Controlling Animal Influenza and Decreasing Animal-to-Human

Transmission

David E. Swayne

Southeast Poultry Research Laboratory

Agricultural Research ServiceU.S. Department of Agriculture

Athens, Georgia

• Studies to assess effectiveness of strategies to control animal influenza

• Studies to assess optimal approaches to use and to assess impact of AI vaccines

• What cross-cutting technologies for control of animal influenza are translatable to human pandemic scenarios

• Studies to determine whether the circulating AI H5N1 virus in Asia is likely to cause a human pandemic

Questions

AR Health 9-04

WildBirds

Indoor Industrial

Poultry

Meat Ducks, Geese & Chickens

VillagePoultry

FightingCocks

Breeders

OutdoorIndustrial

Poultry

Layer Chickens

Meat Turkeys

Other Species

OrganicPoultry

Ducks & Geese

OtherSpecies

CaptiveBirds

Live Poultry Markets

1. Animal Influenza Control StrategiesNo single poultry production system in SE Asia – vary by country

Humans

Thus control will vary with each country & their specific agricultural systems

AR Health 9-04

WildBirds

Indoor Industrial

Poultry

Meat 3 Ducks, Geese & 1-Chickens

1 VillagePoultry

FightingCocks

8 Breeders

OutdoorIndustrial

Poultry

6 -Layer Chickens

Meat Turkeys

Other Species

OrganicPoultry

Ducks & Geese

OtherSpecies

CaptiveBirds

Live Poultry Markets

1. Animal Influenza Control StrategiesEpizootic – S. Korea

Humans

Thus control will vary with each country & their specific agricultural systems

AR Health 9-04

WildBirds

Indoor Industrial

Poultry

Meat Ducks, Geese & Chickens

VillagePoultry

FightingCocks

Breeders

OutdoorIndustrial

Poultry

Layer Chickens

Meat Turkeys

Other Species

OrganicPoultry

Ducks & Geese

OtherSpecies

CaptiveBirds

Live Poultry Markets

1. Animal Influenza Control StrategiesEpizootic – Malaysia

Humans

Thus control will vary with each country & their specific agricultural systems

AR Health 9-04

WildBirds

Indoor Industrial

Poultry

Meat Ducks, Geese & Chickens

VillagePoultry

FightingCocks

Breeders

OutdoorIndustrial

Poultry

Layer Chickens

Meat Turkeys

Other Species

OrganicPoultry

Ducks & Geese

OtherSpecies

CaptiveBirds

Live Poultry Markets

1. Animal Influenza Control StrategiesEpizootic –Thailand (1st wave)

Humans

Thus control will vary with each country & their specific agricultural systems

AR Health 9-04

WildBirds

Indoor Industrial

Poultry

Meat Ducks, Geese & Chickens

VillagePoultry

FightingCocks

Breeders

OutdoorIndustrial

Poultry

Layer Chickens

Meat Turkeys

Other Species

OrganicPoultry

Ducks & Geese

OtherSpecies

CaptiveBirds

Live Poultry Markets

1. Animal Influenza Control StrategiesEpizootic –Thailand (2st wave)

Humans

Thus control will vary with each country & their specific agricultural systems

Household Income. Household Income. Source: UNDP (2003).Source: UNDP (2003).

CountryCountry % Households below poverty line% Households below poverty line

< US$ 1/capita/day< US$ 1/capita/day < US$ 2/capita/day< US$ 2/capita/day

IndonesiaIndonesia 7.27.2 55.455.4

Lao PDRLao PDR 26.326.3 73.273.2

ThailandThailand 2.02.0 32.532.5

VietnamVietnam 17.717.7 63.763.7

CambodiaCambodia 36.1% below National Poverty Line36.1% below National Poverty Line

Dolberg – FAO Study

Many households have poultry – Many households have poultry – few are commercialfew are commercial

0

10

20

30

40

50

60

70

80

90

100

Holdings

* In all countries the large majority of rural households have poultry – even in Thailand.

* How to formulate strategies that have them co-exist with commercial units?

