Avian Influenza

Saad Gharaibeh DVM, PhD, Dip ACPV

Dept. of Pathology and Animal Health

Faculty of Veterinary Medicine

Jordan University of Science and Technology

Irbid 22110, Jordan

saadgh@just.edu.jo

02/720-1000 ext 22059

Avian InfluenzaHistory

1. 1878 Fowl plaque was described (Italy)2. 1901 Fowl plaque is caused by a virus3. 1955 It is type A influenza virus4. 1970 AGP test introduced 5. 1972 Waterfowl is a reservoir 6. 1979 Virulence and hemagglutinin

cleavability was established 7. 1997 Direct transmission of H5 AIV from

birds to humans

Avian Influenza Virus

1. Orthomyxoviridae

2. Pleomorphic RNA viruses, single stranded, negative sense genome.

3. Has glycoprotein projections HA, NA

4. Three antigenic types A, B, C (Avian influenzas are all type A)

5. 8 gene segments code for 10 proteins

6. Vary in pathogenicity

Jong et al., 2000, Journal of Infection

HI test

NI test

ELISA test

AGP test

HI test

NI test

ELISA test

AGP test

Nomenclature

A/chicken/Hong Kong/220/97 (H5N1)1. A: Type of virus A, B, C

2. Chicken: Host of origin

3. Hong Kong: Geographic origin

4. 220: Strain Number (Case number)

5. 97: Year of isolation

6. (H5N1): H & N subtype

Infectious Virus

1. Needs HA0 cleaved into HA1 & HA2

2. Intracytoplasmic:a) Furin-like enzyme (ubiquitous proteases): HP

b) Trypsine-like enzyme: All AIV

Cell Types for Replication

1. All AIV (trypsine-like enzymes): a) Respiratory epithelium

b) GI epithelium

2. HP AIV (Furin-like enzymes): Variety of cells resulting in a systemic infection.

Approaches Used to Characterize AIV Pathogenicity

1. In vivo methods: a) Laboratory Inoculation of chickensb) Chicken embryo lethality

2. In vitro methods (evaluation of HA cleavability):a) Plaques or CPE assays (CEF does not have trypsin)b) Direct detection of cleaved HAc) Nucleotide sequence of HA cleavage site

3. Direct measure of pathogenicity potential:a) Identify pathogenicity increases during virus passage in

chickens under controlled conditionsb) Virulence in mice and ability to infect other mammals

Criteria for HP

1. AIV lethal for 6,7, or 8 / 8 four-to-six-week-old susceptible chickens within 10 days following IV inoculation with 0.2 ml of 1:10 dilution of a bacteria free, infectious allantoic fluid.

2. H5 or H7 has amino acid sequence at the hemagglutinin cleavage site compatible with HPAIV

3. Non-H5 or H7 that kills 1-5 chickens and grows in cell culture w/o added trypsin

Signalment & Clinical Signs(Low pathogenic AI disease)

a) Respiratory signs

b) Diarrhea

c) Drop in egg production 7-10 days 5-30%

d) Mild increase in Mortality (2o bacterial infection will increase mortality)

Gross Lesions (Low pathogenic AI disease)

a) Catarrhal rhinitis / tracheitis

b) Ocular discharge

c) Airsaculitis

d) Ovarian involution and hemorrhage

e) Yolk peritonitis

f) Swollen kidney and urates

Signalment & Clinical Signs(Highly pathogenic AI disease)

a) Sudden onset of high mortality (up to 100%)

b) Depression

c) +/- nervous signs

Gross Lesions (Highly pathogenic AI disease)

a) Edematous to necrotic comb and wattles

b) Edema, necrosis, and hemorrhages in different organs

High Path Avian Influenza Diagnosis1. Clinical features in commercial poultry give a

tentative diagnosticSudden death and high mortality rate

2. RT-PCR and sequencing3. Virus isolation and identification is the gold standard

but very few laboratories in the world can handle such a virus capable of infecting humans.

