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Assessing the impact of low-pathogenicity avian influenza virus on the health of American Black Ducks (Anas rubripes)

Chapter 1. Introduction

1.1 Influenza viruses

Influenza viruses belong to the family Orthomyxoviridae that is divided into 6 genera: Influenzavirus A, Influenzavirus B, Influenzavirus C, Isavirus, Thogotovirus, and Quaranfilvirus (McCauley et al., 2011). Type B and C influenza viruses are human pathogens that typically do not infect other species. Isavirus is exemplified by the fish pathogen infectious salmon anemia virus while thogotoviruses and quaranfilviruses are tick-borne viruses isolated from humans and livestock and humans and birds, respectively. Influenza A viruses (IAVs) are the most important of the group because they are present worldwide and infect a wide range of hosts including a large number of avian and mammalian species. Due to a combination of high mutation rate, caused by an error-prone replicase enzyme that does not have proofreading capability, and a segmented genome, which allows for segment reassortment during mixed infections, they have the ability to rapidly change and adapt to new hosts (Dugan et al., 2008).

1.2 Genetics of influenza A virus

The genomes of IAVs consist of 8 gene segments that encode at least 10 different viral proteins. The proteins present in viral particles can be divided into the surface proteins: hemagglutinin (HA), neuraminidase (NA) and membrane ion channel (M2); and internal proteins: nucleoprotein (NP), matrix protein (M1), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2) and the polymerase acidic protein (PA). Other proteins, such as the non-structural protein (NS), are only found within infected cells. The HA protein can be classified into 18 different subtypes, 16 of which are mostly distributed throughout two orders of wild birds, Anseriformes (e.g. ducks) and Charadriiformes (e.g. shorebirds, gulls and seabirds) (Swayne, 2017). Similarly, 9 of the 11 known subtypes of NA protein are found in viruses infecting these birds (Swayne, 2017). The maintenance of the majority of the diversity of IAVs in wild birds supports their role as the major natural reservoir of this group of viruses. Influenza A viruses from wild birds have been known to switch to novel hosts including horses, swine and even humans, which can then lead to the adaptation of viruses that can be transmitted within the new host (Dugan et al. 2008). A high mutation rate and the ability to reassort seem to be the most common and important factors in these IAV host-switching events and may be the driving force behind the evolution or the virus (Dugan et al. 2008). Additionally, the importance of reassortment is evident with the most recent human influenza pandemic virus, pH1N1(2009), where genes originating from wild bird viruses were identified (reference). The evolutionary properties and large genetic diversity of IAVs present in wild birds severely limits the ability to predict future emergence.

1.3 Avian influenza A viruses in the poultry industry

Avian influenza viruses (AIVs) are important pathogens in the poultry industry. AIVs can be divided into two groups based on their ability to cause disease in birds; high pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI). HPAI viruses, most often of subtype H5 and H7, can cause severe disease and death and are a major threat to commercial poultry farms. LPAI viruses often replicate in the respiratory or gastrointestinal tract and typically cause mild disease, which can be worsened if the host is experiencing other infections or environmental stressors (Alexander, 2000). In some cases, LPAI have evolved into an HPAI form and subsequently cause death (Iqubal et al 2014) and illness in mammals (Plague and Aviaire 2014). HP Viruses Virulence can also be influenced by host age, as very young and very old birds are more susceptible, and egg-production status, as egg production decreases and mortality increases in infected laying turkeys while infected non-layers can show no signs of disease (Swayne, 2016).

