Preventive Veterinary Medicine 145 (2017) 73–77

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Preventive Veterinary Medicine

journal homepage: www.elsevier.com/locate/prevetmed

Thermal Inactivation of avian influenza virus in poultry litter as a

method to decontaminate poultry houses
Christopher B. Stephens, Erica Spackman ∗
Exotic and Emerging Avian Viral Diseases Unit, Southeast Poultry Research Laboratory, U.S. National Poultry Research Center, U.S. Department of
Agriculture, Agricultural Research Service, Athens, GA, USA

a r t i c l e i n f o a b s t r a c t

Article history: Removal of contaminated material from a poultry house during recovery from an avian influenza virus
Received 24 April 2017 (AIV) outbreak is costly and labor intensive. Because AIV is not environmentally stable, heating poultry
Received in revised form 27 June 2017 houses may provide an alternative disinfection method. The objective was to determine the time nec-
Accepted 28 June 2017
essary to inactivate AIV in poultry litter at temperatures achievable in a poultry house. Low pathogenic
(LP) AIV inactivation was evaluated between 10.0◦ –48.9 ◦ C, at ∼5.5 ◦ C intervals and highly pathogenic
Keywords:
(HP) AIV inactivation was evaluated between 10.0◦ –43.3 ◦ C, at ∼11 ◦ C intervals. Samples were collected
Cleaning and disinfection
at numerous time points for each temperature. Virus isolation in embryonating chicken eggs was con-
Virus heat inactivation
Avian influenza virus
ducted to determine if viable virus was present. Each sample was also tested by real-time RT-PCR. Low
Poultry litter pathogenicity AIV was inactivated at 1 day at 26.7 ◦ C or above. At 10.0, 15.6 and 21.1 ◦ C, inactivation times
Animal disease outbreak increased to 2–5 days. Highly pathogenic AIV followed a similar trend; the virus was inactivated after
1 day at 43.3 ◦ C and 32.2 ◦ C, and required 2 and 5 days for inactivation at 21.1 ◦ C and 10.0 ◦ C respectively.
While low pathogenicity AIV appeared to be inactivated at a lower temperature than high pathogenicity
AIV, this was not due to any difference in the strains, but due to fewer temperature points being eval-
uated for high pathogenicity. Endpoints for detection by real-time RT-PCR were not found even weeks
after the virus was inactivated. This provides a guideline for the time required, at specific temperatures
to inactivate AIV in poultry litter and likely on surfaces within the house. Heat treatment will provide
an added level of safety to personnel and against further spread by eliminating infectious virus prior to
cleaning a house.
Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction
Avian influenza virus (AIV) is among the most economically
important viruses that affects poultry worldwide and is potentially
zoonotic (Swayne et al., 2013). Outbreaks of AIV have severe eco-
nomic consequences for the poultry industry. In response to an
outbreak of highly pathogenic (HP) AIV, and often to outbreaks of
the H5 and H7 subtypes of low pathogenic (LP) AIV, infected birds
are quickly depopulated. Unless the carcasses are disposed of by in-
house composting they are removed immediately, then the house
must be cleaned and disinfected. Removal of all organic material
from the house is recommended, which includes the poultry lit-
Abbreviations: AIV, avian influenza virus; Ct, cycle threshold; ECE, embryonating
chicken eggs; EID, egg infectious dose; HM, high moisture; HP, highly pathogenic;
LM, low moisture; LP, low pathogenic; rRT-PCR, real-time reverse transcription
polymerase chain reaction.
∗ Corresponding author.
E-mail address: Erica.Spackman@ARS.USDA.gov (E. Spackman).
ter (USDA, 2016b). Removing contaminated litter from the house
for disposal prior to decontamination is costly and carries some
risk of spreading the virus to other poultry flocks and exposing
personnel. Litter cannot be decontaminated by chemical methods
because of the high organic load (Stringfellow et al., 2009), but AIV
is susceptible to heat inactivation.
Most data on the thermal stability of AIV are based on cooking
conditions (Swayne and Beck, 2004; Isbarn et al., 2007; Thomas
et al., 2008) and composting temperatures (e.g. >56 ◦ C [133 ◦ F])
(Senne et al., 1994; Guan et al., 2009; Elving et al., 2012) or at
lower temperatures on hard surfaces (Guan et al., 2016), in manure
(Chumpolbanchorn et al., 2006) and in water (Stallknecht et al.,
1990a; Stallknecht et al., 1990b; Brown et al., 2009). Currently in
the US, heat treatment at 37.8–48.9 ◦ C (100–120 ◦ F) for 7 days with
3 consecutive days at the maximum temperature, may be used to
help decontaminate surfaces within affected poultry houses (USDA,
2016a). However, there are inadequate data available for AIV inac-
tivation at lower temperatures such as those that can be achieved

