Journal of Medical Virology 87:1436–1440 (2015)
Detection Prevalence of H5N1 Avian Influenza Virus Among Stray Cats in Eastern China Fu-Rong Zhao,1,2 Dong-Hui Zhou,1,2 Yong-Guang Zhang,1,2 Jun-Jun Shao,1,2 Tong Lin,1,2 Yang-Fan Li,1,2 Ping Wei,1,2,3 and Hui-Yun Chang1,2* 1
State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province, People’s Republic of China 2 Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, People’s Republic of China 3 Northeast Agricultural University, College of Veterinary Medicine, Haerbin, Heilongjiang Province, People’s Republic of China
Since 1997, more and more cases of the infectious H5N1 avian influenza virus (AIV) in humans have been reported all over the world but the transmission of H5N1 avian influenza virus to stray cats has been little demonstrated. The objective of this pilot investigation was to determine the prevalence of H5N1 AIV antibodies in stray cats in eastern China where is the dominant enzootic H5N1 highly pathogenic avian influenza virus (HP AIV). A total of 1,020 nasal swab and 1,020 serum samples were collected and tested. Evidence of HPAI H5N1 virus antibodies was present in two of the 1,020 serum samples that were positive by HI assay and NT assay, respectively. The results imply little transmission and that the Clade 2.3.2 HPAIV H5N1 infections in poultry did not significantly affect the rural animal shelters or suburban environment in eastern China. In future studies, these results can be used as baseline seroepidemiological levels for H5N1 AIV among cats in China. J. Med. Virol. 87:1436–1440, 2015. # 2015 Wiley Periodicals, Inc. KEY WORDS:
H5N1; AIV; prevalence; interspecies transmission; stray cat
INTRODUCTION The highly pathogenic H5N1 avian influenza virus (AIV) has posed a serious role in public health since 1997, when the first transmission of the virus from birds to humans was reported in Hong Kong and the virus have established as enzootic viruses in China and other parts of the world [Claas et al., 1998; Enserink and Kaiser, 2004; Keawcharoen et al., 2004; Kuiken et al., 2004; Nguyen et al., 2005; Rimmelzwaan C 2015 WILEY PERIODICALS, INC.
et al., 2006]. 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 [Enserink and Kaiser, 2004; Keawcharoen et al., 2004; Kuiken et al., 2004; Rimmelzwaan et al., 2006]. Moreover, the H5N1 AIV can cross species barriers and adapt to new hosts [Kuiken et al., 2004]. Natural H5N1 AIV infections in several cat species have been reported in Southeast Asia [Claas et al., 1998; Enserink and Kaiser, 2004; Kuiken et al., 2004], and the experimental infection of domestic cats with similar viruses have also been reported [Rimmelzwaan et al., 2006]. Thus, the role of cats has been reconsidered in the transmission and spread of the H5N1 AIV. Direct contact with sick or diseased poultry has been implicated as the major risk factor for feline infection. Three clades, 2.3.4, 2.3.2, and 7.2, cocirculated in mainland China in recent years, and clade 2.3.2 has circulated widely in poultry in China and causing a new wave of cross-continental spreading from Asia to Europe since 2009 [Chen et al., 2006; Li et al., 2010; WHO, 2011]. It has become genetically distinguishable from the viruses isolated before 2007 and antigenically distinguishable from the vaccine strains widely used in China [Chen et al., 2006; Li et al., 2010]. Now, it has been detected in nine countries since 2007, which is highly pathogenic in
Conflict of interest: None. Correspondence to: Hui-Yun Chang, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province 730046, People’s Republic of China. E-mail: [email protected] Accepted 23 March 2015 DOI 10.1002/jmv.24216 Published online 7 May 2015 in Wiley Online Library (wileyonlinelibrary.com).
