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Avian influenza A (H5N1) virus antibodies in pigs and residents of swine farms, southern China
Journal of Clinical Virology 58 (2013) 647–651
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Avian influenza A (H5N1) virus antibodies in pigs and residents of swine farms, southern China Nan Cao a,1 , Wanjun Zhu a,1 , Ye Chen a,1 , Likai Tan a,1 , Pei Zhou a , Zhenpeng Cao a , Changwen Ke b , Yugu Li a , Jie Wu b , Wenbao Qi a , Peirong Jiao a , Guihong Zhang a,∗ a Key Laboratory of Animal Disease Control and Prevention of the Ministry of Agriculture, South China Agricultural University, 483 Wushan Road, Tianhe District, Guangzhou 510642, China b Guangdong Center for Disease Control and Prevention, Guangzhou, Guangdong Province, China
a r t i c l e
i n f o
Article history: Received 10 July 2013 Received in revised form 11 September 2013 Accepted 13 September 2013 Keywords: H5N1 Pigs Swine farm residents
a b s t r a c t Background: Since 1997, the H5 avian influenza viruses (AIVs) circulating in China have become an international concern. Clade 2.3.2 of H5N1 AIVs is genetically distinct from the viruses isolated before 2007 and antigenically different from the vaccine strains widely used in China. Swine farms in rural China are thought to play an important role in AIVs ecology. Objectives: A seroepidemiological study was undertaken among swine farm residents and pigs to understand the prevalence of antibodies against H5N1 AIVs in southern China. Study design: During the period March 24, 2008 to December 25, 2012,serum samples were collected from 1606 swine farm residents on 40 swine farms in southern China. A total of 1980 pigs’ serum samples were collected in the same swine farms where swine workers’ serum samples were collected from March 2009 to March 2013. For a control group, 104 serum samples were collected from healthy city residents in Nanchang. All the serum samples were collected to perform hemagglutination inhibition (HI) and (neutralization) NT assays to investigate the prevalence of H5N1 AIV infections in southern China. Results: Sixteen human samples were positive by HI assay and 10 of these were also positive by NT assay against H5N1. No serum samples from human control and pigs were HI positive for H5N1 AIV. Discussion: Our results demonstrate minimal transmission H5N1 AIV from birds to pigs in the swine farms studied and the risk of poultry-to-human and poultry-to-pig transmission for at least clades 2.3.2 seemed very low. This study provides the first data regarding antibodies against H5N1 AIV in humans and pigs on swine farms in China. The findings of this study can serve as a baseline for additional serologic studies to assess transmission of H5N1 viruses between avian species, pigs and swine workers. © 2013 Elsevier B.V. All rights reserved.
1. Background Influenza A viruses are known to infect a wide variety of sentient beings, including humans, pigs, birds, horses, and sea mammals. Studies have repeatedly shown that the influenza virus can move from one species to another [1–4]. The highly pathogenic H5N1 avian influenza virus (AIV) has posed a serious public health threat since 1997, when the first transmission of the virus from birds to humans was reported in Hong Kong [5–7]. The epidemiology of the H5 subtype AIVs circulating in China is of international concern because it is easy for the viruses to be transmitted across geopolitical boundaries by wild birds and legal or illegal poultry trade [8,9]. Three clades of H5N1 AIV (2.3.4, 2.3.2, and 7.2) have co-circulated in China mainland in recent years. Clade 2.3.2 has
∗ Corresponding author. Tel.: +86 20 85280240; fax: +86 20 85280242. E-mail address: [email protected] (G. Zhang). 1 Equally contributed authors. 1386-6532/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2013.09.017
circulated widely in poultry in China, and since 2009 has caused a new wave transmission of the disease from Asia into Europe [8–10]. Clades 2.3.2 are genetically distinct from the viruses isolated before 2007 and antigenically different from the vaccine strains widely used in China [8,10,11]. On December 29, 2011, a man infected with the H5N1 influenza virus was confirmed in Shenzhen and the virus A/Shenzhen/1/2011 belongs to subclade 2.3.2 [12]. In southern China, pig production is rapidly increasing and some large-scale pig husbandry operations are adjacent to lakes. Various wild and domestic avian species live or are housed in close proximity to pig farms, increasing the possibility of crossspecies influenza A virus transmission between swine workers, wild and domestic avian species and commercially raised-pigs. Swine farms in rural China are thought to play an important role in AIV ecology. However, to date, no serological studies of H5N1 AIV infections have been carried out among pigs and swine farm workers in China. This study was designed to better understand the ecology of H5N1 AIV associated with swine farms in southern China.