http://dx.doi.org/10.1016/j.prevetmed.2017.06.012
0167-5877/Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
74 C.B. Stephens, E. Spackman / Preventive Veterinary Medicine 145 (2017) 73–77
in a poultry house, and for a mixed organic material like litter which
is comprised of wood, manure and decaying feathers,
The objective of this study was to determine the time necessary
to inactivate AIV across a range of temperatures to simulate differ-
ent maximal achievable temperatures for different styles of poultry
houses in different weather conditions. To simulate different litter
conditions within a house, both high moisture (HM) and low mois-
ture (LM) litter were tested. Finally, while all evidence suggests that
LPAIV and HPAIV have the same thermal stability HPAIV was tested
at a subset of temperatures to confirm this assumption.
A secondary objective was to determine if there was any corre-
lation between heat treatment and detection of AIV by the rRT-PCR
test, and compare these results to virus isolation in ECEs. Regardless
of cleaning method, farms are still routinely tested for the absence
of virus before the farm can be restocked. Currently, virus isolation
is used because rRT-PCR can detect inactivated virus (Suarez et al.,
2003). Although rRT-PCR can’t be used to differentiate live from
inactivated virus, if samples are negative by rRT-PCR it could pro-
vide a rapid and more cost effective method for screening saving
substantial time and money.
2. Materials and methods
2.1. Viruses
Both HP and LP AIV isolates were selected for their abil-
ity to grow to high titers in embryonating chicken eggs (ECEs)
in order to maximize the sensitivity of detection from the lit-
ter in downstream applications. The LPAIV strain utilized was
rgA/gyrfalcon/WA/41088/2014x PR8 H5N1, which is a reverse
genetics generated strain with the hemagglutinin gene from
A/gyrfalcon/WA/41088/2014 H5N8 that was engineered to be
LP, with the other 7 gene segments from A/Puerto Rico/8/1934
H1N1, a common laboratory influenza A strain which repli-
cates to high titers in ECE. The HPAIV isolate selected was
A/turkey/Italy/4580/1999 H7N1 (Banks et al., 2001), and was
obtained from the Southeast Poultry Research Laboratory, USDA-
ARS repository. Each isolate was titrated according to standard
procedures in ECE (Spackman and Killian, 2014). The titer of the
LPAIV isolate used was 8.4 log10 50% egg infectious dose per ml
(EID50 /ml), and the titer of the HPAIV isolate used was 8.8 log10
EID50 /1 ml. The viruses were added to the litter undiluted to simu-
late the highest viral loads in the litter possible.
2.2. Litter
Used litter, consisting of kiln dried, medium flake, mixed wood
shavings was obtained from Southeast Poultry Research Laboratory
specific pathogen free (SPF) chicken flocks. High moisture litter was
collected from under the drinkers and LM litter was collected from
dryer areas of the house. Litter pH for both LM and HM litter was
determined to be 7.0–7.5. Moisture content was measured using
soil moisture probes (EC-5 Small Soil Moisture Sensors; Decagon,
Pullman, WA). Low moisture litter had a mean moisture level of
0.025 cubic meters of water per cubic meter of litter (m3 /m3 ) (stan-
dard deviation = ±0.053 m3 /m3 ), which did not change during the
course of any treatment. The HM litter had an average moisture
content of 0.409 m3 /m3 (standard deviation = ±0.110 m3 /m3 ) and
decreased an average of 0.040 m3 /m3 during treatment.
2.3. Experimental design
Litter treatment groups are shown in Table 1. Three replicates of
each moisture level were run at each temperature. Samples were
collected at a minimum of 24hr intervals at which time one vial was
removed, sealed, and immediately stored at −80 ◦ C. Sample collec-
tion times varied by temperature and were based on the expected
endpoint (Table 1).
Individual 5 ml plastic vials were filled with 1.5–2.0 g of LM or
HM litter, and then 0.1 ml of titrated virus was added to each vial.
The vials were used to prevent lateral diffusion of the virus through
the litter so the change in virus titer per gram could be accurately
measured. Each vial served as an individual time-point sample at
each temperature. All litter samples in vials were pre-heated to
ambient temperatures before the virus was added. Individual lit-
ter vials were placed in 1 L canisters that had been filled with the
