Characterization
of Viral Load, Viability and Persistence of Influenza A Virus in Air and on
Surfaces of Swine Production Facilities
Victor
Neira, Peter Rabinowitz, Aaron Rendahl, Blanca Paccha, Shawn G. Gibbs,
Montserrat Torremorell
Indirect
transmission of influenza A virus (IAV) in swine is poorly understood and
information is lacking on levels of environmental exposure encountered by swine
and people during outbreaks of IAV in swine barns. We characterized viral load,
viability and persistence of IAV in air and on surfaces during outbreaks in
swine barns. IAV was detected in pigs, air and surfaces from five confirmed
outbreaks with 48% (47/98) of oral fluid, 38% (32/84) of pen railing and 43%
(35/82) of indoor air samples testing positive by IAV RT-PCR. IAV was isolated
from air and oral fluids yielding a mixture of subtypes (H1N1, H1N2 and H3N2).
Detection of IAV RNA from air was sustained during the outbreaks with maximum
levels estimated between 7 and 11 days from reported onset. Our results
indicate that during outbreaks of IAV in swine, aerosols and surfaces in barns
contain significant levels of IAV potentially representing an exposure hazard
to both swine and people.
Introduction
Influenza A
virus (IAV) causes significant epidemics of respiratory disease in humans that
result in human deaths and raise public health concerns that require a deeper
understanding of IAV epidemiology and control. IAV is shared among animals and
people and novel viruses capable of causing pandemics are the result of
reassortant viruses from different species. Despite evidence that reassortment
can happen in various species, swine is often labeled as the “mixing vessel”
since swine have receptors capable to replicate influenza viruses of avian,
human and swine origin. Because these viruses can infect humans, understanding
transmission of swine-origin IAVs should be a priority.
In addition
to IAV being a major pathogen for humans, IAV is also a serious problem in
swine causing frequent outbreaks that involve both animal illness and zoonotic
infections [1–3]. In swine, IAV is distributed worldwide and is endemic in the
US swine herd [2]. For almost a century, classical H1N1 viruses were the
dominant IAVs until the appearance and subsequent circulation of double and
triple reassortants since 1998 [4–6]. More recently, the 2009 pandemic virus
[7], and the on-going influx of human-origin IAVs in swine [8, 9] has led to a
more complex epidemiologic picture, making control of influenza in swine very
difficult. The 2009 H1N1 pandemic, as well as outbreaks of variant H3N2 (H3N2v)
influenza have demonstrated the potential for swine origin IAVs to cause
significant morbidity and mortality globally, impacting the general public,
swine workers and animal agriculture [10, 11]. Swine workers in particular, and
their non-swine-exposed spouses, have been shown to be at a higher risk of
swine-origin IAV infections than the general public [12], leading to calls for
including such workers in pandemic preparedness and surveillance [13]. Since
both direct and indirect contact exposures in commercial swine and agricultural
fairs have been suspected in IAV zoonotic infections [10, 13], influenza
prevention efforts involving swine production need to address multiple
potential exposure routes.
While it is
known that transmission of IAV occurs by direct contact, IAV can also be
transmitted through indirect routes. Transmission of IAV via contaminated
personnel and fomites has been documented in pigs [14] and aerosol transmission
of IAV has been reported in various species [15–21]. In swine, IAV has been
detected in aerosols from immune swine [22–24] and more recently IAV was
isolated from air samples from inside and outside swine farms [23], and live
animal markets in Minnesota [3].
Despite the
growing evidence of indirect transmission of swine-origin IAV, there is limited
information on the natural dynamics of IAV outbreaks in swine environments
including production facilities. Information is lacking on levels of exposure
encountered by both swine and people exposed to swine aerosols or contaminated
surfaces in swine facilities during outbreaks of IAV. Therefore, our objective
was to characterize viral load, viability and persistence of IAV in the air and
on surfaces during periods of active IAV outbreaks in swine production
facilities. This knowledge would further our understanding of the risk of IAV
transmission between swine and people, and help inform prevention efforts.
Material
and Methods
Procedures
and protocols used in this study were approved by the University of Minnesota
Institutional Animal Care and Use Committee protocol # 1207B17281 and the
Institutional Biosafety Committee protocol # 1208H18341. Prior to the start of
the study signed consent forms were obtained from the participating herds and
forms were signed by herd owners or the production managers. No protected
species were sampled.
