AVIAN DISEASES 52:455-460, 2008

**Using Mean Infectious Dose of High- and Low-Pathogenicity Avian
Influenza Viruses\
Originating from Wild Duck and Poultry as One Measure of Infectivity
and\
Adaptation to Poultry**

> David E. Swayne^AC^ and Richard D. Slemons^B^

^A^Southeast Poultry Research Laboratory, Agricultural Research Service,
U.S. Department of Agriculture, 934 College Station Road,\
Athens, GA 30605\
^B^Department of Preventive Medicine, College of Veterinary Medicine,
The Ohio *State University, 1920* Coffey Road, Columbus, OH 43210

Received 11 February 2008; Accepted and published ahead of print 1 April
2008

> SUMMARY, The mean infectious doses of selected avian influenza virus
> (AIV) isolates, determined in domestic poultry under experimental
> conditions, were shown to be both host-dependent and virus
> strain-dependent and could be considered one measure of the
> infectivity and adaptation to a specific host. As such, the mean
> infectious dose could serve as a quantitative predictor for which
> strains of AIV, given the right conditions, would be more likely
> transmitted to and maintained in a given species or subsequently cause
> an AI outbreak in the given species*.* The intranasal (IN) mean bird
> infectious doses (BID~50~) were determined for 11 high-pathogenicity
> AIV (HPAIV) isolates of turkey and chicken origin for white leghorn
> (WL) chickens, and for low-pathogenicity A1V (LPAIV) isolates of
> chicken *(n =* 1) and wild mallards *(n =* 2) for turkeys, and WL and
> white Plymouth rock (WPR) chickens, domestic ducks and geese, and
> Japanese quail. The BID~50~ for HPAIV isolates for WL chickens ranged
> from 10^1.2^ to 10^4.7^ mean embryo infectious dose (EID~50~) (median
> = 10^2.9^). For chicken-origin HPAIV isolates, the BID~50~ in WL.
> chickens ranged from 10^1.2^ to 10^3.0^ EID~50~ (median = 10^2.6^),
> whereas for HPAIV isolates of turkey origin, the BID~50~ in WL.
> chickens was higher, ranging from 10^2^.^8^ to 10^4.7^ EID~50~ (median
> = 10^3.9^). The BID~50~ of 10^4^.^7^ was for a turkey-origin HPAIV
> virus that was not transmitted to chickens on the same farm,
> suggesting that, under the specific conditions present on that farm,
> there was insufficient infectivity, adaptation, or exposure to that
> virus population for sustained chicken transmission. Although the
> upper BID~50~ limit for predicting infectivity and sustainable
> transmissibility for a specific species is unknown, a BID~50~ \<
> 10^4.7^ was suggestive of such transmissibility. For the LPAIVs, there
> was a trend for domestic ducks and geese and Japanese quail to have
> the greatest susceptible and for WL chickens to be the most resistant,
> but turkeys were susceptible to two LPAIV tested when used at moderate
> challenge doses. [This suggests domestic ducks and geese, turkeys, and
> Japanese quail could serve as bridging species for LPAIVs from wild
> waterfowl to chickens and other gallinaceous poultry. These data do
> provide support for the commonly held and intuitive belief that mixing
> of poultry species during rearing and in outdoor production systems is
> a major risk factor for interspecics transmission of AIVs and for the
> emergence of new AIV strains capable of causing AI outbreaks because
> these situations present a more diverse host population to circumvent
> the natural host dependency or host range of circulating
> viruses.]{.mark}
>
> RESUMEN. Utilizatión de la dosis infecciosa media de virus de
> influenza aviar de alto o baja patogenicidad originados de ayes
> domésticas o patos silvestres como una medida de infectividad y
> adaptatión a las aves domésticas.
>
> Las dosis infecciosas medias de aislamientos seleccionados del virus
> de influenza aviar determinadas bajo condiciones experimentales en
> aves domésticas, demostraron ser dependientes del huésped y de la cepa