same litter as the vials and when a sample vial was removed, an
empty 5 ml vial was added back to the litter canister to maintain
the density. An untreated sample of litter to which virus was added
(time 0 sample) was collected at the time the virus was added to
all the litter sample vials for the same temperature treatment, and
was immediately stored at −80 ◦ C until it was processed for virus
detection. Individual vials and 1L canisters were not sealed so the
litter could evaporate naturally (Supplemental Fig. S1). However, all
material was sealed in a secondary container to contain the virus.
Temperatures were maintained in an environmental chamber.
Changes in moisture content were monitored using data log-
gers with soil moisture probes (EM5B Analog Data Loggers and
EC-5 Small Soil Moisture Sensors; Decagon, Pullman, WA). As a
secondary measure of moisture change, canisters were weighed
before and after each temperature treatment. A separate 5 ml vial
of litter with no virus added was included in each 1 L canister
and was weighed before and after each treatment, to confirm that
moisture loss in the 5 ml vials was equivalent to the 1 L canister.
A data logging thermometer with a probe were placed at a depth
of 2–5 cm into the center of the litter in each canister (SD200 3-
Channel Temperature Data logger; ExTech, Nashua, NH) to verify
the temperature of the litter.
2.4. Extraction of virus from litter
The procedure for extracting virus from the litter was optimized
for virus recovery prior to processing samples. It was determined
that storage at −80 ◦ C with one total freeze-thaw cycle did not
decrease virus titers. Each sample was processed and titrated indi-
vidually as follows: brain heart infusion (BHI) broth (3.5 ml for LM
litter and 3.0 ml for HM litter) was added to each vial, and the
material was then mixed by vortexing and incubated at ambient
temperatures for 10 min. Samples were homogenized with a pestle
by hand to minimize heat generation, then centrifuged at 2900 × g
for 10 min, the supernatant was collected and centrifuged again at
1900 × g for 10 min. The supernatant from the second centrifuga-
tion step was used for virus isolation and RNA extraction.
2.5. Virus isolation and titration
The supernatant was treated with antibiotics (final concentra-
tion: Penicillin G 1000 IU/ml, Streptomycin 200 ␮g/ml, Gentamicin
100 ␮g/ml, Kanamycin 65 ␮g/ml, Amphotericin B 2 ␮g/ml) for 1 h
at ambient temperature which was necessary for the antibiotics
to kill any contaminating bacteria. Each virus isolation was set-up
as a titration to maximize sensitivity and to quantify virus. Titra-
tions were conducted in ECEs by standard methods (Spackman and
Killian, 2014). Briefly, 5 ECEs were inoculated with 0.1 ml of each
dilution by the chorioallantoic sac route using undiluted material
through a dilution of 10−5 in BHI broth. Standard hemagglutination
assay was used to test the fluid from each egg for virus replica-
tion (Killian, 2014). Titers were calculated with the Reed-Meunch
method (Reed and Muench, 1938). The endpoint was defined as
the first time point where all three replicates were negative. Con-
trols collected at time 0 had an average titer of 4.29 ± 0.78 log10
 C.B. Stephens, E. Spackman / Preventive Veterinary Medicine 145 (2017) 73–77
Table 1
Sample collection schedule. Black squares indicate that a sample was collected and tested by virus isolation and rRT-PCR. Gray Squares indicate that a sample was collected
and tested by rRT-PCR only. White squares indicate that no samples were collected. Samples of LM and HM litter were treated simultaneously and were collected at the same
time.
a. LP = low pathogenic avian influenza virus.
b. HP = highly pathogenic avian influenza virus.
c. Day zero of incubation samples were untreated litter with virus added.
EID50 /ml and 4.40 ± 1.05 log10 EID50 /ml for the LM and HM litter
respectively. While some decrease in titer was expected, perhaps
due to absorption of the virus by the litter material, the amount
of reduction was unknown at the start of the study. The time 0
controls revealed an ∼4 log10 reduction in titer.
2.6. Real-time RT-PCR
RNA was extracted from supernatants using established meth-
ods (Das et al., 2009) and the standard USDA M gene AIV rRT-PCR
test was performed (Spackman et al., 2002). The rRT-PCR was run