Farm
identification and selection
Eleven
investigations of IAV outbreaks in six swine farms were conducted from October
2012 to May 2013. Farms with suspected outbreaks were identified by contacting
veterinarians in Southern Minnesota and Northern Iowa. Veterinarians were asked
to alert the investigators upon sudden onset of respiratory clinical signs
suggestive of acute influenza in a swine herd (i.e. rapid onset of widespread
dry hacking cough, sneezing, rhinorrhea, anorexia and lethargy). Each
investigation consisted of visiting a candidate farm multiple times to assess
herd health, collect samples, and gather additional information including
temperature and relative humidity data. Farms were included in the study if the
veterinarian made a presumptive diagnosis of IAV infection in the herd or was
able to collect samples and confirm the diagnosis within 4 days from the onset
of clinical signs, and was able to communicate with the investigators within 3
days from the onset of disease.
The
investigators visited the farm within 1 to 3 days of being contacted and the
clinical history of the outbreak was reviewed after interviews with farm
personnel. During each visit air samples from inside and outside, pig oral
fluids and surface samples were collected. The number of visits in each
investigation varied based on diagnostic results on samples from the prior
visit. If a herd tested negative in oral fluids, the investigation for that
herd was concluded in terms of additional visits for most of the cases. The
number of visits per farm ranged between 1 and 10 and the longest outbreak was
42 days. A summary of the farm characteristics is shown in Table 1.
Sampling
procedures and sampling scheme
Oral
fluids.
Oral fluids
were collected to determine whether IAV was present in the swine at the time of
sampling. Swine oral fluids were sampled using ropes as described previously
[25, 26]. Briefly, two 3-strand twisted cotton ropes (WebRiggingSupply.com,
Barrington, IL 60010, USA) were placed in 2–4 pens for 20 min for the swine to
chew on the ropes. Each rope was estimated to sample approximately between 20 to
50 swine depending on pen size. Oral fluids were extracted from the rope
immediately after collection by wringing the wet portion into a plastic bag and
then the fluid was transferred into a 5 ml plastic sterile tube, and samples
refrigerated at 4°C until processing. Oral fluid samples were processed within
24 hours of collection, centrifuged for 10 min at 5,000 RPM, and stored at
-80°C until tested by RRT-PCR (real time reverse transcriptase polymerase chain
reaction) and virus isolation.
Air
sampling.
Upon
arrival at the farm, the first set of air samples was collected outside the
barn approximately 25 m upwind (n = 2). After that, the second set was
collected downwind (n = 2) from the facility at approximately the same
distance, and lastly the final set was collected in the barn interior (n = 2).
For the air interior samples, air collectors were placed within the barn at
approximately 1/3 and 2/3 the length of the building and 1.5 m above the floor.
Each set of samples was collected simultaneously as duplicates. Swine did not
have direct contact with the air collectors.
Air samples
were collected using a liquid cyclonic collector (Midwest Micro-Tek, Brookings,
SD, USA) capable of processing 200 L / min of air [23, 27]. Briefly, 10 mL of
minimum essential medium (MEM) solution supplemented with 2% bovine serum
albumin (BSA) were added to the collection vessel, and samples collected for 30
minutes. About 4 mL of collection media were recovered for each sample, media
were transferred into a plastic vial with a syringe and stored on ice until
transport within 12 hours to the laboratory.
Surface
sampling.
Surface
samples were collected from areas considered to have high contact by humans
working in the barns including pen railings (n = 2) and door handles from doors
leading into the swine barns (n = 1). Surface samples were collected using a
2”x2” sterile gauze dipped into sterile MEM supplemented with 2% BSA. Sections
of 1 m of pen railing with approximately 0.08 m2 (800 cm2) of surface, were
wiped for 30 seconds using sterile gloves. Door handles were wiped for 15
seconds and both the exterior and interior handles were sampled. Pigs did not
have direct contact with the pen railing as only the top railing was sampled.
Gauzes were placed into individual tubes and samples stored on ice for
transport and processing within 24 hours.
Oral fluids
and surface samples were collected simultaneously at the same time that the air
interior was being sampled.
