Mechanisms of Interspecies
Transmission of Avian Influenza
Viruses at the Human-Animal Interface:
Examination of Animal Factors
David E. Swayne*
Exotic and Emerging Avian Viral Diseases
Research Unit
Southeast Poultry Research Laboratory
USDA/Agricultural Research Service
Athens, Georgia
*Detailed as OFFLU Scientific Officer
World Organisation for Animal Health (OIE)
Paris, France
28 HPAI Epizootics
1. 1959-Scotland, H5N1
2. 1961-S. Africa, H5N3
3. 1963-England, H7N3
4. 1966-Canada, H5N9
5.1975-Australia, H7N7
6. 1979 –Germany, H7N7
7. 1979-England, H7N7
*8. 1983-84 - USA, H5N2
9. 1983-Ireland, H5N8
10. 1985-Australia, H7N7
11. 1991-England, H5N1
12. 1992-Australia, H7N3
13. 1994-Australia, H7N3
*§14. 1994-95-Mexico, H5N2
§15. 1995 & 2004 –
Pakistan, H7N3
16. 1997-Australia, H7N4
17. 1997-Italy, H5N2
**§18. 1996-2010 – Eurasia &
Africa, H5N1
*19. 1999-2000 - Italy, H7N1
*20. 2002 - Chile, H7N3
*21. 2003 – Netherlands, H7N7
*22. 2004 – USA, H5N2
*23. 2004 – Canada, H7N3
24. 2004, 2006 – S. Africa, H5N2
§25. 2005 – N. Korea, H7N7
*26. 2007 – Canada, H7N3
27. 2008 – England, H7N7
28. 2009 – Spain, H7N7
*LPAIV HPAIV
**Largest epizootic in 50 yrs
§Vaccine used in the control strategy
Human-Animal Interface
Portals of Entry (Routes of Exposure)
• Experimental evidence for AIV infections in
various animal models:
• Intranasal, intratracheal & bioaerosol (P-149
[131]; O-813 [p.12]) – various birds, mice, ferrets,
pigs, guinea pigs
• Oral or intragastric – chicken, ducks, gulls, ferrets,
pigs, cats
• Ocular exposure – mice
• Parenteral (e.g. intravenous injection) – chickens,
ducks and turkeys
Human-Animal Interface
Potential Modes of Transmission:
• Inhalation (airborne virus):
• Contaminated dust from rearing or slaughter
• Fine water droplets generated during
household/LPM slaughter process
• Contact with oral/nasal mucus membrane
or conjunctiva:
• Hand-transplantation of virus from
contaminated surface, or dust/feathers to eye
and mucus membranes of nose
• Swimming/bathing in contaminated household
pond (Vong et al. JID 199:1744-52, 2009)
• Consumption of raw or undercooked
infected birds or their products?
• Natural cases in carnivorous mammals, raptors
and scavenging birds – includes consuming
infected wild birds
Transmission
“…What is most important in influenza
transmission is not just the feasible, but the
practical…” Robert Couch
Consumption of Infected Birds/Products
• Natural cases: tigers, domestic cats, dogs,
mustelidae, humans (one case: VN dk blood)
• Experimental cases: cats and red foxes (Reperant et
al. EID 14:1835-41, 2008; Rimmelzwaan et al. AJP 168:176-83, 2006)
Feeding pigs WS/05
infected chicken meat
(100g, 1010 EID50)
No lesions
• No weight loss
• No changes in food
consumption
• Transmission by consumption of • No behavioral
infected meat
abnormalities
• Respiratory infection, no evidence • No virus rectal
swabs or GI tissues
of digestive infection
(Lipatov et al., PLOS Pathogens 7:e1000102, 2008)
Ferret Model: Results
•Meat consumption:
• MDk/Vietnam/05 – mild respiratory infection
• WS/Mongolia/05 – infection & mild respiratory disease: 2 dpe
– virus in tonsil and nasal turbinates, 5 dpe – virus negative
• VN/1203/04 – Lethal systemic disease
5 DPI – virus titer
- = no detection
+ = 1.5-2.5
++ = >2.5-3.5
+++ = >3.5
System
Tonsil
Respiratory
Nasal Turb
Lung
Digestive
SI
LI
Liver
Systemic
Spleen
Brain
Olf bulb
IN
+
IG
-
Meat
+
Meat-IG
-
+++
+++
-
+++
+
++
-
+
+
-
+
+++
+++
+
++
+++
+
++
+
-
+
+
-
+
+
-
(Lipatov et al, JID:717-25, 2009)
Can cooking kill avian influenza
viruses in chicken meat?
