small, reducing the statistical power of the analysis and making 

interpretation of the significance of many variables difficult. There was also a potential for selection bias, as all 

case premises that participated in this study belonged to one integrator and all controls were a subset of the 

remaining premises belonging to the same integrator. Therefore, results may not be representative of the 

entire population of domestic turkey or other poultry production in North and South Carolina. The 

multivariate analysis indicated that variables representing the size of the operation and comingling of 

employees were significantly associated with infection which suggests that biosecurity may have played a role 

in the spread of the virus, although NPIP recommended biosecurity measures were in place. When considered 

in conjunction with additional analyses described in other sections of this report, this information may provide 

insights into management practices in this study population and illuminate opportunities to implement 

additional mitigations to reduce the risk of avian influenza infection in the future.

B. Contact network among case premises

Methods and Results

In order to quickly collect relevant data to assist with contact tracing, owners of the infected farms were asked 

to also complete detailed paper questionnaires that included open ended questions about facility 

Characteristic Level Case Farms Control Farms

Adj.

OR P-value

Number of houses 

Median (range)

5.5 (4-10) 4 (2-8) 3.1 0.011

Workers gather/mingle 

in centralized location 

on farm

Yes 8/10 10/26 15.6 0.04
16

characteristics and biosecurity practices, as well as services and personnel who visited the premises in the 3 

weeks prior to detection of LPAI/HPAI. Twelve of 13 case farms completed the questionnaire. Information 

collected from those questionnaires and follow-up interviews were used to identify a network of common 

connections between case premises (

Figure 2). Eight categories of connections were identified in the data. The most common connection was feed 

source followed by litter source, renderer, garbage hauler, flock supervisor, propane delivery, and manure 

hauler. Two premises were managed by the same grower. Although seven premises identified a common 

renderer, several of those premises indicated that no renderer pickups occurred during the three weeks prior 

to detection of virus because daily mortalities are disposed of on farm until the birds reach 12 weeks of age. 

As indicated in Error! Reference source not found., there is an extensive network of connections among the 

infected premises, which is not surprising given their company relationships and geographic proximity. 

Insufficient information was provided about network relationships to provide strong evidence of a particular 

mechanism of spread or the temporal progression of infection.
17

Figure 2. Categories of connections between LPAI/HPAI infected premises (grey dots) in North Carolina and South 

Carolina that were identified in epidemiologic surveys and/or interviews with premises owners2. 

2 Connections were included in the figure if the data indicated common business or personnel relationships; however not all types of 

contacts identified in the figure occurred during the risk window for LPAI/HPAI infection (e.g., renderer pickup). The spatial 

arrangement of the premises in the figure does not represent true geographic relationships between premises.
18

ESTIMATING THE TIME OF H7N3 LPAI/HPAI INTRODUCTION INTO COMMERCIAL TURKEY 

FLOCKS USING DIAGNOSTIC TEST RESULTS AND PRODUCTION DATA

A. Summary

Determining the time of LPAI or HPAI virus introduction in a flock is an important part of outbreak 

investigations. By narrowing the time window of possible virus introduction, we can better identify the 

potential routes of virus introduction and enhance our understanding of the pattern of disease spread. In this 

analysis, diagnostic testing, egg production data or daily mortality data (where applicable) was used to 

estimate the time of introduction in five premises in North Carolina and South Carolina: two LPAI infected 

turkey breeder flocks, two LPAI infected meat turkey premises, and one HPAI/LPAI infected meat turkey 

premises. 

The analysis was performed using a simulation-based method in which the likelihood of observing the data 

was estimated from a within-house disease transmission model for various candidate times of exposure. Two 

Bayesian estimation approaches were used depending on the types of premises data available. For premises 

that had a drop in egg production or daily mortality above 3 birds per 1,000 on multiple days within two 

weeks of the time of detection, a within-house disease transmission model along with approximate Bayesian 

computation (ABC) was used. For premises that did not have a drop in egg production or mortality above 3 

birds per 1,000, only diagnostic data were used for estimation. The limited number of diagnostic tests made it 

difficult to estimate the contact rate for these latter premises, which is the parameter that determines the 

rate of disease spread. Therefore, the analysis was performed for slow and fast contact rate scenarios. In 

general, slower rates of disease spread are associated with earlier virus introduction times. As such, it is 

important to consider slow rates of spread specifically in order to get an indication of how long the virus could 

have been circulating in a house prior to detection. 

Dates of introduction were estimated for Anson 1, Union 6, Union 7, Union 8, and Chesterfield 2 (Error! 

Reference source not found.). Union 7 had the earliest most likely date of introduction among the premises 

evaluated (6 February 2020; 95% C.I., 29 January 2020-8 March 2020 under the slow spread scenario). Anson 

1 was estimated to have the subsequent most likely date of introduction (26 February 2020; 95% C.I., 23 

February-28 February). Union 6 and Union 8 had similar estimates for the most likely date of introduction (4 

March 2020 under the slow spread scenario and 8 or 9 March 2020 under the fast spread scenario). 

Chesterfield 2 Barn 2 had an estimated LPAI introduction date of 23 March 2020 (95% C.I., 9 March-29 March) 

under the slow spread scenario. Barn 3 of Chesterfield 2 had an estimated virus introduction date of 28 March 

2020 (95% C.I., 24 March-29 March). 

Overall, the time of virus introduction estimates help inform epidemiological investigations together with 

other epidemiological and phylogenetic analyses. For example, the results were used to evaluate whether it 

was conceivable for LPAI to have been introduced to Chesterfield 2 from other infected premises in midMarch and have a sequence of negative test results prior to detection on April 6th

. 
19

Table 4.The most likely time of virus introduction with 95% CI and, when applicable, the most likely contact rate3 with 

the 95% CI for barns infected during the 2020 LPAI/HPAI H7N3 outbreak. 