* Up to 80% no servicesDolberg – FAO Study

Duck systemsDuck systems Of interest because of the ducks’ roles as Of interest because of the ducks’ roles as

silent carriers of the virus, but limited datasilent carriers of the virus, but limited data Ducks are 10-15% of the poultry Ducks are 10-15% of the poultry

population in the five countriespopulation in the five countries Three systems:Three systems:

• CommercialCommercial• Migrating, seasonal and large flocks: rice Migrating, seasonal and large flocks: rice

fields, large water bodiesfields, large water bodies• A household system with a few ducks A household system with a few ducks

mixed with chicken – frequency not mixed with chicken – frequency not knownknown

Dolberg – FAO Study

The marketThe market

Played a role in spreading the Played a role in spreading the diseasedisease• Farmers sell sick animalsFarmers sell sick animals• Women keep birds in more than one Women keep birds in more than one

household as a safety measure for “a household as a safety measure for “a bad day”.bad day”.

Dolberg – FAO Study

1. Animal Influenza Control Strategies

• Veterinary infrastructure

• Adequate GDP

• Low rural population in animal agriculture

• Education on disease control

• Financial incentives to seek control

• Successes (eradication): Japan, S. Korea, Malaysia, (Taiwan)

• Successes in management: Hong Kong, China, (Thailand)

• Country specific epidemiological studies – FAO, OIE, EU, and others

• Surveillance: agricultural systems, captive birds and wild birds

• Understanding pathogenesis in affected bird species – domestic, captive and wild birds

• Could pigs play a future role in pandemic virus generation?

• Creative solutions for smallholder: education, economic incentives, changes in agricultural systems

IOM-NAS 2005

1. Animal Influenza Control Strategies

Potential Modes of Transmission to Humans• Inhalation:

• Contaminated dust from farming operations• Fine water droplets generated during slaughtering,

defeathering, eviscerating and preparing

• Contact with oral/nasal mucus membrane or conjunctiva:

• Hand-transplantation of virus from contaminated surface (poultry feces, respiratory secretions or other contaminated products)

• Direct oral exposure in cleaning fighting cocks?

• Consumption of raw products? • Duck blood pudding & internal organs• No epidemiological evidence at this time

2. Animal-to-Human Transmission

2. Animal-to-Human Transmission

• Exposure Risks for Infection: Assessment of H5N1 HPAI - human cases [HK 1997, Vietnam & Thailand early 2004] (Mounts et al., J. Inf. Dis, 180:505-508, 1999; Tran et al., NEJM 350 [12]:1179-88, 2004; Chotpitayasunondh et al., EID 2005 11(2):201-9)

– Risk: exposure 1 week before illness to live poultry, direct contact w/sick poultry

– Not a risk: travel, preparing or eating poultry meat, or exposure to human AI cases

– Not involved in organized culling or large poultry farms

– Cases were associated with Village (smallholder) poultry or Live Poultry Market

• Occupational risk for exposure & infection (HK 1997): poultry farmers, depopulation crews & processors (Bridges et al., J. Inf. Dis. 185:1005-1010, 2002).

• Some suspected limited human-to-human transmission – family clusters

• January 2004 cases in Hanoi (Liem et al., EID 2005

11(2):210-5): no health careworker cases• Needs:

– Timely epidemiological studies and sharing of data with veterinary medical sector which will assist in focusing control efforts in animal agricultural sector

2. Animal-to-Human Transmission

• Strategies for dealing with poultry disease are developed to achieve one of 3 goals or outcomes:– Prevention: preventing introduction – Management (Control): reducing losses by minimizing

negative economic impact through management practices– Eradication: total elimination

• These goals are achieved through various strategies developed using universal components: – Biosecurity (exclusion and inclusion) including quarantine– Diagnostics and surveillance – Elimination of AI virus infected poultry– Decreasing host susceptibility to the virus (vaccines and host

genetics)– Education

3. Disease Control Basics

IOM-NAS 2005

Avian Influenza Vaccines: Poultry

• Types of Vaccines– Inactivated whole AI virus (C,E)– Recombinant live virus vectors:

Fowl Pox (C), VEE (E), ALV (E), Vaccinia (E), ILT (E), NDV (E)

– Subunit AI proteins (E) - HA, NA: Baculovirus, Yeast, Bacterial, Plant

– Naked DNA vaccines (E)• Critical: safety, purity, potency &

economy

• Vaccination not routine in most of the world• No single vaccine for AI viruses• Anti-HA antibodies are protective, but NA also protective, less effective

