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Avian Influenza Summary
• Worldwide distribution
• Sporadic outbreaks of High Path H5 and H7 in poultry
• Wild aquatic birds are reservoirs for all avian influenza viruses
• Viruses can change quickly (mutation)
• Avian-origin influenza viruses contribute to outbreaks in humans and other animals
• Virus is inactivated by most disinfectants
• Vaccination not done in the US, but is done in countries with HPAI
• Good biosecurity is effective prevention
• Sudden onset, moderate to high
morbidity and mortality.
• Edema of head
• Cyanosis/necrosis of comb/wattle
• Subcutaneous hemorrhage
HP Avian Influenza
Classic Signs & Lesions:
Unique Features
• Unprecedented spread to three continents
• Unprecedented number and variety of
species affected
– Many avian and multiple mammalian
species are susceptible
• Spill-over to wild birds, causing mortality in
wild waterfowl
• Involvement of waterfowl in virus spread
• Unprecedented changes in the virus
• High mortality in humans
HPAI H5N1 Outbreak
HPAI H5N1 Outbreak
Jan 2009. http://www.pandemicflu.gov/
>250 million chickens died/killed
>60 Countries affected (27 in 2008)
391 confirmed human cases (15
countries) -- 247 fatalities
All HPAI Outbreaks from Jan 2011 to Jan 2012
Avian Influenza Virus
HA
PB1
PB2
PA
NP
NAMA
NS
M2
Hemagglutinin
Neuraminidase
M1
Orthomyxovirus • Type A
•Humans are susceptible to A, B, and C
• Enveloped virus
• RNA
• Single stranded (negative sense)
•8 segments, codes 10
proteins
• Major surface proteins
• Hemagglutinin (H1-H16)
• Neuraminidase (N1-N9)
• 144 potential combinations
• H5N1, H7N7, H9N2 etc.
Avian Influenza Viruses Change Frequently
• Antigenic Drift: mutations in H gene cause minor antigenic changes to H protein
– Continuous process
– Limits protective immunity
• Antigenic Shift: Replacement of H or N
– Genetic re-assortment (human and animal viruses)
– Insertion of large section of extraneous RNA near the HA cleavage site – virulence shift
– Emergence of novel (pandemic) strains
Natural Reservoirs of Influenza A Viruses
• Wild aquatic birds
• Majority of influenza isolates comes from two orders:
1. Anseriformes: ducks, geese, swans
-Mallards and other dabbling ducks have highest isolation rates.
2. Charadriiformes: gulls, terns, and shorebirds such as ruddy turnstones
• Typically, no clinical disease except: 1. South Africa in 1961
2. H5N1 infection in wild birds in Asia
Ecology of Influenza A
Genetic
Reservoirs
Intermixing
H1, H3
H1, H2, H3 H3, H7
Commercial, LBMs Others H1-12
H14-15 H1-2, 4-7, H9-13, 15-16
H10
H1, H3, H4, H7, H13
Other Aquatic Birds?
H5N1
Modified from D. Swayne
H3
H1N1
Emergence of HPAI
Reservoir
Genetically stable
and low path in
natural host Most
HPAI
Asian
HPAI H5N1
Mutation- H5/7
may evolve to HP
Exposure/adaptation
to domestic species
that are not natural
hosts
Highly Pathogenic (HPAI)
Low Pathogenicity (LPAI)
Classification of Avian Influenza
• Localized infection
• No to mild disease
• Any subtype (H1-H16)
• Waterfowl (gut)
• Domestic birds (resp)
• LP H5 and H7 in poultry
are reported to OIE
• Acute, systemic disease
• High mortality
• H5 & H7 subtypes only
• Affects primarily poultry
(chickens, turkey, etc)
• Reported to OIE
Avian Influenza
Factors Influencing Pathogenicity:
• Only H5 or H7 subtypes are HP, but the majority of H5 and H7 are LP
• Polygenic trait, but HA plays dominant role
• Multiple basic amino acids at HA cleavage site
• Glycosylation sites near HA cleavage site
Remember: pathogenicity in the field is highly variable depending not only on the virus, but also the species and age of bird infected, concurrent infections, and other factors
1. Any influenza virus that kills 6, 7, or 8 of 8
chickens (75% mortality) or IVPI>1.2
2. Any H5 or H7 subtype that does not meet
the criteria in item 1, but has an amino acid
sequence at the cleavage site of the
hemagglutinin that is compatible with other
HPAI viruses
Note: The OIE also is notified when low path H5
and H7 is found in domestic poultry.