> del virus, pudiendo considerarse como una medida de Ia infectividad y
> adaptatión a un huésped específico. Como tal, las dosis infectivas
> medias podrían servir para predecir cuantitativamenre cuáles cepas del
> virus de influenza aviar, dadas las condiciones adecuadas, sería más
> propensa a ser transmitida y mantenida en una especie determinada o
> subsecuentemente causar un brote de influenza aviar es esa especie. En
> ayes leghorn blancas, se calculó la dosis infecciosa media 50 por ave
> (por sus siglas en Inglés BID~50~) de 11 aislamientos de influenza
> aviar de alta patogenicidad originados en pollos y pavos posterior a
> la inoculación intranasal. A su vez, para aislamientos del virus de
> influenza aviar de baja patogenicidad originados en pollos y dos patos
> silvestres, se calculó el BID~50~ en pavos, aves leghorn blancas y
> plymouth rock blancas, patos domésticos, gansos y codornices
> Japonesas. El BID~50~ para los aislamientos de influenza aviar de alta
> patogenicidad (calculado en ayes leghorn blancas) se present6 en el
> rango de 10^1.2^ a 10^4.7^ dosis infecciosa media de embrión 50
> (DIE~50~) con un promedio de 10^2^.^9^, mientras que para los
> aislamientos de influenza aviar de alta patogenicidad originados de
> pavos, el BID~50~ calculado en aves leghorn blancas fue mayor y en el
> rango de 10^2.8^ a 10^4.7^ D1E~50~ con un promedio de 10^3.9^. El
> BID~50~ de 10^4.7^ correspondió a un aislamiento de influenza aviar de
> alta patogenicidad originado en pavo que no se transmitió a pollos
> presentes en la misma granja, sugiriendo que bajo las condiciones
> específicas presentes en esa granja, hubo adaptación o en su defecto
> infectividad o exposición insuficiente a esa población viral para que
> se produjese la transmisión sostenida a los pollos. Aun cuando el
> límite superior de BID~50~ requerido para predecir infectividad y
> transmisibilidad sostenida se desconoce, un BID~50~ \< 10^4.7^ sugiere
> esa transmisibilidad. Para los virus de influenza aviar de baja
> patogenicidad, se observ6 una tendencia en los patos domésticos,
> gansos y en las codornices a una mayor susceptibilidad y en las aves
> leghorn blancas a ser mas resistentes. Los pavos fueron susceptibles a
> dos virus de influenza aviar de baja patogenicidad cuando se
> utilizaron dosis de desafio moderadas. Esto sugiere que los patos
> domésticos, gansos, pavos y las codornices Japonesas son especies que
> pueden servir como puente para virus de influenza aviar de baja
> patogenicidad provenientes de aves acuáticas hacia pollos y otras
> especies gallináceas domésticas. Estos datos proporcionan soporte para
> la creencia común e intuitiva de que mezclar especies de aves
> domésticas durante la crianza y en los sistemas de producción al aire
> libre es un factor de riesgo importante para la transmisión de virus
> de influenza aviar y para la emergencia de nuevas cepas del virus
> capaces de causar brotes de influenza aviar. Esto debido a que en
> estas situaciones se presenta una población de huéspedes más diversa
> que permite resolver las limitaciones del rango de huésped o
> dependencia al huésped de los virus circulantes.
>
> Corresponding author. [E-mail: david_swayne@ars.usda.gov]{.underline}
>
> Key words: adaptation, avian influenza, infectivity, poultry,
> transmission, wild birds
>
> Abbreviations: AGP = agar gel precipitin; AI = avian influenza;
> BID~50~ = mean bird infectious dose; BLD~50~ = mean bird lethal dose;
> EID~50~ = mean chicken embryo infectious dose; HPAI = high
> pathogenicity avian influenza; HPAIV = high pathogenicity avian
> influenza virus; LPAI = low pathogenicity avian influenza; LPAIV = low
> pathogenicity avian influenza virus; LPM = live poultry market; Ro =
> reproductive ratio; SPF = specific pathogen free; WL = white leghorn;
> WPR= white Plymouth rock