The Prevalence of H5N1 Avian Influenza Virus in Cats
chickens (100% mortality). It is also the dominant prevalence of H5N1 strains, especially in Southern China. The special climate, environment, and lifestyle in eastern China provide more chances for close contact among wild aquatic birds, domestic poultry, dogs or cats, and humans; therefore, creating an opportunity for interspecies transmission. Due to frequent cohabitation and interactions with other animals (dogs or avian) and humans, in southern China, different types of cats are uniquely positioned to act as a reservoir of influenza virus infection both within the household and the larger village farm or suburban environment. Cats are one of the most popular pets in China, so cats carrying AIV can pose a threat to human health. To date, no serological and virological studies about H5N1 AIVs infections have been carried out in stray cats in China. Therefore, this surveillance study is to uncover any evidence of H5N1 AIV, especially clade 2.3.2 viruses transmission in cats in China. MATERIALS AND METHODS Ethics Statement This study was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Permit No. LVRIAEC2011-006). Stray cats from which serum samples were collected were handled with good animal practices required by the Animal Ethics Procedures and Guidelines of the People’s Republic of China. The collection of serum samples was performed as a part of routine process of disease monitoring and surveillance for these cats. Cat Samples Between February 2010 and December 2011, a total of 1,020 nasal swab samples and 1,020 blood samples were collected from 24 different animal shelters in the Shanghai, Jiangsu, and Zhejiang Provinces in eastern China where H5N1 AIV are currently circulating or have been frequently detected in the past. They selected the sites based on their large and dense human, poultry, and pig populations which may be prone to cross-species transmission of influenza virus. In each site, the single largest animal shelters were selected. The cats were selected from the animal shelters based upon several factors: veterinary clinic location for geographical diversity and cats with no history of canine influenza vaccination. At each site, individual cats were selected by a random-number procedure among all the stray cats. Stray cats and bird are housed closely in farming villages, increasing the possibility of contacts among cats and wild animals. The sampling processes were assisted by local authorities and licensed veterinarians. Detection of antibodies to H5N1AIV. Cat nasal swab samples were initially screened for influenza A virus by real-time reverse transcription (RT-PCR)
1437
selective for the matrix gene. The reactions were carried out by using the Quaint Fast Multiplex RTPCR þ R Kit (QIAGEN, Valencia, CA) in a 20-ml reaction mixture containing 10 ml 2 quantitative RT-PCR master mix, 7.5 pmol of forward primer, 2.5 pmol of each reverse primer, 0.125 mmol/L EA minor groove binder probe, 0.0625 mmol/L of each NA minor groove binder probe, 0.4 ml 50 ROX reference dye, 0.2 ml of reverse transcription product, and 5 ml of extracted RNA. The reactions were performed on an M3000P QPCR System (Agilent Technologies, Inc., Santa Clara, CA) under these thermocycling conditions: 50˚C for 20 min, then 95˚C for 5 min, followed by 50 cycles of 97˚C for 2 sec and 60˚C for 40 sec. Samples that were positive by RT-PCR underwent further diagnostics for determination of subtype, including the human H1, H3 viruses, and avian H3, H5, H7, and H9 viruses [Li et al., 2010; WHO, 2011]. Sera were studied with a horse-RBC WHO-recommended hemagglutination inhibition (HI) assay against avian, human, and canine viruses [WHO, 2011]. All serum samples were treated with a receptor-destroying enzyme (RDE) and absorbed with erythrocytes in order to remove nonspecific inhibitors before detection. RDE was added to each serum specimen to inactivate nonspecific reactions and incubated at 37˚C overnight, followed by incubation at 56˚C for 30 min to inactivate RDE. Sera were next incubated and mixed with erythrocytes for 30 min at 4˚C. All serum samples were tested with HI assay as reported in previous studies [Su et al., 2013b]. Briefly, two-fold serial dilutions of serum samples were added in a V-shaped microtitre plate and 4 HA units of virus were added in each well. The mixture was incubated at room temperature for 35 min. Then, 1% (v/v) horse red blood cells (h-RBCs) were added in each well. The plates were left at room temperature for 40 min. The HI titers are expressed as the highest dilution of serum giving complete inhibition of agglutination. Sera from cats that were HI titer 20 by horse-RBC HI assay were confirmed with a microneutralization assay (MN) procedure [Kendal et al., 1982]. Briefly, positive sera were two-fold diluted in serum-free DMEM starting at a dilution of 1:10. Then, 100 TCID50 of virus were added to the serially diluted serum at a 1:1 ratio (v/v) and incubated at 37˚C for 1 hr. Finally, 0.2 ml of the virus–serum mixtures was transferred to 96-well monolayer plates and incubated in 5% CO2 at 37˚C for 72 hr. Three wells were run for each dilution of each serum sample. Cytopathic effects were checked for daily and cell supernatants were tested by HA test to confirm the virus infection. The endpoint for neutralization was expressed as the highest dilution that prevented hemagglutination in all three replicates. In this study, both HI and MN titer of 1:20 or greater was considered positive. For AIV antibody detection by both HI and MN assays, the HPAI H5N1 virus J. Med. Virol. DOI 10.1002/jmv
1438
Zhao et al.