Premises/Barn Data Sources Estimated time of virus 

introduction

(95% CI)

Contact Rate;

(95% CI)

Anson 1/

Barn2

Diagnostic test results, egg 

production, daily mortality

2/26 (2/23-2/28) 4.6 (1.6-5.9)

Union 6/

Barn 2

Diagnostic test results Slow spread: 3/4 (2/28-3/7)

Fast spread: 3/8 (3/2-3/10)

NA

Union 7/

Positive Barn

Diagnostic test results Slow spread: 2/6 (1/29-3/8)

Fast spread: 2/13 (2/4-3/8)

NA

Union 8/

Barn 1

Diagnostic test results Slow spread: 3/4 (2/10-

3/10)

Fast spread: 3/9 (2/17-

3/13)

NA

Chesterfield 2/

Barn 2 (LPAI)

Diagnostic test results Slow spread: 3/23 (3/9-

3/29)

Fast spread: 3/28 (3/14-

3/31)

NA

Chesterfield 2/

Barn 3 (HPAI)

Diagnostic test results, 

daily mortality

3/28 (3/24-3/29) 2.8 (1.7-3.9)

B. Methods

The data used in the analysis included the sequence of available rRT-PCR test results, egg production data or 

daily mortality data. The analysis was based on rRT-PCR test results alone for those barns where the mortality 

levels within 3 weeks prior to detection did not exceed a threshold of 3 per 1,000 birds on multiple days. The 3 

per 1,000 bird threshold was used as a criterion for normal daily mortality in the Secure Turkey Supply plan 

(University of Minnesota, Secure Food Systems Team, USDA-APHIS-VS-Center for Epidemiology and Animal 

Health, 2018). This threshold was developed based on an analysis of mortality data from 116 turkey tom 

houses and 48 turkey hen houses, which indicated that this mortality is exceeded on less than 2% of days, 

resulting in a low frequency of false-positive triggers.

3 For barns where the contact rate could not be estimated, a slow rate of spread scenario and a fast rate of spread scenario were used to e stimate 

the dates of introduction.
20

For premises that had daily mortality that exceeded 3 per 1,000 birds on 2 or more days within 3 weeks of 

detecting LPAI on the premises, mortality data and diagnostic data were both used to estimate the dates of 

introduction. LPAI viruses cause milder disease relative to HPAI and most infected premises in this outbreak 

reported that no clinical signs were observed; however, LPAI viruses can contribute to clinical signs and 

mortality due to concurrent bacterial infections or environmental stress (Beltrán-Alcrudo et al., 2009, 

Halvorson et al., 2003, Mutinelli et al., 2003). In an experimental inoculation study of 12 North American H7 

LPAI strains, 3 strains did not cause any disease mortality while 10-60 percent of the inoculated birds died 

when infected with the other 9 LPAI strains (Spackman et al., 2010). In contrast, there was no mortality among 

experimentally inoculated turkeys with the 2016 LPAI H7N8 strain (Pantin-Jackwood et al., 2017, Garber et al., 

2019). Some of the secondary infections that can cause disease mortality together with LPAI include E. coli, P. 

multicoda, Alcaligenes faecalis, Aspergillus fumigatus, Riemerella anatipestifer, Ornithobacterium 

rhinotracheale, Staphylococcus aureus and Mycoplasma gallisepticum (Umar et al., 2018, Umar et al., 2017). 

An experimental study of LPAI H6N1 and E. coli showed marked synergistic or additive effects, where the 

mortality and clinical score with coinfection was much higher than due to either disease alone. Under field 

conditions, the mortality in LPAI infected flocks may vary significantly due to factors such as age, co-infection, 

or environmental factors such as ventilation and litter condition (Halvorson et al., 2003).

C. Results

The mortality patterns for the flocks involved in the current outbreak were quite variable (Error! Reference 

source not found. and Error! Reference source not found.). There were mortality patterns observed above 3 

per 1,000 birds where LPAI was discounted as a contributing factor. Several premises had mortality above that 

threshold as early as January 2020 (Union 4, Union 7, Union 2, Union 8). Based on previous epidemiological 

analyses for a LPAI H5N2 outbreak and other LPAI studies in the literature, it is unlikely that a flock would still 

be shedding at the time of detection in March if LPAI infection was contributing to mortality as early as 

January (USDA-APHIS, 2019). Therefore, mortality data above the 3 per 1,000 threshold that occurred in 

December and January were not used in the analysis. Similarly, in some cases, mortality data was excluded 

because some barns with mortality above 3 per 1,000 on multiple days within 3 weeks of detection also tested 

negative close to the time of detection (e.g., barns on Union 3, Union 7). For example, the peak mortality in 

barn 3 on Union 3 occurred one day prior to a negative rRT-PCR test result. This result would be unlikely if the 

mortality was due to LPAI since high mortality would be expected to be associated with high prevalence in the 

flock. High prevalence in turn would mean a high likelihood of detection. Likewise, 3 out of 4 barns in Union 7 

tested negative on March 13, 14 and 17. Three barns in Union 7 had peak mortality on March 7, 9 and 10 with 

mortality above 3 per 1,000 birds. Overall, it is unlikely for these barns to have tested negative if the mortality 

was due to LPAI.
21

Figure 3. Variation in daily mortality fraction among 4 barns of Union 7. The data points with mortality above 3 per 

1000 birds are marked in red.

Figure 4.Variation in daily mortality fraction among 4 barns of Union 2. The data points with mortality above 3 per 

1000 birds are marked in red.
22

Ultimately, we included mortality data in the main analysis for two premises, Anson 1-barn 2 and Chesterfield 

2-barn 3. Anson 1-barn 2 had a pattern of mortality above 3 per 1,000 with a simultaneous severe drop in egg 

production and with a low rRT-PCR CT (~22) value for samples collected on 9 March 2020. Chesterfield 2-barn 

3 was a HPAI barn with marked, exponentially increasing disease mortality.