Free-flying aquatic birds, principally of the orders Anseriformes (ducks
and geese) and Charadriiformes (shorebirds, gulls, terns, and auks), are
major reservoirs for biologically and genetically diverse populations of
low-pathogenicity avian influenza viruses (I.PAIVs) (24,36), with the
highest prevalence of infection being reported in juvenile mallards
*(Ands platyrhynchos)* in North America during the late summer-early
fall staging before migration from the northern breeding to southern
winter habitats (24). There is convincing epidemiologic evidence that
the wild aquatic bird LPAIVs cross species harriers and become
established in domestic poultry, but these events are sporadic,
unpredictable, and relatively uncommon. Furthermore, the detection of
the viral introductions in poultry is frequently delayed resulting in
the viral sources seldom being confirmed, which raises questions about
the epidemiologic associations.

Historically, the best documentation of interspecies transmission of
wild waterfowl-origin LPAIVs has been into range-reared turkeys in
Minnesota, with subsequent movement of the viruses into confined
turkeys, where higher population densities were more conducive to
respiratory disease outbreaks. The initial infections were generally
mild or asymptomatic, but if virus replication was permitted to
continue, a more infectious and transmissible lineage of viruses
emerged. Then, if circumstances were appropriate, these better-adapted
LPAIVs caused respiratory disease or drops in egg production in
later-affected flocks (7,9,11,30). At this time, even though the LPAIVs
were still of low pathogenicity, elevated morbidity and mortality
occurred as a result of cofactors, such as bacterial or viral concurrent
infections, which exacerbate the severity of the APAIV infections (17).
In the 1960s, range-rearing accounted for more than 50% of Minnesota
turkey production, which declined to 10% in the early 1990s and to less
than 0.5% after 1998 (9). This decline in range-rearing of turkeys was
associated with a decline in LPAIV infections in turkeys, especially
after 1998, thereby providing additional evidence that such LPAIV
transfers resulted from direct exposure of domestic turkeys to wild
duck-origin LPAIVs. It is possible that under range-production
operations, turkeys may have also served as a bridging species for
LPAIVs of wild ducks to gain access to other domestic poultry in the
United States. Although the probability of this occurring in Minnesota
is markedly reduced, in other geographic regions with similar ecologic
niches and farming systems, turkeys might still serve as bridging
species for LPAIVs entering domestic species. In addition, domestic
turkeys may be more susceptible to some poultry-origin LPAIVs than
chickens. The H7N2 LPAIV, of live poultry market (LPM) origin, which
caused outbreaks in Virginia poultry during 2002, was experimentally
more infectious for turkeys than chickens, and such a biologic feature
may have contributed to the higher proportion of affected turkey than
chicken farms in the region (34).

However, with most other LPAI cases in domestic poultry, the initial
viral source and index infections in domestic species are not always
identifiable and may have occurred at some untraceable time in the
distant past following the initial interspecies transmission. Based on
our current understanding, it is a widely held belief that outdoor
raising, mixing of poultry species during rearing and

distribution, and lack of movement controls and minimal sanitary
standards are apparent risk factors associated with the introduction and
maintenance of LPAIVs in the live poultry market system of the
northeastern United States from 1986 to 2005 (20,32). These production
conditions favor the introduction of wild, aquatic-bird LPAIV into
domestic poultry, but there is incomplete susceptibility, infectivity,
and transmissibility data to determine which domestic species could be
entry points into agricultural systems. In addition, susceptibility,
infectivity, and transmissibility data for poultry are minimal for most
of the high-pathogenicity avian influenza (HPAI) viruses (HPAIVs).

This study was undertaken to quantify the susceptibility of various
poultry species to different HPAIVs and LPAIVs isolated from poultry,
wild ducks, an emu, a tern, and even a human, following intranasal
exposure to a range of virus doses. The intranasal mean bird infectious
dose (BID~50~) is proposed as a quantifiable laboratory assessment of a
virus\'s ability to initiate an infection in a specific species and to
set the stage for virus strain adaptation and transmissibility for a
specific host species (29,34).

MATERIALS AND METHODS

Animals and housing. Three-to-4-wk-old specific pathogen free (SPF)
white Plymouth rock (WPR) and white Leghorn (WL) chickens *(Gallus
gallus domesticus)* were obtained from Southeast Poultry Research
Laboratory flocks. Conventional 3-wk-old turkeys *(Meleagris
gallopavo),* 6-wk-old Japanese quail *(Goturnix japonica),* 3-wk-old
Pekin ducks (Anas *platyrhynchos),* and 3 wk-old Chinese geese *(Anser
cygnoides)* were obtained from various commercial sources. The birds
were nested in negative-pressure, high-efficiency, air-filtered
isolation cabinets with continuous light exposure. The isolation
cabinets were contained within a biosafety level 3 agriculture (BSL-3AG)
facility. Water and feed were provided *ad libitum.*

**Viruses.** The following HPAIVs were used:
A/chicken/Rostock(Ger-many)/1934 (H7N1), A/chicken/Scotland/1959 (H5N1),
A/tern/ SouthAfrica/1961(H5N3), A/turkey/Ontario/7732/1966(H5N9),
A/chicken/Victoria/1975(H7N7),A/turkey/Ireland/1378/1983(H5N8),A/turkey/England/50-92/1991(H5N1),
A/emu/Texas/399924-HP derirarive/1993 (H5N2) (HP virus derived for LP
progenitor by laboratory passage), A/chicken/Queretaro/14588-19/1995
(H5N2), A/Hong Kong/486/1997 (H5N1), and A/turkey/Italy/4580/1999
(H7N1). The following LPAIV were used: A/chicken/Alabama/l975 (H4N8),
A/mallard/Ohio/184/1986 (H5N1), and A/mallard/Ohio/ 338/1986 (H4N8).
Virus stocks from the first---second embryo passage of archived virus
stocks were used. All virus titers were reported as mean embryo
infectious doses (EID50).

