Guidelines for Field Veterinarians and Para-professionalson the Surveillance of Avian Influenza

KOIKE I. (Technical Consultant) 2007

1. Review of Fundamental Information

2. NAI Surveillance Programme and Role of Field Veterinarians (FV) and Para-professionals (PP)

3. Surveillance Strategies: Important Roles of FV and PP for Clinical Surveillance, Serological Surveillance and Virological Surveillance

4. Serological and Virological Surveillance in Vaccinated Populations (Meaning of Sentinel Birds)

5. Endeavour Toward NAI Free Country/Province in Poultry; Case-Study in Some Country

The Surveillance of Avian Influenza

1. Review of Fundamental Information (1)

• Surveillance: ?

The investigation Population

Subpopulation

Detect a pathogen agent / disease

frequency & type (clinical, serological,virological)

Epidemiology Pur

pose

Desired output

Ref.(1)OIE Terrestrial Animal Health Code(TAHC)– 2007, General Definition

1. Review of Fundamental Information (2)

• Monitoring: ?

The continuous investigation Population

Subpopulation

Environment

changes in the prevalence of a disease/ characteristics of a pathogenic agent.

Pur

pose

Detect

Ref.(1) OIE TAHC – 2007, General Definition

1. Review of Fundamental Information (3)

No Country Can Declare Itself Free From Avian Influenza In Wild Birds

Ref.(2) OIE TAHC – 2007, Guidelines for the Surveillance of Avian Influenza (GSAI)

1. Review of Fundamental Information (4)

• NAI ?: One of Notifiable Diseases: listed by the Veterinary Administration

Detected, Suspected in the Field

Ref.(1) OIE TAHC – 2007, General Definition, (4) OIE TAHC – 2007, Avian Influenza

V

Veterinary Authority

Informing

(National Regulation)

NAI: Notifiable Avian Influenza

Influenza A virus

H5 subtype

H7 subtype

Any AI virus (IVPI>1.2 or >75%mortality)

1. Review of Fundamental Information (5)

•　H5 , H7?:

Hemagglutinin (HA:antigenic glycoprotein) on the surface of the influenza virus

Ref: (3) Science 21-April 2006 Vol.312, (5) Wikipedia; Hemagglutinin, Neuraminidase,

Hemagglutinin:16 different antigens at least, Subtypes: H1, H2,H3,H4,H5,H6 H7, H8,H9,H10,H11,H12,H13, H14, H15, H16

(1.biding to the receptors on the target vertebrate cells,. 2. causing the fusion of host endosomal membrane with the viral one)

Neuraminidase:antigenic glycoprotein (enzyme) on the surface of the influenza virus,

Subtype:N1,N2,N3,N4,N5,N6,N7,N8,N91. promoting the release of progeny viruses from infected cells, 2. preventing aggregation of viruses, 3. help the virus to enter the host by cleaving sialic acid from the host glycoprotein

1. Review of Fundamental Information (5)

Mechanism of Hemagglutinin Function

Receptor

Ref: (6) RCSB Protein Data Bank,

1. Review of Fundamental Information (6)

NAI

HPNAI: Highly Pathogenic Notifiable Avian Influenza

LPNAI: Low Pathogenicity Notifiable Avian Influenza

1. Intravenous inoculation (IC) (0.2ml,1/10 dilution of infective A.F )

susceptible 4 to 8 w.o

(lethal at least for six of eight:75%)<10days

Or

2. Intravenous pathogenicity index (IVPI): (0.1ml, 1/10 dilution of A.F.HA>1/16)

SPF, SAN 6 w.o.

(1.2<mean score per bird per observation 24-h,10d)

HPNAI

HPNAI

Ref. (3) OIE TAHC – 2007, Avian Influenza, (7)OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (MDTVTA) NB:

3. H5, H7 : IC mortality test <75 %, IVPI < 1.2

Amino acid sequence at cleavage site of the hemagglutinin to be examined

A.F: allantoic fluid,

Result

(H5 & H7 subtype except HPNAI)

HPNAI

Amino acid motif: non similar to other HPNAI isolates LPNAI

Amino acid motif: similar to other HPNAI isolates

1. Review of Fundamental Information (7)•Amino acid sequence at cleavage site:

HA 1 HA 2

Cleavage site

Ref: (3) Science 21-April 2006 Vol.312, (8) Wikipedia, Genetic code,(9)T.Horimoto et al.

