Inactivation of Avian Influenza Virus Using Common Detergents and Chemicals
M. E. Lombardi,AB. S. Ladman,BR. L. Alphin,Cand E. R. BensonD
ADepartment of Bioresources Engineering, University of Delaware, 264 Townsend Hall, Newark, DE 19716
BDepartment of Animal and Food Sciences, University of Delaware, 315 Worrilow Hall, Newark, DE 19716
CDepartment of Animal and Food Sciences, University of Delaware, 107 C. C. Allen Biotechnology Laboratory, Newark, DE 19716
Received 21 July 2007; Accepted and published ahead of print 26 November 2007
SUMMARY. Six disinfectant chemicals were tested individually for effectiveness against low pathogenic avian influenza virus
(LPAIV) A/H7N2/Chick/MinhMa/04. The tested agents included acetic acid (C2H4O2), citric acid (C6H8O7), calcium
hypochlorite (Ca(ClO)2), sodium hypochlorite (NaOCl), a powdered laundry detergent with peroxygen (bleach), and a
commercially available iodine/acid disinfectant. Four of the six chemicals, including acetic acid (5%), citric acid (1% and 3%),
calcium hypochlorite (750 ppm), and sodium hypochlorite (750 ppm) effectively inactivated LPAIV on hard and nonporous
surfaces. The conventional laundry detergent was tested at multiple concentrations and found to be suitable for inactivating LPAIV
on hard and nonporous surfaces at 6 g/L. Only citric acid and commercially available iodine/acid disinfectant were found to be
effective at inactivating LPAIV on both porous and nonporous surfaces.
RESUMEN. Inactivacio ´n del virus de influenza aviar usando detergentes comunes y agentes quı ´micos.
Seis desinfectantes quı ´micos fueron evaluados individualmente para medir su efectividad contra el virus de influenza aviar de baja
patogenicidad A/H7N2/Pollo/MinhMa/04. Los agentes analizados incluyeron a ´cido ace ´tico (C2H4O2), a ´cido cı ´trico (C6H8O7),
hipoclorito de calcio (Ca(ClO)2), hipoclorito de sodio (NaOCl), un detergente para lavadora en polvo con pero ´xido (blanqueador)
y un desinfectante a ´cido yodado disponible comercialmente. Cuatro de los seis quı ´micos, incluyendo a ´cido ace ´tico (5%), a ´cido
cı ´trico (1% y 3%), hipoclorito de calcio (750 ppm) e hipoclorito de sodio (750 ppm) inactivaron en forma efectiva el virus de
influenza aviar de baja patogenicidad en superficies duras y no porosas. El detergente convencional para lavadora fue usado a
mu ´ltiples concentraciones y se encontro ´ que fue u ´til para la inactivacio ´n del virus en superficies duras y no porosas a una
concentracio ´n de 6 g/L. Unicamente el a ´cido cı ´trico y el desinfectante yodado fueron efectivos tanto en superficies porosas como no
Key words: avian influenza virus, inactivation, decontamination, disinfection
Abbreviations: AIV5avian influenza virus; AMPV5avian metapneumovirus; CAF5chorioallantoic fluid; CRBC5chicken
red blood cell; EID505egg 50% infection dose; EPA5U.S. Environmental Protection Agency; FBS5fetal bovine serum;
H5hemagglutinin; HA5hemagglutination activity; HPAIV5high pathogenic avian influenza virus; LPAIV5low pathogenic
avian influenza virus; N5neuraminidase; NI5neutralizing index; PBS5phosphate-buffered solution; PC5positive control;
SPF5specific pathogen free
The risk of an avian disease outbreak is always a concern for the
poultry industry. High pathogenic avian influenza virus (HPAIV)
H5N1 emerged in Hong Kong in 1996–1997, and by July 2007 it
had caused 191 human deaths and .300 outbreaks in domestic
poultry, water fowl, or wild birds in Asia, central Europe, Africa, and
parts of the Middle East (23). Although the U.S. poultry industry
has not been affected by the H5N1 virus yet, one of the worst avian
influenza (AIV) outbreaks in recent U.S. history occurred in
Pennsylvania in 1983, in which an outbreak of H5N2 HPAIV
resulted in the destruction of 17 million birds at a cost of .$60
million (9,11). More recently, a low pathogenic avian influenza virus
(LPAIV) H7N2 outbreak in Virginia in 2002 resulted in the
destruction of 4.7 million birds at a cost of $160 million (3). The
detection of two separate H7N2 LPAIV strains in the Delmarva
region in 2004 resulted in the destruction of 328,000 birds on two
farms in Maryland and 85,000 birds on two farms in Delaware
Avian influenza virus is a lipid-enveloped virus of the family
Orthomyxoviridae and genus Influenzavirus A (18). The virus occurs
in two forms in domestic poultry (chickens and turkeys): low
pathogenic and high pathogenic. LPAIV is less severe and causes
mild respiratory disease. HPAIV, however, causes rapid increases in
mortality. Two genes in the AIV single-stranded sequence code for
envelope proteins (hemagglutinin [H] and neuraminidase [N]).