Experimental design. For the HPAIV experiment, groups of 10 WL chickens
were intranasally inoculated with 10-to-50-fold dilutions of HPAIVs
ranging from 100.5 to 108.0 E1D50. For the first LPAIV experiment,
groups of 10 chickens and turkeys were intranasally inoculated with
10-to-50-fold dilutions of LPAIV ranging from 10^2.^° to 10^8^\'^5^
EID~50~, In both experiments, serum was collected on day 14 af~t~er
inoculation, and infections were determined by detection of antibodies
against the nucleoprotein and matrix protein by the agar gel precipitin
(AGP) test. For the second LPAIV experiment, groups of four domestic
ducks, five Japanese quail, and four domestic geese were intranasally
inoculated with virus doses ranging from 10^1^ to 10^7^ EID~50~. Because
the

> 457\
> Table 1. The BLD~50~ and BID~50~ for 10 field and 1 laboratory-derived
> HPAIVs in WL chickens following intranasal inoculation.
>
> Virus isolate
>
> A/chicken/Rostock(Gerrnany)/1934
>
> A/chicken/Scotland/1959 A/tern/SouthAfrica/1961
> A/turkey/Ontario/7732/1966 A/chicken/Victoria/ 1975
> A/turkey/lreland/1378/1983 A/turkey/England/50-92/1991
>
> A/ernu/Texas/39924-HP derivative/1993
> A/chicken/Queretaro/14588-19/1995
>
> A/Hong Kong/486/1997 A/turkey/Italy/4580/ 1999
>
> AGP test is unreliable at detecting antibodies in ducks and geese and
> is of unknown sensitivity in Japanese quail, AIV infections were
> determined based on virus isolation from oropharyngeal and cloacal
> swabs 4 days after inoculation. In all experiments, birds receiving
> the same challenge dose were housed together in the same isolation
> cabinet.
>
> **Detection of infection and lethality.** Determination of infection
> was by the AGP serologic test for chickens and turkeys, and virus
> isolation in 9-to-11-day-old, embryonating chickens eggs for ducks,
> geese, and quail, both using procedures previously described (2,31).
> The mean BID~50~ and the mean bird lethal dose (BLD~50~) were
> calculated by the method of Spearman and Kärber (6).

# RESULTS

> All 11 HPAIV isolates produced high mortality in WL chickens. For
> eight HPAIV isolates, the BID~50~ and BLD~50~ were equal; i.e., all
> infected chickens died. However, with three HPAIV isolates (A/
> chicken/Scotland/1959, A/turkey/Ontario/7732/1966, and A/emu/
> Texas/39924-HP derivative/1993), the BID~50~ was slightly less than
> the respective BLD~50~; i.e., a few WL chickens survived and produced
> antibodies. The BID~50~ for ~t~he HPAIV isolates in WL chickens ranged
> from 10^1.2^ to 10^4.7^ **B**ID~50~, with a median of 10^2^-^9^
> BID~50~ (Table 1). The five chicken-origin HPAIV isolates had BID~50~
> in WL chickens ranging from 10^1.2^ to 10^3.0^ BID~50~ (median =
> 10^2.6^). Three turkey-origin HPAIV isolates had a higher median
> BID~50~ (10^3.9^) in WL chickens, with a range of 10^2-8^ to 10^4.7^.
> The human (A/Hong Kong/486/1997) and tern (A/tern/South Africa/1961)
> influenza A isolates, and the emu laboratory derivative
> (A/emu/Texas/39924-HP derivative/1993) HPAIV isolate had BID~50~ of
> 10^2.4^, 10^3^-^4^, and 10^3.0^, respectively.
>
> For the LPAIVs, none of the bird groups exhibited clinical signs
> following intranasal inoculation. Susceptibility to subclinical
> infections varied by both host species and strain of AIV isolate.
> Although the numbers of LPAIVs examined were limited, a few general
> trends were observed (Table 2). First, based on the BID~50~ values for
> all three isolates, domestic ducks had low and least variability
> between BID~50~ values (10^1.9^ to 10^3.3^). Second, WL

chickens were the most resistant (BID~50~ = 10^5.8^ to 10^7.7^). Third,
for individual strains, domestic ducks (10^1.9^) and quail (10^1.5^ and
10^2^.6) had the lowest BID~50~ values for single isolates, but for one
virus, domestic geese were also very susceptible (BID~50~=10^1^.^7^).
Fourth, for the two wild duck viruses, turkeys had BID~50~ values
(10^4.0^ to 10^4.2^) within the range of susceptibility and
transmissibility as compared with previous poultry outbreak viruses
(i.e., 10^1^-^2^ to 10^4^-^7^; Table 1). Fifth, among chickens, a
difference in host-strain susceptibility was seen; i.e., WPR chickens
were more susceptible than WL chickens to the three viruses.