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Table 1 Survey structure in southern China where the sero-epidemiological study was conducted. Locations
No.
No. Demographic description
Guangdonga
794
40 (13–65)
Zhejianga
489
29 (12–70)
Fujiana
323
Jiangxib
104
Median of age (range)
No. of female (%)
Occupation (%) Swine farm Veterinarian Farm owners Swine farm employee, spouses and children
26 (3.27) 49 (6.18) 719 (90.55)
93 (19.02)
Swine farm Veterinarian Farm owners Swine farm employee, spouses and children
11 (2.25) 19 (3.89) 459 (93.86)
37 (8–67)
96 (29.72)
Swine farm Veterinarian Farm owners Swine farm employee, spouses and children
15 (4.64) 32 (9.91) 276 (85.45)
31 (9–87)
49 (47.12)
Students Teachers Industrial workers
58 (55.76) 23 (22.12) 23 (22.12)
201 (25.3)
Unvaccinered swine Farm rosdens and caplplogees. a Survey sites in southern China where the sero-epidemiological study was conducted. b Control serum samples were collected from unvaccinated healthy city residents in Nanchang, the capital city of Jiangxi province. Table 2 Pigs in southern China where the sero-epidemiological study was conducted. Locations
Pig blood samples
a
Guangdong (21 swine farms) Zhejiang (8 swine farms)b Fujian (11 swine farms)c Total (1980 pigs from 40 swine farms) a b c
Weaning pigs
Growing-finishing pigs
Sow pigs
216 129 103 448
305 201 301 807
391 105 229 725
21 farms collected three times in 2009, 2010 and 2012. 8 farms collected twice in 2009 and 2013. 11 farms collected four times in 2009, 2010, 2011 and 2012.
2. Objectives To investigate the prevalence of H5N1 AIV infection in swine farm workers and pigs in southern China, an active influenza surveillance program was conducted on pig farms in this region during March 2008 to March 2013. 3. Study design materials and methods 3.1. Clinical samples collection Human clinical samples collection in rural areas of southern China was provided by Center for Disease Control and Prevention (CDC) staff in the area. To take part in the study, participants were required to complete a questionnaire regarding their general health and the history of their contact with avian species and pigs. Participation was also extended to other employees on the swine farm as well as farm veterinarians. During the period March 24, 2008 to December 25, 2012, a total of 1606 participants were enrolled, including 1554 persons from 100 pig farms, as well as 52 pig farm veterinarians (Table 1). Peripheral blood samples (5 mL each) were obtained by trained healthcare workers. Participants’ sera were stored at −20 ◦ C until use. Most participants admitted having close contact with avian species in swine farms, and many participants indicated they sometimes bathed or swam in swine farm ponds, putting them in close contact with waterfowl and
ducks. As a control, during the period from March 2, 2009 to May 5, 2012, a total of 104 serum samples were collected from healthy city residents in Nanchang (the capital city of Jiangxi) who reported rarely having close contact with avian species. Each participant was assigned an ID number so that laboratory samples could be assayed without knowledge of personal identifying information. In addition, to investigate the prevalence of H5N1 AIV infections in pigs in southern China, a total of 1980 pig blood samples were collected from March 2009 to March 2013 from 40 swine farms distributed throughout Guangdong, Zhejiang and Fujian Provinces. Pig clinical samples collection in pig farms were provided by local veterinarians. These farms were the same swine farms where swine workers’ serum samples were collected (Table 2). These provinces are the major pig, chicken, and duck breeding regions. This study protocol was reviewed and approved by the Institutional Review Board at the Guangdong Center for Disease Control and Prevention. An informed consent form was provided to and signed by each participant. 3.2. HI and NT assays The influenza virus used in serological assays was a clade 2.3.2 A/chicken/Guangdong/178/04(H5N1) (Genbank Accession: AY737296) isolated by the College of Veterinary Medicine, South China Agricultural University [13]. The 2.3.2 clade has been the most prevalent lineage since 2009 and
N. Cao et al. / Journal of Clinical Virology 58 (2013) 647–651
649
Fig. 1. Phylogenetic trees for the H5N1 HA gene of avian influenza A viruses. The analysis was based on the nucleotide sequences from the open reading frame of the HA gene. The phylogenetic trees were generated using the MEGA program (version 5.0) by neighbor-joining analysis.