4. Commercial antigen capture ELISA (lack sensitivity and will cross react with other endemic subtypes)

5. Serology: AGP, ELISA, HI, NI

RT-PCR

1. Testing can be performed in one day for multiple agents.

2. Sensitivity is very high and comparable to virus isolation.

3. Can be applied on samples from any species.

4. Decrease the chance of contamination with live virus.

RT-PCR Diagnostics in JUSTSafety Considerations

AIAI APVAPVIBIB NDNDMWMMWM

RT-PCR Diagnostics in JUSTChicken Respiratory Disease viruses

Serologic Testing and Surveillance

1. AGPT: Type specific (available at JUST)

2. ELISA: Type specific (available at JUST)

3. HI: Subtype specific (available at JUST)

4. NI: Subtype specific

5. Antigen capture ELISA (available at JUST)

6. RT-PCR: Surveillance and diagnosis (available at JUST)

Agar Gel Precipitation Agar Gel Precipitation

Negative Flock Positive Flock

ELISA Readings

Hemagglutination Inhibition

Control & Prevention

1. Biosecurity

2. Stamping out infected flocks

3. Vaccination of flocks at high risk :a) Killed vaccines

b) Viral vector vaccines

c) Live attenuated vaccines are not licensed for poultry

www.animalactivism.org/ documents

Drastic measures in some Asian countries

AI crossed Species Barrier into HumansA/chicken/Hong Kong/220/97 (H5N1)

What conditions favor AI spread?

Densely populated countries

Very popular Live-birdmarkets

Free Range Poultry

Is H5N1 AI bad for economies?

Disease Country Year Impact Cost ($US)‘mad cow’ UK 1990-98 Beef export 9 billionCholera Peru 1991 Seafood export 770 millionPlague India 1995 Tourism, trade 2 billionH5N1 flu Hong Kong 1997 Loss of poultry

(1.5 million birds)22 million

Cholera Tanzania 1998 Seafood export 36 millionNipah Malaysia 1999 Loss of swine

(0.9 million pigs)540 million

West Nile US 1999 400 millionSARS Worldwide 2003 Tourism, trade 80 billionH5N1 flu SE Asia 2003-05 Loss of poultry

(~140 million birds so far)~10 billion? (and growing)

Sources: WHO, Institute of Medicine, FAO, OIE, Asian Development Bank, World Bank

High Risk Areas in Jordan

Should we start vaccinating poultry against H5N1?

Killed Vaccines

1. Will result in only humoral antibody response against all viral proteins except NS1.

2. Will significantly reduce shedding of the challenge virus.

3. Will interfere with AGP, ELISA, HI, and NI if (homologous).

4. If sequence of the HA gene is identical to the challenge virus it may eliminate shedding completely.

Swayne et al., 2000, Veterinary Microbiology

Advantages of Vaccines

• Reduces the number of chickens from which AI challenge virus could be reisolated.

• Decreased the titers of virus detected in the cloaca and oropharynx (up to 99.99%)

• Reduced environmental contamination and prevented subsequent bird to bird transmission.

• The use of killed H5N2 vaccine in the face of HPAI H5N1 virus challenge was able to protect chickens from disease and interrupt virus transmission.

DIVADifferentiating Infected from Vaccinated Individuals

• The use of killed vaccine and unvaccinated sentinels: leaving 0.5-1% of the flock unvaccinated and marked (wing band) and these individuals will be subjected to serological monitoring.

• Heterogonous killed vaccine: Screen for field infection using NI.

• Measuring serological response to NS1 by ELISA or western blot.

Vaccine / Industry / Politics• The use of vaccine to aid in the control of AI

is a political issue and different people have a different say on this.

• In some countries financial constrains preclude stamping out policy.

• In some countries, export markets are not an issue to prevent vaccination.

• In some countries, stamping out attempt may be unsuccessful.

• “With the ubiquitous nature of AI in wild birds it may be vaccination the most feasible tool to soften the sting of AI” Beard 1981

Vaccine / Industry / Politics• “Field results have not shown vaccine to increase the

risk of undetected infection; in fact, field experience has shown that vaccination greatly enhances a control program.” Halvorson, 2002, Avian Pathology

• There is no way a vaccinated flock can be a greater threat to disease control than a non-vaccinated flock that breaks with AI. Halvorson, 2002, Avian Pathology

• Epidemiological observations have shown that serologically positive birds are not associated with AI transmission. (Kradel, 1992)

• Should the government set the rules when no indemnity (compensation) is paid?