Estimating the time of LPAI introduction for Anson 1

On this turkey breeder premises, only barn 2 tested positive via 2 pooled samples tested by rRT-PCR with 

samples collected on 9 March and 12 March 2020. There was a severe drop in egg production and increased 

mortality in this barn. The model fits for the increased mortality and drop in egg production are shown in 

Error! Reference source not found. and Error! Reference source not found.. The model fit indicated a 

significant delay of 6.53 days (95% C.I., 4.96-6.96 days) between when birds were infected and when the drop 

in egg production occurred. Other studies in the literature also indicated a similar interval from the LPAI 

infection of turkeys to when drops in egg production occurred (Pillai et al., 2010, Samadieh et al., 1970).

Figure 5. Observed egg production and model fitted egg production for Barn 2, Anson 1
23

Figure 6.Observed daily mortality and model fitted daily mortality for Barn 2, Anson 1.

Estimating the time of LPAI introduction for Union 6, Union 7, and Union 8

Union 6 was a meat type turkey premises with three barns. Barn 2 on Union 6 first tested positive by rRT-PCR 

performed on a pooled sample of 11 swabs taken on 15 March 2020. A pooled sample taken 17 March from 

barn 2 also tested positive. The remaining test samples (a 13 March sample from barn 2, 13 March and 15 

March samples from barn 1, and 14 March and 15 March samples from barn 3) all tested negative. Due to low 

daily mortality in barn 2, only testing data was considered in the estimation of the time of virus introduction. 

Furthermore, in order to reduce uncertainty in the estimates for barn 2, the test results from all barns on the 

premises were included in the estimation procedure (Error! Reference source not found.).

Figure 7. The likelihood of observing individual and combined test results assuming different times of LPAI virus 

introduction for Union 6 under the slow rate of spread scenario.

Union 7 was a meat turkey premises with five barns. The barns were tested by rRT-PCR on 13 March with one 

pooled sample of 11 swabs per barn, 14 March with two pooled samples of 10 swabs per barn, and on 17 

March also with two pooled samples of 10 swabs per barn. One of the samples from an unspecified barn 

tested positive on 14 March, all other samples tested negative. All barns were assumed in the analysis to have 

been equally likely to have been the barn with the positive test result. Daily mortality was low in the barns on 

this premises, leading to only the diagnostic test results being used to estimate the time of virus introduction 

(Error! Reference source not found..
24

Figure 8. The likelihood of observing individual and combined test results assuming different times of LPAI virus 

introduction for Union 7.

The six barns on Union 8, also a meat type turkey premises, all tested negative on 13 March and then all 

tested positive on 19 March by rRT-PCR performed on a pooled sample of 11 swabs. The barns on Union 8 had 

low daily mortality during the observation period, leading to only testing data being used in the analysis. As all 

barns on Union 8 had the same test results and similar flock sizes, the estimated time of virus introduction 

would be expected to be nearly identical for each barn (Error! Reference source not found.). There was 

substantial uncertainty in the estimates for barn 1, Union 8 due to the limited amount of information present 

in the data for this premises. 
25

Figure 9. The likelihood of observing individual and combined test results assuming different times of LPAI virus 

introduction for barn 1, Union 8

Estimating the time of HPAI and LPAI introduction for Chesterfield 2

Each of the five barns on Chesterfield 2 were negative on testing by pooled rRT-PCR on 15 March, 20 March,

and 1 April before testing positive on 6 April. Only in barn 3 was heightened mortality observed (Error! 

Reference source not found.). In this analysis, the most likely dates of introduction were estimated for HPAI in 

barn 3 and LPAI in barn 2. Barns 1, 4, and 5 would be expected to have nearly identical results to barn 2 given 

similarities in mortality and test results. Due to the low levels of mortality in barn 2, only testing data was used 

to estimate the time of virus introduction in this barn.

Under the slow spread scenario, LPAI was introduced into barn 2 as late as 23 March 2020 and possibly as 

early as 9 March 2020 (Error! Reference source not found.). The range of dates of introduction for barn 2 

overlap with the detection dates of earlier LPAI infected farms in the current outbreak suggesting that lateral 

spread from one of those premises to Chesterfield 2 was possible.
26

Figure 10. Observed daily mortality and model fitted daily mortality for barn 3, Chesterfield 2.

Figure 11.The likelihood of observing individual and combined test results assuming different times of LPAI virus 

introduction for barn 2, Chesterfield 2 under the slow rate of spread scenario.

Estimating the time of LPAI introduction for Union 2 and Union 3

The results for Union 2 and Union 3 are presented here for exploratory purposes but were not included with 

the other premises in Error! Reference source not found. due to a higher degree of uncertainty in the 

estimates (Error! Reference source not found.). Mortality exceeding the 3 per 1,000 bird threshold was 

observed in barns on both of these premises. However, it cannot be ruled out that this mortality was due 

primarily to another pathogen, in which case it would not be representative of the transmission of LPAI but 

rather the other pathogen.
27

Table 5. The most likely time of virus introduction with the 95% C.I. and the most likely contact rate with the 95% C.I. 

for barns infected on Union 2 and Union 3 during the 2020 LPAI/HPAI H7N3 outbreak.

4

Premises Barn Data sources

Estimated Day of Virus Introduction

(95% C.I.)

Contact rate

(95% C.I.)

Union 2

Barn 6

Diagnostic test 

results

daily mortality

21 January 2020 (15 Jan-1 Feb) 0.35 (0.27-0.46)

Barn 5

Diagnostic test 

results

daily mortality

20 February 2020 (13 Feb-23 Feb) 1.04 (0.63-1.94)

Union 3

Barn 4

Diagnostic test 

results

daily mortality

12 February 2020 (7 Feb-16 Feb) 0.7 (0.5-2.76)

Barn 3

Diagnostic test 

results

daily mortality

21 February 2020 (19 Feb-25 Feb) 3.6 (1.1-6.2)

Union 2 was a 10-barn commercial LPAI infected meat turkey premises. The premises was sampled on 10 

March (5 houses) and 12 March 2020 (9 houses) and all the samples were positive by IAV Matrix gene rRTPCR. For Union 2, data to link test results to individual barns were not available. We considered partial 

information from the test results from samples collected on 12 March by calculating the likelihood of a barn 

being sampled (9/10 barns were sampled). The magnitude of daily mortality exceeding the 3 bird per 1,000 

threshold was quite variable in this premises with some barns showing a rapid increase in mortality (Error! 