[A/duck/Guangdong/1/2013(H5N1)] was used in this study. Virus A/duck/Guangdong/1/2013(H5N1) belonging to Clade 2.3.2 is the most dominant lineage from 2009 and suitable for a detection virus. To control for result quality, the HI assay was also performed for all serum with a virus derived from a recently circulating canine influenza virus (CIV) A/ canine/Lanzhou/1/2013 (H3N2) and a human pdm 09 virus A/California/7/2009(H1N1). Statistical Analysis The statistical tests about the association between serological outcome selected HI titer 40. Bivariate c2 test of independence or Fisher’s exact test were used to examine the association between the demographic characteristics and serological outcome. Covariates that had a P < 0.25 in the bivariate analyses were entered into a multivariable logistic regression model. The analysis was performed according to backward elimination of the covariates, keeping covariates in the model which had a P < 0.05. Final covariates were tested for goodness-of-fit. RESULTS From the total of 1,020 nasal swab samples collected, 19 (1.8%) were positive for influenza A virus by RT-PCR, and no viruses were isolated from these samples. Of the 19 nasal swab samples with RT-PCR-positive results for influenza A virus, 10 samples were H3N2 subtype CIV, 8 were H1N1 subtype human influenza viruses, and 1 were H9 subtype AIVs. None of 1,020 nasal swab samples were positive for influenza A (H5N1) virus by RTPCR. A total of 1,020 feline serum samples (461 from females and 559 from males) were examined using the MN and HI assays for H5N1 AIV antibodies. The serological screening revealed that only two of the sera (0.2%; OR ¼ 0.04; 95%CI [0.02–0.06], P < 0.05) from stray cats was positive according to the MN test by the Clade 2.3.2 virus, although 32 (3.1%; 95%CI [2.2–4.4], P < 0.05) samples were detected HI titers of 20 or more. All samples were also tested with other antigens (H1, H3) for the HI test (Table I), HI titers of 20 or more were detected in 101 (9.9%) of 1,020
serum (OR ¼ 9.9; 95%CI [8.1–11.9], P < 0.05) samples by used the H1N1 pdm09 antigens, twenty of the 101 samples were positive by the MN assay (MN > 1:20) (data not shown). However, a total of 108 (108/1020) sera were found HI antibody titer 1:20, 62 of these 108 samples were also positive by the MN assay (MN > 1:20) (data not shown). None of the three provinces studied had statistically significant different rates of cats infection with H5N1 AIV. However, for HI assay, samples from 2011 showed an increased risk of elevated antibodies against avian influenza virus H7N9 as compared to samples from 2010. Also, cats without signs of influenza-like-illness had a significant increased risk of elevated antibodies to canine H3N2 and pdm09 H1N1 as compared to cats with signs of influenzalike-illness. Influenza-like-illness had no significant effect on antibodies against H5N1 virus (Table II). DISCUSSION Stray cats are often seen living near live poultry markets and in rural areas of China. The special climate environment and lifestyle provide more chances for wild aquatic birds, domestic poultry, stray dogs, and humans to have close contact, and creates an opportunity for interspecies pathogens transmission [Su et al., 2013b]. Compared with pets, the stray cats in rural have more possibility to contact with the sick avian. Therefore, they were more easily infected with AIV. In addition, the stray cats are generally weaker due to these poor surroundings and they have a great possibility to be infected by wild avian influenza virus and are a threat to both veterinary and human public health. All of these indicate the importance of performing investigations of AIV infection among stray cats in different environments. In this study, we first investigated whether subclinical cat occurred infections with the H5N1 AIV among stray cats in eastern China. None of 1,020 nasal swab samples were positive for influenza A (H5N1) virus by RT-PCR and only two of the 1,020 serum samples were positive by both the HI assay (HI titer > 1:20) and MN assay (MN titer > 1:20). Thus, we found China’s first evidence of H5N1 AIV
TABLE I. Antibody Titers of H5N1 AIV; H1N1 pdm09 and H3N2 CIV Infections Using Hemagglutination Inhibition (HI) Assay HI titer Viral strainsa H3N2 H1N1 H5N1
No.