Type A influenza viruses can be divided into 16 hemagglutinin (HA;
H1–H16) and nine neuraminidase (N1–N9) subtypes based on the
genetic sequence for the envelope proteins (7). H5 and H7 are the
most virulent strains to cause HPAIV in domestic poultry. Although
most H5 or H7 viruses are low pathogenic, H5 has been
demonstrated to mutate from LPAIV to HPAIV (4).
Viruses can be grouped in terms of resistance to disinfection
strategies. Avian influenza is relatively easy to disinfect due to the
lipid envelope that increases its sensitivity to dehydration, detergents,
and surfactants (1). Acids, for example, are highly virucidal and can
be used under a wide range of conditions. Acetic acid has been
shown to effectively disinfect surfaces contaminated with Salmonella
typhi and Escherichia coli; however, acetic acid did not effectively
eliminate some more resistant bacteria, and it is not recommended
for use as a broad-spectrum antibiotic (2,12). Citric acid is effective
at inactivating acid-sensitive viruses, it can be combined with
detergents, and it is typically safe for use on personnel and clothing.
The AUSVETPLAN (1) recommends using citric acid at 0.2%
(weight per volume) for 30 min to effectively inactivate viruses and
eliminate harmful microorganisms.
Oxidizers are recommended for many disinfection applications
(1). Sodium hypochlorite is a strong oxidizer, and it has been used as
DCorresponding author. Department of Bioresources Engineering,
University of Delaware, 242 Townsend Hall, Newark, DE 19716. E-mail:
AVIAN DISEASES 52:118–123, 2008
a disinfectant since World War I when it was used to prevent
infection in open wounds. Sodium hypochlorite reacts in water,
causing the hypochlorite compounds to partially split, releasing
hypochlorous acid and hypochlorite anions. The resulting hypo-
chlorous acid has strong antimicrobial properties, even at very low
concentrations. The exact antimicrobial mechanism of chlorine is
unknown, but it most likely causes inhibition of key enzymatic
reactions within the cell and protein denaturation (15). The
disinfecting efficiency of chlorine increases with decreasing pH.
Water hardness has no effect on the ability of chlorine to inactivate
viruses and kill bacteria, but chlorites have reduced efficacy in the
presence of organic matter, because some of the available acid binds
to the organic matter leaving less available to react with
microorganisms (6). In general, the use of hypochlorite solutions
should be discouraged when the organic matter concentrations
exceeds 10%. However, even with the reduced efficacy, chlorine is
effective at inactivating many viruses, including hepatitis B and
human immunodeficiency virus, in the presence of organic matter
(14). Calcium hypochlorite and sodium hypochlorite are both
effective broad-spectrum disinfectants, even against highly resistant,
hydrophilicviruses, at 2–3%
30,000 ppm). For influenza A, a minimum of 200 ppm concentra-
tion of sodium hypochlorite is required for inactivation within
10 min (13). Sodium hypochlorite, however, can be corrosive to
Evaluation of virus inactivation is a critical component of
disinfectant testing. In vivo testing relies on the H surface
glycoprotein to bind to receptors on a variety of mammalian and
avian erythrocytes if the virus is active. This produces hemaggluti-
nation activity (HA), or clumping of cells that is visible to the naked
eye (19). The method of virus inactivation varies based on the
disinfectant tested. For example, phenolic disinfectants and
quaternary ammonia compounds denature the surface proteins on
AIV, preventing the virus from fusing to and infecting host cells, and
resulting in inactivation. However, denaturing the surface proteins
does not alter the RNA. Other disinfecting agents such as chlorine
and peroxygen compounds inactivate the AIV in addition to
denaturing the viral RNA, but both disinfecting agents require
higher concentrations to denature viral RNA than they do to
inactivate the virus (20). Ma et al. (10) found similar results with
hydrochloric acid and sodium hydroxide. The purpose of this
research was to evaluate the efficacy of common, commercially
available detergents, chemicals, and disinfectants to inactivate
MATERIALS AND METHODS
Chicken embryos. Specific-pathogen-free (SPF) White Leghorn
embryonated chicken eggs and chickens were purchased from SPAFAS,
Inc. (Norwich, CT). Embryos were used for the titration of the viral
stock and virus isolation.