1.4 Influenza A viruses in wild waterfowl

Wild waterfowl, including species such as American Black Duck (Anas rubripes), are the major natural reservoir for a wide range of IAVs. Infection rates in dabbling ducks are higher than in any other bird, primarily due to their behaviour and the transmission route of the virus. Dabbling ducks feed on surface waters, which increases the chances of fecal-oral viral transmission, and also exhibit a pattern of abmigration (switching to different breeding grounds between years) that allows the spread of infection to different subpopulations (Olsen et al., 2006). Infection rates typically peak in early fall before migration begins. Juvenile birds within these populations can have a high rate of infection (maybe a couple of references on this?) caused by their immunologically naive status and a period of lowered immune response as their T lymphocyte system is not fully developed until they fully mature (Causey and Edwards, 2008). Within the respiratory and digestive tracts, replication occurs primarily in the enterocytes that line the small and large intestines and the epithelium of the bursa of fabricius (Brown et al., 2012). Once the virus replicates and is released from infected cells it spreads by the excretion of fecal matter throughout the animals’ habitat. The virus can be excreted for up to 10 days, however, in some cases LPAI virus can be detected for up to 28 days (Hénaux and Samuel, 2011; Brown et al., 2012). Once viruses have been introduced into the environment, they can remain infectious in fresh water at 0°C for more than 30 days, which allows for a high rate of fecal-oral transmission via contaminated surface water (Olsen et al., 2006; Lage Ferreira et al., 2015). Migratory waterfowl can travel thousands of kilometers each year and will form large feeding flocks in wetland environments at stop-over sites and at breeding and wintering habitats, allowing the virus to spread quickly and into new areas (Causey et al. 2008).

1.5 Low pathogenic AIVs and their virulence in wild birds

Only type ‘A’ influenza viruses are known to cause infection in birds, and most of the viruses circulating in wild birds are in the low pathogenic (LP) category.

Most infections of wild birds by LPAI appear to be asymptomatic, but the exact effects on infected birds have not been well investigated and are not well understood. Once an individual is infected, the virus replicates in the gastrointestinal tract, producing large amounts of virus with no apparent clinical signs, making infected individuals impossible to identify through field observations. In 2008, Latorre-Margalef et al. found that Mallards (Anas xxxxx) were negatively affected by infection, where body mass of AIV-positive ducks was approximately 18 g lower than the body mass of AIV-negative ducks (Latorre-Margalef et al. 2008). Body mass is dependent on many variables, including health status, age, sex, food, and environmental stressors, and these must be considered when using mass as an indicator of effects due to viral infection. Despite this, there is still no easy way to evaluate the LPAI infection status of birds in the field. In order to explore the virulence of LPAI in waterfowl, some research has been done employing experiments in controlled laboratory settings (references?), but these may not simulate the natural external stressors wild birds experience, such as migration and food and mate competition, and therefore may not be representative of what occurs in wild populations.

Diseases in American Black Ducks

Avian influenza virus is not the only pathogen that American Black Ducks are susceptible to. Other infections, such as duck virus enteritis, herpesvirus, avian botulism and avian cholera are prevalent among black duck populations. In particular, duck virus enteritis and herpesvirus have caused some large-scale mortality events across the U.S., explained partially by their ability to spread quickly throughout large aggregations of birds in limited habitats (Michael et al., 2002; Longcore et al., 2000). Duck enteritis virus causes one of the most contagious and fatal diseases of ducks and due to ducks’ migratory natures, this virus has spread worldwide.

1.7 Low pathogenic avian influenza A virus and the health of wild American Black Ducks

Although LPAI infections appear to be asymptomatic, a recent analysis of available data indicated that there may be effects on the respiratory and digestive tract function in LPAIV-infected birds (Kuiken, 2013). In some cases, tracheitis, pneumonia, and air-sacculitis were observed in the respiratory tracts of mallards. Each of these illnesses was characterized by the infiltration of lymphocytes and macrophages in the tissues. In this same article, Kuiken reported that the body weight of mallards was significantly lower for LPAIV-positive birds and for juvenile birds that were found to be shedding more of the virus. A major caveat to these findings is that they were based on research conducted on captive birds kept in a laboratory in a temperature-controlled environment with adequate food and water. It is therefore expected that the observed virulence in wild birds will be higher due to the high energy expenditure and stressors (e.g. predation, food and mate competition, migration) and additional energy expenditure required for immune response to infection (Kuiken, 2013). AIVs circulate within the Newfoundland population of American black ducks (Huang references), but their role in the global AIV cycle has not been extensively studied. This population forms large aggregations, which can spread AIV infection rapidly, allow for high virus turnover and act as local disease amplifiers (Verhagen et al., 2014), but is primarily resident on the island as most individuals do not migrate to other locations in North America (Longcore et al., 2000).