as per standard protocol on the AB 7500 FAST platform (Thermo
Fisher Scientific, Waltham, MA).
3. Results
3.1. Virus inactivation
No differences between LM and HM litter were observed unless
noted. For LPAIV, all treatments from 26.7–48.9 ◦ C were inactivated
at 1 day (Table 2). At 21.1 ◦ C inactivation occurred at 2 days. At
15.6 ◦ C, inactivation occurred at 4 days of incubation for the HM
litter, and 5 days for the LM litter. At 10 ◦ C LPAIV was inactivated in
LM at 4 days and at 5 days in the HM litter.
Inactivation of HPAIV in litter followed the same trend as LPAIV
(Table 2) and no differences between LM and HM litter were
observed unless noted. Highly pathogenic AIV was inactivated at
1 day of incubation at 43.3 ◦ C and 32.2 ◦ C and at 2 days at 21.1 ◦ C
at 2 days. At 10 ◦ C inactivation occurred at 3 days for the HM litter,
and at 5 days for the LM litter.
3.2. Real-Time RT-PCR
RNA was detected by rRT-PCR in all samples at the endpoint of
virus viability (Supplemental Table 1). Additional time points from
the LPAIV samples were then tested by rRT-PCR to evaluate RNA
decay with heat treatment. No endpoints were reached in the sam-
ples available from the inactivation testing and since data on later
times would not contribute to an improved diagnostic approach
testing was discontinued. A trend where Ct values increased more
rapidly at higher temperatures than at lower temperatures was
observed indicating that RNA was decaying more rapidly at higher
temperature (Supplemental Table 1).
75
4. Discussion
Improved approaches to decontaminating farms that improve
safety and biosafety are beneficial because physically cleaning a
premises after an AIV outbreak always carries some risk of spread-
ing the virus and exposing workers to viral aerosols. Our results
have shown that both LPAIV and HPAIV can be inactivated at
similar temperatures and incubation lengths. Other studies have
performed direct comparisons on the thermal inactivation of both
LPAIV and HPAIV, however these studies were performed at pas-
teurization temperatures, which as expected, inactivated AIV in a
shorter amount of time when compared to the lower temperatures
used in this study (Swayne and Beck, 2004; Chmielewski et al.,
2011, 2013). Heating a poultry house to inactivate viral pathogens
is not a new concept (Halvorson, 1986; Giambrone et al., 2008)
since most poultry houses have heaters because of the need to keep
young birds warm.
One common variable that is found in poultry litter is moisture
content, so our study evaluated both low and high moisture con-
tent. Treatments for litter can likely be extrapolated to hard surfaces
as our data correlate with previous reports that looked at porous
and non-porous hard surfaces that would be in a poultry house
(Guan et al., 2016) although the conditions were not identical.
A short-coming of this study is that the time 0 samples revealed
approximate 4 log10 reduction in virus titer just by the addition of
the virus to litter. This decrease could be attributed to absorption
of the virus by the litter material; although the extraction method
had been optimized for the best recovery of virus from the litter
samples some binding may be irreversible. Practically, this means
that a 4–5 log10 titer reduction is what could be reliably be mea-
sured here. There are no data on what virus levels could be in litter
during an AIV outbreak, as every flock and premises are different.
Therefore, in practical application, a minimum of 24 h of treatment
should be added to any target temperature as an added margin of
safety. The temperature of the litter should be monitored at a vari-
ety of depths and locations throughout the house, and the “clock”
for inactivation should not start until the litter throughout the
house has reached the target temperature. As expected, higher tem-
peratures inactivated virus faster and temperatures of 32.2–48.9 ◦ C
range are achievable in many poultry houses, but lower tempera-
tures can also be used if necessary due to the structure of the house
or weather conditions. If lower temperatures are used, adding addi-
tional time beyond the 24hr safety margin would be recommended
as inactivation times for AIV seems to be more variable among dif-
76 C.B. Stephens, E. Spackman / Preventive Veterinary Medicine 145 (2017) 73–77