Virus. LPAIV strain A/H7N2/Chick/MinhMa/04, isolated from a
commercial broiler flock during the 2004 Delmarva outbreak, is
maintained as a low-embryo-passage challenge virus at the University of
Delaware. A seed stock of A/H7N2/Chick/MinhMa/04 was prepared
from the sixth chorioallantoic sac passage in SPF embryonated eggs and
was titrated as described previously (19,22). The seed stock was
determined to have a titer of 106.8egg 50% infection dose (EID50)/
0.1 ml, and it was used for all tests at this titer. To increase the organic
load of the tested sample, 5% fetal bovine serum (FBS) (Sigma Aldrich,
St. Louis, MO) was mixed with virus.
Virus reisolation from coupons. Virus isolation attempts were
performed by inoculating 0.2 ml of the solution in question into 9-to-
11-day-old embryonated SPF chicken eggs that were incubated for 5
days and candled daily for viability. Embryo mortality observed within
the first day postinoculation was considered nonspecific and discarded.
Recorded mortalities between days 2 and 5 postinoculation were held at
4 C for subsequent screening. At 5 days postinoculation, all remaining
embryos were place at 4 C for 24 hr. Chorioallantoic fluid (CAF) was
collected from each egg and tested for HA as described previously (19).
Briefly, chicken red blood cells (CRBCs) were obtained by mixing whole
blood with 50% Alsever’s solution, centrifuging at 1000 3 g for 5 min,
and washing the cells three times in phosphate-buffered solution (PBS;
Fisher Scientific, Hanover Park, IL). A final dilution was made in PBS
to produce a solution to contain approximately 25% CRBCs. HA tests
were performed by combining 60 ml of CAF from one egg and 36 ml of
CRBCs. The mixture of chorioallantoic fluid and CRBCs was gently
agitated for 3 to 5 min at room temperature. A positive result indicated
the presence of the LPAIV strain A/H7N2/Chick/MinhMa/04 in the
CAF sample tested. The HA test was performed on each egg
Building materials. Materials typically present in a poultry house
were used in this study. Thin squares of metal (14-gauge, galvanized
steel; A & H Metals Inc., Newark, DE), plastic (polyvinylidene
difluoride Kynar Film; Arkema, Inc., Philadelphia, PA), and wood
(basswood; Midwest Products Company, Inc., Hobart, IN) were cut
into 2.2- 3 2.2- 3 ,0.2-cm coupons. Metal and plastic coupons were
triple washed, and wood coupons were soaked in double-distilled
(deionized) water containing 2% skim milk powder (15). All coupons
were thoroughly dried and sterilized at 121 C for 30 min before use.
Chemical compounds. Six agents at various concentrations were
tested individually for effectiveness against LPAIV for a 10-min contact
time. Test combinations included 5% acetic acid (C2H4O2); 1% citric
acid (C6H8O7); 3% citric acid (C6H8O7); 750 ppm calcium hypochlo-
rite (http://en.wikipedia.org/wiki/Molecular_formula) (Ca(ClO)2); 750
ppm sodium hypochlorite (NaOCl); 2 g/L, 4 g/L, and 6 g/L powdered
laundry detergent with peroxygen; and a commercial iodine/acid
disinfectant diluted 300:1.
The test agents were diluted into 400 ppm hard water solution on the
day of use. The hard water solution was prepared by combining 40 ml
of hard water (1 mg of CaCO3/ml) solution with 60 ml of double
distilled (deionized) water per 100 ml of 400 ppm CaCO3solution
Acetic acid solution was prepared by diluting USP grade acetic acid to
produce a solution of 5% acetic acid, the same as distilled vinegar. The
solution was made by combining 10 ml of acetic acid with 190 ml of
prepared 400 ppm CaCO3hard water solution.