**DISCUSSION**

A central premise in the development and sustainability of an AI
outbreak is the presence of a strain of AIV adapted to a specific host
species, which suggests easy infecting of the host following low-dose
exposure, adequate replicating of the virus in the host, and sufficient
shedding of the virus into the environment to infect the next host,
thereby sustaining transmission between birds *via* environmental
exposure (26). In a quantitative study, van derGoot *et al.* (35) noted
in a stochastic, epidemiologic, susceptible-infectious-removed
con-tact-transmission model that the HPAIV A/chicken/PA/1370/83 (H5N2)
was easily transmitted between *in contact* chickens as evident by a
high range of reproductive ratios (Ro=5.3-31.7) (35). A Ro \> 1 was
indicative of sustainable transmission, and the field data from the
northeastern United States during the 1983-84 outbreak indicated this
virus lineage was well adapted to chickens and turkeys, spreading to
infect millions of poultry on farms in three states. In addition, the
intranasal mean BID~50~ For this chicken-origin virus was 10^3.0^ (28),
suggesting a laboratory test value that is predictive of infectivi~t~y
for chickens with adequate adaptation for sustained field and laboratory
transmission.

This intranasal BID~50~ is proposed as a quantifiable laboratory
assessment of a virus\'s ability to initiate an infection and the stage
of adaptation and transmissibility for a specific host *species*
(29,34). The H5N1 HPAIVs from Asian outbreaks since 1997 had BID~50~

Table 2. The BID~50~ For three LPAIVs in chickens, turkeys, domestic
ducks and geese, and Japanese quail following intranasal inoculation.

BID~50~ for specific experimental host species (E1D~50~ log~10~)

  ------------------------- ---------- ------- ------- ------- ------- --------
                                                                       

  A/chicken/Alabama/1975    3.0        5.8     6.0     3.3     4.0     2.6
  (H4N8)                                                               

  A/mallard/Ohio/338/1986   4.4        8.3     4.0     3.1     1.7     1.5
  (H4N8)                                                               

  A/mallard/Ohio/18411986   6.7        7.7     4.2     1.9     5.6     5.4
  (H5N1)                                                               
  ------------------------- ---------- ------- ------- ------- ------- --------

> ^A^Infection defined by positive serologic reaction.
>
> ^B^Infection defined by positive virus isolation from oropharyngeal or
> cloaca! swabs.
>
> Table 3. The published BID~50~ for various LPAIVs and HPAIVs following
> intranasal challenge in chickens.

+---------------------------+--------+-------+----------+-------------+
| > Viruses                 | S      | Host  | BID~50~  | References  |
|                           | ubtype | breed | (EID~50~ |             |
|                           |        |       | log~10~) |             |
+---------------------------+--------+-------+----------+-------------+
| HPAIV                     |        |       |          |             |
+---------------------------+--------+-------+----------+-------------+
| > A/chick                 | 115N2  | WL    | 3.0      | > \(28\)    |
| en/Pennsylvania/1370/1983 |        |       |          |             |
+---------------------------+--------+-------+----------+-------------+
| > A/Hong Kong/491/97      | H5N1   | WL    | 2.3      | > \(25\)    |
+---------------------------+--------+-------+----------+-------------+
| > A/chicken/Korea/ES/2003 | H5N1   | WL    | > 2.5,   | > \(28\)    |
|                           |        |       | > 3.1    |             |
+---------------------------+--------+-------+----------+-------------+
| > A                       | H5N1   | WL    | 2.5      | > \(33\)    |
| /chicken/Yamaguchi/7/2004 |        |       |          |             |
+---------------------------+--------+-------+----------+-------------+
| > A/Whooper               | H5N1   | WL    | 2.8      | > \(3\)     |
| > Swan/Mongolia/7/2005    |        |       |          |             |
+---------------------------+--------+-------+----------+-------------+
| > A/                      | H5N1   |       | 2.5      | > \(37\)    |
| chicken/Miyazaki/K11/2007 |        |       |          |             |
+---------------------------+--------+-------+----------+-------------+
| LPAIV                     |        |       |          |             |
+---------------------------+--------+-------+----------+-------------+
| > A/emu/Texas/ 39924/1993 | H5N2   | WI.   | 4.2      | > \(29\)    |
+---------------------------+--------+-------+----------+-------------+
| > A/rhea/North            | H7N1   | WL    | 6.9      | > \(29\)    |
| > Carolina/39482/93       |        |       |          |             |
+---------------------------+--------+-------+----------+-------------+
| > A/chicken/Hong          | H9N2   | WL    | \<2.9^A^ | > \(5\)     |
| > Kong/G9/97              |        |       |          |             |
+---------------------------+--------+-------+----------+-------------+
| > A/cinnamon              | H7N3   | WI.   | 6.2      | > \(21\)    |
| > teal/Bolivia/4537/2001  |        |       |          |             |
+---------------------------+--------+-------+----------+-------------+
| > A/t                     | H7N2   | WL    | 2.8      | > \(34\)    |
| urkey/Virginia/15821/2002 |        |       |          |             |
+---------------------------+--------+-------+----------+-------------+
| > A/                      | H5N2   | WL    | \>5.3^B^ | > \(23\)    |
| mallard/Maryland/791/2002 |        |       |          |             |
+---------------------------+--------+-------+----------+-------------+
| > A/parrot                | II5N2  | WL    | > 4.1    | > (D.       |
| /California/D0406032/2004 |        |       |          | > Swayne    |
|                           |        |       |          | > unpubl.   |
|                           |        |       |          | > data)     |
+---------------------------+--------+-------+----------+-------------+
| > A/ruddy turnstone/New   | H5N7   | WL    | \        | > \(23\)    |
| > Jersey/1148676/2004     |        |       | >7.5^13^ |             |
+---------------------------+--------+-------+----------+-------------+
| > A/duck/P                | H5N1   | WL.   | 7.5      | > \(23\)    |
| ennsylvania/454069-9/2006 |        |       |          |             |
+---------------------------+--------+-------+----------+-------------+
| > A/mute                  | H5N1   | WL    | 6.3      | > \(23\)    |
| > sw                      |        |       |          |             |
| an/Michigan/451072-2/2006 |        |       |          |             |
+---------------------------+--------+-------+----------+-------------+

