Summary To better understand the transmission route of H9N2 subtype avian influenza virus (AIV), two duplicate trials were conducted to observe the process of aerosol infection and direct contact in specific pathogen free chickens. Fifteen chickens (G1) were inoculated with H9N2 subtype AIV and housed together with another 15 chickens (G2) in the same positive-negative pressure isolator (A). Fifteen chickens (G3) were bred in another isolator (B) which was connected with A. Air, oropharyngeal and cloacal swabs, and blood samples were collected for the detections of aerosol forming, virus shedding, and seroconversion. AIV aerosols were initially detected at the day 2–3 post inoculation (dpi), reaching peak concentrations at 7 dpi. Virus shedding was detected in all chickens of G2, but only in 80–87% chickens of G3. Antibodies were initially detected at 4–5 dpi, peaking at 14–21 dpi. The results showed that H9N2 AIV could transmit by both aerosol exposure and direct contact.
Keywords: Transmission, H9N2 Subtype Avian Influenza Virus, Aerosol Infection, Specific Pathogen Free Chickens, Positive-negative Pressure Isolator
Introduction
Avian influenza (AI) is caused by viruses that are members of the family orthomyxoviridae and placed in the genus influenza virus A. Highly pathogenic AI may cause great loss to poultry husbandry and great harm to public health, and is listed internationally as a major zoonotic disease. Although H9N2 subtype belonged to mild pathogenic AI, together with other pathogens, it may cause increased morbidity and mortality （Nili and Asasi, 2002; Brown et al, 2006）. Human cases infected by H9 subtype have continuously occurred since 1999 (Guo et al, 1999; Guo et al, 2000; Lin et al, 2000; Butt et al, 2003). Although no evidence of efficient person-to-person transmission has yet been reported，being a member of influenza A virus, H9N2 AIV could create a reassortant by genetic recombination and then became a potential pandemic strain. This influenza subtype attracted more and more public attentions.
In recent years, researches about AI were focused on etiology, diagnostics and prevention (Li et al, 2003; Ong et al, 2007; Pereda et al, 2008; Xing et al, 2008). With respect to transmission mode of AI, Landman and Schrier (2004) clarified that the airborne transmission of AI was also important, and AIV might transmitted through aerosols among populations. Bridges et al (2003) emphasized that although droplet transmission was the main mode by which influenza virus infection was acquired, there was limited evidence of infection through droplets or aerosols. Webster et al (2002) confirmed that AIV (H5N1) could be transmitted to quail by either aerosol or faeces. However, there was little experiment concerning occurrence and transmission of AIV aerosol. In this study, two duplicate trials were performed to trace the process of direct contact and aerosol infection of H9N2 AIV in specific pathogen free (SPF) chickens in order to better understand the transmission mechamisms of this virus.
Materials and Methods
Experimental Design
Two positive-negative pressure isolators A and B (size: 2350 mm ×900 mm ×1750 mm) were connected with an enclosed tube (length: 1.5 m; diameter: 8 cm), where air flowed from A to B (Fig. 1). Two duplicate trials were performed: Trial 1 (T1) and Trial 2 (T2). In each trial 30 SPF chickens were bred in the isolator A, among which 15 chickens were designed as inoculation group (G1) and the other 15 served as direct contact group (G2). Fifteen SPF chickens were bred in the isolator B and exposed to AIV aerosol and served as aerosol contact group (G3). All chickens were supplied with enough sterilized feed and water through automatic feeders to sustain the chickens throughout the experiment. Routine operations were strictly according to the requirements for breeding SPF chickens.
Animals and Eggs
SPF white Leghone chickens and 10-day-old embryonating chicken eggs were purchased from Shandong Agriculture Science Academy. All chickens were seronegative to AIV antibody (AIV-Ab) and negative to AIV (H9N2) before the trials. Chickens were acclimated for one week and then marked with shank tags and randomly assigned to different groups. The chickens were approximately four weeks on day 0 and were housed in positive-negative pressure isolators. Embryonating eggs were used for detection of Egg Lethal Dose 50 percent (ELD50).
Inoculation
Each chicken in G1 was inoculated with H9N2 AIV (A/chicken/Shandong/1/08/ (H9N2)) diluted in sterile PBS in a dose of 107 ELD50. The chickens were inoculated ocularly and nasally at 0 day. Upon inoculation, the isolator A was fully flushed with clean air to remove the AIV aerosols formed from inoculation and then was connected with the isolator B.
