Volume 16, Number 4—April 2010
Limited Susceptibility of Chickens, Turkeys, and Mice to Pandemic (H1N1) 2009 Virus
To determine susceptibility of chickens, turkeys, and mice to pandemic (H1N1) 2009 virus, we conducted contact exposure and inoculation experiments. We demonstrated that chickens were refractory to infection. However, oculo-oronasally inoculated turkeys and intranasally inoculated mice seroconverted without clinical signs of infection.
The current outbreak of pandemic (H1N1) 2009 continues to expand in humans, with occasional spillover into domestic pigs. Pandemic (H1N1) 2009 virus causes only mild disease compared with pandemic influenza viruses from the 20th century. However, this characteristic might change if pandemic (H1N1) 2009 viruses acquire virulence markers by reassorting with influenza viruses that cause severe disease in humans, such as highly pathogenic avian influenza viruses (HPAIVs) of the H5N1 subtype. Such reassortment might occur in humans but appears more likely in so-called mixing vessels. Pigs, which had been described as potential mixing vessels (1), are highly susceptible to infection with pandemic (H1N1) 2009 virus (2,3). In addition, pigs have been infected subclinically by HPAIV (H5N1) in countries to which HPAIV (H5N1) is endemic (4). However, whether poultry can be infected with pandemic (H1N1) 2009 virus is not well understood. Therefore, we determined the susceptibility of chickens, turkeys, and mice to pandemic (H1N1) 2009 virus.
All animal experiments were reviewed and approved by the responsible state ethics committee (LALLF M-V/TSD/7221.3–2.1.-014/09). Five chickens (12 weeks of age, specific pathogen–free [spf]) were inoculated oculo-oronasally by dripping a 106 50% tissue culture infectious dose (TCID50) of virus A/Regensburg/D6/09/H1N1 on the cornea, nares, and oropharynx. Ten chickens (3 months of age, spf) were inoculated intravenously with 104 TCID50. Five chickens (15 weeks of age, spf) were housed (permanent contact behind bars and daily direct contact for 30 min) with pigs infected with pandemic (H1N1) 2009 virus (3).
A second transmission experiment was performed with the same contact exposure experimental setup, which included 8 infected pigs and 5 turkeys (4 weeks of age). In addition, 28 chickens (1 week of age) and 28 turkeys (1 week of age) were inoculated with 200 μL of virus suspension (106 TCID50) directly into the left air sac. Six fattening turkeys (16 weeks of age) from a local flock were inoculated oculo-oronasally with 106 TCID50 of A/Regensburg/D6/09 (H1N1) virus. Six fattening turkeys were inoculated oculo-oronasally with 108 TCID50 of the more recently isolated pandemic (H1N1) 2009 A/Bayern/74/2009 virus. Cloacal and oropharyngeal swab samples from poultry infected oculo-oronasally, through the air sac, or by contact with pigs were collected daily. Samples were tested by using a real-time reverse transcription–PCR with subtype H1N1–specific primers (http://offlu.net) (Table).
Additional studies were conducted to determine the 50% lethal dose of strain A/Regensburg/D6/2009 pandemic (H1N1) 2009 virus for mice. Sixteen BALB/c mice (6 weeks of age) were inoculated intranasally with 102–105 TCID50/animal (30 μL), and 4 mice were inoculated intraperitoneally with 105 TCID50/animal (30 μL).
None of the inoculated animals became ill after infection by any tested route; the intravenous pathogenicity index of the virus for chickens was 0. All swab samples from poultry after contact with infected pigs or from poultry inoculated through the air sac were negative for virus (Table). Virus excretion by pigs was detected (3); real-time reverse transcription–PCR cycle threshold values >26 (contact experiment with chickens) or >17 (contact experiment with turkeys). Virus RNA was detected (cycle threshold values 27–39) in swab samples obtained 1–6 days postinoculation from the oropharynx of turkeys inoculated with 108 TCID50 of the A/Bayern/74/2009 (H1N1) strain. Small amounts of virus RNA also were detected in the lung and left air sac from a few chicks and poults early after infection through the air sac (Table), which most likely represent residual inoculum. Although contact exposure pigs were infected (3), seroconversion was not detected in any of the tested contact exposure poultry species. Poultry inoculated intravenously or through the air sac and chickens inoculated oculo-oronasally were negative for antibodies against influenza A virus nucleoprotein.