Calcium hypochlorite solution was prepared by diluting technical
grade calcium hypochlorite with 65% available chlorine into prepared
400 ppm CaCO3 hard water solution. The solution tested at
approximately 750 ppm with test strips.
Citric acid solution was prepared by combining 1% and 3% by
weight to volume USP grade citric acid into 200 ml of prepared
400 ppm CaCO3hard water solution.
The commercial iodine/acid disinfectant solution was prepared as a
300:1 dilution combining 3 ml of disinfectant concentrate with 897 ml
of prepared 400 ppm CaCO3hard water solution. The active ingredient
in the commercial iodine/acid disinfectant is 0.42% iodine from
polyoxyethylene-polyoxyprpylene block polymer iodine complex.
Sodium hypochlorite solution was prepared by diluting 5 ml of
reagent grade sodium hypochlorite solution, reagent grade with greater
than or equal to 4% available chlorine into 400 ml of prepared
400 ppm CaCO3 hard water solution. The solution tested at
approximately 750 ppm with test strips.
Due to the nonhomogeneous nature of the powdered laundry
detergent, the powder was mixed in a cross-flow micro-ingredient mixer
for 15 min to produce a more uniform mixture. To prepare the laundry
detergent test solutions, the specified weight of powdered laundry
detergent was mixed into a solution of 1000 ml of prepared 400 ppm
CaCO3hard water solution. Due to the nonhomogeneous nature of
powdered laundry detergent, a 1.0-liter solution was prepared,
increasing the probability of a representative sample.
Inactivation of AIV using common chemicals
PBS was prepared adding 8.5 g of sodium chloride, 1.18 g of dibasic
sodium phosphate, and 0.22 g of monobasic sodium phosphate to 1
liter of double-distilled water. Cold, sterile PBS was used to dilute a
penicillin/streptomycin antibiotic solution (Lonza Walkersville, Inc.,
Walkersville, MD) 10,000 units/ml and 10,000 mg/ml, respectively.
This PBS antibiotic mixture was used for all necessary dilutions.
Efficacy of tested chemical compounds to inactivate LPAIV.
Fourteen coupons of each of metal, plastic, and wood were coated with
0.1 ml of the virus/5% FBS mixture. Additionally, six plastic coupons
were placed on the foil to be used as chemical compound cytotoxic
control. This coupon set was coated with 0.1 ml of the prepared
cytotoxic control solution consisting of 1.9 ml of PBS containing 0.53
antibiotic solution (described above) combined with 0.1 ml of FBS to
produce a solution of 5% FBS. All coupons were allowed to dry for
approximately 1.5 hr at room temperature. Once dry, the coupons were
placed into six-well, flat-bottomed, nontissue culture plates (BD
Biosciences, Franklin Lakes, NJ). Two plates, A and B, were used for
each material containing either metal, plastic, or wood. The remaining
two coupons of each material were placed in a separate plate to serve as
the virus control group. An additional six-well plate containing six
plastic coupons coated with cytotoxic control solution served as the
chicken embryo cytotoxic control.
Each disinfecting agent was prepared as specified above. 2.0 ml of
each solution was placed into each of the six wells of the treatment plates
and cytotoxic control plate containing the metal, plastic, and wood
coupons. Each plate was then gently agitated on a horizontal plate shaker
for 10 min at room temperature. After agitation, each coupon was
scraped with a pipette, and the fluid was aspirated from the well and
jetted back onto the coupon three times to dislodge virus from the
coupon. The fluids from the six wells of the plate were pooled into a
single tube. The virus control plate was handled in a manner similar to
the other test plates; however, 2.0 ml of PBS was used to dislodge virus
from the coupons, rather than the disinfecting agent. The fluid from the
two wells containing like materials was pooled into a single tube,
resulting in a virus control for each material.
The pooled fluid from the plates containing metal, plastic, and wood
coupons was diluted by making three 10-fold serial dilutions, resulting
in dilutions from 1021to 1023. The pooled fluid for the metal, plastic,
and wood positive controls was diluted using six 10-fold serial dilutions,
resulting in dilutions from 1021to 1026. The cytotoxic control was
diluted once resulting in a 1021dilution. Difco D/E neutralizing broth
(BD Biosciences) was used for the first dilution for each group to
inactivate the chemical compounds in question, with subsequent
dilutions occurring in PBS. Virus reisolation attempts were made using
Neutralizing index (NI). A numerical method was used to express
the ability of a pc2tadisinfectant agent to inactivate virus. An NI of
virus inactivation was used to evaluate the efficacy of each agent. This
method was a modification of the classical avian serologic virus-
neutralization test (19). The NI of virus inactivation is calculated using
the following equation:
where tais titer of the recovered virus from the disinfectant-treated plates
and tpcis the titer of the positive control plate. For viruses, it is often
only practical to measure a 3 to 4 log10reduction in titer, and no
detectable infectious virus in the highest dilution of the virus–
disinfectant mixture tested. For this reason, inactivation of AIV was
considered effective when NI $ 2.8, the positive control titer was $4.0,
and there was no recoverable virus from any treated coupon. No
recoverable virus equals a titer of ,1.2 via the method of Reed and
Mu ¨ench (17).