A/chicken/Guangdong/178/04(H5N1) is suitable for a detection virus (Fig. 1). All human and pig serum samples were treated with receptor-destroying enzyme (RDE) and absorbed with erythrocytes to remove nonspecific inhibitors before the assays [14,15]. Our HI assay used horse erythrocytes instead of fresh Turkey red blood cells, in order to increase the assay sensitivity [16]. This assay was carried out following the WHO influenza laboratory procedures [17]. All samples were tested by hemagglutination inhibition (HI) according to previous study [16–18] and HI-positive sera were tested for neutralizing antibodies by using a traditional neutralization method [19] and supplementary HI titer ≥ 80 and NT titer ≥ 80 were considered as having positive evidence of previous H5N1 infection [19]. 4. Results The total number of subjects enrolled included 794 people from Guangdong, 489 from Zhejiang and 323 from Fujian (Table 1). Most of these participants reported direct exposure to avian species around lakes adjacent to their homes. Sixteen of these human samples were positive by HI assay and 10 of these were also positive by NT assay (Table 3) against A/chicken/Guangdong/178/04(H5N1). None of the 104 human control serum samples from Jiangxi were HI positive. None of the 1980 pig serum samples were positive by HI assay. To rule out non-specific cross-reactivity, all 1710 human serum samples were titrated against seasonal influenza viruses (H1N1 and H3N2). A total of 522 human samples (522/1710) were positive by HI assay against H1N1 and a total of 293 human samples (293/1710) were positive by HI assay against H3N2 (data not shown). Interestingly, only three of the H5-positive samples had dual reactivity toward H5 and season influenza viruses (data not shown).
5. Discussion The HI test has been widely used to detect antibodies of animal and human sera. However, the traditional HI assay which uses chicken erythrocytes, is not sufficiently sensitive for detecting HI antibodies specific to avian influenza viruses. Previously, it was demonstrated that employing an assay using horse erythrocytes increase the sensitivity of HI assay [18]. The NT detects the specific antibody is often carried out in parallel with the HI test. In this study, we combined the two methods and found the NT assay to validate a number of the HI positive specimens. Based on questionnaire data, age, gender and history of pig and bird contact were not statistically associated with the only 10 seropositive subjects (P > 0.05, by Chi-square test). Because we identified only 10 seropositive subjects among 1606 swine workers and no seropositive cases among 1980 pigs, we argue that the risk of poultry-to-human transmission and poultry-to-pig transmission for at least this highly pathogenic avian influenza (HPAI) H5N1 seems very low (we identified only 10 seropositive human subjects and the seropositive subjects which may be due to heterotypic antibodies from a previous human or swine influenza virus infection, so we do not confirm this 10 seropositive human subjects were previously infection with H5N1 AIV). This study provides the first data regarding antibodies against H5N1 AIV in humans and pigs on swine farms in China. While we agree that pigs have the potential to serve as mixing vessels for generation of novel influenza virusese, [20], our results demonstrate minimal transmission from birds to pigs in the swine farms studied. The lack of evidence of H5 infection in the pig population of southern China suggests, to some extent, that domestic pigs and swine farm workers have low susceptibility to H5N1 infection.
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N. Cao et al. / Journal of Clinical Virology 58 (2013) 647–651
Table 3 Sixteen from 1606 swine-exposed study subjects with positive serological assay result, China from 2008 to 2012. Province
Guangdong Guangdong Guangdong Guangdong Guangdong Guangdong Guangdong Guangdong Fujian Fujian Fujian Fujian Fujian Zhejiang Zhejiang Zhejiang
Age (yrs)
52 43 40 39 34 29 25 21 38 29 29 22 19 38 29 22
Gender
Serological assays results gainst A/chicken/Guangdong/178/04 (H5N1)
Male Male Male Female Male Female Male Male Female Male Male Female Male Male Male Female
HI
NT
80 320 80 160 640 80 80 160 80 320 160 640 160 160 80 160
40 320 80 40 320 20 80 320 40 320 160 320 160 80 40 40
HI: hemagglution inhibition assay; NT: neutralization assay.