Reference source not found.), while other barns had a more gradual increase (Error! Reference source not 

found.). 

4

. Note that the results should be viewed cautiously due to uncertainty that the daily mortality exceeding 3 birds per 1,000 in these barns was 

due to LPAI.
28

Figure 12. Observed daily mortality and model fitted daily mortality for Union 2, barn 5.

Figure 13. Observed daily mortality and model fitted daily mortality for Union 2, barn 6.

Union 3 was a meat turkey premises with birds in two out of six barns at the time of detection. Barn 4 tested 

positive via samples taken on 13 March while barn 3 tested negative. The daily mortality observed in the 

barns with a model fit is given in Error! Reference source not found. and Error! Reference source not found.. 

Both barns experienced elevated mortality; however, the peak mortality in barn 3, which exceeded the 3 bird 

per 1,000 threshold, occurred one day prior to the negative test result on 13 March (Error! Reference source 

not found. and Error! Reference source not found.). This result would be unlikely if the mortality was due to 

LPAI. Furthermore, the CT value of the positive rRT-PCR result in barn 4 was quite high. In the 2018 LPAI H5N2 

outbreak in Minnesota CT values close to 40 were generally observed when the flock was close to recovered 

(USDA-APHIS, 2019). Based on this observation, introduction of the virus into barn 4 could have occurred 
29

much earlier than the heightened mortality, in which case the mortality exceeding the threshold could be due 

to a pathogen other than LPAI. 

Figure 14. Observed daily mortality and model fitted daily mortality for Union 3, barn 4.

Figure 15. Observed daily mortality and model fitted daily mortality for Union 3, barn 3.

Data Limitations/Sources of Uncertainty

There were several data limitations contributing to uncertainty in the analysis. First, information linking the 

rRT-PCR pooled samples to individual barns were not available for all the premises. While the premises-level 

status is most important during an outbreak, the barn-level test results can be very helpful in improving 

understanding of the outbreak progression. In addition, serology tests were not performed in the current 
30

outbreak on infected premises. Serology tests performed after a rRT-PCR positive result can provide 

information on the recovery status of the flock and therefore on how long the infection has been circulating, 

reducing uncertainty in epidemiological investigations.

There is considerable uncertainty in the transmission model parameters for the current LPAI H7N3 outbreak 

strain. Transmission model inputs, such as the length of the latent and infectious period, while estimated from 

data on other North American H7 LPAI virus strains, may not capture the behavior of the H7N3 strain involved 

in the current outbreak. Data from transmission experiments performed with the current outbreak strain, 

such as those being done at the USDA ARS Southeast Poultry Research Laboratory (SEPRL), can help inform 

these parameters.

Finally, in some premises (e.g., Union 2 and Union 3), there was uncertainty in the nature of the daily 

mortality exceeding the threshold. For example, a coinfection could have occurred when a flock was close to 

recovering from LPAI. In this scenario, mortality patterns that look characteristic of LPAI could be indicative of 

another bird health issue. Information regarding disease investigations for other concurrent pathogens may 

help reduce this uncertainty in some cases.

Discussion

Estimating the time of virus introduction into barns provides a valuable piece of information for epidemiologic 

investigations and outbreak response. The estimated parameters can improve understanding of disease 

spread patterns together with other epidemiological and phylogenetic analyses. For example, the results from 

this analysis were used to evaluate whether it was conceivable for LPAIV to have been introduced onto the 

HPAI premises from other infected premises in mid-March. The overlap in the estimated dates of LPAIV

introduction onto the HPAI premises with the interval where other flocks are likely infectious supports the 

possibility of lateral spread among infected premises despite biosecurity measures. Of note, the NPIP program 

requires all commercial flocks to be tested prior to slaughter. Although the virus may have been circulating in 

these flocks for a time prior to detection, no infected birds were sent to slaughter.

Some of the premises had wide intervals for the LPAIV introduction date given the minimal disease mortality 

and rRT-PCR test results available. Additional diagnostic testing performed after an rRT-PCR positive result 

(e.g., serology tests) can provide information on the recovery status of the flock, reducing uncertainty in 

epidemiological investigations.

This work is dependent on the quality of production records and access to laboratory diagnostic results. 

Adding serologic testing to PCR testing can provide additional information to improve the precision of time of 

introduction estimates. Quickly sharing this information early in an outbreak can help target response 

resources for traceability and improve our understanding of how avian influenza viruses may spread between 

flocks. This work also highlights the value of closely monitoring egg production, mortality, and/or feed and 

water consumption to quickly identify disease issues in the flock. These factors may vary across flocks and 

between barns, so understanding the trends within each production setting is important. The presence of 

other illness in the flock can make it difficult to determine whether the observed mortality is related to LPAI.
31

PHYLOGENETIC ANALYSIS AND DIAGNOSTICS

A. North American H7N3 HPAI and LPAI from poultry (AM H7N3 2020)

This section of the report describes AM H7N3 HPAI and LPAI from poultry confirmed by the National 

Veterinary Services Laboratories (NVSL) during March and April 2020. AM H7N3 LPAI was confirmed in two 

commercial meat-type turkey operations in Union County, North Carolina and one commercial breeder turkey 

operation in Anson County, North Carolina. Samples were collected from the breeder turkey operation on 09 

March 2020 (Anson County) due to a drop in egg production. Samples were collected on 10 March 2020 from 

the two meat turkey operations (Union County) for routine pre-slaughter surveillance. Enhanced surveillance 

identified ten additional H7N3 LPAI-infected premises in North Carolina and one premises in South Carolina, 

followed by confirmation of H7N3 HPAI in a single barn of a second premises in South Carolina on 08 April 

2020 (the LPAI virus was detected in all other houses). Intravenous pathogenicity index (IVPI) was 0.0 for LPAI 

virus isolates (n = 5), and 2.47 for the HPAI virus.