Fattening turkeys seroconverted after oculo-oronasal inoculation with 106 TCID50 or 108 TCID50 (Table). In addition, 7 of 8 mice inoculated with 104 TCID50 or 105 TCID50 by the oculo-oronasal route seroconverted; 4 mice inoculated with 105 TCID50 by the intraperitoneal route showed no detectable antibody response to the virus (Table).
Our results demonstrate lack of susceptibility of chickens and minor susceptibility of turkeys for infection by pandemic (H1N1) 2009 virus. Transmission of swine influenza viruses to poultry, particularly to turkeys, has been demonstrated (5), and experimental infection of chickens with virus subtype H3N2 resulted in a low replication rate, primarily in the alimentary tract (6). In contrast, our data indicate that pandemic (H1N1) 2009 virus cannot productively infect chickens at the ages of 1 week (air sac inoculation), 12 weeks (contact exposure, intravenous inoculation), or 15 weeks (oculo-oronasal inoculation), or turkeys at the ages of 1 week (air-sac inoculation) and 4 weeks (contact exposure) because neither virus excretion nor seroconversion was observed during the 10-day observation period. Fattening turkeys from a local flock seroconverted after oculo-oronasal inoculation, but virus RNA was detected in respiratory samples only in turkeys inoculated with a high dose of the A/Bayern/74/2009 (H1N1) virus (Table).
On the basis of our experiments, risk for transmission of pandemic (H1N1) 2009 virus strain to chicken and turkeys and subsequent possible reassortment with other avian influenza viruses should be low. However, we observed a slightly higher susceptibility of older turkeys to high doses of pandemic (H1N1) 2009 virus, which is consistent with recent reports of infected layer turkey flocks in Chile and Canada (7,8). Analysis of specific virus strains involved in these outbreaks would be useful for confirming these observations and analyzing different strains. Host factors (age, physiologic state, stress levels, and concurrent infections) influencing susceptibility of turkeys should be investigated. Our data, which demonstrate absence of illness in poultry after inoculation with pandemic (H1N1) 2009 virus, are consistent with those of recent experimental infections of poultry, including turkeys (9–11). Seroconversion was detected only in turkeys by using a hemagglutination-inhibition test in 1 study (9), which might indicate higher sensitivity of competitive ELISAs used in our study.
As in other studies (12,13), characterization of pandemic (H1N1) 2009 virus strains in BALB/c mice showed differences in lethal dose and clinical signs dependent on the virus strain. None of the infected BALB/c mice in our study showed clinical signs. These findings may have resulted from the fact that mice were infected intranasally without anesthesia and with a dose <106 TCID50/animal or because of a different phenotype of the virus strain used. However, replication competence of pandemic (H1N1) 2009 virus in mice without prior adaptation was indicated by seroconversion, at least for the higher infectious doses (Table). Intraperitoneal inoculation did not cause development of influenza virus–specific antibodies. This finding cannot be explained by receptor specificity of pandemic (H1N1) 2009 virus because Childs et al. (14) showed that representative pandemic (H1N1) 2009 viruses bound not only to most α2–6-linked sialyl moieties, irrespective of the backbone chain length and type, but also to a considerable range of α2–3-linked sialyl residues. Although virus replication may be reduced after intraperitoneal inoculation, development of neutralizing antibodies after inoculation by this route has been reported (15).
Our results demonstrate lack of susceptibility and absence of serologic responses of chickens and turkeys to contact infection with an early human-origin isolate of pandemic (H1N1) 2009 virus. However, direct inoculation of fattening turkeys resulted in seroconversion and detection of virus RNA in oropharyngeal swab samples. Generation of new variants and reassortants caused by double infections with other influenza A viruses and pandemic (H1N1) 2009 virus in poultry other than turkeys appears unlikely. However, further adaptation of pandemic (H1N1) virus strains in turkeys cannot be excluded.
Dr Kalthoff is a veterinarian at the Friedrich-Loeffler-Institut in Greifswald-Insel Riems, Germany. Her research interests are virus pathogenesis and vaccine development.