HA

- - -P Q R R R K T R / G - - -

Alanine:A, Arginine: R, Asparagine: N, Aspartic acid: D, Cysteine: C, Glutamine: Q, Glutamic acid: E,

Glycine: G, Histidine: H, Isoleucine : I, Leucine: L, Lysine: K, Methionine: M, Phenylalanine: F, Proline: P, Serine: S Threonine: T, Tryptophan: W, Tyrosine: Y, Valine: V,

Abbreviation of amino acid

HPNAI motif: X-X-R/K –X- R/K-R, or R-R/K- R-K-T- R,

e.g.--E R S R R K K R / G---

--R E R R R K K R / G---

A/muscovy duck/Jakarta/Sum 106/2006 (H5N1)

HPNAI

A/goose/Cambodia/28/2004 (H5N1)

HPNAI

--P Q R K R K T R / G---

A/chicken/Queretaro/14588-19/1994(H5N2)

HPNAI

--N V P Q R E T R / G--- LPNAIA/chicken/Jalisco/14585-660/1994(H5N2)

X: nonbasic residues

1. Review of Fundamental Information(8)

Ref. (10) Wikipedia, Influenza

1. Review of Fundamental Information (9)

• Access to OIE Terrestrial Animal Health Code,

• Access to OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals,

Through Web Site

OIE Home Page

http://www.oie.int

OIE Publications1st

2nd Online Publications

Terrestrial Code

Terrestrial Manual

Aquatic Code

Aquatic Manual

Disease Information

etc

For Latest Information

2. NAI Surveillance Programme and Role of Field Veterinarians (FV) and Para-professionals (PP)

•The Purpose of NAI Surveillance To Detect NAI in Poultry

Ref.(2) OIE TAHC – 2007, GSAI

farmer/worker FV , PP’s communication with farmers

Veterinary Authority

V

Chief of village

Sample collection

To Show NAI Free Country etc.

Laboratory Testing

Early warning system

2. NAI Surveillance Programme and Role of Field Veterinarians (FV) and Para-professionals (PP)

•Free

<Status> <Surveillance> <Target> <Duration> <Verification>In accordance

with OIE TAHC – 2007, GSAI

Susceptible poultry population (non-vaccinated, vaccinated)

Preceding 12 months

Absence of NAIV Infection

HPNAI infection found in a previously free country/zone

LPNAI infection found in a previously free country/zone

In accordance with OIE TAHC – 2007, GSAI

In accordance with OIE TAHC – 2007, GSAI

Susceptible poultry population

3 months after a stamping-out policy, disinfection of all affected establishments

Absence of NAIV Infection

Regain

Susceptible poultry population

3 months after a stamping-out policy, disinfection of all affected establishments

Absence of NAIV Infection

Active surveillance: at least every 6 months Ref. (3) OIE TAHC – 2007, Avian Influenza,

Active

Passive

3.Surveillance Strategies: Important Roles of FV and PP for Clinical Surveillance, Serological Surveillance and Virological Surveillance

Clinical Surveillance * HPNAI, LPNAI

Serological Surveillance *Detection of antibodies against NAIV

Virological Surveillance * Monitor at risk population * Confirm clinically suspect cases * Follow up positive serological results * Test daily mortality * Ensure early detection of infection in the

face of vaccination or in establishment epidemiologically linked to an outbreak

FV / PP

Information through communication,

Sample collection,

Liaison between veterinary authority and farmers

Ref.(2) OIE TAHC – 2007, GSAI

bacteria

virus

protozoa mold other parasites

heating

humidity

ventilation

premise temperature

raining

Major Factors Affecting Health of Fowls

premise structure

vectors

feed

mice etc.

wild birds

personnel

drinking water litter

lighting

predators

Clinical Surveillance

Mor

tali

ty

Economic Loss

100%

100%

Understanding of Major Viral Diseases

50

Newcastle (V

VND) Dise

ase

Virulen

t In

fectio

us B

ursal

D

iseas

e

Marek’s D

isease

(Loss of Chickens, Egg Production, Growth Rate, Feed Efficiency etc.)