A summary of the NI indices for the common detergents and
disinfectants tested is shown in Table 1. The United States
Environmental Protection Agency (EPA) considers a disinfectant
agent to be effective if the product can demonstrate complete
Table 1. Summary of recovered titers by material and chemical agent.
Acetic acid (5%)Metal
Calcium hypochlorite (750 ppm)
Citric acid (1%)
Citric acid (3%)
Iodine/acid (300:1 dilution)
Sodium hypochlorite (750 ppm)
Laundry detergent with peroxygen (2 g/L)
Laundry detergent with peroxygen (4 g/L)
Laundry detergent with peroxygen (6 g/L)
M. E. Lombardi et al.
inactivation of the virus at all dilutions while at least 4 logs of virus
particles per milliliter must be recovered from the nonvirucidal-
treated control carrier. When the EPA criteria for effectiveness is
applied, multiple common chemicals including acetic acid (5%),
citric acid (1% and 3%), calcium hypochlorite (750 ppm), and
sodium hypochlorite (750 ppm) were effective at inactivating
LPAIV on hard and nonporous surfaces. The conventional laundry
detergent with peroxygen detergent tested was also effective at
inactivating LPAIV on hard and nonporous surfaces at concentra-
tions of 6 g/L or greater. Only citric acid and the commercial iodine/
acid disinfectant were found to be effective at inactivating LPAIV on
both porous and nonporous surfaces.
Acetic acid (5%) had a modest virus neutralization potential as
measured by NI, with NI values from 2.3 (wood) to 3.3 (plastic).
The wood NI was reduced due to the relatively low positive control
(PC 5 3.5) value. The recovered virus titer on all treated surfaces
was less than 1.2. There was no positive HA on any of the treated
Citric acid (1%) had the highest NI values tested on hard and
nonporous surfaces (metal: NI 5 5.7, plastic: NI 5 4.7). Strong
positive control values ranging from 4.1 (wood) to 6.9 (metal)
improved the NI values. The recovered virus titer on all treated
surfaces was less than 1.2. There was no positive HA on any of the
treated surfaces tested.
Citric acid (3%) seemed to have reduced efficacy; however, the
reduced efficacy was related to lower positive controls. The recovered
virus titer on wood was less than 1.2, whereas the metal (PC 5 4.9)
and plastic (PC 5 4.2) positive controls were also reduced. With 3%
citric acid, the resulting NI values for metal (NI 5 3.7) and plastic
(NI 5 3.0) meet the criteria for successful inactivation. There was no
positive HA on any of the treated surfaces tested.
Calcium hypochlorite (750 ppm) was effective at inactivating
LPAIV on hard and nonporous surfaces. Virus recovery from wood
reduced the positive control (PC 5 3.1), whereas modest recovery
on positive control metal (PC 5 4.9) and plastic (PC 5 5.1) surfaces
improved NI values. The recovered virus titer on all treated surfaces
was less than 1.2. There was no positive HA on any of the treated
Sodium hypochlorite (750 ppm) was also effective at inactivating
LPAIV on hard and nonporous surfaces. The recovered virus titer on
wood was less than 1.2, but the metal (PC 5 4.9) and plastic (PC 5
5.1) positive controls were higher. The recovered virus titer on all
treated surfaces was less than 1.2. There was no positive HA on any
of the treated surfaces tested.