Sampling
Air samples were collected with the AGI-30 Air Sampler (All Glass Impinger) (Brachmann et al, 1964) from the isolator A at 1, 2, 3, 4, 5, 7, 9, 11, 14, 17, 21, and 28 days post inoculation (dpi) for detection of airborne AIV. The sampler was operated for 20 min with a flow rate at 12.5 l/min. The sample solution was 20 ml sterile PBS (pH 7.2) containing penicillin and streptomycin in the final concentrations of 1000 U/ml and 1 mg/ml, respectively. Two samples were collected each time. The collected air samples were filtered with Millex Syringe Filters (Millipore, Billerica, USA) in a pore diameter of 0.45 m to remove bacteria and other impurities.
Oropharyngeal and cloacal swabs were collected from chickens by sterilized coton swabs at 1, 2, 3, 4, 5, 7, 9, 11, 14, 17, and 21 dpi for detection of virus excretion by haemagglutination inhibition test (HI-test) and reverse transcription-polymerase chain reaction (RT-PCR). After a sample was collected, the swab was dipped into 1–1.5 ml of the sample solution (sterile PBS containing antibiotics) in a centrifuge tube, and the swab tail was discarded. The centrifuge tube was adequately shaken to rinse the swab, and then the swab was repeatedly squeezed against the tube wall and took out. The solution was filtered with Millex Syringe Filter in a pore diameter of 0.45 m.
Blood samples were collected via the wing vein of chickens at 0, 4, 5, 7, 9, 11, 14, 17, 21, and 28 dpi for evaluation antibody response by HI-test as recommended by the OIE standard (2008). 0.5–1 ml each time and transferred to a 1.5–ml centrifuge tube. The separated serum was stored at –20℃ until detection.
Tests
Cell culture and HI-test. Madin-Darby Canine Kidney (MDCK, Chinese Academy of Sciences, Beijing, China) cells grown into monolayer were used for virus isolation. 1 ml of the sample solution was inoculated on to the cells and incubated at 35℃ in a humid incubator with 5 percent CO2 atmosphere, and observed for any cytopathic effects (CPE) in the next 5 days. The maintenance solutions (Dulbecco’s modified Eagle medium, DMEM) were harvested when CPE occurred, and detected for haemagglutination activity. Specimens with haemagglutination activity were subjected to HI-test with standard positive serum (China Animal Health and Epidemiology Centre, Qingdao, Shandong, China). All maintenance solutions were harvested and centrifuged at 1000 ×g, and the supernatants were collected for virus identification by RT-PCR.
RT-PCR. Viral RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, California, USA), according to the manufacturer’s protocol.
The primer sequences were referred to the detection criteria issued by the Ministry of Agriculture of the People’s Republic of China: RT-PCR test for avian influenza. The primer sequences were as follow: P1: 5′-TCAACAAACTCCACCGAAACTGT-3′; P2: 5′-TCCCGTAAGAACATGTCCATACCA-3′. RT-PCR was performed with a BioRT two step RT-PCR kit (BIOER Technology Co., Hangzhou, China) and analyzed with agarose gel electrophoresis.
Plaque assay. AIV concentrations were quantitatively determined with plaque assay (Yin and Liu, 1997) in air samples that were identified as AIV positive by HI-test and RT-PCR. 1 ml of the sample filtrate was subjected to continuous 10-fold serial dilutions with DMEM. MDCK cells (rinsed twice prior to inoculation) in a one-off 6-well cell culture plate were inoculated with 1 ml of the diluted filtrate, 3 wells for each dilution, and then adsorbed at 35℃ for 1 h. During this period, the cell plate was shaken gently every 15 min to adsorb adequately viruses, and then the viral solution was pipetted out. The cells were overlaid with 2 mm thick DMEM containing 0.7% agarose, and incubated at 35℃ in a humid CO2 incubator. When plaques began to appear, the cell monolayer was stained with neutral red and plaques were subsequently enumerated. The results were expressed as plaque forming units (PFU) per cubic meter of air (PFU/m3 air).
Statistical Analysis
To evaluate the difference between two independent methods for detection of AIV, the x2 test was used. Antibody titers (log 2) in each group were evaluated using SPSS 14.0 (Statistical Package for the Social Science, Chicago, IL, USA). The Student’s t-test (two-tailed) determined the level of significance for the differences between groups of sera and statistical significance was calculated at the 1% level of probability.
Results
Detection and Quantification of AIV Aerosols in the Isolator A
The detection rates of air samples for airborne AIV in isolator A by HI-test and RT-PCR were similar, and there was no significant difference of the results by statistical analysis (p>0.05). In the duplicate trials, viruses were detectable initially in air samples at 2 dpi (T2) or 3 dpi (T1), in the concentrations of 240 PFU/m3 air (T2) and 560 PFU/m3 air (T1), respectively. The concentrations peaked at 4880 PFU/m3 air (T1) and 7200 PFU/m3 air (T2) at 7 dpi, respectively. Later, the concentrations were decreased slowly, and airborne AIV was undetectable at 21 dpi (Tab. 1). The results showed that SPF chickens were able to shed viruses after inoculated with AIV and form viral aerosols.