• Cooking 70 C will kill HPAI virus in less than 5 sec
8
80
70
6
60
Temperature Curve
Korea/03 - Thigh
Korea/03 - Breast
PA/83 - Thigh
PA/83 - Breast
Virus Isolation Limits
5
4
50
40
3
30
2
1
20
-40
-20
0
20
40
Time (s)
Swayne, Int. J. Food Microbiol, 2006
60
Temperature (oC)
Virus Titer (Log10EID50/gram)
7
Chicken Eggs and HPAIV
• HPAIV and chicken eggs
– High incidence of HPAI virus in eggs laid by infected hens
before death
– Moderate concentration of HPAI virus in such eggs
– Standard pasteurization temperatures and times kill HPAI
virus in liquid and dried egg products
– Cooking kills HPAI virus
• Conclusions:
• Most poultry products are
consumed cooked or
pasteurized
• H5N1 HPAI has not been a
Food Safety issue for humans
Swayne & Beck, Av. Pathol. 33(5):512-518, 2004
Portals of Entry: Exposure Dose
BID50, SPF WL chickens, A/Whooper
Swan/Mongolia/244/2005 (H5N1) HPAIV
1.
2.
3.
4.
5.
Intranasal - 103.3 and 102.8 EID50
Crop Gavage of liquid - 106.2 and 106.3 EID50
Crop Gavage of Meat – > 108 and 107.1
Oral Feeding of Meat – > 106.0, 107
Oral Water - approx. 106.7
Conclusion: Upper respiratory exposure requires
lower dose of virus than oral or digestive tract
exposure in chickens
Individual verses group housed
Kwon and Swayne, Avian Diseases, in press
Corneal Scarification
Harvest
Tissues
Challenge
106 EID50
Day 0
Day 3
Observe Daily
Lung, Nose,
Eye
Day 6
Viral Titers
(EID50/mL)
Day 14
Morbidity/Mortality
• H7N7 and H7N3 HPAIV replicated to significant titer
in mouse eye , but some H5N1 HPAIV, also
replicated in the eye, but usually to lower titers
• Pronounced morbidity and mortality w/NL/219
(H7N7), HK/483 (H5N1), & Thai/16 (H5N1)
(Belser et al., J. Virology 83:7075-84, 2009)
• Growth in human respiratory and ocular epithelium
(Belser et al., O-882 [p.66])
Mechanism: Inhalation of
contaminated airborne particles
Preliminary Study: Air Sampling at Necropsy
1. IN inoculated 4 wk-old SPF Chickens with
WS/Mongolia/05 HPAIV
2. Three birds that died (2DPI) necropsied
3. Air sampled – negative air ionizing sampler
a. 60 cm from birds
b. 10 min
c. 104.1 EID50 of virus
Emphasizies need for respiratory protection
for necrospy of H5N1 HPAIV suspect birds
LPM: inhalation or contact exposure
Exposure to Wet Markets in Developing World
LPM: airborne virus study
Experimental Approach:
1. Simulated home slaughter (exception anesthetized
the birds before slaughter process began)
2. 10 asymptomatic H5N1 HPAI virus infected
chickens in 1 hr (inoculated 24hrs previously)
3. Measurement of virus in the air (negative air
ionizing sampler)
4. Determine particle size and number (noneinfected)
5. Transmission to caged chickens
Home Slaughter Simulation: Airborne Virus Generation
Isolators
3
2
1.
2.
3.
4.