Diagnostic
procedures
Oral fluid
samples were first screened at the University of Minnesota Veterinary
Diagnostic Laboratory for influenza A RNA by a RRT-PCR targeting the matrix
gene [28]. Samples with a cycle threshold (ct) value <35 35="" and="" considered="" positive="" suspect="" were="">40 negative. Samples with ct < 40 were
further tested using a quantitative RRT-PCR test as described previously [23].
RRT-PCR positive samples were cultured for virus isolation using Madin-Darby
Canine Kidney (MDCK) cells [29, 30] and subtyped using the Path-ID Multiplex
One-Step RRT-PCR kit (Applied Biosystems, Foster City, CA, USA) and custom
subtyping assay primers and probes (Life Technologies) [31].
Swine
clinical scores
Swine were
visually inspected during each visit by a veterinarian member of the study
team. Clinical scores consisted of coughing and sneezing and were measured
following previously described procedures [32]. Briefly, the number of cough
and sneeze episodes observed in 4 pens during 3 minutes were recorded. A cough
or sneeze episode was defined as one or several coughs or sneezes in a sequence
by an individual pig. The percentage of coughing or sneezing swine was
calculated by dividing the number of swine observed coughing or sneezing by the
total number of animals observed in the pens. The total number of swine evaluated
in each visit ranged from 100 to 400 depending on pen and barn size.
Environmental
conditions
Temperature
and relative humidity inside and outside the barns were recorded at the time of
collection using a weather meter (Kerstrel 3000, Nielsen-Kellerman, PA, USA).
Statistical
and influenza modeling in indoor air and statistical analysis
Statistical
analyses were conducted using R programming language [33].
To look for
associations between the count of positive samples of each type compared with
each other type, we performed pair-wise Kendall’s rank correlation tests,
corrected for multiple comparisons with the Bonferroni-Holm adjustment.
Correlations between quantity of IAV RNA copies between samples of oral fluids,
surface and air inside the barns were also computed using Kendall’s
correlation. Correlations of these samples with clinical scores were computed
as well. Correlations between quantity of IAV in indoor air with recorded
measurements of relative humidity and temperature were also determined.
To compute
correlations and modeling of IAV in indoor air, data from four investigations
with at least 5 days of samples were used (investigations 5, 9, 10 and 11).
Data were limited to the first 21 days after the reported onset given that most
of the farms tested negative after that, and the mean indoor air IAV quantity
was calculated for each visit. The concentration of IAV in the air inside the
barn as a function of time over the outbreak was modeled using a quasipoisson
model with log link to appropriately handle both the days at which zeros were
recorded and the fact that the variance increased with the quantity of virus
detected. Additionally, as the reported day of onset may have been early or
late relative to the true progress of the infection, the reported day of onset
was allowed to shift relative to the estimated maximum for each investigation
to minimize the deviance of the fit.
A quadratic
effect was used for day and an additive blocking effect was used to allow each
investigation to have a different maximum value; the fitted equation was:
Indoor air
quantity = M*exp(-0.035*day—0.082*day^2), where M is the maximum value for that
investigation, and day is relative to the day of the maximum.
Results
Clinical
signs
Clinical
scores of coughing and sneezing were recorded in both IAV positive and negative
investigations. Mean scores ranged from 0.83% to 36.71% and 0.33% to 10.27% for
sneezing and coughing, respectively (Table 2) and there was variation in the
scores throughout the course of the investigations (results not shown).
Eleven
suspected IAV outbreak investigations in barns corresponding to 6 farms were
identified during the study. There were a total of 49 farm visits, which took
place between 2 to 8 days apart for 4 to 42 days after the initial visit. Six
of the 11 barn investigations, corresponding to three different farms, were
confirmed positive for IAV by RRT-PCR testing in aerosols, surfaces, and/or
swine oral fluid samples.
Forty-seven
out of 98 (48%) oral fluid samples tested were RRT-PCR positive for IAV while
32 of 84 (38%) pen railing samples, and 35 of 82 (43%) indoor air samples
tested positive for IAV (Table 3). There were two door handle samples that
tested positive at low levels. All air samples collected outdoors tested negative.
There was a significant positive correlation of 0.69 between the count of oral
fluid positive samples and air (p = 0.0001), of 0.47 between oral fluids and
pen railing (p = 0.009) and 0.42 between indoor air and pen railing (p = 0.01).