Virus Shedding
The results of virus shedding was showed in Table 2. In the duplicate trials, AIV shedding was detected in the chickens of G1 at as early as 2 dpi, and in all chickens of this group at 3 dpi (T2) or 4 dpi (T1). The AIV shedding period lasted for approximately 7–10 days. Virus was initially detected in the chickens of G2 at 4 dpi, and successively in all chickens of this group. AIV was detected in the SPF chickens of G3 at as early as 7 dpi, and virus was detectable in 80% (T2) or 87% (T1) of those chickens. AIV was undetectable at 17 dpi.
AIV-Ab Titration
AIV-Ab was initially detected at 4 dpi (T2) or 5 dpi (T1) in chickens of G1 (Tab. 3). In the duplicate trials, the increases of antibody titers were different among the chickens of G1. The antibody titers reached to a peak level at approximately 21 dpi in Trial 1, and at approximately 14 dpi in Trial 2. Antibodies were detected in the chickens of G2 at as early as 9 dpi, and then the antibody titers were slowly increased and maintained at a low level. AIV-Abs were detected in the chickens of G3 at 14 dpi, and the antibody titers were maintained at a low level all the time.
Discussion
This study observed dynamically the occurrence and transmission of airborne H9N2 AIV from infected chickens. It was initially detected at 2 or 3 dpi in the isolator A (Tab. 1). Correspondingly, emitting of AIV by infected chickens started at 2 or 3 dpi (Tab. 2). It indicated that AIV emitted from the infected chickens might be readily aerosolized. AIV aerosols peaked at 7 dpi in the concentrations of 4880 PFU/m3 air (T1) or 7200 PFU/m3 air (T2) after which the concentrations decreased gradually (Tab. 1). The viral aerosol concentrations are dependent on the air flow, flock density, the number of infected chickens and the activity of chickens (Alexander, 1995, Jones and Harrison, 2004). In the present study, the stocking density in the isolator A was correlated with that in the field, and the chickens were treated gently and were not disturbed while sampling.
The air flowed through the isolators were filtered and clean, and the biosecurity was ensured. The air in the isolator B came completely from the isolator A so that the chickens of the aerosol contact group were infected with AIV only via the airborne transmission excluding any other routes. The airborne concentrations in the isolator B were not measured directly. The chickens in isolator B were demonstrated infection with AIV by virus detection and seroconversion, so it could be concluded that airborne transmission did occur.
All the chickens in G2 were able to emit viruses via throat and cloaca, while only part of the chickens in G3 (T1: 87%, T2: 80%) emitted viruses. It indicated that all the chickens in G2 were infected with AIV, while only a part of the chickens in G3 were infected with AIV. The authors suggested that such results were first associated with the amount of viruses the chickens received. The chickens in G2 might have become infected with a higher dose of viruses through direct contact as well as possible aerosols, while the AIV aerosol level in the isolator B did not reach the dose enough to infect all the chickens in G3. Lu et al (2003) reported that the infectious doses of AIV subtype H7N2 in SPF chickens ranged between 104.7 and 105.7 ELD50 per bird. The minimum infectious dose of AIV (H9N2) was still unknown and would be investigated in another study.
According to Table 3, antibody titers of infected chickens in G2 and G3 were not high, and their peak levels were of significant difference compared with those of the chickens in G1 (p<0.01). This was possibly associated with antigen amount absorbed by chickens of different groups, and other reasons remained yet to be further investigated.
In this study, the air samples were collected with the AGI-30 microorganism sampler (Brachmann et al, 1964). Being recommended as a standard sampler in an International Air Biology Symposium, the AGI-30 could collect air samples in great amount for a long time. In recent years, many researchers have successfully collected virus aerosols by AGI-30 (Lin et al, 1997, Lin et al, 1999, Hogan et al, 2005, Tseng and Li, 2005). The air samples were cultured by MDCK cell, and the concentrations of airborne H9N2 AIV were evaluated by plaque assay that ensured the infective viral particles detected.
Shi et al (2007) demonstrated that A/chicken/Shanghai/F/98(H9N2) could be transmitted through aerosols by putting a group of healthy chickens near another group of infected chickens in a same room and detecting virus from oropharyngeal and cloacal swabs. In the present study, the aerosol generation and airborne transmission of infectious AIV (H9N2) were measured directly. It could be concluded that in addition to direct contact infection, chickens in the same or adjacent henhouses might also infect AI by aerosols. That might be used to predict airborne spread of AIV during outbreaks.