This survey’s findings are subject to several limitations. Confirmation of H5N1 AIV infection of poultry on the swine farms where swine workers’ and pigs’ serum samples were collected was limited. Human antibodies to H5N1 titers wane over time and our serological assay cut points may have missed a number of true HPAI H5N1 infections. Although only three of the positive samples had dual reactivity toward H5 and season influenza viruses (data not shown) .It is also possible that some of our positive assay findings may be due to heterotypic antibodies from human influenza virus infection, [21], also, as we did not collect symptom data, we do not know if the study subjects with positive serologies had any clinical evidence of H5N1 AIV infection. The special environment and lifestyle on swine farms in southern China provide many opportunities for wild aquatic birds, domestic poultry, pigs, and humans to come in close contact, increasing the likelihood of interspecies transmission and generation of novel influenza viruses through reassortment [3,6,7]. Although we identified only 10 seropositive subjects among 1606 swine workers and some of the seropositive subjects which may be due to heterotypic antibodies from a previous human influenza virus infection. The findings of this study can serve as a baseline for additional serologic studies to assess transmission of H5N1 viruses between avian species, pigs and swine workers. In a special pig farming model in southern China, the staff and residents are in close contact with infected birds, and pigs may be among the first to be infected. By spreading the illness to their families, these workers may serve as a bridging population to the general populace, [22]. Therefore such studies will be crucial in the future, as the prevalence of novel influenza A viruses may be increasing among domestic birds and swine in southern China [7,8,13].
Competing interests None declared. Ethical approval This study protocol was reviewed and approved by the Institutional Review Board at the Guangdong Center for Disease Control and Prevention. Acknowledgements We thank Professor Gregory C. Gray of the University of Florida for his contributions in the research design and editorial review of this report. We thank Mr. Charlie Kidd of the University of Florida for his technical editing assistance. We also thank Dr. Shuo Su of the South China Agricultural University for his technical editing assistance. We acknowledge the scientific support from professor Guangzhi Tong, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Professor Fu Gao, Institute of Microbiology, Chinese Academy of Sciences; Professor Ming Liao, South China Agricultural University; Professor Hualan Chen and Associate Professor Chuanling Qiao, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences; Professor Henry Wan, Mississippi State University. The reference strains used in this article were kindly provided by Professor Peirong Jiao and Professor Wenbao Qi, College of Veterinary Medicine, South China Agricultural University. Appendix A. Supplementary data
Funding This work was supported by the Science and Technology Projects of Guangdong province (2012B020306005, the National Key Basic Research Program (Project 973) of China (grant no. 2011CB504700G), the International Science & Technology Cooperation Program (2010DFB33920) and the Modern Agricultural Industry Technology System (CARS-36). The funding organizations had no role in the study design, data collection and analysis, ownership of the materials or preparation of the manuscript.
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcv.2013.09.017. References [1] Wright PF, Webster RG. Orthomyxoviruses. Philadelphia, PA: Lippincott William & Wilkins; 2001. p. 1533–9. [2] Kida H, Ito T, Yasuda J, Shimizu Y, Itakura C, Shortridge KF, et al. Potential for transmission of avian influenza viruses to pigs. J Gen Virol 1994;75: 2183–8. [3] Peiris JSM, Guan Y, Markwell D, Ghose P, Webster RG. Shortridge KF. Cocirculation of avian H9N2 and contemporary human H3N2 influenza A viruses
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in pigs in Southeastern China: Potential for genetic reassortment. J Virol 2001;75:9679–86. Su S, Chen JD, Qi HT, Zhu WJ, Xie JX, Huang Z, et al. Complete genome sequence of a novel avian-like H3N2 swine influenza virus discovered in southern China . J Virol 2012;86:9533. World Health Organization Situation updates – avian influenza; 2007. http://www.who.int/csr/disease/avian influenza/updates/en/index.html Bridges CB, Lim W, Hu-Primmer J, Sims L, Fukuda K, Mak KH, et al. Risk of influenza A (H5N1) infection among poultry workers, Hong Kong, 1997–1998. J Infect Dis 2002;185:1005–10. Li H, Yu K, Xin X, Yang H, Li Y, Qin Y, et al. Serological and virologic surveillance of swine influenza in China from 2000 to 2003. Int Congr Ser 2004;1263:754–7. Li Y, Shi J, Zhong G, Deng G, Tian G, Ge J, et al. Continued evolution of H5N1 influenza viruses in wild birds, domestic poultry, and humans in China from 2004 to 2009. J Virol 2010;84(17):8389–97. Guan Y, Shortridge KF, Krauss S, Chin PS, Dyrting KC, Ellis TM, et al. H9N2 influenza viruses possessing H5N1-like internal genomes continue to circulate in poultry in southeastern China. J Virol 2000;74:9372–80. Jiang W, Liu S, Chen J, Hou G, Li J, Cao Y, et al. Molecular epidemiological surveys of H5 subtype highly pathogenic avian influenza viruses in poultry in Chinaduring 2007–2009. J Gen Virol 2010;91(10):2491–6. Zhao G, Zhong L, Lu XL. Characterisation of a highly pathogenic H5N1 clade 2.3.2 influenza virus isolated from swans in Shanghaim, China. Virus Genes 2012;44:55–62. Chunli Wu, Xing Lu, Xin Wang. Clinical symptoms, immune factors, and molecular characteristics of an adult male in Shenzhen, China infected with influenza virus H5N1. J Med Virol 2013;85:760–8.