IAV was
isolated by culture from 19 oral fluid and 18 indoor air samples (Table 4)
representing five and four investigations, respectively. H1N1, H1N2 and H3N2
subtypes, and mixtures of these, were identified in both, oral fluid and indoor
air samples. Virus isolation of surface samples did not yield positive results.
Viral
quantification
Influenza
RNA levels in oral fluids, indoor air and pen railing varied between farms and
throughout the course of the clinical outbreaks (Table 5). Individual sample
viral levels ranged from 0 to 4.03x107 RNA copies/ml in oral fluids, 0 to
4.16x107 RNA copies/m2) in pen railing surface and 0 to 1.25x106 RNA copies/m3
of air in indoor air samples. There was a significant positive correlation of
0.4 between quantity of IAV in the air and oral fluids (p = 0.015) and of 0.372
between quantity of IAV in oral fluids and coughing (p = 0.023) (Table 6).
Correlations between quantity of IAV on the pen railing with both the air and
oral fluids were not significant (p>0.05).
Environmental
conditions
Measured
mean indoor temperatures ranged between 19°C and 25°C while relative humidity
ranged between 19°C and 25°C. Both of these ranges were within the expected
ranges for swine commercial facilities. Correlations between quantity of IAV in
indoor air samples with relative humidity and temperature were 0.26 (p = 0.12)
and 0.01 (p = 0.93) respectively.
Influenza
level modeling
Results of
modeling of indoor air levels of IAV are shown in Fig 1 based on results from 4
investigations. The best fit for the reported day of onset relative to the
estimated maximum was -11, -11, -7 and -9 days for investigations 10, 11, 5 and
9, respectively. These results indicated a short spread between farms in the
duration of IAV detection in the air. The model also showed the best fit for
the estimated mean maximum RNA copy viral load (x10^4 RNA copies/m3) with 95%
confidence interval for each investigation at 12 (4.5, 31), 38 (21, 68), 60
(38, 97), and 71 (49, 103) for investigations 11, 5, 10, and 9 respectively,
indicating differences in the modeled levels of airborne IAV between farms and
throughout the duration of an outbreak. Data from oral fluids and surfaces
could not be modeled because of a lack of a common pattern in the data
obtained.
Discussion
Despite the
common occurrence of IAV infections in swine, there is limited information on
the levels and persistence of IAV in the air and environment of swine
production facilities. This is the first study, to our knowledge, that has
quantified and characterized the level of IAV in samples of aerosols and
surfaces of swine environments during acute outbreaks of influenza infections
in swine. We found that IAV could be isolated from indoor air of commercial
swine production facilities, that airborne IAV levels were sustained for
periods of 20 days and that there was a correlation between the number of positive
samples of each type and the quantity of virus in the swine oral fluids and in
the air. Our results provide a first estimation on levels of environmental IAV
in swine commercial production facilities, and thus an assessment of potential
sources of IAV exposure to swine workers or other pigs.
Detection
of IAV in air was sustained throughout the course of the acute outbreaks and
lasted approximately 20 days across the studied barns. The peak of detection of
virus in air samples occurred between 7 and 11 days into the outbreaks. Maximum
airborne levels varied between affected facilities and were in the order
magnitude of 104 to 107 RNA copies/m3 of air. We allowed our model to shift
relative to the estimated maximum for each investigation given that the
reported day may not have been when the infection truly started. Interestingly
the IAV detection curves were similar across farms with limited variations in
duration which suggests a similar course of disease between farms and, that
similar measures could be implemented to minimize risk of IAV infections. There
were differences in the levels of IAV found in the air between farms and we
speculate that these differences could be due to varying levels of infection
and immunity in the swine, type of IAV, number of pigs in the barn, barn
volume, ventilation rates and farm and management characteristics. Thus,
information from our model can be used to estimate risk of exposure to swine
workers, other pigs and help target intervention strategies to mitigate the risk
of IAV transmission between pigs and from pigs to people.