Phylogenetic analysis of viruses from these events support a single introduction of North American wild bird 

lineage H7N3 LPAI into turkey farms in North Carolina followed by lateral spread to other farms, with a single 

mutation event resulting in HPAI in a single barn on a commercial turkey operation in South Carolina (Youk et 

al., 2020). Analysis suggests the precursor virus likely emerged in wild waterfowl from the Mississippi flyway. 

The insertion at the cleavage site responsible for the mutation to HPAI likely originated from host 28S rRNA; 

this 27-nucleotide sequence is highly conserved across many avian and mammalian species. An identical 

insertion occurred during the 2017 AM H7N9 LPAI, HPAI event, demonstrating a repetitive pathway by which 

H7s may acquire insertion at the cleavage site to mutate to HPAI. 

Detailed information on the evolution and clustering of the H7N3 viruses involved in this outbreak is available 

online5. 

NOTE: The outcomes of phylogenetic analysis should be interpreted in context of all available 

virus and epidemiologic information and should not be used directly to infer transmission.

5 Youk S, Lee D, Killian ML, et al. Highly Pathogenic Avian Influenza A(H7N3) Virus in Poultry, United States, 

2020. Emerging Infectious Diseases. 2020;26(12):2966-2969. doi:10.3201/eid2612.202790.
32

Figure 16. Phylogenetic analysis using a maximum-likelihood tree data suggests that the LPAI introduction was of wild 

bird origin, and that the H7N3 is distinct from other recent H7 viruses from poultry, including 2016 IN H7N8, 2017 

TN H7N9, and Mexico H7N3 HPAI.6

Comparison to Other Viruses/Lineages 

The 2020 H7N3 virus is distinguishable from other recent H7 poultry detections (2016 H7N8, 2017 H7N9, 2018 

H7N1, and 2018-2019 H7N3) across the entire genome (Error! Reference source not found.). 2020 H7N3 was 

also genetically distinct from H7N3 HPAI viruses present in Mexico since 2012, as well as the Anhui lineage 

6 Slide courtesy of ML Killian NVSL and colleagues at USDA ARS Southeast Poultry Research Laboratory.
33

H7N9 LPAI/HPAI originating in China which has infected poultry since 2013 with transmission to humans. 

Although no recent single wild bird origin precursor with all 8 gene segments was identified, highly similar 

progenitor genes were identified from wild bird surveillance efforts, particularly from the Mississippi flyway. 

Analysis also suggests that this H7 HA clade may represent a repetitive threat to poultry.

B. Public Health Aspects

There were no reports of the 2020 H7N3 virus infection in humans. State-level efforts to monitor the health of 

response workers and on-farm personnel were conducted.

The virus sequences have been shared with CDC for analysis which indicated that the viruses lack key amino 

acid substitutions associated with human-like receptor binding or substitutions in the polymerase or other 

internal genes associated with increased virulence and transmission in mammals; no known markers of 

neuraminidase inhibitor (Oseltamivir) resistance have been identified. 

C. Diagnostics and Characterization for Influenza A Viruses

The NVSL rapidly shares genetic and biological materials in collaboration with the Southeast Poultry Research 

Laboratory, the Influenza Division of the Centers for Disease Control and Prevention, Wildlife Services, as well 

as other key partners. Consensus data from whole genome sequencing are used to monitor virus evolution 

and assess the risk to veterinary and public health based upon the presence/absence of specific amino acid 

substitutions or protein motifs. Analysis of sequence data includes phylogeny of all eight segments and 

determination of amino acid substitutions across the HA1 protein. Genetic data are also used to confirm that 

diagnostic assays are fit for purpose. In silico analysis confirmed high identity between the H7N3 virus 

sequences and the primers and probes used for the IAV and H7 diagnostic rRT-PCR tests.

General Information

Avian influenza subtypes H5 and H7 are reportable worldwide because of their potential for mutation to high 

pathogenicity during replication in poultry. The presence of basic amino acids at the cleavage site contribute 

to the mutation from low to high pathogenicity. Mechanisms by which H5/H7 mutate from LPAI to HPAI 

include the gradual accumulation of basic amino acids (AA), insertion of repeated basic AA, and insertion of 

non-homologous genetic material (only reported for H7 viruses). 

Molecular diagnostic tests for influenza A virus (IAV) are used across the U.S. National Animal Health 

Laboratory Network (NAHLN). The most sensitive and specific tool for influenza A detection is the Type Aspecific rRT-PCR, which targets at least the matrix gene (IAV-M); this is the primary surveillance tool used and 

provides a semi-quantitative result. The NAHLN tests samples first by the IAV-M test and further by the 

NAHLN H5 and H7 tests where IAV is detected. 

All poultry samples with a non-negative test result for IAV (serology or PCR) are forwarded to NVSL for 

confirmatory testing. The NVSL uses Sanger sequencing protocols to generate partial HA/NA gene sequence 

directly from the sample for subtype and pathotype determination, when sufficient viral RNA is present. 