Fowl

P

ox

M ty

pe

I. BEDS

Highly Pathogenic

Notifiable

Avian Influ

enza

(HPNAI)

Y C

Adult Birds

A. E

I L TOthers

Reo

CA

AE: Avian EncephalomyelitisReo: Reovirus InfectionILT: Infectious LaryngotrachitisCA: Chicken AnemiaEDS: Egg Drop SyndromesYC: Young Chickens CasesM. type: Mucous-membrane typeLPAI: Low Pathogenicity A I (except H5 & H7)LPNAI: Low Pathogenicity Notifiable A I VV: Velogenic Viscerotropic (Asian type)

ND, Mesogenic

LPAILPNAI

H5 & H7

Mutation

Clinical Surveillance

Development of AI Outbreaks in Mexico (1)

Reference: (11)Laureano(CPA), (12)M.Garcia(SPRL)

< Review > 1980-1981: field investigation by Dr.Lorenzo T. & Dr.Benzamine L. (AGP test all negative)< Harbinger > 1993 : Mexico Central Region(6 states) (decreased egg production, respiratory disorder etc.) 1994 (suspected: ND, Coryza, IB, Swelling syndrome etc.) Mar-Aug: Campaign for strengthening biosecurity May : Mexico Central Region—AI virus isolation from broiler farms (AIV: H5N2, LPAI)

Development of AI Outbreaks in Mexico (2)<Epidemic> 1994 15th Dec:Puebla State (Tehuacan district; AIV isolation from layer farms) AIV (H5N2) of HPAI,(10-15% mortality in layer farms) 1995 1st Jan : Queretaro State(Marques, Atongo town) AIV (H5N2) of HPAI from parents stock farms 1st Feb: Vaccine introduced (homologous type:local isolate) HPAI : 9 farms infected, LPAI: 424 farms (HPAI mortality :20-30% in breeders, 50% in broilers) Amount of suffered birds: about 49 million ones,

1995 5th Jun: Last case of HPAI 2000 Guatemala 2001 El Salvador Sporadic occurrence of LPAI 2006 12 States: free, 20 States: eradication stage,

LPAI only

Approximate No. of birds infected/culled so far: >1,000,000,000 in Mexico, Guatemala and El Salvador

Reference: (11)Laureano(CPA), (13)I.Capud(OIE) (14)John Summers (PIC,Canada)

LPNAI

Lessons from the NAI Outbreaks in Mexico

1. Less Attention to LPNAI (H5) in the First Half of 1990s

2. Less Seriousness to LPNAI Because of Little Manifestation and Little Economic Loss in Management and Public Society

3. Less Seriousness to LPNAI in All Poultry Sectors (Lack of Public Awareness)

Particularly for Biosecurity and Workers’ Stance

4. Easily Spread from One Farm to Another Farm (State)

5. Passages of the AI Virus in the Poultry Flocks

6. Mutation from LPNAI to HPNAI in Some Poultry Flocks

7. Crisis in the Poultry Sectors and Public Society

Serological Surveillance

• Detection of Type A : • Nucleoprotein/Matrix(NP/M)

•ELISA test

•Agar gel immunodiffusion (AGID) test

• Detection of Subtype: • HA, NA, Serum Neutralization (SN)

• HI test

• NI test(FPS:NG)

•SN test

FV / PP

Blood collection with filter paper strip(FPS) / syringe-needle

FPS:NG, serum from FPS is not suitable for NI test because hemolytic phenomenon affects OD measure

Serological Surveillance for the Detection of Antibodies Against NAIV

Positive reactors mean;

• Natural Infection with NAIV

• Vaccination against NAIV

• Maternal antibodies derived from a vaccinated or infected parent flock are usually found in the yolk and can persist in progeny for up to 4 weeks• Positive result due to the lack of specificity of the test

<Interpretation of Characteristics of Serological Tests as Reference Information>