Table 2
Results of virus isolation for each replicate in embryonating chicken eggs by treatment. Data past day 6 of treatment were all negative and are not shown.

Temperature Moisture Untreated Day 1 Day 2 Day 3 Day 4 Day 5

Low pathogenicity AIV

48.9 ◦ C Low 3/3 (4.2)a 0/3 NTb NT NT NT
(120 ◦ F) High 3/3 (3.3) 0/3 NT NT NT NT

43.3 ◦ C Low 3/3 (5.5) 0/3 NT NT NT NT

(110 ◦ F) High 3/3 (4.8) 0/3 NT NT NT NT

37.8 ◦ C Low 3/3 (5.3) 0/3 NT NT NT NT

(100 ◦ F) High 3/3 (4.9) 0/3 NT NT NT NT

32.2 ◦ C Low 3/3 (4.4) 0/3 NT NT NT NT

(90 ◦ F) High 3/3 (4.0) 0/3 NT NT NT NT

26.7 ◦ C Low 3/3 (4.4) 0/3 NT NT NT NT

(80 ◦ F) High 3/3 (5.4) 0/3 NT NT NT NT

21.1 ◦ C Low 3/3 (4.4) 2/3 (0.9) 0/3 NT NT NT

(70 ◦ F) High 3/3 (4.2) 2/3 (0.9) 0/3 NT NT NT

15.6 ◦ C Low 3/3 (5.4) 3/3 (1.6) 3/3 (0.9) 2/3 (0.9) 1/3 (0.9) 0/3
(60 ◦ F) High 3/3 (5.1) 3/3 (3.3) 3/3 (3.4) 2/3 (1.4) 0/3 0/3

10.0 ◦ C Low 3/3 (3.3) 3/3 (1.5) 3/3 (1.1) 3/3 (1.2) 0/3 0/3
(50 ◦ F) High 3/3 (4.3) 3/3 (2.0) 3/3 (0.9) 3/3 (1.3) 1/3 (0.9) 0/3

High Pathogenicity AIV

43.3 ◦ C Low 3/3 (3.8) 0/3 NT NT NT NT
(110 ◦ F) High 3/3 (3.9) 0/3 NT NT NT NT

32.2 ◦ C Low 3/3 (4.9) 0/3 NT NT NT NT

(90 ◦ F) High 3/3 (5.0) 0/3 NT NT NT NT

21.1 ◦ C Low 3/3 (3.9) 2/3 (1.1) 0/3 NT NT NT

(70 ◦ F) High 3/3 (3.1) 3/3 (1.2) 0/3 NT NT NT

10.0 ◦ C Low 3/3 (4.4) 3/3 (1.3) 1/3 (1.2) 2/3 (0.9) 1/3 (0.9) 0/3
(50 ◦ F) High 3/3 (4.4) 3/3 (2.2) 3/3 (0.9) 0/3 0/3 0/3

a. Results shown as number of positive replicates/total replicates (mean log10 titer of positive replicates). When titers were <101 50% egg infectious doses they were given
the value of 100.9 to calculate the mean.
b. NT = not tested.

ferent matrices and media at these temperatures (Stallknecht et al., Acknowledgements

1990a; Chumpolbanchorn et al., 2006; Guan et al., 2016).
Virus detection after clean-up at an infected premises is another
critical element of outbreak recovery. Reducing the use of virus
isolation is important because of the time (up to 2 weeks) and
resources it can take versus rRT-PCR which can provide a result in a
few hours. Here the rRT-PCR results demonstrated that AIV RNA can
still be detected well past the point of virus inactivation, so samples
at any temperature have a high possibility of producing a false pos-
itive result. Although a lack of correlation between rRT-PCR and
virus isolation was expected at lower temperatures, if there was
better correlation with inactivation at higher temperatures the use
of rRT-PCR as a screening test would have been viable. Since no cor-
relation was evident, rRT-PCR is not appropriate for prescreening
samples that were heat treated for the presence of viable virus. In
this paradigm screening samples treated at any temperatures with
rRT-PCR offers little advantage to predicting whether viable virus
is present.
The authors would like to thank Dr. David Suarez for provid-
ing the rgA/gyrfalcon/WA/41088/2014x PR8 H5N1 LPAIV strain
used in this study. The authors would also like to thank Scott Lee,
Alliyah Byrd, Jesse Gallagher, Kyle Dyson, Bill Gagnon, and Marisela
Rodriguez, for their technical assistance. Funding for this work was
provided by US Poultry and Egg Association Project #BRU005, and
USDA CRIS Project #6040-32000-063-00D. Contents are solely the
responsibility of the authors and do not necessarily represent the
official views of the USDA. Mention of trade names or commercial
products in this manuscript is solely for the purpose of provid-
ing specific information and does not imply recommendation or
endorsement by the USDA. USDA is an equal opportunity provider
and employer.
Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at http://dx.doi.org/10.1016/j.prevetmed.2017.
5. Conclusions 06.012.

These studies show that AIV can be inactivated with heat prior
to cleaning a poultry house during outbreak recovery, which will
improve the safety for workers from a potential zoonotic infection
with AIV and it will reduce the likelihood of lateral spread of virus
during clean-up. Unfortunately, it could not be demonstrated that
rRT-PCR would correlate with heat inactivation at any temperature,
so testing strategies that would reduce the need for virus isolation
still need to be developed and will be the focus of future work.
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