> ^A^The endpoint was not determined but was below the lowest dilution.

^B^The maximum titer of the virus in allantoic fluid of embryonating
chickens eggs was inadequate to produce infection in 50^0^/s or more of
inoculated chickens.

values for WL chickens ranging from 10^2^\'^3^ to 10^3^\'^1^ (Table 3),
supporting the observed, easy field transmission of this virus lineage
and widespread outbreaks of the H5N1 HPA1 (3,4,28,33; D. Swayne, unpubl.
data). Such adaptation to WL chickens even included H5N1 HPAIV isolates
that were obtained from a human and a whooper swan (BID~50~, 10^2.3^ and
10^2.8^, respectively), suggesting these isolates had previously
inflected and adapted to chickens followed by infection of other animal
species but with retention of adaptation to chickens (Table 3). The
current experimental study reports BID~50~ values for eight H5 and three
H7 HPAIV isolates and a slightly broader range of BID~50~ values, i.e.,
10^1^-^2^ to 10^4.7^. This broader range is suggestive of variations in
the length of circulation and extent of adaptation of the virus strains
to chickens. For example, the A/chicken/Rostock/1934 (H7N1) HPAIV had
the lowest BID~50~ (10^1^\'^2^) and represented the European endemic
HPAIVs (i.e., fowl plague viruses) that circulated in chickens within
Europe from the late-1800s to mid-1930s. By contrast, the
A/turkey/Ireland/1378/1983 (H5N8) had the highest BID~50~ (10^4.7^) and
represented a virus that was isolated only from turkeys in a limited
outbreak and did not circulate in or infect chickens on the same farm.
By contrast, the chicken-origin HPAIVs in this study had a median
BID~50~ of 10^2^-^6^, which suggested well-adapted HPAIVs for chickens
that are corroborated by field outbreaks in chickens. From these
studies, the upper limit for BID~50~ that would predict a virus with
sustainable transmission and adaptation for chickens was not determined.
However, because of the lack of chicken cases on a 1983 Ireland turkey
outbreak farm (1), the experimental BID~50~ of 10^4^-^7^ EID~50~ is
suggestive of a virus that is insufficiently adapted to chickens to be
transmissible in the field and to produce sustained outbreaks.

In addition to intranasal BID~50~ studies, laboratory
contact-transmission studies have been used to assess the transmission
potential of individual strains of HPAIV and LPAIV. Such contact
transmission is affected not only by the virus strain and specific host
species but also by experimental conditions such as air flow within the
isolator or room, humidity, density of birds, amount of environmental
contamination, type of housing, temperature, and duration and quantity
of virus shedding. Typically, these conditions are not

standardized between laboratories and have a profound effect on the
outcome of transmission studies. For example, in 2004, an H5N1 HPAIV
caused outbreaks in three chicken farms in Japan, which suggests
efficient transmission between chickens in the field (15), and the virus
(A/chicken/Yamaguchi/7/2004 \[H5N1\]) had a intranasal BID~50~ of
10^2^\'^5^ EID~50~ (33). However, in experimental contact‑