A key
aspect of determining the risk of IAV transmission is the relationship between
number of RNA copies and infectivity. We estimated the ratio of viral particles
to TCID50 (tissue culture infectious dose) at 3,000 RNA copies/TCID (results
not shown) and based on the mean airborne IAV concentration, our results
corresponded to 47 TCID50/m3. Similar TCID50 estimates were obtained in a
health center [34] and although it is unclear how our results relate to
transmission to swine or people, we speculate that they represent a significant
risk to both people and swine, since IAV was readily isolated from the air
multiple times throughout the duration of the outbreaks. Overall, our results
provide evidence that air can be an important route of IAV transmission in
swine production facilities. Furthermore there was an association between the
levels of IAV in oral fluids and the air indicating a direct relationship
between level of virus in the swine and potential exposure through aerosols.
However, further studies are needed to fully understand the relationship
between airborne IAV levels and transmission.
We isolated
a mixture of genetically diverse IAV from swine and air samples representing
the three most common IAV subtypes in swine, H1N1, H1N2 and H3N2. However, in
contrast to a prior study [23], we did not detect IAV in air samples collected
outside swine facilities. This difference is probably a result of sampling
frequency and our investigations being carried out under colder environmental
conditions and larger distances from the air exhaust site that likely
negatively impacted both distribution of the virus and viral survivability.
Therefore more research is needed to fully characterize the risk of IAV
transmission outside swine production facilities.
IAV genetic
material was also detected in surfaces, in particular on pen railings although
we could not isolate IAV from surfaces. Source of IAV genetic material in the
surfaces may be the result of deposition of airborne IAV particles. Whether
inability to culture IAV from surfaces was due to lack of viable IAV in
surfaces or conditions of sampling or limited sensitivity of the culture
technique could not be assessed, but we speculate that viable IAV can still be
present on surfaces from swine barns although with less quantity than in the
air due to environmental conditions such as desiccation or preservation in
dust. In deed surface contamination with viable IAV was shown in a live animal
market housing swine [3]. Therefore, precautions to prevent exposure to
contaminated surfaces should still be followed.
Clinical
signs of coughing and sneezing can be an indicator of IAV infections and IAV
can be found in both clinically and subclinically infected swine [2]. We found
an association between coughing and levels of IAV in the swine but not in air
or on surfaces. We did not have a common pattern on the presentation and
evolution of clinical signs across farms. This could be due to presence of
concomitant infections or farm factors not measured in this study. Overall our
results indicate that clinical signs in swine cannot be used as a reliable
indicator of the levels of IAV present in the environment and thus they should
not be used to predict risk of exposure to people.
The farm
investigations in this study were selected by convenience based on recognized
acute clinical signs in the swine herds, thus results from this study should be
interpreted carefully when extrapolating them to endemically infected farms.
Furthermore data in this study was obtained from a limited number of farms
which do not represent the full spectrum of types of production facilities and
management conditions encountered in the swine industry. Thus further research
is needed to characterize the levels and risk of IAV environmental exposure in
non-outbreak situations and production facilities representative of different
management and environmental conditions.
Lastly,
although environmental conditions of relative humidity and temperature have
been associated with IAV viability [35], in this study we did not see an
association between quantity of IAV in air or on surfaces and relative humidity
or temperature. This lack of association could be the result of the relatively
stable indoor conditions throughout our study. Producers put a great deal of
effort into maintaining a relatively stable temperature and relative humidity
within these facilities to maintain the health and safety of the animals.
In summary,
our results indicate that during outbreaks of IAV in swine, the air and
surfaces in barns contain significant levels of IAV potentially representing an
exposure hazard to both swine and people. Further studies are needed to
evaluate the viability of IAV in the environment, evaluate strategies to
mitigate the risk of indirect transmission of IAV, confirm the impact of
personal protective equipment on exposure risk to people and explore strategies
to prevent bidirectional transmission of IAV between humans and swine.
Information from this study should help to develop evidence-based guidelines to
minimize the impact of IAV infections on swine production.
Acknowledgments
Funding was
provided by the National Pork Board. The authors would like to acknowledge the
contributions of My Yang, Andres Diaz and Macarena Cortez for technical
assistance.
Author
Contributions
Conceived
and designed the experiments: MT SGG PR. Performed the experiments: VN MT.
Analyzed the data: AR VN MT. Contributed reagents/materials/analysis tools: SGG
PR BP AR. Wrote the paper: MT VN. Reviewed critically the manuscript: SGG PR BP
AR.