Highly Pathogenic Avian Influenza USDA/OIE definition:
Transmission Sources of virus Routes of transmission
Major
• Infected poultry flocks and
live bird markets
• Feces/resp secretions
• Infected wild waterfowl
Minor
• Uncooked infected poultry
products
• H1/H3 from swine to
turkeys
• Pet birds
• Oral - feces or cannibalism
• Aerosol
• IN, IC, IP, IM, IV, IC
• Fomites
– People
– Equipment
– Vehicles
– Feed and water
– Rodents, sparrows, etc
– Wind (less than 0.5mi)
Incubation and shedding of virus
• Incubation period of hours to 3 days
• Duration of virus shedding is highly variable
• More important than individual shedding is
flock shedding of virus
– AI viruses can be maintained for long
periods in domestic and wild flocks via
individual, susceptible birds
Days at Condition
Stability of Hong Kong H5N1 Virus in Feces at Environmental Temperatures
Shortridge et al. 1998
0 1 2 3 4 5 8 17 21 28 40 0
1
2
3
4
5
Vir
us
Tit
er
(lo
g 1
0)
Wet Feces
(25 C)
Dry Feces
(25 C)
Wet Feces
(35 C)
Wet Feces
(4 C)
Common Findings with Low Path AI
• Flocks with respiratory disease
• Flocks with decreased egg production
• Flocks with decreased food/water intake
• Increased daily mortality >3 bird deaths/1000 for 2
days
• These characteristics are not specific to AI
– Many possible differential diagnoses at this
stage
• These signs may only be identified retrospectively
after routine serologic surveillance detects LPAI
LP Avian Influenza
I.
Capua
D. Halvorson
•Coughing, sneezing, rales
•Sinusitis, tracheitis, air sacculitis
•Bronchopneumonia w/ secondary
pathogens (P. mult, E. coli)
•Epiphora
•Depression and huddling
•Ruffled feathers
D. Halvorson
LP Avian Influenza
• Rough, misshapen eggs
• Hemorrhage in ovary
• Oviduct edema
• Egg yolk peritonitis
• Catarrhal enteritis
• Urates in kidney
D. Swayne D. Swayne
Common Findings with HPAI • Sudden onset and rapid spread
• Mod to high morbidity and
mortality (>20 dead/1000/day)
• Sudden death
• Severe depression, drop in
feed/water consumption
• Cough, sneeze, nasal discharge
• Ataxia, tremors, torticollis
• Dramatic decreased egg
production and abnormal eggs
HPAI
HPAI
Dr. H K Wong, Hong Kong Zoo
HPAI
Dr. A. Wilson, NVSL Asian H5N1
HPAI
HPAI
HPAI
HPAI
HPAI Differential diagnosis
• Infectious laryngotracheitis (ILT)
• Infectious bronchitis (IB)
• Fowl cholera (P. multocida)
• Mycoplasma gallisepticum
Acute high mortality
• Newcastle disease
• Water deprivation
• Heat exhaustion
• Toxins
Respiratory disease
Sample collection
1. Serology (Important diagnostic specimen)
• AGID, ELISA (type-specific), and HI/NI
(subtype-specific)
– 0.5 to 1 ml of serum, plasma, egg yolk
– 4°C or minus 20°C
– Typically sample 30 birds per flock
+
-
+
AG
AS AS
AS
Sample collection for VI or rRT-PCR
2. Tissue
– Lung and spleen
– Ship samples in plastic bag, and then double bagged
– Do not pool tissues from different birds
– Typically sample 3-5 sick birds per flock
3. Swabs: oropharyngeal (OP) and cloacal (CL)
– Use sterile polyester swabs on plastic shafts
– Poultry: can pool 5/tube, but do not mix OP with CL swabs
– Water fowl use individual cloacal swabs
– Place swabs in 2-4ml brain-heart infusion (BHI) broth
• Shipping/Storage of tissue and swabs
– Short term: 4°C (up to 72 hours)
– Long term: minus 70°C (minus 20°C not recommended)
Virus Isolation
• Embryonating chicken eggs
• Expensive and labor intensive
• Special facilities (BSL-2, -3Ag)
• Test all sample types
• Sensitivity – high (all subtypes), may have low
sensitivity to some wild bird influenza viruses
• Specificity – low (grow many viruses, bacteria)
– Detection of AI is by HA
– Differentiation from other HA agents by HI
e – 2 days-2 weeks
Diagnostics
Real Time RT-PCR (rRT-PCR) • Target
– Influenza A virus (matrix gene)