The efficacy of the powdered laundry detergent with peroxygen
was concentration dependent. At low concentrations (2 g/L or 4 g/
L), laundry detergent was more inconsistent than the other
compounds tested. At concentrations at or higher than 6 g/L,
powdered laundry detergent with peroxygen was effective at
inactivating LPAIV on hard and nonporous surfaces. Screening
tests with laundry detergent indicated that concentrations greater
than 6 g/L did not increase the efficacy of the detergent. At lower
concentrations (2 g/L or 4 g/L), satisfactory positive controls were
achieved; however, virus was recovered. At 6 g/L, however, the metal
(PC 5 6.2) and plastic (PC 5 4.1) positive controls were higher,
and no virus was recovered on any treated surfaces for a titer of less
than 1.2. At 6 g/L, there was no positive HA on any of the treated
surfaces tested. For all dilutions, the recovered virus titer on wood
was less than 1.2.
Cytotoxic controls were collected for each chemical. There was
100% survivability of the cytotoxic controls except for laundry
detergent with peroxygen (6 g/L) and 1% citric acid. However,
when repeated in 10 additional eggs, there was 100% survivability
and no visible lesions, indicating any egg death was not due to any
cytotoxic affect due to the presence of the chemical agent.
NI values were used as one of the primary means of
determining whether a disinfection agent was effective. The NI
value, however, is heavily dependent on the positive control titer
for a given test. A low positive control titer does not directly indicate
that a disinfection agent was ineffective. Early testing resulted in
low positive control titers due to a loss of virus during the drying
process. This loss was verified by comparing the positive control titer
to a wet positive control titer. As a result, the initial virus titer of
106.8EID50/ml was increased to 107.8EID50/ml. The results
presented in this article are from the increased initial virus working
titer of 107.8EID50/ml applying 0.1 ml/coupon for a titer of
In the United States, all pesticides including disinfectants are
required to be either registered or exempted by the EPA before their
sale, distribution, or use. This project was performed to provide
virucidal data for EPA exemption of certain detergents and
disinfectants. Because these products will be used for public health,
the EPA requires efficacy data that support the general level of claim
made on the product label (e.g., sanitizers, disinfectants, and
sterilants). For virucidal claims, the EPA may accept data developed
by any virologic technique that is recognized as technically sound,
and simulates to the extent possible, in the laboratory, the conditions
under which the product is intended for use (EPA DIS/TSS-7).
Carrier-based efficacy test methods must be used in the development
of virucidal efficacy data for virucides recommended for use on dry,
inanimate environmental surfaces (e.g., floors and tables). For
virucidal efficacy data to be acceptable, the product must
demonstrate complete inactivation of the virus at all dilutions while
at least 4 logs of virus particles per milliliter must be recovered from
the nonvirucidal-treated control carrier. Claims of virucidal activity
for a product may only be made for those viruses that have actually
been tested. The procedures outlined in this article were reviewed by
In general, lower NIs were recorded on porous surfaces
(basswood). Poultry houses include significant amounts of exposed
wood surfaces, which raises concern because there are no current
EPA-approved testing procedures for use on porous surfaces.
Recovery of viruses from nonporous carriers is a common problem
in virus testing (5,21). Lower NI values for wood may be due to
recovery from the media rather than poor inactivation on the
surface. For most agents tested, the NI was higher on hard plastic
and metal surfaces than on wood surfaces. For this reason, lower NIs
on wood in connection with documented efficacy on other surfaces
may be acceptable for porous surfaces.
All disinfecting agents were effective at the maximum concentra-
tion tested, although not all of the tests on porous surfaces were
conclusive. Without further testing, the iodine/acid disinfectant
(1:300 dilutions) and calcium hypochlorite (750 ppm) are the only
two disinfecting agents with an NI value greater than or equal to 2.8
on porous wood surfaces. However, both undiluted iodine/acid
disinfectant and calcium hypochlorite are toxic to fish, and they
should not be used near lakes, ponds, streams, or sewer systems.
Both disinfecting agents are also highly corrosive, and they cause eye
damage and severe skin irritation. In case of an outbreak, iodine/acid
disinfectant and calcium hypochlorite would be effective at
eliminating AIV from contaminated poultry facilities, but care
Inactivation of AIV using common chemicals
should be taken when using both agents to ensure they are diluted
properly, greatly reducing the environmental risks.
Many successful test agents have corrosion concerns. Acetic acid
can be corrosive to austenitic stainless steel, brass, and mild steel.
Sodium hypochlorite and calcium hypochlorite are both commonly
available, but they have potential corrosion concerns. Sodium
hypochlorite, in particular, is highly corrosive on aluminum.