Haptoglobin (Hp) is an acute infection phase protein that will bind to free hemoglobin, limiting the available nutrients for pathogens (Quaye, 2008), and typically is present in the bloodstream in low concentrations under normal conditions. Its concentration increases in response to infection, inflammation or trauma. In 2014, van Dijk et al. found some associations between AIV infection and Hp levels in free-living mallards, however it varied according to age, sex and migratory strategy. Haptoglobin concentrations were higher in AIV-infected juveniles and females, and also local migrants. These differences were explained by immune status (juvenile birds have naïve immune systems), behaviour (males tend to use more energy preparing for and during the mating season) and migration strategy (migrants have the added stressor of long migrations in search of food and habitat in comparison to resident birds) (van Dijk et al., 2014). Although the findings from this study reveal some immune response to AIV infection, there were no symptomatic associations observed between Hp concentrations and AIV infection, and it was therefore suggested this was evidence of mallards’ coevolution with the virus. Aside from these findings, there is very little known about the effects on the immune system caused by influenza A virus infection (Causey et al., 2008) in waterfowl, and similar studies have not been carried out on American Black Ducks.

The tissues of wild birds can also be used as indicators of internal health of birds infected with LPAIV. Experimental studies have reported histological lesions, and even pneumonia and tracheitis, in wild birds experimentally infected with LPAIV (Kuiken. 2013, Latorre-Margalef et al., 2009).

1.8 Biology, trends and disease prevalence in American Black Ducks

American Black Ducks are widely distributed throughout North America, with a large portion of its breeding population found in Canada. Black ducks are known as short-distance migrants and in some cases can be sedentary, between inland and coastal areas. In addition to short migrations, black ducks also show high fidelity to coastal overwintering areas, which has been attributed to more predictable food resources available in these habitats (Longcore et al., 2000 and Diefenbach et al., 1988). The American Black Duck was once one of the most abundant duck species in eastern North America and it remains an important harvest species. The population suffered a major decline from the 1950s to the 1980s (Conroy et al., 1989), has since stabilized, but numbers remain below those targeted by the North American Waterfowl Management Plan (North American Waterfowl Management Plan [NAWMP] Plan Committee 2004) (Figure 1.1). Although the species has been intensively studied over its range, there is a general lack of agreement about the causes of its decline. It is speculated that the decline, followed by its failure to increase may be a result of loss of breeding and wintering habitat, over-harvest, and/or the competition and hybridization with mallards (Conroy et al., 2002). The loss of coastal wetland habitats throughout the Mississippi and Atlantic flyways has been extensive and researchers have been working to manage these habitats with consideration of bioenergetics modeling. This type of modelling allows researchers to calculate the energetic carrying capacity of important wintering habitat. With this value, researchers can inform on habitat management goals which can ultimately improve food availability for black ducks using these habitats. Through extensive research of the Atlantic flyway habitats (Cumberland, Cape May, Atlantic, and Southern Ocean, and Burlington counties in New Jersey, USA), researchers have found that mudflat habitats provide the greatest food availability for black ducks (invertebrates and vertebrates) (Cramer et al., 2012). Given that these areas are disappearing, black ducks are either forced to forage in limited space leading to competition or forced to forage in habitats where less food is available. Although understanding food resource availability is important when considering black duck population changes, it is also important to accurately quantify other variables that may play a role (e.g. diseases). Despite ongoing research efforts and speculations, the cause of the major decline and slow population growth has not been determined, and therefore identifying and understanding current limiting factors remain critical to future black duck management.

Newfoundland as an important area to monitor disease

Although black duck distribution is limited to North America, they are highly susceptible to the spread of disease from other parts of the world. American Black Ducks that spend time breeding and/or overwintering in Newfoundland are exposed to other bird species that are highly migratory and potentially carrying disease from other parts of the world. For example, gull species such as the Slaty-backed Gull and the Black-tailed Gull endemic to Japan and Russia have been recorded in St. John’s, Newfoundland as vagrants (willie). These types of long-distance movements indicate that Newfoundland is an important site for AIV transmission.

Species like gulls travel internationally and spread influenza (refs). Newfoundland is situated in such a way that it receives birds from all over the world via different migration paths. These species share habitat and interact with ducks at common foraging sites such as onshore waterbodies and coastal areas. Therefore, although the Newfoundland black ducks might not leave the island, they can still be exposed to infectious disease agents from around the globe.