• HI test: Reliable in Avian Species, but not in Mammals

• AGID:

Reliable for Detection of NP/M Antibodies in Chickens and Turkeys, but not in Other Avian Species

• ELISA: (blocking)

Detect NP/M Antibodies in All Avian Species

• SN: Preferred Test for Mammals and Some Avian Species

Ref.(2) OIE TAHC – 2007, GSAI

No. of farms surveyed

(No. of chickens tested)

No. of positive ｆarms

(No. of positive chickens)

No. of negative ｆarms

(No. of negative chickens)

+ ratio in farms(%)

( + ratio in chickens

CB 22 (44) 10 (17) 12(27) 45 (38)

BS 11(31) 8(10) 3(21) 73 (32)

BG 3(17) 3(6) 0(11) 100 (35)

DJ 12(21) 1(1) 11(20) 8.3 (4 .7)

Farm

Site

Test method: ELISA test kit for Detection of Antibody to AI (Type: A) Matrix/Nucleoprotein antigens (made in USA)

Survey Year: 2002

AI Serological Surveillance in Some Country

Target: Village chickens of free range under extensive rearing

Factors: Bringing Pathogens, Transmitting Factors,

4. Serological and Virological Surveillance in Vaccinated Populations (Meaning of Sentinel Birds)<Type of Vaccine>

1. Inactivated whole AI virus

•Homologous NA to Field Virus

•Heterologous NA to the Field Virus

<Surveillance>

•Setting of Sentinel Birds: Check of Infection

• virus isolation, detection of virus specific genome material or proteins

• detection of antibody to the field virus NA

• virus isolation, detection of virus specific genome material or proteins2. Hemagglutinin expression-

based vaccine (recombinant vaccine-fowl pox base)

• detection of antibody to NP/M

• virus isolation, detection of virus specific genome material or proteins

<Sentinel birds on flat rearing>

<Sentinel birds in cage rearing>

Endeavour Toward NAI Free Country / Province: Case-Study in Some Country

Development of AI Outbreaks in Mexico

<Spatial Distribution of AI Outbreaks in 1994-1995>

Reference: (15)Chang-Won Lee(SPRL)

Affected: 13/32

Current Situation of AI Map in Mexico

Reference:(11) Laureano(CPA)

Year Outbreaks (Detection of LPNAI) 2000 3 2001 4 2002 15 2003 11 2004 9 2005 49

(without vaccine)

Free: 12/32, Eradication stage: 20/32

Countermeasure against AI

　 Year 　　 HPAI 　　　　 Countermeasure 　　　　　　　 Vaccine (dose)

１９９５ ＋ Pa, Pn, Qi, S, te, V 383.2 million

１９９６ 0 Pa, Pn, Qi, S, te, V 331.3 million

１９９７ 0 Qf, Te, M, Su, V, Z 240.8 million

1998 0 Qf, Te, M, Su, V, Z Unknown

1999 0 Qf, Te, M, Su, V, Z Unknown

２０００ 0 Qf, Te, M, Su, V, Z 86.91 million

２００１ 0 Qf, Te, M, Su, V, Z 87 million

２００ 2 0 Qf, Qi 　 Te, M, Su, V,Z 260.242 million

２００３ 0 V, Su, M, S, Qi, Z, 281.494 million

２００４　 ０　　　　 Su, M, S, Qi (Jalisco State case)

Pa: control programme for some areas of the country only or for certain types of

breeding only, Pn: control programme for the whole country, Qf: Quarantine, precautions

at the border, Qi: Movement control inside the country, S: stamping out, Te: testing,

V: vaccination, Su: Surveillance, M: monitoring, Z: Zoning,Reference:(11), (16)OIE, Handistatus II Multiannual disease status

Nati onw

i de

Countermeasure against AI<Vaccine> 1. Inactive vaccine with oil adjuvant Seed virus: CK/Mexico/232/94(H5N2) controlled by CPA* (field isolate of LPAI) 2. Recombinant vaccine ( Vector: Fowl Pox) in Mexico -1. H5 hemagglutinin gene from AI virus A/turkey/Ireland/83, -2. Developed in the Lab. of Enzo Paoletti, Canada, during mid-1980’s (AI gene insert being provided by Robert G.Webster of St. Jude Children's Research Hospital) -3. Merial Co. purchased the vaccine and is licensed by USDA. -4. 850 million doses have been used in Mexico, Guatemala, and El Salvador since 1997