IOM-NAS 2005

Vaccines in AI Control LPAI outbreaks -

• Waterfowl - origin viruses: Meat Turkeys (Minnesota: 22 million doses over 20 years) –

• Swine influenza (H1N1, H1N2, H3N2): Turkey Breeders (2.6 million USA 2001): other subtypes in world used

• H7 & H5 in Italy – use in areas high risk since 2000• H9N2 Middle East and Asia: billions (?) doses• Layers - rare use USA (inactivated H6N2 & H7N2)

HPAI – outbreaks Mexico (1995-2001) - H5N2: >1.3 billion doses inactivated &

>1 billion doses Fowlpox recombinant• Pakistan (1995-04) - H7N3: inactivated (? Doses)• Hong Kong (2002-04) – H5N1: inactivated; China &

Indonesia for unknown period (2 billion doses)IOM-NAS 2005

Avian Influenza Vaccines in Asia

• Inactivated vaccine strains:• A/turkey/England/73 (H5N2) LPAIV• A/chicken/Mexico/94 (H5N2) LPAIV• A/chicken/Indonesia/03 (H5N1) HPAIV• A/turkey/Wisconsin/68 (H5N9) LPAIV• Infectious clone: H5 & N1 genes of A/goose/Guangdong/96, 6 internal genes PR8

• Fowlpox recombinants with cDNA inserts of AI viral genes

• H5 gene - A/turkey/Ireland/83• H5 & N1 - A/goose/Guangdong/96

IOM-NAS 2005

Components of Effective Inactivated AI Vaccines

Proper adjuvant system (major) High antigen mass in each dose (major) Proper transportation, storage &

administration in high proportion of population to get effective immunization (major)

Proper vaccine strain – homologous hemagglutinin and sufficient sequence similarity (minor contribution)

IOM-NAS 2005

Priorities for Vaccination

1. High risk situations; e.g. in an outbreak zone as ring or suppressor vaccination

2. Valuable genetic stock such as pure lines or grandparent stocks whose individual value is high

3. Rare captive birds4. Long-lived birds, such as egg layers or parent

breeders5. Meat birds

Decreasing Order of Priority

IOM-NAS 2005

Properly Used AI Vaccines

Increase resistance to AIV infection Prevent clinical signs and death Reduced shedding of field virus when infected Prevent or reduce contact transmission Provide long protection from single vaccination Protect against high exposure dose of field virus Protect against a changing virus, but vaccine

strains will have limited life span NO STERLIZING IMMUNITY outside the

laboratory

Protection

IOM-NAS 2005

Fowlpox recombinant

(103 TCID50)* 0/12A 0/12A

Fowlpox recombinant

(104 TCID50)* 1/12A 1/12A (6.0)

Diluent 9/10B 9/10B (4.7)TW/68 Oil Emusified (2-

3 μg HA protein)** 0/12A 0/12A

Vaccine GroupMortality (Mean

Death Time in days)Morbidity

*H5 gene of A/turkey/Ireland/83** A/turkey/Wisconsin/68 (H5N9)

Chickens vaccinated SQ 1d with fowlpox-AIV-H5 recombinant* or inactivated whole AIV vaccine** and IN challenged at 3 wks with low challenge dose (103.3

EID50 of HPAIV A/chicken/South Korea/2003 [H5N1]).

Recombinant Fowlpox & Inactivated H5N9 AI Vaccine Protection Against H5N1

Swayne, Develop. Biol. 119:219-228, 2004

IOM-NAS 2005

Chickens vaccinated SQ 1d with fowlpox-AIV-H5 recombinant* or inactivated whole AIV vaccine** and IN challenged at 3 wks with low challenge dose (103.3

EID50 of HPAIV A/chicken/South Korea/2003 [H5N1]).