1.8 Study objectives

This study was designed to investigate the possible effects of AIV infection on wild black duck health status. I captured and collected samples from 53 wild American Black Ducks to: 1) test for the presence of active AIV infection; 2) genetically characterize the detected AIVs; 3) analyze tissue samples from infected birds to look for histological lesions and cell abnormalities that could be an indicator of lowered health; 4) correlate these findings with measurements of bird body mass; and 5) evaluate possible ongoing responses to AIV infection by quantifying haptoglobin levels.

OTHER SECTIONS TO ADD

Thesis overview

American Black Ducks in Newfoundland and Labrador allow for an interesting look into AIV in a short distance migrant species.

Co-authorship statement

Chapter 2. Methods

2.1 Ethical and regulatory oversight

The American Black Ducks in this study were caught by bait trapping under permit 10559 from Environment Canada at public locations requiring no access permits. This work was carried out under the guidelines specified by the Canadian Council on Animal Care with approved protocol number 14-01-AL from the Memorial University Institutional Animal Care Committee, and biosafety permit S-103 from the Memorial University Biosafety Committee.

2.2 Location

2.2 Bird sampling

From September to November of 2014, 53 American Black Ducks (Anas rubripes) were caught by bait-trapping in the duck pond located along the Waterford River at Bowring Park in the city of St. John’s, Newfoundland and Labrador . Sample collection timing was based on previous observation of peak AIV infection in this bird population occurring in the autumn months (Huang et al., 2013). Ducks were captured and evaluated for age and sex based on cloacal examination and plumage characteristics (Pyle et al., 2008). Once aged and sexed, oropharyngeal and cloacal swabs were collected from each bird using independent sterile cotton-tipped applicators. The paired swabs were placed into a single tube containing Multi-trans viral transport medium (Starplex Scientific Inc., Etobicoke, Canada). Tubes containing samples were kept cool in the field and were then stored at -80°C within 24 hours of collection.

In addition to swab samples, blood samples were collected from every duck. Two mL of blood was drawn from the right brachial vein using a needle and this was separated into two pre-labelled 2.0-mL microcentrifuge tubes (1 mL of blood in each). One tube was used for collection of serum (see below) and the other was frozen and stored at -80°C.

After collection of swab and blood samples, birds were euthanized by cervical dislocation. Sections of trachea, duodenum, ileum and colon were immediately collected, fixed in 15 mL of 10% formalin solution and embedded in paraffin blocks.

ADD morphometric info

2.3 Virus screening

Oropharyngeal and cloacal swab samples were screened for the presence of AIV by real time reverse transcriptase polymerase chain reaction (RT-qPCR) as described for Canada’s Inter-agency wild bird influenza survey (REF?) at the Veterinary Diagnostics and Food Safety Laboratory, St. John’s, NL, Canada. Once RNA was extracted, screening of each sample for the presence of AIV targeted the M gene using the M52C and M253R primers and SuperScript III One-Step RT-PCR System with Platinum Taq using XXXXX with 45 cycles. Samples were considered positive when the cuttoff threashold (Ct) was <40 and inconclusive if 40-45.

Positive results were independently verified by re-extraction of RNA from the swab samples using Trizol-LS (Life Technologies) following manufacturer’s instructions. RT-PCR was performed using the primers M52C and M253R (ref) and SuperScript III One-Step RT-PCR System with Platinum Taq. The amplicons were cleaned using the Wizard SV Gel and PCR Clean-up System (Promega) and Sanger sequencing was performed at The Centre for Applied Genomics (Toronto, Canada). Sequences were visually inspected using XXX, looking for mixed peaks. Primers were trimmed from sequences and forward and reverse reads were aligned using XXX. To verify sequences were influenza A virus, they were subjected to a BLASTn search against the NCBI nonredundant nucleotide database as reference with the default settings. The top 5 matches for each sequence were recorded.

2.4 Full viral genomic sequencing

The amplicons from the RT-PCR reactions were cleaned using Wizard SV Gel and PCR Clean-up System (Promega) and sent for Sanger sequencing at The Centre for Applied Genomics (Toronto, Canada). The relationship between the identified viruses and those previously characterized were evaluated using Blast searches of the GenBank sequence database.