Reference:(11), (17)D. Swayne,

*Comision Mexico-Estados Unidos para Prevencion de la Fiebre Aftosa y otras Enfermedades Exoticas en los Animals

Countermeasure against AI

Vaccine maker

Strain　 (H5N2) Product

Laboratorio Avimex

A/Chicken/Mexico/232/94/CPA

*Avian Influenza (mono: inactive)*Newcastle-Influenza (duo: inactive)

Boehringer Ingelheim

A/Chicken/Mexico/232/94/CPA

*Enza-vac (mono:inactive)*Enza-vac+NDV (duo:inactive)

IASA(IASA-Biommune)

A/Chicken/Mexico/232/94/CPA

*Influenza Aviar-Newcastle (duo:inactive)

Intervet A/Chicken/Mexico/232/94/CPA

*Nobilis IA Inac (mono:inactive)*Nobilis IA+ND Inac(duo:inactive)*Nobilis IA Coryza Inac(duo:inactive)

<Major Vaccine Domestic Makers>

HPAI

Vaccine Strain

HPAI

Guatemala,El Salbador

(12)M.Garcia, (15)Chang-Won Lee(SPRL)

Antigen Drift

*Endeavor for Eradication of AI in Jalisco State

*Diagnosis in The Laboratory

J a l i s c o

Surveillance ProcedureKinds of Birds Sample

Commercial birds

(broiler, layer,etc)

Living birds, Cloaca swabs, Trachea swabs,

Organs, Feeder swabs,(trachea/cloaca swab:60 pcs/flock, whole body:15-20/flock)

Back yard birds Cloaca swabs, Trachea swabs, Organs,

(trachea/cloaca swab: all birds/flock, organs:case by case)

Fighting cock Cloaca swabs, Trachea swabs (trachea/cloaca swab:30 - 60/flock,

Ostrich Cloaca swabs, Trachea swabs (trachea/cloaca swab:60 pcs /flock, )

Hobby/Pet,

Others

Cloaca swabs, Trachea swabs, Organs (trachea/cloaca swab: all birds/flock, organs:case by case)

Frequency of SurveillanceCommercial Production Sector

• Broiler Industry

• Layer Industry

• Breeding Stock Farms for Broilers/Layers/Others

•Parents Stock Farms

Frequency Inspection Fee

Every time of marketing

Payment by themselves

At least 6 months

At least 6 months

At least 6 months

Payment by themselves

Payment by themselves

Payment by themselves

•Backyard Raising/ Fighting Cock/ Pet Bird/Zoo Animal etc. At least 6 months

Covered by Regional Livestock Committee

Virological Surveillance

<Living birds>

< Organs to be inspected for ND and AI >

•Trachea•Lung•Spleen•Cerebrum

The same organs of thesame flock shall pool asone sample respectively

Homogenized suspension of each sample shall inoculate into5 CE eggs,9-10 d.o.,respectively.Incubate eggs in a hatcher for4 – 5 days. Observe eggs every day.

< Swabs to be inspected for ND and AI >

The same procedure as the above

Achievement of Monitoring from 2002 to 2005

Year

Item2002 2003 2004 2005

No. of districts monitored to

total districts

25

124

37

124

113

124

?

124

Ratio (%) 20 29 91 ?

Administrative Districts Monitored

Total birds inspected with virological surveillance per year: 15,000 on the average

Relacion de Muestras Serologicas por Enfermedad Rumiantes y Aves

0

2000

4000

6000

8000

10000

12000

14000

16000

Sospechosos 57 14

Negativos 9122 740 351 58 18 13486

Positivos 737 805 225 48 132 572

Brucela(Tar) Brucela(Riv) Leptospira IBR DVB Influenza Aves

Nov. 2003 - Jul.2004

Total: 26,365

Review of AI Virus(H5) Isolation through Monitoring from 2002 to 2005 in Jalisco State

No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15Year 02 03 03 03 03 03 03 03 04 04 04 04 04 04 04Farm A B B C C D D E F G H H I J KDistrict TZ ES ES CC CC ZP ZP ZP ZP TN EZ EZ IX GF TIPlaque

Test_ _ _ _ _ _ _ _ _ _ _ _ _ _ _

PCR*

Matrix ＋＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋

PCR*

H5＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋

Type LP LP LP LP LP LP LP LP LP LP LP LP LP LP LP

LP: low pathogenic,

* Ref (17)Erica Spackman et al.,

All of positive farms were commercial broiler ones.