Whole genome sequencing is conducted on all isolated viruses, and select viruses are further characterized by 

pathotype assay in specific pathogen-free chickens. 
34

NVSL confirms the virus HA and NA subtype through molecular sequencing and/or antibody subtyping, and 

the pathotype (LPAI vs HPAI); if no virus is recovered nor sequence obtained directly from the sample, the 

pathotype is determined by the clinical presentation of the flock compared to the USDA HPAI case definition.
35

USING GEOSPATIAL METHODS TO MEASURE THE RELATIVE RISK OF ENVIRONMENTAL 

PERSISTENCE OF AVIAN INFLUENZA VIRUS IN NORTH CAROLINA AND SOUTH CAROLINA

A. Summary

Introduction of avian influenza virus (AIV) into domestic poultry can be initiated by exposures to infectious 

wild birds or to virus surviving in the environment. This analysis aimed to evaluate the risk of AIV 

environmental persistence in North Carolina and South Carolina both spatially and temporally. Environmental 

factors known to influence AIV survival were identified through a review of the published literature. Using Esri

GIS software, temporal and static spatial data were weighted based on their influence on virus survivability 

and then combined to produce weekly results at a 1km resolution from 29 January to 30 June 2020. This 

period encompasses the avian influenza outbreak in domestic poultry in these States, 11 March to 6 April 

2020. Five categories defined in the World Organization for Animal Health (OIE) Risk Assessment Guidelines 

were assigned to each 1km grid cell ranging from very low/negligible to very high. Maps and charts showing 

the relative risk of AIV persistence in the North Carolina and South Carolina environment were created from 

these results. Maps were also produced showing the numbers of commercial poultry premises in relation to 

areas at risk. These results can be used to improve surveillance activities and to inform biosecurity practices 

and emergency preparedness efforts within the Carolinas.

B. Model Inputs and Development

Factors known to influence AIV introduction and survival in the ambient environment, focusing on H5 and H7 

subtypes, were identified through an evaluation of the peer-reviewed literature. This information was 

combined with an assessment of data availability and five factors (water presence, water temperature, 

wetland cover, presence of wildlife refuges, and presence of wild birds) were selected as inputs to develop 

weekly risk models. These factors, their corresponding data sources, trends relative to AIV survival, and 

rationale for model inclusion are summarized in Error! Reference source not found.. Detailed descriptions of 

data sources and processing for each factor are available in Appendix B.
36

Table 6. Environmental factors selected as model inputs, data sources, trends relative to AIV survival, and rationale for 

model inclusion.

Factor Data Source Trends with AIV Rationale

Water 

Presence

USGS Gap Analysis 

Program

AIV particles survive well in 

water compared to air or other 

dry media

AIV survives in water more 

prominently than dry land

Weekly 

Water

Temperature

USGS Moderate 

Resolution Imaging 

Spectroradiometer

(MODIS) remote 

sensing data

Inverse association between 

persistence and increasing 

temperature: optimal

temperatures are near freezing 

and sub-optimal temperatures 

are 17-28°C

Temperature of water sources 

greatly impacts the rate of 

survival of AIV

Weekly Wild 

Bird 

Presence

Dabbling Duck 

Occurrence and 

Abundance model 

(USGS Patuxent 

Wildlife Research 

Center)

Wild birds are a main reservoir 

for AIV, and introduce the virus 

to their surrounding 

environment

When carrying the virus, wild 

birds deposit viral particles 

into water and other habitat 

locations

Wetland 

Cover United States Fish 

and Wildlife Service

Wetland cover provides ideal 

habitat for migratory birds, and 

contains aquatic zones where 

AIV can thrive for long periods

Previous studies show that 

wetland areas are associated 

with AIV presence

Wildlife

Refuges

United States Fish 

and Wildlife Service

Wildlife refuges are preservation 

zones of wild bird habitat

Refuges provide ideal habitat 

for AIV reservoir birds

C. Model Execution

Risk of environmental AIV persistence was calculated from the inputs using the Esri Spatial Analyst toolset 

within ArcGIS 10.6. Weights were determined based on their relative contribution to the persistence of AIV, 

based upon subject matter expert consultation and literature review (Error! Reference source not found.). 

Weighted grids for each environmental factor and week were created using spatial analyst tools and then 

summed using a simple raster calculator additive expression.
37

Table 7. Environmental factors, spatial layer values, and weights used to develop the final predictive model for the 

persistence of avian influenza virus in the environment.

Factor Spatial Layer Values Weighted Values

Wild Birds 1 = Presence 3

0 = Absence 0

Water Temperature 1 = < 10oC 3

2 = 10oC ≤ > 20oC 2

3 = ≥20oC 1

Water Presence 1 = Fresh water 3

0 = No water 0

Wetlands 1 = Presence 2

0 = Absence 0

Wildlife Refuges 1 = Presence 3

0 = Absence 0
38

D. Results and Discussion

The raw model output values (ranging from 0 – 14) were reclassified to illustrate relative risk (OIE categories 

ranging from 0 – 5). The five risk categories displayed correspond to the OIE Risk Assessment Guidelines for 

describing risk: negligible/very low, low, moderate, high, and extremely high risk (OIE, 2017). Reclassification 

followed the scheme outlined in Error! Reference source not found.. The results by week for each State and 

North Carolina and South Carolina combined are illustrated in Error! Reference source not found.. The results 

are also displayed for the three affected counties graphically (Error! Reference source not found.) and 

spatially (Error! Reference source not found.).

Table 8. Raw model output values (0-14) and reclassification into OIE risk categories.

Original model value Risk category (value)

0 Negligible/Very low risk (0)

1-2 Low risk (1)

3-4 Moderate risk (2)

5 High risk (3)

6-14 Very high risk (4)
39

Figure 17. Percent of geographic area in each risk category by week (1 km resolution) in North Carolina, South Carolina, 

and both North and South Carolina (29 January – 30 June 2020).
40

Figure 18. Percent of geographic area in each risk category by week (1 km resolution) in HPAI and LPAI affected 

counties (29 January 29 – 30 June 2020).
41

Figure 19. Weekly risk (1 km resolution) of environmental persistence of AIV in counties affected by AI in 

spring 2020 in North Carolina and South Carolina (29 January – 30 June 2020).

Figures 17, 18, and 19 illustrate an increasing level of weekly relative environmental risk from the 

beginning of the study period through the week ending with April 7th. From the week beginning 

April 8th through the end of the study period, weekly relative risk remains similar. This result may be 

related to observed warmer water temperatures not supportive of AIV persistence in the later part 

of the study period and annual migration of dabbling duck species out of the study area. Small areas 

with High and Very High risk persisted in all weeks of the study period. 