References
1. Myers
KP, Olsen CW, Gray GC. Cases of swine influenza in humans: A review of the
literature. Clinical Infectious Diseases. 2007;44(8):1084–8. doi:
10.1086/512813 pmid:WOS:000244928200012.
View
Article PubMed/NCBI Google Scholar
2. Corzo
CA, Culhane M, Juleen K, Stigger-Rosser E, Ducatez MF, Webby RJ, et al. Active
Surveillance for Influenza A Virus among Swine, Midwestern United States,
2009–2011. Emerging Infectious Diseases. 2013;19(6):954–60. doi:
10.3201/eid1906.121637 pmid:WOS:000328173500015.
View
Article PubMed/NCBI Google Scholar
3. Choi MJ,
Torremorell M, Bender JB, Smith K, Boxrud D, Ertl JR, et al. Live animal
markets in Minnesota: a potential source for emergence of novel influenza A
viruses and interspecies transmission. Clin Infect Dis. 2015. Epub 2015/08/01.
doi: 10.1093/cid/civ618 pmid:26223994.
View
Article PubMed/NCBI Google Scholar
4. Zhou NN,
Senne DA, Landgraf JS, Swenson SL, Erickson G, Rossow K, et al. Genetic
reassortment of avian, swine, and human influenza A viruses in American pigs.
Journal of Virology. 1999;73(10):8851–6. pmid:WOS:000082554300105.
View
Article PubMed/NCBI Google Scholar
5. Webby
RJ, Rossow K, Erickson G, Sims Y, Webster R. Multiple lineages of antigenically
and genetically diverse influenza A virus co-circulate in the United States
swine population. Virus Research. 2004;103(1–2):67–73. doi:
10.1016/j.virusres.2004.02.015 pmid:WOS:000222065200012.
View
Article PubMed/NCBI Google Scholar
6. Karasin
AI, Schutten MM, Cooper LA, Smith CB, Subbarao K, Anderson GA, et al. Genetic
characterization of H3N2 influenza viruses isolated from pigs in North America,
1977–1999: evidence for wholly human and reassortant virus genotypes. Virus
Research. 2000;68(1):71–85. doi: 10.1016/s0168-1702(00)00154-4
pmid:WOS:000088282200008.
View
Article PubMed/NCBI Google Scholar
7. Forgie
SE, Keenliside J, Wilkinson C, Webby R, Lu P, Sorensen O, et al. Swine Outbreak
of Pandemic Influenza A Virus on a Canadian Research Farm Supports
Human-to-Swine Transmission. Clinical Infectious Diseases. 2011;52(1):10–8.
doi: 10.1093/cid/ciq030 pmid:WOS:000286214200003.
View
Article PubMed/NCBI Google Scholar
8. Nelson
MI, Gramer MR, Vincent AL, Holmes EC. Global transmission of influenza viruses
from humans to swine. Journal of General Virology. 2012;93:2195–203. doi:
10.1099/vir.0.044974–0 pmid:WOS:000310043400012.
View
Article PubMed/NCBI Google Scholar
9. Nelson
MI, Wentworth DE, Culhane MR, Vincent AL, Viboud C, LaPointe MP, et al.
Introductions and Evolution of Human-Origin Seasonal Influenza A Viruses in
Multinational Swine Populations. Journal of Virology. 2014;88(17):10110–9. doi:
10.1128/jvi.01080-14 pmid:WOS:000341232300051.
View
Article PubMed/NCBI Google Scholar
10. Jhung
MA, Epperson S, Biggerstaff M, Allen D, Balish A, Barnes N, et al. Outbreak of
Variant Influenza A(H3N2) Virus in the United States. Clinical Infectious
Diseases. 2013;57(12):1703–12. doi: 10.1093/cid/cit649
pmid:WOS:000327720400008.
View Article
PubMed/NCBI Google Scholar
11. Smith
GJD, Vijaykrishna D, Bahl J, Lycett SJ, Worobey M, Pybus OG, et al. Origins and
evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic.
Nature. 2009;459(7250):1122–U107. doi: 10.1038/nature08182 pmid:WOS:000267636700042.
View
Article PubMed/NCBI Google Scholar
12. Gray
GC, McCarthy T, Capuano AW, Setterquist SF, Olsen CW, Alavanja MC. Swine
workers and swine influenza virus infections. Emerg Infect Dis.