Due to the potential toxicity of iodine/acid disinfectant and
calcium hypochlorite, additional testing on porous surfaces of some
of the other disinfecting agents that are less of an environmental
hazard should be retested on porous surfaces. Citric acid, acetic acid,
and powdered laundry detergent with peroxygen are less hazardous
when undiluted, and they would be ideal agents.
Acetic acid and citric acid are both relatively mild acids with few
environmental concerns. Similarly, the commercial powdered
laundry detergent is used for clothes, and it is gentle on skin. These
three disinfecting agents are ideal, and they can safely be used on
poultry farms. However, citric acid was the only mild disinfecting
agent that was effective on porous and nonporous surfaces.
Other criteria to consider including in further experimentation
include temperature and degree of agitation. Detergents generally
work best with hot water and significant scrubbing (1); so,
commercial laundry detergents may be more effective at inactivating
AIV in a washing machine with hot water and significant agitation
than it was in the plate test. Powdered laundry detergent is likely
more effective at disinfecting when used in a washing machine to
treat clothes or other fabrics that have been contaminated with AIV
than it is at inactivating virus in a poultry house. Other disinfecting
agents are most likely more successful when applied at higher
temperatures as well, because AIV is sensitive to heat (8). Raising the
temperature of the environment, both in the laboratory and in the
poultry house, will most likely increase the virus inactivation.
Because of the poor results on the porous surfaces, further testing
is needed to re-evaluate acetic acid, calcium hypochlorite, sodium
hypochlorite, and laundry detergent for their efficacy at inactivating
AIV on porous surfaces. It is unclear why some wood positive
controls worked, but others did not. The discrepancy raises
questions on the validity of the procedure, and it may be necessary
to adjust the protocol used for nonporous surfaces so that it is also
effective on porous surfaces. Previous tests were performed using
concrete in addition to wood, but results (not shown) were also
inconclusive, even when the porous surface was crushed in an
attempt to increase virus recovery. Chen (5) notes that this is a
common problem in virus carrier tests, and Tiwari et al. (21) found
similar results with avian metapneumovirus (AMPV) and AIV when
testing survivability on multiple porous and nonporous surfaces.
Both viruses consistently survived longer on nonporous surfaces, and
AIV was not recoverable on several porous surfaces (including an egg
tray and polyester fabric) after only 24 hr, although it survived on
surfaces such as latex and tile for 72 hours. Interestingly, Tiwari et al.
(21) reported no detectible AIV on an eggshell at 24 hr, but low
virus titers were detectible at 48 and 72 hr, and similar results were
reported for AMPV on the plastic surface. This demonstrates there
can be inconsistencies with virus carrier tests, especially on porous
surfaces. New methods need to be developed for testing inactivation
and survivability on porous surfaces.
Accurate measures for detecting live virus on porous surfaces are
especially important during an outbreak. In 2001–2002 when
H7N2 avian influenza outbreaks occurred in Pennsylvania, swabs
were used to sample the floor, dust, nests, and manure in the houses
where the outbreaks occurred. The 2001/02 H7N2 outbreak in
Pennsylvania was successfully controlled in 1 mo. AIV-positive
flocks were depopulated 14 days after clinical signs developed, and
the carcasses were removed from the house after an additional 3 to 4
days. The affected chicken houses were tested for the presence of
AIV 23 days after depopulation. If test results were negative, the
house was cleaned and disinfected, and additional testing was
completed before repopulation (9). However, as seen in the carrier
tests, it is difficult to determine the amount of live virus on a porous
surface. Environmental sampling could therefore result in false
negatives, leading to prematurely repopulating a poultry house
following an outbreak. When a new flock of birds is placed in a
contaminated house, the outbreak could re-emerge.
The scientific community currently has no method of detecting
the full impact of porous surfaces on the survivability of viruses, and
a method needs to be developed to test the true survivability of virus
on any porous surface. Virus recovery could potentially be increased
by changing the procedure slightly. One change to try is soaking the
material contaminated with dried virus for longer than 10 min in
the PBS solution to see whether increased saturation would lead to
increased viral recovery. Another option would be to test materials
with larger pores, such as woven burlap fabric, to see whether the
virus recovery increased. Because several porous positive control
groups had a titer .104.0, the current carrier method has potential
to be improved upon rather than creating an entirely new method.
However, virus recovery was inconsistent on the porous surface; so,
the current carrier method needs to be evaluated and improved
achieve consistent and conclusive results.
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This project was funded by USDA–APHIS.
Inactivation of AIV using common chemicals