*H5 gene of A/turkey/Ireland/83** A/turkey/Wisconsin/68 (H5N9)

Oral swab Cloacal swabFowlpox recombinant

(103 TCID50)* 3/5AB (1.67) 0/5A (<0.97)

Fowlpox recombinant

(104 TCID50)* 0/5A (<0.97) 0/5A (<0.97)

Diluent 4/5B (3.06) 4/5B (1.98)TW/68 Oil Emusified (2-

3 μg HA protein)** 0/5A (<0.97) 0/5A (<0.97)

Vaccine Group

Virus Isolation, 2 days Post-challenge (Log10 EID50 titer/ml)

Swayne, Develop. Biol. 119:219-228, 2004

Recombinant Fowlpox & Inactivated H5N9 AI Vaccine Protection Against H5N1

IOM-NAS 2005

Vaccine Protection Against Asian H5N1

Virus Isolation, 2 DPC (Log10 EID50 titer/ml)

Group Vaccine Morbidity

(3-4+)* Mortality (MDT)** oral cloacal

1 Nobilis Hepatitis + ND Inac (Control)

10/10A

10/10A (2.2)

10/10A (6.16 a)

10/10A (5.82 a)

2

Nobilis I.A. Inactivated H5N2 (Mexican Strain)

0/10B 0/10B

5/10B (1.23 b)

3/10B (1.00 b)

3

Nobilis Influenza, H5N2 (European Strain)

1/10B 1/10B (2.0)

6/10AB (1.78 b)

3/10B (1.53 b)

Chickens vaccinated SQ 3 wks with inactivated whole AIV vaccine and IN challenged 3 wks later with 106.0 EID50 of HPAIV

(A/chicken/Indonesia/7/2003 [H5N1])• 1994 North American vaccine virus

•1986 Eurasian vaccine virus

HA1 A.A. similarity with challenge virus, Mexican Strain 84.8% & European 91.9%IOM-NAS 2005

Ck/Hong Kong/156/97 *CK/Hong Kong/915/97Hong Kong/483/97CK/Hong Kong/220/97CK/Hong Kong/728/97

CK/Italy/1485/97Peking Duck/Singapore/645/97

TK/England/50-92/91 *DK/Hong Kong/342/78DK/Ireland/113/83TK/Ireland/1378/83 *CK/Scotland/59 *Tern/South Africa/61 *

CK/Pennsylvania/1/83 *TK/WI/68TK/Ontario/7732/66 *Emu/Tx/39442/93 *CK/Pennsylvania/13609/93 Ruddy Turnstone/DE/244/91CK/Mexico/31381-1/94CK/Queretaro/7653-20/95 *

Eurasian

North American

(Swayne et al., Vaccine 18:1088-1095. 2000)

* challenge viruses

Protect Against a Changing Virus

• Fowl pox with H5 AIV gene insert• Different challenge viruses (87.3-100% aa sequence similarity)

IOM-NAS 2005

Fowlpox Recombinant Virus: Protection Against Changing Virus

Vaccine Virus HA A.A. Similarity

Fowlpox controls

Fowlpox-AI HA*

TK/Ireland/83 100% 10/10 0/10

TK/England/91 94.2% 10/10 0/10

Tern/SAfrica/59 93.1% 10/10 0/10

CK/Scotland/59 92.0% 9/10 0/10

Hong Kong/156/97 90.2% 8/10 0/10

CK/Queretaro/14588/95 89.3% 10/10 0/10

TK/Ontario/77322/66 89.1% 9/10 0/10

Emu/TX/399924/93 88.8% 7/10 0/10

CK/PA/1370/83 87.3% 10/10 0/10

*Fowlpox-AI HA had TK/Ireland/83 as insert(Swayne et al., Vaccine 18:1088-1095. 2000)IOM-NAS 2005

• Variable reduction in shedding of challenge virus

(Swayne et al., Vaccine 18:1088-1095. 2000)

Oropharangeal Swabs - Peak Shedding

1 1.5 2 2.5 3 3.5 4 4.5

Cloacal Swabs - Peak Shedding

1 1.5 2 2.5 3 3.5 4 4.5

rs = 0.783, P = 0.009 rs = -0.100, P = 0.780

Reduction in Titer (Log 10)

Protection in the Face of Changing AIV

• 100% protection from clinical signs and death

IOM-NAS 2005

Broad and longer-term protection efficacy of poultry AI vaccines

• Proprietary oil-emulsion-adjuvant technology → intense & long-lived immune response