2007;13(12):1871–8. Epub 2008/02/09. doi: 10.3201/eid1312.061323 pmid:18258038;
PubMed Central PMCID: PMCPMC2876739.
View
Article PubMed/NCBI Google Scholar
13. Myers
KP, Olsen CW, Setterquist SF, Capuano AW, Donham KJ, Thacker EL, et al. Are
swine workers in the United States at increased risk of infection with zoonotic
influenza virus? Clinical Infectious Diseases. 2006;42(1):14–20. doi:
10.1086/498977 pmid:WOS:000233698900009.
View
Article PubMed/NCBI Google Scholar
14.
Allerson MW, Cardona CJ, Torremorell M. Indirect Transmission of Influenza A
Virus between Pig Populations under Two Different Biosecurity Settings. Plos
One. 2013;8(6):9. doi: 10.1371/journal.pone.0067293 pmid:WOS:000320846500134.
View
Article PubMed/NCBI Google Scholar
15.
Brankston G, Gitterman L, Hirji Z, Lemieux C, Gardam M. Transmission of
influenza A in human beings. Lancet Infectious Diseases. 2007;7(4):257–65. doi:
10.1016/s1473-3099(07)70029-4 pmid:WOS:000245120700027.
View
Article PubMed/NCBI Google Scholar
16.
Blachere FM, Lindsley WG, Pearce TA, Anderson SE, Fisher M, Khakoo R, et al.
Measurement of Airborne Influenza Virus in a Hospital Emergency Department.
Clinical Infectious Diseases. 2009;48(4):438–40. doi: 10.1086/596478
pmid:WOS:000262585700011.
View
Article PubMed/NCBI Google Scholar
17. Tellier
R. Aerosol transmission of influenza A virus: a review of new studies. Journal
of the Royal Society Interface. 2009;6:S783–S90. doi:
10.1098/rsif.2009.0302.focus pmid:WOS:000271957900009.
View
Article PubMed/NCBI Google Scholar
18.
Mubareka S, Lowen AC, Steel J, Coates AL, Garcia-Sastre A, Palese P.
Transmission of Influenza Virus via Aerosols and Fomites in the Guinea Pig
Model. Journal of Infectious Diseases. 2009;199(6):858–65. doi: 10.1086/597073
pmid:WOS:000263677900013.
View
Article PubMed/NCBI Google Scholar
19. Munster
VJ, de Wit E, van den Brand JMA, Herfst S, Schrauwen EJA, Bestebroer TM, et al.
Pathogenesis and Transmission of Swine-Origin 2009 A(H1N1) Influenza Virus in
Ferrets. Science. 2009;325(5939):481–3. doi: 10.1126/science.1177127
pmid:WOS:000268255100058.
View
Article PubMed/NCBI Google Scholar
20. Yee KS, Cardona CJ, Carpenter TE. Transmission of low-pathogenicity avian
influenza virus of subtype H6N2 from chickens to Pekin ducks and Japanese quail
(Coturnix coturnix japonica). Avian Pathology. 2009;38(1):59–64. doi:
10.1080/03079450802632023 pmid:WOS:000262514600010.
View
Article PubMed/NCBI Google Scholar
21.
Schulman JL. Experimental transmission of influenza virus infection in mice.
IV. Relationship of transmissibility of different strains of virus and recovery
of airborne virus in the environment of infector mice. J Exp Med.
1967;125(3):479–88. Epub 1967/03/01. pmid:6016901; PubMed Central PMCID:
PMCPMC2138293.
View
Article PubMed/NCBI Google Scholar
22. Loeffen
WLA, Stockhofe N, Weesendorp E, van Zoelen-Bos D, Heutink R, Quak S, et al.
Efficacy of a pandemic (H1N1) 2009 virus vaccine in pigs against the pandemic
influenza virus is superior to commercially available swine influenza vaccines.
Veterinary Microbiology. 2011;152(3–4):304–14. doi: 10.1016/j.vetmic.2011.05.027
pmid:WOS:000294937500012.
View
Article PubMed/NCBI Google Scholar
23. Corzo
CA, Culhane M, Dee S, Morrison RB, Torremorell M. Airborne Detection and
Quantification of Swine Influenza A Virus in Air Samples Collected Inside,
Outside and Downwind from Swine Barns. Plos One. 2013;8(8). doi:
10.1371/journal.pone.0071444 pmid:WOS:000323124000058.