Detection Prevalence of H5N1 Avian Influenza Virus Among Stray Cats in Eastern China Fu-Rong Zhao,1,2 Dong-Hui Zhou,1,2 Yong-Guang Zhang,1,2 Jun-Jun Shao,1,2 Tong Lin,1,2 Yang-Fan Li,1,2 Ping Wei,1,2,3 and Hui-Yun Chang1,2* 1
State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province, People’s Republic of China 2 Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, People’s Republic of China 3 Northeast Agricultural University, College of Veterinary Medicine, Haerbin, Heilongjiang Province, People’s Republic of China
Since 1997, more and more cases of the infectious H5N1 avian influenza virus (AIV) in humans have been reported all over the world but the transmission of H5N1 avian influenza virus to stray cats has been little demonstrated. The objective of this pilot investigation was to determine the prevalence of H5N1 AIV antibodies in stray cats in eastern China where is the dominant enzootic H5N1 highly pathogenic avian influenza virus (HP AIV). A total of 1,020 nasal swab and 1,020 serum samples were collected and tested. Evidence of HPAI H5N1 virus antibodies was present in two of the 1,020 serum samples that were positive by HI assay and NT assay, respectively. The results imply little transmission and that the Clade 2.3.2 HPAIV H5N1 infections in poultry did not significantly affect the rural animal shelters or suburban environment in eastern China. In future studies, these results can be used as baseline seroepidemiological levels for H5N1 AIV among cats in China. J. Med. Virol. 87:1436–1440, 2015. # 2015 Wiley Periodicals, Inc. KEY WORDS:
H5N1; AIV; prevalence; interspecies transmission; stray cat
INTRODUCTION The highly pathogenic H5N1 avian influenza virus (AIV) has posed a serious role in public health since 1997, when the first transmission of the virus from birds to humans was reported in Hong Kong and the virus have established as enzootic viruses in China and other parts of the world [Claas et al., 1998; Enserink and Kaiser, 2004; Keawcharoen et al., 2004; Kuiken et al., 2004; Nguyen et al., 2005; Rimmelzwaan C 2015 WILEY PERIODICALS, INC.
et al., 2006]. 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 [Enserink and Kaiser, 2004; Keawcharoen et al., 2004; Kuiken et al., 2004; Rimmelzwaan et al., 2006]. Moreover, the H5N1 AIV can cross species barriers and adapt to new hosts [Kuiken et al., 2004]. Natural H5N1 AIV infections in several cat species have been reported in Southeast Asia [Claas et al., 1998; Enserink and Kaiser, 2004; Kuiken et al., 2004], and the experimental infection of domestic cats with similar viruses have also been reported [Rimmelzwaan et al., 2006]. Thus, the role of cats has been reconsidered in the transmission and spread of the H5N1 AIV. Direct contact with sick or diseased poultry has been implicated as the major risk factor for feline infection. Three clades, 2.3.4, 2.3.2, and 7.2, cocirculated in mainland China in recent years, and clade 2.3.2 has circulated widely in poultry in China and causing a new wave of cross-continental spreading from Asia to Europe since 2009 [Chen et al., 2006; Li et al., 2010; WHO, 2011]. It has become genetically distinguishable from the viruses isolated before 2007 and antigenically distinguishable from the vaccine strains widely used in China [Chen et al., 2006; Li et al., 2010]. Now, it has been detected in nine countries since 2007, which is highly pathogenic in
Conflict of interest: None. Correspondence to: Hui-Yun Chang, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province 730046, People’s Republic of China. E-mail: [email protected] Accepted 23 March 2015 DOI 10.1002/jmv.24216 Published online 7 May 2015 in Wiley Online Library (wileyonlinelibrary.com).