Error! Reference source not found. illustrates the typical variation in risk categories within a small 

area in the three affected counties. This result shows that a wide diversity of risk can exist within a 

small geographic area even within states or counties that are generally low risk. Individual poultry 

operations may be in close proximity to moderate and/or high-risk geographic areas at 1km spatial 

resolution.
42

Figure 20. Relative risk (1 km resolution) of environmental persistence of AIV in an undisclosed area within 

an AI-affected county.

Error! Reference source not found. illustrates the number and location of commercial poultry 

facilities in combination with relative risk for AIV persistence, with model results for the week of 

February 26th to March 3rd chosen by way of example. Counties in the central portion of the twostate area including those affected by the outbreak generally have lower risk of AIV persistence than 

other areas such as the coast plains of North Carolina and the river valleys in South Carolina. 

Counties in higher risk areas such as the coastal plains in North Carolina have relatively low numbers 

of commercial poultry operations.
43

Figure 21. Commercial poultry facilities in relation with relative risk for AIV environmental persistence for 

the week of 29 February - 3 March 2020.
44

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48

ACKNOWLEDGEMENTS

We greatly appreciate the cooperation and support of the poultry industry and farm owners for 

providing information on their biosecurity and production practices and for their full 

cooperation with this investigation. 

We thank Peter Bonney and Sasidhar Malladi from the University of Minnesota for modeling 

support. We thank Erica Spackman and Mary Pantin-Jackwood for providing experimental 

infection data.

This report would not have been possible without a large group of researchers, epidemiologists, 

laboratory staff, state animal health officials and data scientists who participated in the various 

analyses, drafting, and review of this report. We appreciate their dedication and professionalism 

in providing the best information possible to help producers reduce the risk of AIV introduction 

into their operations. 
49

APPENDIX A: TABLE OF VARIABLES INCLUDED IN THE CASE-CONTROL 

QUESTIONNAIRE

Table 9. Variables evaluated in the case-control study. All variables included in the table were not 

statistically significant at the p<0.1 level by univariate analysis. Variables with p-values<0.1 can be 

found in Table 2.