Attempts were made to get full genome sequences from all positive samples using ViDiT amplicon-based viral genome sequencing method (Verhoeven et al., 2018) and sequence on Ion Torrent PGM. RNA was isolated from 200µl VTM using the ZR Viral RNA Kit™ (Zymo Research) according to the manufacturer’s protocol. An RT-PCR was performed that could simutlantious amplify all eight viral segments using SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA Polymerase (ThermoFisher Scientific) in a reaction mix of 25 μL and tailed primers, at previously described conditions (Verhoeven et al., 2018). Random amplification with addition of sequencing barcodes and production of sequencing library with required sequencing tails for Ion Torrent PGM were preformed using Dream-Taq Green PCR Master Mix, primers, and conditions previously described (Verhoeven et al., 2018). All PCR clean-ups and size selections were preformed using Agencourt AMPure XP beads and previously described ratios. Samples were pooled at equimolarity, emulsion PCR was performed using Ion PGM™ Hi-Q™ View OT2 kit on the Ion OneTouch™ 2 System (Life Technologies), and sequencing was performed on the Ion PGM™ Hi-Q™ View Sequencing kit and an Ion 316™ Chip run on the Ion PGM System (Life Technologies).

Add how sequences were quality controlled, primers and barcodes removed. Sequences were assembled to produce contigs, or maps. This resulted in eight viral segements per genome.

2.5 Serology

One tube of the collected blood sample was centrifuged at 12,000 rpm for 10 min to separate red blood cells. The serum was transferred into a clean 2.0-mL microcentrifuge tube. Each serum sample was screened for antibodies that recognize the influenza A virus nucleoprotein (NP) using the AI MultiS-Screen ELISA Kit (IDEXX) at the Veterinary Diagnostics and Food Safety Laboratory, St. John’s, NL, Canada. A sample to negative control ratio (S/N) of <0.5 was considered positive.

2.6 Haptoglobin analysis

Haptoglobin concentrations (mg mL-¹) were quantified in all serum samples using a functional assay (TP801; Tri-Delta Diagnostics, NJ, USA) that colorimetrically quantifies the heme-binding capacity of the plasma. The assay was performed according to the manufacturer’s instructions with a few modifications as described previously (reference). Absorbance was measured at two different wavelengths (450 nm and 630 nm) before the addition of the final reagent that initiates the color-change reaction. To determine the standard curve, a total of 7 calibration dilutions were prepared (see appendix for dilution details). From these calibrations, a standard curve was constructed and the concentration for each sample was determined.

2.7 Tissue analysis

Tissue sampling was performed immediately after individuals were euthanized. Sections of trachea, duodenum, ileum and colon were collected, fixed in 15 mL of 10% formalin solution and embedded in paraffin blocks. Tissue samples were sliced and inspected for abnormalities (performed by Dr. Laura Rogers, Chief Veterinary Officer, Veterinary Diagnostics and Food Safety Laboratory, St. John’s, NL, Canada). The ileum sample from each AIV rRT-PCR-positive individual was sent for influenza A viral antigen detection by immunohistochemistry (Prairie Diagnostic Services Inc., SK, Canada).

any stats on the data?

2.8 Statistical analysis

All statistics on prevalence were performed using R XXX (R core team). Prevalence of AIV within American Black Ducks according to XXXXX was compared using a generalized linear model (GLM) with a binomial distribution. Seroprevalence was compared using a similar analysis. A comparison between prevalence and seroprevalence between birds was completed using correlation.

2.9 Phylogenetic analysis

THIS WILL BE MOSTLY Marta that WILL BE OF A BIG HELP.

Chapter 3. Results

Prevalence of AIV detection

A total of 53 American Black Ducks were sampled between September 2014 and November 2014. Of these, 12 swab samples (22.6%) were collected in September, 31 samples (58.5%) in October and 10 samples (18.9%) in November. The highest capture rates occurred in September with an average of 6 birds/day, while it was 4/day in October and 3/day in November (Figure 2.1). Of the 53 swab samples collected, five samples (9.4%) tested positive for avian influenza (n=2 in September, n=2 in October and n=1 in November). Most positive samples were isolated from female birds (n=4, 80%) and there was a higher AIV prevalence in adult females (2/14, 14.3% versus #/##, ###%; p = #####) (Table 2.1). There was no difference in AIV prevalence rate in juveniles (2/25, 8.0%) and adults (3/28, 10.7%) (p = ####). The same is true when comparing prevalence of males (1/20, 5.0%) to females (4/33, 12.1%) (Table 2.2).