The Prevalence of H5N1 Avian Influenza Virus in Cats
chickens (100% mortality). It is also the dominant prevalence of H5N1 strains, especially in Southern China. The special climate, environment, and lifestyle in eastern China provide more chances for close contact among wild aquatic birds, domestic poultry, dogs or cats, and humans; therefore, creating an opportunity for interspecies transmission. Due to frequent cohabitation and interactions with other animals (dogs or avian) and humans, in southern China, different types of cats are uniquely positioned to act as a reservoir of influenza virus infection both within the household and the larger village farm or suburban environment. Cats are one of the most popular pets in China, so cats carrying AIV can pose a threat to human health. To date, no serological and virological studies about H5N1 AIVs infections have been carried out in stray cats in China. Therefore, this surveillance study is to uncover any evidence of H5N1 AIV, especially clade 2.3.2 viruses transmission in cats in China. MATERIALS AND METHODS Ethics Statement This study was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Permit No. LVRIAEC2011-006). Stray cats from which serum samples were collected were handled with good animal practices required by the Animal Ethics Procedures and Guidelines of the People’s Republic of China. The collection of serum samples was performed as a part of routine process of disease monitoring and surveillance for these cats. Cat Samples Between February 2010 and December 2011, a total of 1,020 nasal swab samples and 1,020 blood samples were collected from 24 different animal shelters in the Shanghai, Jiangsu, and Zhejiang Provinces in eastern China where H5N1 AIV are currently circulating or have been frequently detected in the past. They selected the sites based on their large and dense human, poultry, and pig populations which may be prone to cross-species transmission of influenza virus. In each site, the single largest animal shelters were selected. The cats were selected from the animal shelters based upon several factors: veterinary clinic location for geographical diversity and cats with no history of canine influenza vaccination. At each site, individual cats were selected by a random-number procedure among all the stray cats. Stray cats and bird are housed closely in farming villages, increasing the possibility of contacts among cats and wild animals. The sampling processes were assisted by local authorities and licensed veterinarians. Detection of antibodies to H5N1AIV. Cat nasal swab samples were initially screened for influenza A virus by real-time reverse transcription (RT-PCR)
1437
selective for the matrix gene. The reactions were carried out by using the Quaint Fast Multiplex RTPCR þ R Kit (QIAGEN, Valencia, CA) in a 20-ml reaction mixture containing 10 ml 2 quantitative RT-PCR master mix, 7.5 pmol of forward primer, 2.5 pmol of each reverse primer, 0.125 mmol/L EA minor groove binder probe, 0.0625 mmol/L of each NA minor groove binder probe, 0.4 ml 50 ROX reference dye, 0.2 ml of reverse transcription product, and 5 ml of extracted RNA. The reactions were performed on an M3000P QPCR System (Agilent Technologies, Inc., Santa Clara, CA) under these thermocycling conditions: 50˚C for 20 min, then 95˚C for 5 min, followed by 50 cycles of 97˚C for 2 sec and 60˚C for 40 sec. Samples that were positive by RT-PCR underwent further diagnostics for determination of subtype, including the human H1, H3 viruses, and avian H3, H5, H7, and H9 viruses [Li et al., 2010; WHO, 2011]. Sera were studied with a horse-RBC WHO-recommended hemagglutination inhibition (HI) assay against avian, human, and canine viruses [WHO, 2011]. All serum samples were treated with a receptor-destroying enzyme (RDE) and absorbed with erythrocytes in order to remove nonspecific inhibitors before detection. RDE was added to each serum specimen to inactivate nonspecific reactions and incubated at 37˚C overnight, followed by incubation at 56˚C for 30 min to inactivate RDE. Sera were next incubated and mixed with erythrocytes for 30 min at 4˚C. All serum samples were tested with HI assay as reported in previous studies [Su et al., 2013b]. Briefly, two-fold serial dilutions of serum samples were added in a V-shaped microtitre plate and 4 HA units of virus were added in each well. The mixture was incubated at room temperature for 35 min. Then, 1% (v/v) horse red blood cells (h-RBCs) were added in each well. The plates were left at room temperature for 40 min. The HI titers are expressed as the highest dilution of serum giving complete inhibition of agglutination. Sera from cats that were HI titer 20 by horse-RBC HI assay were confirmed with a microneutralization assay (MN) procedure [Kendal et al., 1982]. Briefly, positive sera were two-fold diluted in serum-free DMEM starting at a dilution of 1:10. Then, 100 TCID50 of virus were added to the serially diluted serum at a 1:1 ratio (v/v) and incubated at 37˚C for 1 hr. Finally, 0.2 ml of the virus–serum mixtures was transferred to 96-well monolayer plates and incubated in 5% CO2 at 37˚C for 72 hr. Three wells were run for each dilution of each serum sample. Cytopathic effects were checked for daily and cell supernatants were tested by HA test to confirm the virus infection. The endpoint for neutralization was expressed as the highest dilution that prevented hemagglutination in all three replicates. In this study, both HI and MN titer of 1:20 or greater was considered positive. For AIV antibody detection by both HI and MN assays, the HPAI H5N1 virus J. Med. Virol. DOI 10.1002/jmv
1438
Zhao et al.