transmission studies, when 50% of the chickens in the isolator were
intranasally inoculated with HPAIV and housed at a density of 1.13
ft^2^/bird, a moderately successful contact-transmission rate of 50% was
observed for the remaining in-contact birds, and if the bird density~,~
was increased to 0.5 ft^2^/bird, a highly successful
contact-transmission rate of 100% resulted, but no contact transmission
occurred if only 20% of the birds in the isolator were intranasally
inoculated and housed at a density of 0.9 ft^2^/bird. Airborne exposure
was less efficient at transmission than was contact exposure.
Standardization of laboratory contact-transmission models are needed to
more satisfactorily reproduce the field situation, for use in predicting
transmission risk and outbreaks, and for evaluation of the effectiveness
of control measures.

[The wild bird hosts of origin for LPAIVs are much more diverse than
HPAIVs. Free-living aquatic birds, especially ducks and shorebirds,
serve as the primary reservoir for LPAIVs, with the potential for
transfer to domestic poultry following environmental exposure.]{.mark}
Previous studies (21,23) with five LPAI wild birds viruses
(A/Mallard/MD/791/2002 \[H5N2\], A/Duck/PA/454069-9/2006 \[H5N1\],
A/RuddyTurnstone/NJ/1148676/2004 \[1-15N7\], A/Mute
Swan/MI/451072-2/2006 \[H5N1l, and A/cinnamon teal/Bolivia/ 4537/2001
\[H7N3}) indicated WL chickens were resistant to infection by these
viruses, as evidenced by high BID~50~ values (\>10^5.3^, 10^7-5^,
\>10^7-5^, 10^6^\'^3^, and 10^6-2^, respectively). Such indications of
low infectivity suggest these LPAIVs were not naturally adapted for WL
chickens. Furthermore, in our current study, \'WL chickens were
resistant to infection with an additional two wild duck LPAIVs,
A/mallard/Ohio/184/1986 (H5N1) (BID~50~ = 10^7.7^) and
A/mallard/Ohio/338/1986 (H4N8) (BID~50~ = 10^83^). However, broiler-type
chickens (WPR chickens) were moderately susceptible to one virus,
A/mallard/Ohio/338/1986 (H4N8) (BID~50~ = 10^4.4^), suggesting
differences in susceptibility

to wild bird LPAIV infection between different chicken types or strains.

By contrast, compared to WPR and WL chickens, turkeys in the current
study were infected by both wild duck LPAIV isolates at lower challenge
doses (BID~50~ of 10^4.2^ and 10^4^, respectively), but interestingly,
they were more resistant to a chicken-adapted LPAIV than WPR and WL
chickens (even though turkeys were more closely genetically related to
chickens) or than ducks, geese, and quail. In a previous study, turkeys
were shown to he very susceptible to the wild duck LPAIV, A/cinnamon
teal/Bolivia/4537/2001 (H7N3) (BID~50~ = 10^1^-^1^) (22), suggesting
turkeys may be more naturally susceptible to wild bird LPAIV infections
than chickens and even to some chicken-adapted LPAIVs. The field data
corroborate this concept because seasonal LPAIV infections have been
reported in domestic turkeys raised on ranges in Minnesota between the
1960s and mid-1990s (8,10). These infections resulted from both outdoor
exposure to free-ranging, infected ducks during the late summer and fall
migration and the apparent natural susceptibility of turkeys to wild
duck LPAIVs (8,10). The change in turkey production in the mid-1990s
from outdoor range to predominantly indoor systems has nearly eliminated
the seasonal outbreaks of LPAI in commercial Minnesota turkeys. The rare
occurrence of LPAI in Minnesota chickens suggests at least one of the
following: a lack of exposure from confinement rearing, a lower
susceptibility compared with turkeys, or a proportionately lower premise
density. Our BID~50~ data suggest that chickens are more resistant to
the wild duck-origin LPAIVs. Finally, turkeys appear to be more
susceptible to H1 and H3 swine influenza A viruses than chickens are, as
indicated by the greater number of field cases reported in turkeys than
chickens within the United States (18,19). Collectively, these findings
suggest that domestic turkeys may he inherently more susceptibility than
chickens to infection by both wild duck LPAIVs and swine influenza A
viruses.