Acknowledgements
This study was sponsored by the following foundation programs: National Natural Science Foundation of China (30871865); Chinese International Cooperation Program (2009DFA32890); National Doctoral Station Foundation (20060434007); and Special Fund of Shandong Postdoctoral Innovation Program (200703043).
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TABLE 1: Detection and quantification of AIV aerosol in the isolator A
dpia Trial 1 Trial 2
C (PFU/m3)b HI-Test RT-PCR C (PFU/m3) HI-Test RT-PCR
1 0 -c – 0 – –
2 0 – – 240 + +
3 560 +d + 2320 + +
4 1200 + + 4560 + +
5 3600 + + 6400 + +
7 4880 + + 7200 + +
9 4640 + + 5040 + +
11 3760 + + 4240 + +
14 2080 + + 2560 + +
17 480 + + 640 + +
21 0 – – 0 – –
28 0 – – 0 – –
a dpi: days post-inoculation of chickens in G1; b C: concentration of AIV aerosol in the isolator A (PFU/m3 air); c _: negative result; d: +: positive result.

TABLE 2: Results of AIV excretion
dpia Trial 1 Trial 2
G1 G2 G3 G1 G2 G3
HI-Test RT-PCR HI-Test RT-PCR HI-Test RT-PCR HI-Test RT-PCR HI-Test RT-PCR HI-Test RT-PCR
1 0/15b 0/15 0/15 0/15 0/15 0/15 0/15 0/15 0/15 0/15 0/15 0/15
2 0/15 0/15 /c / / / 4/15 4/15 / / / /
3 7/15 7/15 0/15 0/15 / / 15/15 15/15 0/15 0/15 / /
4 15/15 15/15 0/15 0/15 0/15 0/15 15/15 15/15 5/15 5/15 0/0 0/0
5 15/15 14/15 0/15 0/15 0/15 0/15 15/15 15/15 15/15 15/15 0/0 0/0
7 12/15 12/15 6/15 6/15 0/15 0/15 11/15 12/15 13/15 13/15 5/15 5/15
9 4/15 5/15 15/15 15/15 11/15 11/15 5/15 5/15 8/15 8/15 12/15 12/15
11 0/15 0/15 11/15 11/15 13/15 13/15 2/15 2/15 5/15 5/15 8/15 9/15
14 0/15 0/15 6/15 6/15 6/15 6/15 0/15 0/15 0/15 0/15 6/15 5/15
17 / / 0/15 0/15 0/15 0/15 0/15 0/15 0/15 0/15 0/15 0/15
21 / / 0/15 0/15 0/15 0/15 / / / / 0/15 0/15
a dpi: days post-inoculation of chickens in G1; b Number positive out of all experimental chickens by RT-PCR and HI-Test; c Samples were not tested at this time point.

TABLE 3: Results of antibody titration( , log2, n = 15)
dpia Trial 1 Trial 2
G1 G2 G3 G1 G2 G3
0 0 0 0 0 0 0
4 0 / / 2.07±0.80 / /
5 1.13±0.74 / / 4.00±0.93 / /
7 2.20±1.08 0 0 6.73±0.59 0 0
9 2.93±1.28 0 0 6.93±0.80 0.72±1.22 0
11 3.73±1.16 0.63±0.82 0 7.00±0.65 1.66±0.82 0
14 4.73±1.16 1.28±0.75 0.53±0.64 7.20±0.86 2.25±0.81 0.67±0.72
17 6.20±0.86 2.14±0.74 1.27±0.83 6.93±0.70 2.33±0.72 1.57±0.88
21 7.07±0.59 2.26±0.86 2.10±1.12 7.07±0.70 2.29±0.84 2.24±1.26
28 7.13±0.64 2.23±0.72 2.13±1.37 7.20±0.77 2.30±0.76 2.27±1.44
a days post-inoculation of chickens in G1.

FIGURE 1: Arrangement of the isolators. Chickens in G1 and G2 were housed in the isolator A, and chickens in G3 were housed in the isolator B. The two isolators were connected with a tube which allowed the air flowed from A to B. F1 is a positive pressure fan and F2 is a negative pressure fan. The air flowed through the two fans and filtered to remove microorganisms.

Address of correspondence:
Dr. Tongjie Chai
College of Animal Science and Veterinary Medicine
Shandong Agricultural University
271018 Taian
P.R. China
chaitj117@163.com