Table 1.1 Demographics of American Black Ducks captured in 2014 at Bowring Park in St. John’s, Newfoundland, Canada

Adult (>1 yr. old)

Juvenile (<1 yr. old)

Male

14 (26.4%)

19 (35.8%)

Female

14 (26.4%)

6 (11.3%)

Sequencing of positive samples showed a strong relationship between viruses isolated in Newfoundland and viruses isolated in the United States.

In 2014, serum samples were collected from 52 birds between September and November. The prevalence of anti-NP antibodies in American Black Ducks increased marginally from September (3/12, 25.0%) to November (4/10, 40.0%) (Figure 2.2). Of the 52 screened serum samples, 16 samples (30.8%) were positive for anti-NP antibodies. Prevalence of previous infection was highest in adult males (6/14, 42.9%) with almost half of the captured birds testing positive for anti-NP antibodies. Results also show that the prevalence of previous infection was higher in adult birds (10/28, 35.7%) than in juvenile birds (6/24, 25.0%).

Figure 1.2 Capture rates for American Black Ducks from September to November in Newfoundland, Canada

Table 2 Prevalence of avian influenza virus infection for each sex separated by age classes in American Black Ducks captured at Bowring Park in St. John’s, Newfoundland, Canada.

Juvenile

Adult

Male

Female

Male

Female

Samples

Positives

10.5%

7.1%

14.3%

Table 3 Prevalence of avian influenza virus infection in each age and sex class in American Black Ducks captured at Bowring Park in St. John’s, Newfoundland, Canada.

Age

Sex

Juvenile

Adult

Male

Female

Samples

Positives

8.0%

10.7%

5.0%

12.1%

Prevalence of anti-AIV antibodies in sampled birds

Figure 3 Prevalence of anti-nucleoprotein (NP) antibodies in American Black Ducks from September to November in Newfoundland, Canada.

Table 4 Prevalence anti-nucleoprotein antibodies for each age class and sex in American Black Ducks in St. John’s, Newfoundland, Canada.

Juvenile

Adult

Male

Female

Male

Female

Samples

Positives

16.7%

27.8%

42.9%

28.6%

Table 5 Serological detection of avian influenza virus anti-nucleoprotein antibodies within age class and sex in American Black Ducks in St. John’s, Newfoundland, Canada.

Age

Sex

Month

Juvenile

Adult

Male

Female

September

2/7

1/4

2/7

October

2/13

7/18

5/12

4/19

November

2/4

2/6

1/4

3/6

Total

6/24,25.0%

10/28,35.7%

7/20,35.0%

9/32,28.1%

Genetic characterization of the detected viruses

Sequencing of positive samples showed a strong relationship between viruses isolated in Newfoundland and viruses isolated in the United States.

Relationship of haptoglobin concentrations to infection status

A total of 52 samples were assayed to determine haptoglobin (Hp) concentrations. Hp concentrations were higher in juveniles compared to infected adults (Fig. 2a), and Hp concentrations were higher in females compared to males (Fig. 2b). Haptoglobin levels in birds were higher in September than in November and showed a progressive decline between those months (Fig. 2e). No relationship was found between Hp levels and LPAIV infection or anti-NP antibody presence (Fig. 2c and d).

Figure 4 Immune status (mean ± SE) of resident wild American Black Ducks from September to November 2014 in St. John’s, Newfoundland, Canada. Relationship between haptoglobin (Hp) concentrations and (a) age and sex, (b) sex, (c) LPAIV infection, (d) anti-NP antibodies presence, and (e) month of sample collection.

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Appendix 1

Field data collection in 2014 at Bowring Park pond, St. John’s, Newfoundland.