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[13] Wan XF, Ren T, Luo KJ, Liao M, Zhang GH, Chen JD, et al. Genetic characterization of H5N1 avian influenza viruses isolated in southern China during the 2003-04 avian influenza outbreaks. Arch Virol 2005;150:1257–66. [14] Myers KP, Setterquist SF, Capuano AW, Gray GC. Infection due to 3 avian influenza subtypes in United States veterinarians. Clin Infect Dis 2007;45:4–9. [15] Kendal AP, Pereira MS, Skehel JJ. Concepts and procedures from laboratorybased influenza surveillance. Atlanta: Centers for Disease Control and Prevention; 1982. http://stacks.cdc.gov/view/cdc/12251/ [16] Jia N, Wang SX, Liu YX, Zhang PH, Zuo SQ, Lin Z, et al. Increased sensitivity for detecting avian influenza-specific antibodies by a modified hemagglutination inhibition assay using horse erythrocytes. J Virol Methods 2008;153:43. [17] World Health Organization. Manual for the laboratory diagnosis virological surveillance of influenza; 2011, whqliband doc.who.int/publications/2011/9789241548090 eng.pdf. [18] Su S, Ning Z, Zhu W, Jiao P, Ke C, Qi W, et al. Lack of evidence of avian-tohuman transmission of avian influenza A (H5N1) virus among veterinarians, Guangdong, China, 2012. J Clin Virol 2013;56(4):365–6. [19] Jung Kwonil Song DS. Serologic surveillance of swine H1 and H3 and avian H5 and H9 influenza A virus infections in swine population in Korea. Prev Vet Med 2007;79:294–303. [20] Brown IH. The epidemiology and evolution of influenza viruses in pigs. Vet Microbiol 2000;74:29–46. [21] Couch RB. An overview of serum antibody responses to influenza virus antigens. Dev Biol (Basel) 2003;115:25–30. [22] Munster VJ, Baas C, Lexmond P. Spatial, temporal, and species variation in prevalence of influenza A viruses in wild migratory birds. PLoS Pathog 2007;3:61.
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Avian influenza A (H5N1) virus antibodies in pigs and residents of swine farms, southern China Nan Cao a,1 , Wanjun Zhu a,1 , Ye Chen a,1 , Likai Tan a,1 , Pei Zhou a , Zhenpeng Cao a , Changwen Ke b , Yugu Li a , Jie Wu b , Wenbao Qi a , Peirong Jiao a , Guihong Zhang a,∗ a Key Laboratory of Animal Disease Control and Prevention of the Ministry of Agriculture, South China Agricultural University, 483 Wushan Road, Tianhe District, Guangzhou 510642, China b Guangdong Center for Disease Control and Prevention, Guangzhou, Guangdong Province, China
a r t i c l e
i n f o
Article history: Received 10 July 2013 Received in revised form 11 September 2013 Accepted 13 September 2013 Keywords: H5N1 Pigs Swine farm residents
a b s t r a c t Background: Since 1997, the H5 avian influenza viruses (AIVs) circulating in China have become an international concern. Clade 2.3.2 of H5N1 AIVs is genetically distinct from the viruses isolated before 2007 and antigenically different from the vaccine strains widely used in China. Swine farms in rural China are thought to play an important role in AIVs ecology. Objectives: A seroepidemiological study was undertaken among swine farm residents and pigs to understand the prevalence of antibodies against H5N1 AIVs in southern China. Study design: During the period March 24, 2008 to December 25, 2012,serum samples were collected from 1606 swine farm residents on 40 swine farms in southern China. A total of 1980 pigs’ serum samples were collected in the same swine farms where swine workers’ serum samples were collected from March 2009 to March 2013. For a control group, 104 serum samples were collected from healthy city residents in Nanchang. All the serum samples were collected to perform hemagglutination inhibition (HI) and (neutralization) NT assays to investigate the prevalence of H5N1 AIV infections in southern China. Results: Sixteen human samples were positive by HI assay and 10 of these were also positive by NT assay against H5N1. No serum samples from human control and pigs were HI positive for H5N1 AIV. Discussion: Our results demonstrate minimal transmission H5N1 AIV from birds to pigs in the swine farms studied and the risk of poultry-to-human and poultry-to-pig transmission for at least clades 2.3.2 seemed very low. This study provides the first data regarding antibodies against H5N1 AIV in humans and pigs on swine farms in China. The findings of this study can serve as a baseline for additional serologic studies to assess transmission of H5N1 viruses between avian species, pigs and swine workers. © 2013 Elsevier B.V. All rights reserved.