We thank Mareen Grawe for excellent technical assistance; Stephan Becker, Jennifer Uhlendorf, Mikhail Matrosovich, and Markus Eickmann for isolating and providing virus strain A/Regensburg/D6/2009; and Brunhilde Schweiger and Barbara Biere for providing virus strain A/Bayern/74/2009.
This study was supported by European Union FP7 project European Management Platform for Emerging and Re-emerging Infectious Disease Entities (no. 223498).
- Scholtissek C, Burger H, Kistner O, Shortridge KF. The nucleoprotein as a possible major factor in determining host specificity of influenza H3N2 viruses. Virology. 1985;147:287–94.
- Brookes SM, Irvine RM, Nunez A, Clifford D, Essen S, Brown IH, Influenza A (H1N1) infection in pigs. Vet Rec. 2009;164:760–1.
- Lange E, Kalthoff D, Blohm U, Teifke JPT, Breithaupt A, Maresch C, Pathogenesis and transmission of the novel swine origin influenza virus A/H1N1 after experimental infection of pigs. J Gen Virol. 2009;90:2119–23.
- Takano R, Nidom CA, Kiso M, Muramoto Y, Yamada S, Shinya K, A comparison of the pathogenicity of avian and swine H5N1 influenza viruses in Indonesia. Arch Virol. 2009;154:677–81.
- Choi YK, Lee JH, Erickson G, Goyal SM, Joo HS, Webster RG, H3N2 influenza virus transmission from swine to turkeys, United States. Emerg Infect Dis. 2004;10:2156–60.
- Thomas C, Manin TB, Andriyasov AV, Swayne DE. Limited susceptibility and lack of systemic infection by an H3N2 swine influenza virus in intranasally inoculated chickens. Avian Dis. 2008;52:498–501.
- PRO/AH/EDR. Influenza pandemic (H1N1) 2009, animal health (03): Chile, avian, RFI [cited 2010 Jan 14]. http://www.promedmail.org, archive no. 20090821.2961.
- PRO/AH/EDR. Influenza pandemic (H1N1) 2009, animal health (14): Canada (ON), avian [cited 2010 Jan 14]. http://www.promedmail.org, archive no. 20091020.3602.
- Terregino C, De Nardi R, Nisi R, Cilloni F, Salviato A, Fasolato M, Resistance of turkeys to experimental infection with an early 2009 Italian human influenza A(H1N1)v virus isolate. Euro Surveill. 2009;14:19360.
- Swayne DE, Pantin-Jackwood M, Kapczynski D, Spackman E, Suarez DL. Susceptibility of poultry to pandemic (H1N1) 2009 virus. Emerg Infect Dis. 2009;15:2061–3.
- Russell C, Hanna A, Barrass L, Matrosovich M, Núñez A, Brown IH, Experimental infection of turkeys with pandemic (H1N1) 2009 influenza virus (A/H1N1/09v). J Virol. 2009;83:13046–7.
- Itoh Y, Shinya K, Kiso M, Watanabe T, Sakoda Y, Hatta M, In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses. Nature. 2009;460:1021–5.
- Maines TR, Jayaraman A, Belser JA, Wadford DA, Pappas C, Zeng H, Transmission and pathogenesis of swine-origin 2009 A(H1N1) influenza viruses in ferrets and mice. Science. 2009;325:484–7.
- Childs RA, Palma AS, Wharton S, Matrosovich T, Liu Y. Chai Wet al. Receptor-binding specificity of pandemic influenza A (H1N1) 2009 virus determined by carbohydrate microarray. Nat Biotechnol. 2009;27:797–9.
- Bertram EM, Lau P, Watts TH. Temporal segregation of 4–1BB versus CD28-mediated costimulation: 4–1BB ligand influences T cell numbers late in the primary response and regulates the size of the T cell memory response following influenza infection. J Immunol. 2002;168:3777–85.
Suggested citation for this article: Kalthoff D, Grund C, Harder TC, Lange E, Vahlenkamp TW, Mettenleiter TC, et al. Limited susceptibility of chickens, turkeys, and mice to pandemic (H1N1) 2009 virus. Emerg Infect Dis [serial on the Internet]. 2010 Apr [date cited]. http://wwwnc.cdc.gov/eid/article/16/4/09-1491.htm
Comments to the Authors
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