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 Influenza
History
1. 1878 Fowl plaque was described (Italy)
2. 1901 Fowl plaque is caused by a virus
3. 1955 It is type A influenza virus
4. 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
 NI test

HI test

AGP test

ELISA test

Jong et al., 2000, Journal of Infection

HI test

ELISA test

NI 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 chickens
b) 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 HA
c) Nucleotide sequence of HA cleavage site
3. Direct measure of pathogenicity potential:
a) Identify pathogenicity increases during virus passage in
chickens under controlled conditions
b) 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 Diagnosis
1. Clinical features in commercial poultry give
a tentative diagnostic
Sudden death and high mortality rate
2. RT-PCR and sequencing
3. 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 JUST
Safety Considerations
RT-PCR Diagnostics in JUST
Chicken Respiratory Disease viruses

MWM AI IB ND APV
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
 ELISA Readings

Negative Flock Positive Flock

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
Drastic measures in some Asian countries

www.animalactivism.org/ documents
AI crossed Species Barrier into Humans
A/chicken/Hong Kong/220/97 (H5N1)
What conditions favor AI spread?
Densely populated countries
Very popular Live-bird
markets
Free Range Poultry
 Is H5N1 AI bad for economies?

Disease Country Year Impact Cost ($US)

‘mad cow’ UK 1990-98 Beef export 9 billion
Cholera Peru 1991 Seafood export 770 million
Plague India 1995 Tourism, trade 2 billion
H5N1 flu Hong Kong 1997 Loss of poultry 22 million
(1.5 million birds)
Cholera Tanzania 1998 Seafood export 36 million
Nipah Malaysia 1999 Loss of swine 540 million
(0.9 million pigs)
West Nile US 1999 400 million
SARS Worldwide 2003 Tourism, trade 80 billion
H5N1 flu SE Asia 2003-05 Loss of poultry ~10 billion?
(~140 million birds so far) (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.
 DIVA
Differentiating Infected from Vaccinated Individuals

1. 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.

2. Heterogonous killed vaccine: Screen for field infection

using NI.

3. Measuring serological response to NS1 by ELISA or

western blot.
 Vaccine / Industry / Politics
1. The use of vaccine to aid in the control of AI is
a political issue and different people have a
different say on this.
2. In some countries financial constrains
preclude stamping out policy.
3. In some countries, export markets are not an
issue to prevent vaccination.
4. In some countries, stamping out attempt may
be unsuccessful.
5. “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
6. “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
7. 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
8. Epidemiological observations have shown that
serologically positive birds are not associated with AI
transmission. (Kradel, 1992)
9. Should the government set the rules when no
indemnity (compensation) is paid?