1. Background Influenza A viruses are known to infect a wide variety of sentient beings, including humans, pigs, birds, horses, and sea mammals. Studies have repeatedly shown that the influenza virus can move from one species to another [1–4]. The highly pathogenic H5N1 avian influenza virus (AIV) has posed a serious public health threat since 1997, when the first transmission of the virus from birds to humans was reported in Hong Kong [5–7]. The epidemiology of the H5 subtype AIVs circulating in China is of international concern because it is easy for the viruses to be transmitted across geopolitical boundaries by wild birds and legal or illegal poultry trade [8,9]. Three clades of H5N1 AIV (2.3.4, 2.3.2, and 7.2) have co-circulated in China mainland in recent years. Clade 2.3.2 has
∗ Corresponding author. Tel.: +86 20 85280240; fax: +86 20 85280242. E-mail address: [email protected] (G. Zhang). 1 Equally contributed authors. 1386-6532/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2013.09.017
circulated widely in poultry in China, and since 2009 has caused a new wave transmission of the disease from Asia into Europe [8–10]. Clades 2.3.2 are genetically distinct from the viruses isolated before 2007 and antigenically different from the vaccine strains widely used in China [8,10,11]. On December 29, 2011, a man infected with the H5N1 influenza virus was confirmed in Shenzhen and the virus A/Shenzhen/1/2011 belongs to subclade 2.3.2 [12]. In southern China, pig production is rapidly increasing and some large-scale pig husbandry operations are adjacent to lakes. Various wild and domestic avian species live or are housed in close proximity to pig farms, increasing the possibility of crossspecies influenza A virus transmission between swine workers, wild and domestic avian species and commercially raised-pigs. Swine farms in rural China are thought to play an important role in AIV ecology. However, to date, no serological studies of H5N1 AIV infections have been carried out among pigs and swine farm workers in China. This study was designed to better understand the ecology of H5N1 AIV associated with swine farms in southern China.
648
N. Cao et al. / Journal of Clinical Virology 58 (2013) 647–651
Table 1 Survey structure in southern China where the sero-epidemiological study was conducted. Locations
No.
No. Demographic description
Guangdonga
794
40 (13–65)
Zhejianga
489
29 (12–70)
Fujiana
323
Jiangxib
104
Median of age (range)
No. of female (%)
Occupation (%) Swine farm Veterinarian Farm owners Swine farm employee, spouses and children
26 (3.27) 49 (6.18) 719 (90.55)
93 (19.02)
Swine farm Veterinarian Farm owners Swine farm employee, spouses and children
11 (2.25) 19 (3.89) 459 (93.86)
37 (8–67)
96 (29.72)
Swine farm Veterinarian Farm owners Swine farm employee, spouses and children
15 (4.64) 32 (9.91) 276 (85.45)
31 (9–87)
49 (47.12)
Students Teachers Industrial workers
58 (55.76) 23 (22.12) 23 (22.12)
201 (25.3)
Unvaccinered swine Farm rosdens and caplplogees. a Survey sites in southern China where the sero-epidemiological study was conducted. b Control serum samples were collected from unvaccinated healthy city residents in Nanchang, the capital city of Jiangxi province. Table 2 Pigs in southern China where the sero-epidemiological study was conducted. Locations
Pig blood samples
a
Guangdong (21 swine farms) Zhejiang (8 swine farms)b Fujian (11 swine farms)c Total (1980 pigs from 40 swine farms) a b c
Weaning pigs
Growing-finishing pigs
Sow pigs
216 129 103 448
305 201 301 807
391 105 229 725
21 farms collected three times in 2009, 2010 and 2012. 8 farms collected twice in 2009 and 2013. 11 farms collected four times in 2009, 2010, 2011 and 2012.