Processing site
Sample point – 300 cm
Sample point – 150 cm
Sample point – 75 cm
8.3 air changes/hr (340 m3/hr)
1
Table
4
Home Slaughter Simulation: Airborne
Virus Generation
LPM Slaughter Simulation: Airborne
Virus Generation
LPM Slaughter Simulation: Airborne
Virus Generation
Droplet Distribution – non-infected layers
Particle
size
(microns)
>25
10-24.9
5-9.9
1-4.9
0.5-0.9
0.3-.049
2.5cm
ft
75
(log10)
3.5
4.6
5.4
6.6
6.7
7.5
5 ft (log10)
10ft
300
cm
(log10)
3.8
4.9
5.7
6.7
6.7
6
LPM Slaughter Simulation: Airborne
Virus Generation
WL Adult Layers, 106 IN Mongolia/05,
Slaughtered 1 DPI (preclinical)
• Ferrets exposed at
Dead Birds 150 cm for 1 hr to
5/5 (4.4)
slaughter of VN/1203/04
5/5 (3.6)
infected chickens; 3 of 4
5/5 (5)
ferrets became infected
& died
Conclusions:
• Home slaughter of chickens generates aerosols & large droplets
• Home slaughter of infected chickens generates airborne virus
• Can transmit H5N1 HPAIV to chickens & ferrets when exposed
in same air space as slaughter of infected asymptomatic chickens
• Takes lower dose to infect than via IN inoculation (IN-CID50 =
103 EID )
Sampler
(Ft)
2.5cm
75
1505cm
10cm
300
Virus
(log10)
2.1
1.4
1.1
Intervention Strategies
Determine what interventions will reduce H5N1
HPAIV in air samples and reduce transmission
of the virus to ferrets in relation to the location
where slaughter of asymptomatic H5N1 HPAIVinfected chickens is performed:
•Standard home slaughter process versus the kill
step in a plastic bag (Andrew Clark and
Mohammad Hassan)
• Non-vaccinated versus vaccinated chickens
Study Design
• White leghorn chickens, 24 hr post-H5N1
HPAIV inoculation intranasally (106 EID50,
A/VN/1203/2004): do home slaughter
•1 hr, 5 chicken study per treatment group
• Measure virus in air – high volume air
sampler
•Repeat and expose 3 ferrets in same airspace
Study Design
• 5 steps in halal slaughter process:
• Kill (anesthetized)
• Hard-scald
• Defeathering
• Evisceration
• Clean-up
Study Design
• Kill step:
• Tie feet
• Place in bag
• Incise carotid and jugular
• Retract head, place in can
& allow muscle contractions
• Contains blood & feathers
HEPA Isolators with infected chickens
BSL-3Ag Certified Room
3
1
Table
1.
2.
3.
4.
2
4
Processing site
Scald site
Kill can
Air sampler/particle
sizer/ferret cage (<75 cm)
5. 8.3 air changes/hr (340 m3/hr)
Results
Air Samples During Slaughter
• Non-vax cks: average oropharyngeal titer 105.25, 5.75 EID50/ml,
cloacal titer 104.0, 4.1 EID50/ml at 24 hrs post-challenge
• Vax cks: ave. oropharyngeal titer 103.15 EID50/ml, cloacal titer
<100.97 EID50/ml at 24 hrs post-challenge
Group
Non-vax
Vax
Method
Kill*
Scald*
Defea.*
Evisc*
Clean-up*
Standard
5/5 (2.2)
5/5 (1.9)
5/5 (1.8)
4/5 (1.4)
4/5 (1.5)
Bag
2/5 (1.3)
2/5 (1.4) 2/5 (0.97)
0/5
1/5 (0.97)
Standard
0/5
0/5
0/5
0/5
0/5
Bag
0/5
0/5
0/5
0/5
0/5
* Number positive/total tested (virus titer, EID50/ml)
Results
Ferret Exposure During Slaughter
Group
Non-vax
chickens
Vax ck
* Number infected/total exposed
Process
Method
Ferret
Infected*
Standard
3/3
Bag
1/3
Standard
0/3
Bag
0/3
LPM Simulation: Mitigation Strategies
• Egyptian Islamic Council
approval
• Miniaturize air sampler
LPM Simulation: Mitigation Strategies
• Future: Repeat using a 5 gallon plastic bucket
and kitchen pan/lid (sustainable and consistent)
• Developing a 12 panel cartoon for CDC/WHO
to communicate the new process
• Examining the LPM processes that produce airborne
virus and develop mitigation steps
Conclusions
• Transmission occurs through various routes of
exposure, mechanisms of infection and vehicles
• In mouse model, conjunctival infection has been
demonstrated, especially feasible with H7 AIV
• In the pig model, consumption of WS/05 H5N1
infected meat caused respiratory infection via tonsil
and pharynx exposure
• In ferret model, consumption of two H5N1 HPAIV
caused non-fatal respiratory infection while one
H5N1 HPAIV caused lethal infection with initiation
of infection in both respiratory and digestive tracts
• When processing H5N1 HPAI infected chickens,
home slaughter process produces aerosols & large
droplets which contain virus
Conclusions
• Using Bag technique for the kill step on
asymptomatic H5N1 HPAIV-infected chickens, there
was a reduction in number of virus+ air samples,
reduced quantity of virus in air samples and reduced
transmission of HPAIV to ferrets
• Using H5N1 HPAIV-infected but vaccinated chickens,
prevented virus+ air samples and prevented
transmission of H5N1 HPAIV to ferrets
• Simple intervention strategies may prevent
transmission of HPAIV from birds to humans
Contributions
USDA/SEPRL:
USDA/APHIS:
Joan Beck
Kira Moresco
James Doster
Justin Brown
Grady Witcher
Y.B. Kwon
A. Lipatov
Andrew Clark
AHRI (Egypt):
Mohammed Hassan
CDC:
Terry Tumpey
FUNDING:
DHS
CDC
USDA/ARS