[A/duck/Guangdong/1/2013(H5N1)] was used in this study. Virus A/duck/Guangdong/1/2013(H5N1) belonging to Clade 2.3.2 is the most dominant lineage from 2009 and suitable for a detection virus. To control for result quality, the HI assay was also performed for all serum with a virus derived from a recently circulating canine influenza virus (CIV) A/ canine/Lanzhou/1/2013 (H3N2) and a human pdm 09 virus A/California/7/2009(H1N1). Statistical Analysis The statistical tests about the association between serological outcome selected HI titer 40. Bivariate c2 test of independence or Fisher’s exact test were used to examine the association between the demographic characteristics and serological outcome. Covariates that had a P < 0.25 in the bivariate analyses were entered into a multivariable logistic regression model. The analysis was performed according to backward elimination of the covariates, keeping covariates in the model which had a P < 0.05. Final covariates were tested for goodness-of-fit. RESULTS From the total of 1,020 nasal swab samples collected, 19 (1.8%) were positive for influenza A virus by RT-PCR, and no viruses were isolated from these samples. Of the 19 nasal swab samples with RT-PCR-positive results for influenza A virus, 10 samples were H3N2 subtype CIV, 8 were H1N1 subtype human influenza viruses, and 1 were H9 subtype AIVs. None of 1,020 nasal swab samples were positive for influenza A (H5N1) virus by RTPCR. A total of 1,020 feline serum samples (461 from females and 559 from males) were examined using the MN and HI assays for H5N1 AIV antibodies. The serological screening revealed that only two of the sera (0.2%; OR ¼ 0.04; 95%CI [0.02–0.06], P < 0.05) from stray cats was positive according to the MN test by the Clade 2.3.2 virus, although 32 (3.1%; 95%CI [2.2–4.4], P < 0.05) samples were detected HI titers of 20 or more. All samples were also tested with other antigens (H1, H3) for the HI test (Table I), HI titers of 20 or more were detected in 101 (9.9%) of 1,020
serum (OR ¼ 9.9; 95%CI [8.1–11.9], P < 0.05) samples by used the H1N1 pdm09 antigens, twenty of the 101 samples were positive by the MN assay (MN > 1:20) (data not shown). However, a total of 108 (108/1020) sera were found HI antibody titer 1:20, 62 of these 108 samples were also positive by the MN assay (MN > 1:20) (data not shown). None of the three provinces studied had statistically significant different rates of cats infection with H5N1 AIV. However, for HI assay, samples from 2011 showed an increased risk of elevated antibodies against avian influenza virus H7N9 as compared to samples from 2010. Also, cats without signs of influenza-like-illness had a significant increased risk of elevated antibodies to canine H3N2 and pdm09 H1N1 as compared to cats with signs of influenzalike-illness. Influenza-like-illness had no significant effect on antibodies against H5N1 virus (Table II). DISCUSSION Stray cats are often seen living near live poultry markets and in rural areas of China. The special climate environment and lifestyle provide more chances for wild aquatic birds, domestic poultry, stray dogs, and humans to have close contact, and creates an opportunity for interspecies pathogens transmission [Su et al., 2013b]. Compared with pets, the stray cats in rural have more possibility to contact with the sick avian. Therefore, they were more easily infected with AIV. In addition, the stray cats are generally weaker due to these poor surroundings and they have a great possibility to be infected by wild avian influenza virus and are a threat to both veterinary and human public health. All of these indicate the importance of performing investigations of AIV infection among stray cats in different environments. In this study, we first investigated whether subclinical cat occurred infections with the H5N1 AIV among stray cats in eastern China. None of 1,020 nasal swab samples were positive for influenza A (H5N1) virus by RT-PCR and only two of the 1,020 serum samples were positive by both the HI assay (HI titer > 1:20) and MN assay (MN titer > 1:20). Thus, we found China’s first evidence of H5N1 AIV
TABLE I. Antibody Titers of H5N1 AIV; H1N1 pdm09 and H3N2 CIV Infections Using Hemagglutination Inhibition (HI) Assay HI titer Viral strainsa H3N2 H1N1 H5N1
No.