2. Objectives To investigate the prevalence of H5N1 AIV infection in swine farm workers and pigs in southern China, an active influenza surveillance program was conducted on pig farms in this region during March 2008 to March 2013. 3. Study design materials and methods 3.1. Clinical samples collection Human clinical samples collection in rural areas of southern China was provided by Center for Disease Control and Prevention (CDC) staff in the area. To take part in the study, participants were required to complete a questionnaire regarding their general health and the history of their contact with avian species and pigs. Participation was also extended to other employees on the swine farm as well as farm veterinarians. During the period March 24, 2008 to December 25, 2012, a total of 1606 participants were enrolled, including 1554 persons from 100 pig farms, as well as 52 pig farm veterinarians (Table 1). Peripheral blood samples (5 mL each) were obtained by trained healthcare workers. Participants’ sera were stored at −20 ◦ C until use. Most participants admitted having close contact with avian species in swine farms, and many participants indicated they sometimes bathed or swam in swine farm ponds, putting them in close contact with waterfowl and
ducks. As a control, during the period from March 2, 2009 to May 5, 2012, a total of 104 serum samples were collected from healthy city residents in Nanchang (the capital city of Jiangxi) who reported rarely having close contact with avian species. Each participant was assigned an ID number so that laboratory samples could be assayed without knowledge of personal identifying information. In addition, to investigate the prevalence of H5N1 AIV infections in pigs in southern China, a total of 1980 pig blood samples were collected from March 2009 to March 2013 from 40 swine farms distributed throughout Guangdong, Zhejiang and Fujian Provinces. Pig clinical samples collection in pig farms were provided by local veterinarians. These farms were the same swine farms where swine workers’ serum samples were collected (Table 2). These provinces are the major pig, chicken, and duck breeding regions. This study protocol was reviewed and approved by the Institutional Review Board at the Guangdong Center for Disease Control and Prevention. An informed consent form was provided to and signed by each participant. 3.2. HI and NT assays The influenza virus used in serological assays was a clade 2.3.2 A/chicken/Guangdong/178/04(H5N1) (Genbank Accession: AY737296) isolated by the College of Veterinary Medicine, South China Agricultural University [13]. The 2.3.2 clade has been the most prevalent lineage since 2009 and
N. Cao et al. / Journal of Clinical Virology 58 (2013) 647–651
649
Fig. 1. Phylogenetic trees for the H5N1 HA gene of avian influenza A viruses. The analysis was based on the nucleotide sequences from the open reading frame of the HA gene. The phylogenetic trees were generated using the MEGA program (version 5.0) by neighbor-joining analysis.
A/chicken/Guangdong/178/04(H5N1) is suitable for a detection virus (Fig. 1). All human and pig serum samples were treated with receptor-destroying enzyme (RDE) and absorbed with erythrocytes to remove nonspecific inhibitors before the assays [14,15]. Our HI assay used horse erythrocytes instead of fresh Turkey red blood cells, in order to increase the assay sensitivity [16]. This assay was carried out following the WHO influenza laboratory procedures [17]. All samples were tested by hemagglutination inhibition (HI) according to previous study [16–18] and HI-positive sera were tested for neutralizing antibodies by using a traditional neutralization method [19] and supplementary HI titer ≥ 80 and NT titer ≥ 80 were considered as having positive evidence of previous H5N1 infection [19]. 4. Results The total number of subjects enrolled included 794 people from Guangdong, 489 from Zhejiang and 323 from Fujian (Table 1). Most of these participants reported direct exposure to avian species around lakes adjacent to their homes. Sixteen of these human samples were positive by HI assay and 10 of these were also positive by NT assay (Table 3) against A/chicken/Guangdong/178/04(H5N1). None of the 104 human control serum samples from Jiangxi were HI positive. None of the 1980 pig serum samples were positive by HI assay. To rule out non-specific cross-reactivity, all 1710 human serum samples were titrated against seasonal influenza viruses (H1N1 and H3N2). A total of 522 human samples (522/1710) were positive by HI assay against H1N1 and a total of 293 human samples (293/1710) were positive by HI assay against H3N2 (data not shown). Interestingly, only three of the H5-positive samples had dual reactivity toward H5 and season influenza viruses (data not shown).