• AI virus immune response in poultry appears to be broader than in humans

• Greater genetic homogeneity in poultry gives more consistent immunity

• Young, healthy poultry population are immunized verses in humans with emphasis on population at highest risk of severe illness and death

• Limit to how long a vaccine strain can be used – evaluated biennially

IOM-NAS 2005

Limitations and Disadvantages of Avian Influenza Vaccines

• Best protection is in experimental studies with specific pathogen free chickens

• Field protection less than in laboratory• High challenge exposure• Improper vaccination technique• Reduced vaccine dose• Immunosuppressive viruses• Improper storage & handling of vaccines• Unable to vaccinate 100% of poultry

population

IOM-NAS 2005

Cross Protection of Commercially Vaccinated Birds to Different LPAI Challenge

Oropharyngeal Viral Titers Challenge virus DPIa Vaccinated Control

3DPI 1.66 (5/10) 4.5 (5/5) Vaccine strain Jalisco Lineage 5DPI 0.98 (4/10) 3.1 (5/5)

3DPI 4.44 (10/10) 4.2 (5/5) CK/AG/124-3705/98 Lineage A 5DPI 2.14 (8/10) 2.4 (5/5)

3DPI 4.86 (10/10) 4.9 (5/5) CK/Guatermala/194573/02 Lineage B 5DPI 3.62 (10/10) 3.4 (5/5)

• Mexico: use of vaccination to control both LPAI and HPAI started in 1995 using killed and in 1998 using recombinant Fowl poxvirus (TK/Ireland/83 H5 gene insert)

• Has field strain drifted from vaccine? Is protection still adequate?

IOM-NAS 2005(Lee et al., J. Virol. 78:8372-8381, 2004)

CK/Jalisco/14585-660/94CK/Jalisco/28159-600/95CK/Hidalgo/28159-460/95

CK/Guanajuato/28159-331/95CK/Michoacan/28159-530/95

CK/Queretaro/14588-19/94CK/Queretaro/7653-20/95

CK/Queretaro/22019-853/96CK/Mexico/31381-1/94

CK/Mexico/31381-2/94CK/Mexico/31381-4/94CK/Mexico/31381-3/94CK/Mexico/31382-1/94CK/Mexico/31381-5/94

CK/Hidalgo/28159-232/95 (VACCINE)CK/Queretaro/26654-1373/94CK/Morelos/28159-538/95

CK/Hidalgo/26654-1368/94CK/Mexico/26654-1374/94

CK/VeraCruz/28159-398/95CK/Mexico/31381-6/94

CK/Mexico/31381-8/94CK/Mexico/31381-7/94

CK/Mexico/15407/97CK/Mexico/28159-541/95

CK/Chiapas/28159-488/95CK/Mexico/37821-771/96CK/Chiapas/15406/97

CK/Chiapas/15408/97CK/Vera Cruz/232-6169/98CK/Puebla/231-5284/98

CK/Morelos/FO22189/98CK/Morelos/227-4353/98CK/Jalisco/229-4592/98

CK/Aguascalientes/124-3705/98CK/Puebla/14585-622/94CK/Puebla/14586-654/94

CK/Puebla/8623-607/94CK/Puebla/8624-604/94

CK/Puebla/28159-474/95CK/Chiapas/15224/97

CK/Chiapas/15405/97CK/Tabasco/234-8289/98

CK/FO/22066/98CK/Guatemala/45511-1/00CK/Guatemala/45511-2/00CK/Guatemala/45511-3/00

CK/Guatemala/45511-4/00CK/El Salvador/102711-1/01

CK/El Salvador/102711-2/01CK/Guatemala/45511-5/00CK/Guatemala/194573/02

5 changes

HA

Jalisco

Puebla

A

B

Mexican H5 Phylogenetic Lineage

A

B

IOM-NAS 2005(Lee et al., J. Virol. 78:8372-8381, 2004)

Limitations and Disadvantages of Avian Influenza Vaccines

• Must be able to differentiate infected from vaccinated animals (DIVA):• Must detect “silent” infections and eliminate

immediately• All vaccinated flocks must have surveillance

• Specific serological tests, or

• Unvaccinated sentinel animals – serology and virus detection, and

• Virus detection (virus isolation or RT-PCR) on dead birds

IOM-NAS 2005

Interference of AI Vaccination with Surveillance

AI Field VirusHomologous NA inactivated AIV vaccineHeterologous NA inactivated AIV vaccineRecombinant Fowlpox,

subunit HA & DNA HA vaccines

Unvaccinated sentinels

Serological Test

NP/M(AGP/ELISA)

X

X

X

--

HA(HI)

X

X

X

X-

NA(NI)

X

X

-

--

NA(NI)

-

-

X

--

Homo. Hetero.