Characteristic Level n

Case Control

Other animals present on farm

Beef cattle 1/10 9/26

Horses 0/10 1/26

Prearranged depopulation plan 6/10 20/26

Is the plan exercised 0/6 0/20

House with family on premises 5/10 9/26

Common drive entrance 3/5 7/9

Number of farm entrances • 1 6/10 20/26

• 2 3/10 6/26

• 3 1/10 0/26

Biosecurity signage 10/10 26/26

Farm fenced in 2/10 10/26

Double perimeter fencing 0/2 0/10

Gate to premises 2/2 1/10

Gate locked 1/2 0/10

Are the following vehicle types provided access

Garbage truck • Not at all 8/10 20/26

• Perimeter of farm 1/10 3/10

• On farm away from barns 1/10 2/10

• On farm near barns 0/10 1/10

• Enter barns 0/10 0/10

Propane delivery • Not at all 8/10 21/26

• Perimeter of farm 0/10 2/26

• On farm away from barns 2/10 3/26
50

• On farm near barns 0/10 0/10

• Enter barns 0/10 0/10

Feed delivery • Not at all 0/10 0/26

• Perimeter of farm 0/10 0/26

• On farm away from barns 0/10 0/26

• On farm near barns 10/10 25/26

• Enter barns 0/10 1/26

Renderer • Not at all 8/10 22/26

• Perimeter of farm 1/10 3/26

• On farm away from barns 1/10 1/26

• On farm near barns 0/10 0/26

• Enter barns 0/10 0/26

Company personnel • Not at all 0/10 0/26

• Perimeter of farm 0/10 0/26

• On farm away from barns 0/10 0/26

• On farm near barns 0/10 0/26

• Enter barns 10/10 26/26

Other business visitors • Not at all 10/10 17/25

• Perimeter of farm 0/10 1/25

• On farm away from barns 0/10 3/25

• On farm near barns 0/10 2/25

• Enter barns 0/10 2/25

Number of times property mowed in last 6 

months

• 6 or fewer 0/10 2/26

• 7-12 2/10 4/26

• 13-18 3/10 6/26

• 19-24 3/10 13/26

• > 24 2/10 1/26

Facility free of debris 8/10 24/26

Vehicle wash station 5/10 9/26
51

Located on farm 5/10 9/26

Organic material removed 2/5 7/9

Vehicle exterior washed 5/5 9/9

High pressure spray wash 3/10 6/26

Frame cleaned and disinfected underneath 3/10 9/26

Interior cleaned 0/5 0/9

Designated parking area for workers and visitors 8/10 26/26

Changing area for workers 7/10 17/26

Workers shower at premises before entering barns 0/7 1/17

Workers don dedicated laundered coveralls before entering barns 2/9 7/26

Workers wear rubber boots or covers in poultry houses 10/10 26/26

Barn/pen doors lockable 6/10 20/26

Barn/pen doors routinely locked 0/6 0/20

Feed covers kept closed 10/10 26/26

Foot pans available at barn/pen entrances 2/10 9/26

Dry disinfectant 0/2 3/9

Liquid disinfectant 2/2 6/9

Foot pans in use prior to February 19th 1/2 5/9

Barns/pens have entry areas 8/10 20/26

Biosecurity audits on premises 10/10 26/26

Workers assigned to entire farm 10/10 26/26

Workers employed by other poultry premises 0/10 1/26

Family members employed by other poultry premises 2/10 1/25

Family members employed by other processing plants 0/10 0/26

Biosecurity training per year = 1 10/10 26/26

Hire extra part-time workers or extended family 4/10 9/25

Workers restricted from contact with backyard poultry 10/10 26/26

Communicated verbally 8/10 22/26

Communicated in writing 0/10 0/10

Communicated both ways 2/10 4/26
52

Grower or workers visited other poultry premises 4/10 0/26

Workers ever interact with workers from other poultry premises 3/10 1/26

Visitor log to sign 2/10 1/26

Visitor log current 2/2 0/1

Outer clothing provided to visitors 2/10 4/26

Service visits after February 19th

Flock supervisor 10/10 26/26

Feed delivery 7/10 26/26

Processing plant 1/10 18/26

Rendering 1/10 1/26

Cleanout 4/10 0/26

Litter 0/10 3/26

Service visits with access to birds

Flock supervisor 6/10 17/26

Feed delivery 0/10 1/26

Processing plant 1/10 10/26

Rendering 0/10 0/26

Cleanout 0/10 0/26

Litter 0/10 0/26

Poult delivery 0/10 6/26

Service visit equipment shared/rented/borrowed 2/10 3/26

Shared equipment had contact with birds 2/10 3/26

Equipment used jointly on other premises

Company vehicles 4/10 2/26

Feed trucks 3/10 2/26

Lawn mowers 4/10 0/26

Live haul loaders 3/10 2/26

Poult trailers 3/10 2/26

Pressure washers/sprayers 2/10 0/26

Skid-steer 2/10 0/26
53

Tillers 4/10 0/26

Trucks 4/10 0/26

Other equipment 1/10 1/26

Litter type = wood shavings 10/10 26/26

Litter shed present 4/10 16/26

Litter disposal

Off farm 8/10 25/26

On farm 0/10 1/26

Both 2/10 0/26

How is daily mortality handled

Rendering 9/10 20/26

Composting 0/10 5/26

Landfill 2/10 1/26

Burial 3/10 6/26

Incinerator 4/10 2/26

If burial or composting pits used, covered with soil daily 4/5 11/13

Carcass bin have a cover 9/9 22/22

Carcass bin routinely kept closed 9/9 22/22

Observe wild birds around farm 8/10 25/26

Types of wild birds observed

Small perching birds 8/10 25/26

Waterfowl 3/10 1/26

Other birds 0/10 1/26

Fly control used 4/10 13/26

Raccoons, possums, coyotes, foxes, rabbits observed around poultry houses 5/10 6/26
54

APPENDIX B: DATA FACTORS AND PROCESSING FOR GEOSPATIAL ANALYSIS 

PREDICTING AVIAN INFLUENZA VIRUS PERSISTENCE IN THE ENVIRONMENT

A. Data Preparation

All data were prepared using tools within ArcGIS 10.6, and final layers were projected to Universal 

Transverse Mercator (UTM) Zone 17 North in the datum World Geodetic System 1984 (WGS1984).

Predictive Factors and Data Sources

Water Presence 

Avian influenza viruses (AIV) have been shown to have improved survival in water compared to dry 

land (Brown et al., 2009; USGS, 2011). United States Geological Survey (USGS) Gap Analysis Project 

hydrography data collected between 1994 and 2004, derived from satellite imagery at a 30m 

resolution, were downloaded and reclassified into two categories of surface water based on 

suitability for AIV survival: presence of fresh water (high suitability), or no water present (low 

suitability). The surface water presence layer was aggregated to a one-kilometer (1km) resolution.

Water Temperature 

Water temperature is inversely associated with the rate of AIV survival (Brown et al., 2009; Keeler et 

al., 2012; Lang et al., 2008; Stallknecht et al., 1990a; 1990b). USGS Moderate Resolution Imaging 

Spectroradiometer (MODIS)-derived 8-day land surface and emissivity scenes were downloaded for 

2015-2017. Using R version 3.3.3, individual scenes were masked by quality indicators (i.e., cloud 

cover) and recombined to create summary temperature surfaces by season, at a spatial resolution of 

1km (Grim and Knievel, 2013; Ke and Song, 2014; NASA, 2012). Refined MODIS data were then 

masked with the water presence layer to reflect locations only where surface water was present, 

and reclassified to represent high suitability (< 10° C), moderate suitability (≥ 10°C and < 20°C), or 

low suitability (≥ 20°C) for AIV survival (Brown et al., 2007; Brown et al., 2009; Keeler et al., 2014; 

Nazir et al., 2010).

Wetlands and Wildlife Refuges 

U.S. Fish and Wildlife Service (USFWS) National Wetland Inventory data were used to identify 

locations of wetlands and wildlife refuges, which are considered favorable for AIV persistence 

(USFWS, 2016; Keeler et al., 2012; Belkhiria, et al., 2016; Iglesias et al., 2010; Fuller et al., 2010). 

Locations classified as ‘freshwater emergent wetland’ and ‘freshwater forested/shrub wetland’ were 

extracted; data were reclassified based on presence or absence of either wetland type and 

resampled to 1km. For wildlife refuges, USFWS Cadastral data were obtained and reclassified at a 

1km resolution based on presence or absence of National Wildlife Refuge (NWR) land.

Wild Bird Presence 

Data for wild bird presence were provided by the USGS Patuxent Wildlife Research Center. These 

data were derived using a spatio-temporal model estimating seasonal occurrence (presence) and 
55

abundance of 13 dabbling duck species (Error! Reference source not found.) throughout the 

conterminous United States at a 1km resolution (Humphreys, 2019). The occurrence model data for 

each species was combined then reclassified to create a presence/absence dataset for North 

Carolina and South Carolina.

Table 10. Common and scientific names of dabbling duck species that were included in the wild bird model.

Common Name Scientific Name

American black duck Anas rubripes

American wigeon Anas americana

Blue-winged teal Anas discors

Cinnamon teal Anas cyanoptera

Gadwall Anas strepera

Green-winged teal Anas carolinensis

Mallard Anas platyrhynchos

Mottled duck Anas fulvigula

Northern pintail Anas acuta

Northern shoveler Anas clypeata

 Ruddy duck Oxyura jamaicensis

 Ring-necked duck Aythya collaris

 Wood duck Aix sponsa

Commercial Poultry Operations 

Poultry operation data were extracted from the USDA APHIS Emergency Management Response 

System (EMRS). The data included location information for 4,416 commercial poultry operations. 

These data were summarized by county to illustrate the number of commercial premises in relation 

to areas at high risk for AIV introduction from wild birds.