ID #

Date (2014)

Sex

Age

Mass

Head-bill

Culmen

Total Tarsus

Tarsus

Wing

Notes (Band #)

1507

9-Oct

1300

114.9

53.7

270

1512

7-Oct

1125

107.4

48.9

43.3

269

1927-07860

1567

19-Sep

1250

111.5

49.2

58.6

263

1569

19-Sep

1150

109.6

60.1

54.3

275

1574

3-Oct

1150

112.2

54.3

57.8

273

1578

3-Oct

1100

107.6

48.7

55.1

42.7

268

1580

10-Oct

1300

107.7

47.7

56.6

47.5

260

1827-40379

1619

5-Nov

1475

114

50.5

47.2

285

1629

28-Oct

1450

110

50.8

57.2

44.8

273

1506

7-Oct

1050

108

50.6

55.3

42.2

257

1509

9-Oct

1050

106.8

264

1513

7-Oct

950

111.4

52.5

57.4

257

1561

8-Sep

1175

107.5

48.6

58.5

53.3

270

1564

8-Sep

1100

109.2

53.1

53.4

254

1568

19-Sep

875

100.3

45.5

54.2

47.5

229

1575

3-Oct

950

104.5

48.9

53.7

40.2

254

1576

3-Oct

1050

110.5

55.5

43.7

256

1577

3-Oct

1025

107.1

47.9

56.7

43.5

267

1579

3-Oct

1050

111.5

51.5

57.2

260

1581

10-Oct

1000

109

52.1

56.5

260

1584

14-Nov

1300

108.2

50.3

56.5

44.3

263

1620

5-Nov

1475

109.5

50.2

57.2

273

1511

9-Oct

1400

115.6

54.2

59.5

48.5

285

1547-39183

1562

8-Sep

1325

117.8

53.3

60.9

56.2

312

1927-07877

1582

10-Oct

1250

114.3

48.2

282

1927-07879

1614

7-Nov

1350

114.3

50.2

57.7

290

1927-07809

1615

7-Nov

1800

122.8

54.5

62.1

48.9

282

1867-31(5/3)42

1626

28-Oct

1300

114.9

54.8

287

1797-51215

1630

28-Oct

1200

116.8

53.2

58.5

278

1510

9-Oct

1275

117

47.6

277

1563

8-Sep

1225

115.8

56.2

55.3

269

1566

19-Sep

1175

110.5

60.7

46.9

274

1572

3-Oct

1150

108.2

50.3

56.9

44.8

267

1583

10-Oct

1200

113.9

53.7

58.1

46.2

280

1515

7-Oct

1225

115

55.5

57.3

46.1

302

1565

19-Sep

1075

108.4

51.5

57.2

43.2

272

1621

5-Nov

1350

107.5

57.3

45.5

273

1617

14-Nov

1050

105

50.5

264

1622

7-Nov

1300

105.9

49.3

56.5

44.3

267

1624

29-Oct

1150

111.6

49.5

57.1

269

1625

29-Oct

1050

105.7

48.5

55.5

44.2

270

1505

10-Oct

1450

117.1

54.2

60.9

47.2

290

1508

9-Oct

1250

114.4

51.4

60.2

52.2

285

1827-40381

1514

7-Oct

1400

115.8

46.6

292

1927-07564

1573

3-Oct

1250

116.5

54.5

58.5

42.5

272

1616

7-Nov

1450

115.5

51.5

58.5

44.7

282

1628

23-Oct

1350

114.8

52.6

58.6

45.5

283

1927-07755

1560

8-Sep

1250

116.9

53.9

60.3

280

1631

28-Oct

1400

107.5

48.4

44.6

271

1927-07681

1571

19-Sep

released

1623

7-Nov

1550

122.7

57.4

46.6

291

1632

28-Oct

1300

110

52.7

55.9

44.7

261

1570

19-Sep

1025

103.6

58.5

44.5

260

Avian Influenza presence in both oropharyngeal and cloacal swabs.

AIV #

Swab Positive

Serum Positive

1507

1512

1567

1569

1574

1578

1580

1619

1629

1506

1509

1513

1561

1564

1568

1575

1576

1577

1579

1581

1584

1620

1511

1562

1582

1614

1615

1626

1630

1510

1563

1566

1572

1583

1515

1565

1621

1617

1622

1624

1625

1505

1508

1514

1573

1616

1628

1560

1631

1571

1623

1632

1570

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