– H5, H7, N1 subtypes validated
• Moderate cost/test ($8)
• Expensive equipment ($25-90K)
• Sensitivity – high (similar to VI)
(assuming using right primers)
• Specificity – high, Time - rapid (3-5 hrs)
• Potential for high throughput
• Live virus not required
Diagnostics
Antigen Capture Immunoassays
• Commercially available (several sources)
• Detection of influenza A virus
• Pen-side – no special facilities required
• Sensitivity – low/moderate (70-80% compared to
VI, need 3-5 logs of virus)
– Good for testing sick or dead birds
– Low sensitivity for vaccinated populations,
healthy birds, and live wild bird surveillance
• Specificity – high
• Moderate cost ($7-8) and rapid (10-15 minutes)
Synbiotics – Flu Detect Test
Diagnostics
AI Control • Strategies:
–Prevention – prevent introduction of AIV
–Control – reduce economic impact – limit spread
–Eradication – total elimination of AIV
• Achieved through…
–Biosecurity (exclusion and containment)
–Diagnostics and surveillance – early detection
–Elimination of AI virus in infected poultry
–Reduce susceptibility through vaccination
–Education of producers and public
National Biosecurity • Import restrictions on poultry and poultry
products countries with HPAI
• Heighten border surveillance for illegally
imported poultry and poultry products
• Wild bird surveillance programs
– 2006 (>160,000 specimens)
– 2007 (>95,000 specimens)
– 2008 (>50,000 specimens)
– 2009 (>19,000 specimens)
• Imported live birds
– 30-day quarantine
– Laboratory testing for AIV
Van Borm S, EID 2005
Farm Biosecurity • Avian influenza is a preventable disease
• Protect poultry, feed and water from contact with
reservoir hosts/feces
• All-in, all-out management ideally, with a single species
and single age group
• Purchase birds from reputable, tested supplier and/or
quarantine and test incoming birds
• Limit access to farm
• Provide clean clothing and boots to workers
• Clean and decontaminate all vehicles and equipment
coming on to the farm
• Don’t bring in equipment from other farms
• Avoid visiting other farms and live bird markets
Personal Biosecurity for HPAI
• Educate staff on disinfection, hygiene, etc.
• Disposable nitrile or vinyl gloves
• Disposable coverall with impermeable apron
• Shoe covers or boots, which can be disinfected
• Safety goggles
• Respirator (N-95, N-99, N-100), requires fit testing
• Prophylactic antiviral medications
• Seasonal flu vax and medical surveillance of workers
AI PPE recommendations from the CDC http://www.cdc.gov/flu/avian/professional/protect-
guid.htm See website for complete details
Iodophores
Phenolics
Quaternary ammoniums
Ethanol
Bleach
Peroxygens (Virkon-S)
Food Safety
• Eating properly handled and cooked
poultry and eggs is safe
• The chance of HPAI infected meat getting
into the human food chain in the US is
extremely low
• Wash hands with warm, soapy water for 20
seconds
• Wash utensils with hot, soapy water
• Sanitize cutting boards with dilute bleach
• Cook poultry to an internal temperature of
165°F
Control and Eradication
• Vaccination – not a stand alone control method
– Advantages • Reduce viral shedding
• Prevent illness and death (does not prevent infection)
– Disadvantages • No cross protection between subtypes
• Can interfere with diagnostics and surveillance
• Can hide clinical signs
• Impairment to international trade unless DIVA is used
• Vaccine bank
US Surveillance Programs Federal, State and Private laboratories
• Diagnostic surveillance
– Unexplained respiratory disease, decreased
egg production, and increased mortality
– FADD investigations
• Live bird market surveillance
• Commercial producers surveillance via NPIP
and national H5 and H7 LPAI control program
• Wild Migratory Bird surveillance