NS

X

-

-

--

IOM-NAS 2005

Needs in Vaccines and Vaccination

1. Standards in purity, safety & potency of AI vaccines

2. Studies to confirm efficacy of AI vaccines in ducks, geese and other minor poultry species

3. Effective vaccines that can be applied by mass immunization method

4. Metabolizable oil adjuvant systems5. Sterilizing immunity?6. Effective DIVA strategies that will be used to

identify infected flocks for elimination7. Periodic evaluation of vaccine strains for efficacy

against predominate circulating strains

IOM-NAS 2005

4. Will the H5N1 Be the Next Pandemic Virus?

Virus Mort. %BW

Italy/97 0/8 -3

Scot/59 0/8 5

Eng/91 1/8 -18

Q20/95 0/8 -2

HK/156 8/8 -26

HK/220 8/8 -25

HK/728 6/8 -10

Sham 0/8 7(Dybing et al., 2000)

• Experimentally, some AIV cause infection and disease in mice, but not all AIV do!

• Lesion distribution and infection in mice similar to humans

Dybing et al., J. Virol 74(3):1443-1450, 2000

• Value of Ferret and Primate models

IOM-NAS 2005

Not all Asian H5N1 AIV have same potential to infect and cause disease in humans

Virus Mortality Lung Lesions Virus location220/97 100% 33-80% lung, trachea, brain, kidney317.5/01 0-10% 20-33% lung, trachea Anyang/01 22-33% 0-10% lung, trachea

Three H5N2 HPAIV in intranasally inoculated BALB/c mice

• 2003-2004: Cases only in Thailand, Vietnam and Cambodia, but not other Asian countries with H5N1 poultry cases

• Differences in virus strains• Exposure differences

(Tumpey et al., J. Virol. 76:6344-6355, 2002)

IOM-NAS 2005

Do LPAIV have the potential to infect and cause disease in humans?

Groups Mouse Strain

BALB/c (Mx1-) CAST/Ei (Mx1+ & Mx1-)

Mortality Virus Isolationb Mortality Virus Isolationb

Trachea Lung Trachea Lung

Control 0/2 0/2 0/2 0/2 0/2 0/2

PA/11767/97(H7N2)

0/5 1 /2(102.5) 0/2 0/5 0/2 0/2

PA/19241/97 (H7N2)

0/5 2/2(106.2) 2/2(105.2) 0/5 0/2 0/2

HK/156/97 (H5N1) 5/5 2/2(106.0) 2/2(107.8) 5/5 2/2(104.0) 2/2(107.2)

(Henzler et al., Avian Diseases 47:1022-1036, 2003)IOM-NAS 2005

Human Pandemic Influenza

• Which one of the avian influenza viruses could contribute genes to the next human pandemic virus?• LP verses HPAI as contributor of genes• Asian H5N1 HPAI• Asian H9N2 LPAI• H7N3 HPAI• H7N7 HPAI• H7N2 LPAI• H2N2 – the old nemesis?

Needs: Determine What AI Virus(es) Have Greatest Potential to be the Next Pandemic

Virus!

1. Develop, evaluate and use animal models to predict and understand human infection and transmission potential for circulating AI viruses including H5N1 Asian viruses

2. Determining in vitro anti-viral susceptibility and in vivo vaccine protection against circulating H5N1 AI viruses as prelude to human prevention

3. Reverse genetic studies to identify avian genes from specific strains with greatest pandemic potential (reassortants) - Caution should be exercised as to the appropriate biosafety level for reassortant studies to generate a potentially pandemic strain by reverse genetics

IOM-NAS 2005

Thank You For Your Attention!

IOM-NAS 2005