Although, chickens and turkeys are the major poultry species reared
commercially around the world, other poultry species are significant in
agricultural production, especially for village, backyard, and the LPM
systems. [In these production systems, rearing of mixed species on one
premise is common, and within LPM system, birds in some countries travel
between wholesalers, dealers, and retailers with minimal regulatory
control, which affects pathogen transmission, including A1V
transmission.]{.mark} From our studies, susceptibility to LPAIV
infection varied with both the individual bird species and the LPAIV
isolate. There was a general tendency for domestic ducks and geese and
Japanese quail to have the greatest susceptible to one or more LPAIV and
for WL chickens to be the most resistant (Table 2). However, turkeys
were susceptible to two LPAIV rested at moderate challenge doses. This
suggests domestic ducks and geese, turkeys, and Japanese quail could
serve as bridging species for IPAIVs from wild waterfowl to WL chickens
and possibly other gallinaceous poultry. Previously, Japanese quail and
pheasants were proposed to be bridging species for introduction of
LPAIVs from wild birds to poultry (13,14,16). [Mixing of poultry species
during raising, combined with outdoor rearing, could favor introduction
and adaptation of wild bird AIVs to gallinaceous poultry.]{.mark}

Some LPAIVs have been introduced into poultry, with these viruses being
adapted and maintained in chickens and other poultry, such as H7N2
LPAIVs in LPM system of the northeastern United Stares, H5N2 LPAIVs in
commercial poultry in Central America, and H9N2 LPAIVs in commercial
poultry throughout Asia and the Middle East (24,27). WL chickens have
been shown to be susceptible to A/chicken/Hong Kong/G9/97 (H9N2), which
had a BID~50~ of \<10^2.9^ (5). An H7N2 LPAIV lineage circulated in the

LPM system from 1993 to 2006 (20,32) and was well-adapted to both
chickens and turkeys, as evidenced by transmission from LPM system to
commercial WL chickens and turkeys in Pennsylvania (1996-98);
broiler/broiler breeders in Pennsylvania (2001-02); WL chickens in
Connecticut (2003); mixed, laying thickens in Rhode Island (2003); and
broilers in Maryland and Delaware (2004) (27). In experimental studies,
WL and WPR chickens were susceptible to A/turker/Virginia/158512/2002
(H7N2) LPAIV (BID~50~ of 10^3^\'^2^ and 10^2.8^, respectively), but
turkeys were most susceptible to this virus (BID~50~ = 10°^.8^),
suggesting a higher degree of adaptation to turkeys than chickens, which
may be responsible for an overrepre-sentation of affected turkey *vs.*
chicken flocks in the outbreak zone (34).

Although the upper limit for BID~50~ in predict infectivity and
sustainable transmissibility for a specific species is unknown, from the
previous HPAI outbreaks, viruses with BID~50~ \<10^4.7^ were indicative
of such transmissibility (Table 1). Recently, we examined the
infectivity of an H5N2 LPAIV isolated from an illegally imported
red-lored Amazon parrot in Southern California (12). The virus was
phylogenetically of the Mexican-lineage of H5N2 LPAIV, which has been
circulating and maintained in chickens in Central America since 1993,
implying adaptation to chickens with sustained transmission. Although
the recent H5N2 virus was isolated from a psittacine bird with an
unknown passage history, this LPAIV isolate retained its ability to
infect chickens, as evidenced by a BID~50~ of 10^4.1^ EID~50~ for WL
chickens (D. Swayne, unpubl. data). By contrast, the LPAIVs isolated
from ratites, A/emu/Texas/39924/ 1993 (H5N2) and A/rhea/North
Carolina/39924/93 (H7N1), had variable susceptibility for WL chickens,
as evidenced by an BID~50~ of 10^4^-^2^ and 10^6.9^, respectively,
suggesting the emu virus might produce sustained infections in chickens,
whereas chickens would be resistant to the rhea virus (29). Finally, the
chicken-origin LPAIV isolate A/ chicken/Alabama/1975 (H4N8) had a
BID~50~ \<10^4.7^ EID~50~ for Japanese quail, broiler chickens, and
domestic ducks but \>10^5^ EID~50~ for turkeys and layer-type chickens.
From field data, this virus caused a limited outbreak in only three
layer farms despite the high density of broiler and layer chicken
poultry farms in the geographic region, suggesting the virus was poorly
adapted to chickens or that good biosecurity existed in the region.
[Collectively, the BID~50~ data suggest that poultry-origin and wild
bird--origin LPAIVs express broad variations in susceptibility to
various poultry species, which leads to complexity and unpredictability
of the virus for infecting birds in various production systems.]{.mark}

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> **ACKNOWLEDGMENTS**
>
> We thank J. Beck, J. Doster, and K. Moresco for excellent technical
> support. Mention of trade names or commercial products in this
> publication is solely for the purpose of providing specific
> information and does not imply recommendation or endorsement by the
> U.S. Department of Agriculture. This project was supported by CRIS
> project 6612-32000-048-00D.