5. Discussion The HI test has been widely used to detect antibodies of animal and human sera. However, the traditional HI assay which uses chicken erythrocytes, is not sufficiently sensitive for detecting HI antibodies specific to avian influenza viruses. Previously, it was demonstrated that employing an assay using horse erythrocytes increase the sensitivity of HI assay [18]. The NT detects the specific antibody is often carried out in parallel with the HI test. In this study, we combined the two methods and found the NT assay to validate a number of the HI positive specimens. Based on questionnaire data, age, gender and history of pig and bird contact were not statistically associated with the only 10 seropositive subjects (P > 0.05, by Chi-square test). Because we identified only 10 seropositive subjects among 1606 swine workers and no seropositive cases among 1980 pigs, we argue that the risk of poultry-to-human transmission and poultry-to-pig transmission for at least this highly pathogenic avian influenza (HPAI) H5N1 seems very low (we identified only 10 seropositive human subjects and the seropositive subjects which may be due to heterotypic antibodies from a previous human or swine influenza virus infection, so we do not confirm this 10 seropositive human subjects were previously infection with H5N1 AIV). This study provides the first data regarding antibodies against H5N1 AIV in humans and pigs on swine farms in China. While we agree that pigs have the potential to serve as mixing vessels for generation of novel influenza virusese, [20], our results demonstrate minimal transmission from birds to pigs in the swine farms studied. The lack of evidence of H5 infection in the pig population of southern China suggests, to some extent, that domestic pigs and swine farm workers have low susceptibility to H5N1 infection.
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Table 3 Sixteen from 1606 swine-exposed study subjects with positive serological assay result, China from 2008 to 2012. Province
Guangdong Guangdong Guangdong Guangdong Guangdong Guangdong Guangdong Guangdong Fujian Fujian Fujian Fujian Fujian Zhejiang Zhejiang Zhejiang
Age (yrs)
52 43 40 39 34 29 25 21 38 29 29 22 19 38 29 22
Gender
Serological assays results gainst A/chicken/Guangdong/178/04 (H5N1)
Male Male Male Female Male Female Male Male Female Male Male Female Male Male Male Female
HI
NT
80 320 80 160 640 80 80 160 80 320 160 640 160 160 80 160
40 320 80 40 320 20 80 320 40 320 160 320 160 80 40 40
HI: hemagglution inhibition assay; NT: neutralization assay.
This survey’s findings are subject to several limitations. Confirmation of H5N1 AIV infection of poultry on the swine farms where swine workers’ and pigs’ serum samples were collected was limited. Human antibodies to H5N1 titers wane over time and our serological assay cut points may have missed a number of true HPAI H5N1 infections. Although only three of the positive samples had dual reactivity toward H5 and season influenza viruses (data not shown) .It is also possible that some of our positive assay findings may be due to heterotypic antibodies from human influenza virus infection, [21], also, as we did not collect symptom data, we do not know if the study subjects with positive serologies had any clinical evidence of H5N1 AIV infection. The special environment and lifestyle on swine farms in southern China provide many opportunities for wild aquatic birds, domestic poultry, pigs, and humans to come in close contact, increasing the likelihood of interspecies transmission and generation of novel influenza viruses through reassortment [3,6,7]. Although we identified only 10 seropositive subjects among 1606 swine workers and some of the seropositive subjects which may be due to heterotypic antibodies from a previous human influenza virus infection. The findings of this study can serve as a baseline for additional serologic studies to assess transmission of H5N1 viruses between avian species, pigs and swine workers. In a special pig farming model in southern China, the staff and residents are in close contact with infected birds, and pigs may be among the first to be infected. By spreading the illness to their families, these workers may serve as a bridging population to the general populace, [22]. Therefore such studies will be crucial in the future, as the prevalence of novel influenza A viruses may be increasing among domestic birds and swine in southern China [7,8,13].
Competing interests None declared. Ethical approval This study protocol was reviewed and approved by the Institutional Review Board at the Guangdong Center for Disease Control and Prevention. Acknowledgements We thank Professor Gregory C. Gray of the University of Florida for his contributions in the research design and editorial review of this report. We thank Mr. Charlie Kidd of the University of Florida for his technical editing assistance. We also thank Dr. Shuo Su of the South China Agricultural University for his technical editing assistance. We acknowledge the scientific support from professor Guangzhi Tong, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Professor Fu Gao, Institute of Microbiology, Chinese Academy of Sciences; Professor Ming Liao, South China Agricultural University; Professor Hualan Chen and Associate Professor Chuanling Qiao, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences; Professor Henry Wan, Mississippi State University. The reference strains used in this article were kindly provided by Professor Peirong Jiao and Professor Wenbao Qi, College of Veterinary Medicine, South China Agricultural University. Appendix A. Supplementary data
Funding This work was supported by the Science and Technology Projects of Guangdong province (2012B020306005, the National Key Basic Research Program (Project 973) of China (grant no. 2011CB504700G), the International Science & Technology Cooperation Program (2010DFB33920) and the Modern Agricultural Industry Technology System (CARS-36). The funding organizations had no role in the study design, data collection and analysis, ownership of the materials or preparation of the manuscript.
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