View
Article PubMed/NCBI Google Scholar
24. Corzo
CA, Allerson M, Gramer M, Morrison RB, Torremorell M. Detection of Airborne
Influenza A Virus in Experimentally Infected Pigs With Maternally Derived
Antibodies. Transboundary and Emerging Diseases. 2014;61(1):28–36. doi:
10.1111/j.1865-1682.2012.01367.x pmid:WOS:000329310800005.
View
Article PubMed/NCBI Google Scholar
25.
Prickett J, Simer R, Christopher-Hennings J, Yoon KJ, Evans RB, Zimmerman JJ.
Detection of Porcine reproductive and respiratory syndrome virus infection in
porcine oral fluid samples: a longitudinal study under experimental conditions.
Journal of Veterinary Diagnostic Investigation. 2008;20(2):156–63.
pmid:WOS:000262533700003.
View
Article PubMed/NCBI Google Scholar
26.
Romagosa A, Gramer M, Joo HS, Torremorell M. Sensitivity of oral fluids for
detecting influenza A virus in populations of vaccinated and non-vaccinated
pigs. Influenza and Other Respiratory Viruses. 2012;6(2):110–8. doi:
10.1111/j.1750-2659.2011.00276.x pmid:WOS:000300689300006.
View
Article PubMed/NCBI Google Scholar
27. Dee S,
Otake S, Oliveira S, Deen J. Evidence of long distance airborne transport of
porcine reproductive and respiratory syndrome virus and Mycoplasma
hyopneumoniae. Veterinary Research. 2009;40(4):13. doi: 10.1051/vetres/2009022
pmid:WOS:000267963400009.
View
Article PubMed/NCBI Google Scholar
28. Slomka
MJ, Densham ALE, Coward VJ, Essen S, Brookes SM, Irvine RM, et al. Real time
reverse transcription (RRT)-polymerase chain reaction (PCR) methods for
detection of pandemic (H1N1) 2009 influenza virus and European swine influenza
A virus infections in pigs. Influenza and Other Respiratory Viruses. 2010;4(5):277–93.
doi: 10.1111/j.1750-2659.2010.00149.x pmid:WOS:000280976100005.
View
Article PubMed/NCBI Google Scholar
29. Detmer
SE, Patnayak DP, Jiang Y, Gramer MR, Goyal SM. Detection of Influenza A virus
in porcine oral fluid samples. Journal of Veterinary Diagnostic Investigation.
2011;23(2):241–7. pmid:WOS:000288838300007.
View
Article PubMed/NCBI Google Scholar
30. Detmer
S, Gramer M, Goyal S, Torremorell M, Torrison J. Diagnostics and Surveillance
for Swine Influenza. In: Richt JA, Webby RJ, editors. Swine Influenza. Current
Topics in Microbiology and Immunology. 370. Berlin: Springer-Verlag Berlin;
2013. p. 85–112.
31. Zhang
JQ, Harmon KM. RNA Extraction from Swine Samples and Detection of Influenza A
Virus in Swine by Real-Time RT-PCR. In: Spackman E, editor. Animal Influenza
Virus, Second Edition. Methods in Molecular Biology. 1161. Totowa:
Humana Press Inc; 2014. p. 277–93.
32. , , . Monitoramentos sanitarios. Goiania: Canone
Editorial; 2007.
33. The R
Project for Statistical Computing. Vienna, Austria: Foundation for Statistical
Computing; 2008.
34. Yang W,
Elankumaran S, Marr LC. Concentrations and size distributions of airborne
influenza A viruses measured indoors at a health centre, a day-care centre and
on aeroplanes. Journal of the Royal Society Interface. 2011;8(61):1176–84. doi:
10.1098/rsif.2010.0686 pmid:WOS:000292083300010.
View
Article PubMed/NCBI Google Scholar
35. Irwin
CK, Yoon KJ, Wang C, Hoff SJ, Zimmerman JJ, Denagamage T, et al. Using the
Systematic Review Methodology To Evaluate Factors That Influence the
Persistence of Influenza Virus in Environmental Matrices. Applied and
Environmental Microbiology. 2011;77(3):1049–60. doi: 10.1128/aem.02733-09
pmid:WOS:000286597100041.