Plaque Test: Chicken Embryo Fibroblast Culture without Trypsin

Surveillance Toward NAI Free Country/Province in Mexico

<Administrative Stage> <Situation> Control stage: Epidemic, Stamping out/Vaccination etc. Strengthen Biosecurity,

Eradication Strengthen Biosecurity stage : 1St Sporadic, partly vaccination, 2nd Rarely occur, vaccination prohibited No occurrence in the field during 1-2 years

Free stage : No occurrence without vaccination under strengthened biosecurity

Monitoring supported by field vet., para-prof.

(Monitoring, Surveillance)

Carry on Monitoring

Reference: (18) NOM-044-1995,

Surveillance: clinical, serological, virological (supported by field vet., para-prof.)

(field vet., para-prof.)

Monitoring

Others<Background of AI Invading>

Aquatic migratory birds

Reference: (19) Horimoto, (20) R.G. Webster

Lessons from the Countermeasures in Mexico

1. Adoption of VaccinationPartly success for control

Antigen drift has taken place

Less effect in vaccination

2. Thorough Serological Surveillance and Virological One

3. All Birds (Poultry & Pet Birds) Reared by Public

The Targets of Surveillance

5. Vaccination Shall be Ceased in Accordance with the Progress of Control/Eradication Stage

4. Virological Positive Flock Stamping Out

Thank you very much

October 2007

The Dawn Comes Surely

(1)OIE Terrestrial Animal Health Code(TAHC)– 2007, General Definition

(2) OIE TAHC – 2007, Guidelines for the Surveillance of Avian Influenza

(3) Science, Vol. 312, 2006 April 21,

(4) OIE TAHC – 2007, Avian Influenza

(5) Wikipedia; Hemagglutinin, Neuraminidase, http://en.wikipedia.org/wiki/Hemagglutinin,

http://en.wikipedia.org/wiki/Neuraminidase,

(6) RCSB Protein Data Bank, http://www.pdb.org./pdb/static.do?p=education_discussion/molecule_of_the_month/p--

(7)OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Avian Influenza

(8) Wikipedia, Genetic code, http://en.wikipedia.org/wiki/Genetic code,

(9) T.Horimoto, Y.Kawaoka, Journal of Virology, May,1994,p.3120-3218

(10) Wikipedia, Influenza, http://en.wikipedia.org/wiki/Influenza

(11) Laureano Vazquez M., Panel Discussion about AI, 2005, 11,3, CUCBA, Guadalajara, Jalisco, Mexico

(12) M.Garcia et al. Journal of General Virology (1996),Vol. 77,1493 – 1504,

(13) I. Capud et al.; Currently available tools and strategies for emergency vaccination in case of avian influenza, http://library.wur.nl/frontis/avian_influenza/idex.html

<Reference List >

<Reference List>

(15) Chan-Won Lee, et al., Journal of Virology(2004) Vol.78, No.15, 8372 – 8381,

(16) OIE, Handistatus II Multiannual disease status, 2005(17) D. Swayne et al. Av Dis. (1997) 41, 910 – 922,(18) Erica Spackman et al., Journal of Clinical Microbiology (2002) Vol.40, No.9, 3256-3260,(19) NOM-044-1995, Mexico(20) Horimoto et al. Virology (1995) Vol.213, 1, 223 – 230,(21) R.G. Webster , Influenza: An Emerging Disease, http://cdc.gov/ncidod/eid/vol4no3/webster.htm

(14) John Summers, http://poultryindustrycouncil.ca/Factsheets/fact59.htm