Virological and Epidemiological Evidence of Avian Influenza Virus Infections Among Feral Dogs in Live Poultry Markets, China: A Threat to Human Health?

To theEditor— Since its first detection in March 2013, the novel H7N9 avian influenza virus (AIV) has quickly spread among poultry and people in China. As of 16 February 2014, a total of 348 laboratory-confirmed human H7N9 infections in China have been confirmed by the World Health Organization [1–3]. The H7N9 virus has spread widely with little sign of infection among poultry [4]. Epidemiologic studies have identified poultry exposure as an important risk factor for human infections with H5N1 and H7N9, especially for those individuals associated with live poultry markets (LPMs) [5–8]. As dogs in China have been shown to be infected with AIVs, we sought to investigate whether dogs living in close proximity to LPMs and H7N9-affected farms might have been infected with the novel H7N9 virus or other influenza viruses. From August 2011 to August 2013, we studied a total of 2357 dogs that lived in close proximity to LPMs and poultry farms in the rural areas of Shanghai, Guangdong, Zhejiang, and Jiangsu provinces in China where novel H7N9 AIV had been previously detected (for Materials and Methods, see Supplementary Data). Overall, 68.18% (n = 1607) of the 2357 stray dog samples were collected in rural areas, with the remaining samples collected in LPMs (Table 1). Of the 2357 nasal swab samples collected, 93 (3.9%) were positive for influenza A virus by real-time reverse transcription polymerase chain reaction (PCR), and 11 viruses were isolated from these samples (see Supplementary Data). Hemagglutination inhibition (HI) assays and hemagglutinin antigen–specific enzyme-linked immunosorbent assays against H7N9 viral antigens revealed no evidence of H7N9 infection. Results of the HI and microneutralization (MN) assays are reported in Table 1 and in the Supplementary Data. A total of 19 serum samples had HI antibody titers of ≥1:20 against H5 antigen (Table 1), and 3 of these 19 samples were also positive by MN assay. Dogs that were sampled in LPMs had a greater probability of having elevated HI antibodies against avian H9N2, avian H5N1, and canine H3N2 viruses (Table 2), compared with the dogs that were raised in poultry farms. Characteristics and Influenza Serological Assay Results of Feral Dogs Sampled With Nasal Swabs and Serum Specimen Collections, August 2011–August 2013, by Sampling Site Abbreviations: H3N2, A/canine/Guangdong/2/2011(H3N2); H5N1, A/chicken/Guangdong/178/04(H5N1); H9N2, A/chicken/Guangdong/V/2008(H9N2); HI, horse red blood cell hemagglutination inhibition assay; ILI, influenza-like-illness; LPM, live poultry market; MN, microneutralization assay. Characteristics and Influenza Serological Assay Results of Feral Dogs Sampled With Nasal Swabs and Serum Specimen Collections, August 2011–August 2013, by Sampling Site Abbreviations: H3N2, A/canine/Guangdong/2/2011(H3N2); H5N1, A/chicken/Guangdong/178/04(H5N1); H9N2, A/chicken/Guangdong/V/2008(H9N2); HI, horse red blood cell hemagglutination inhibition assay; ILI, influenza-like-illness; LPM, live poultry market; MN, microneutralization assay. Risk Factors for Elevated Antibody Against Influenza A Viruses by Horse Red Blood Cell Hemagglutination Inhibition Assay (Titer ≥1:20) Among Feral Dogs Sampled in Shanghai, Guangdong, Zhejiang, and Jiangsu Provinces, China Dogs were studied during the period August 2011 to August 2013. Abbreviations: CI, confidence interval; OR, odds ratio. Risk Factors for Elevated Antibody Against Influenza A Viruses by Horse Red Blood Cell Hemagglutination Inhibition Assay (Titer ≥1:20) Among Feral Dogs Sampled in Shanghai, Guangdong, Zhejiang, and Jiangsu Provinces, China Dogs were studied during the period August 2011 to August 2013. Abbreviations: CI, confidence interval; OR, odds ratio. Our study supports this premise in that, although we failed to find evidence of previous H7N9 infections among the dogs, we found the world's first evidence of previous H5N1 and H9N2 infection among dogs by real-time PCR, HI, and MN assay. These findings were unexpected but biologically plausible. In LPMs and farms in rural China, stray dogs and cats have considerable contact with poultry or poultry products. This can occur indirectly through aerosol and fecal transmission or directly through the consumption of dead bird carcasses or entrails. LPMs are particularly problematic as they offer a mixing of animal species from often diverse geographical areas, frequent venues for contact with the public, and often nonhygienic behavior of workers who handle and process the birds for sale. Both rural farms and LPMs provide opportunities for wild aquatic birds, domestic poultry, stray dogs, and humans to closely interact and potentially share pathogens (Supplementary Data). Additionally, compared with pets, feral dogs or cats living in LPMs or rural farms would logically have more potential to come into contact with sick or dead avian species. Moreover, feral dogs and cats may be immunocompromised due to poor diet, harsh environmental conditions, open wounds, etc, which may make them more susceptible to cross-species pathogen transmission. Hence, such feral animals may increase the risk of the emergence and transmission of novel influenza A viruses and serve as a threat to both veterinary health and human public health. These data support the importance of periodically conducting influenza A surveillance among feral dogs in China. As humans live in very close contact with dogs in many areas of the world, we posit that surveillance for novel viruses among feral dogs living in close proximity to LPMs or poultry farms could serve as an early warning system of viral threats to humans. Acknowledgments. We greatly thank Professor Pamela P. McKenzie of the St Jude Children's Research Hospital and Professor Kwok-yung Yuen, University of Hong Kong, for their helpful critical review and suggestions regarding this manuscript. 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; and Professor Henry Wan, Mississippi State University. Financial support. This work was supported in part by the National Natural Science Foundation of China (grant numbers 30972233 and 31372448), the Natural Science Foundation of Guangdong Province (grant numbers 8251064201000008 and S2012020011111), and the Basic Conditions for Science and Technology Projects of Guangdong Province (grant number 2011B060400015), Special Fund for Agro-scientific Research in the Public Interest (grant number 201303042). Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.