BACKGROUND
among the  <dig> hemagglutinin  subtype viruses detected among domestic birds, wild birds and bats, h <dig> subtypes have occasionally infected humans and generated significant public health concerns.
in  <dig>  a novel h7n <dig> avian influenza virus  was isolated from ill humans in shanghai and anhui province, china  <cit> .since then, the virus has spread quickly throughout the country.
as of august  <dig>   <dig>  a total of  <dig> human h7n <dig> infections  in china had been reported to the world health organization   <cit> .
recent research has shown that the novel h7n <dig> aiv may be transmissible between mammals   <cit> , <cit> .
the h7n <dig> aiv has also been isolated from one retail pork worker in shanghai.
h7n <dig> zoonosis was not preceded by overt epizootics in domestic poultry or other avian species in the wild, and the source of human infection remains to be definitively established  <cit> .
in response to the reported human infections with h7n <dig> virus, the ministry of agriculture of the people’s republic of china expanded and enhanced surveillance in live bird markets and poultry farms, as well as in swine farms and slaughterhouses, across the whole country, particularly in the affected region and surrounding provinces of southern china.
within six weeks of the initial case report, testing of tens of thousands of samples from poultry and their environment resulted in the identification of  <dig> h7n <dig> virus isolates from the provinces of anhui, guangdong, zhejiang, fujian, and jiangsu, as well as the shanghai municipality, mostly from live poultry markets  <cit> .
for multiple reasons, it seems biologically plausible that pigs could be involved in the ecology of this emergent h7n <dig> virus in southern china.
some large-scale swine farms are adjacent to lakes that are home to multiple species of wild birds.
in southern china, many farms raise pigs and poultry in close proximity.
recently, avian-origin h7n <dig> influenza viruses have been isolated from pigs in south korea  <cit> .
the isolation of swine h7n <dig> virus suggests zoonotic significance, highlighting the probable transmission of h <dig> subtype aivs through pigs and prompting us to perform surveillance in this species.
prior to the recent zoonosis of h7n <dig>  there were very few studies on the seroprevalence of the h <dig> subtypes in pigs.
serologic surveys are urgently needed to help us understand outbreaks of influenza a  virus.
thus, we conducted surveillance from january  <dig> to november  <dig> in the jiangsu, zhejiang, guangdong and fujian provinces in eastern china to evaluate whether pigs were infected with the novel h7n <dig> virus.
methods
swine farms in southern china vary in size from large-scale farms  to backyard farms .
farms often raise pigs indoors, have little biosafety and seldom use swine influenza vaccines.
many pigs on swine farms have contact with wild birds or domestic poultry.
we worked with local veterinary department leaders who regularly inspect swine farms in jiangsu, zhejiang, guangdong and fujian provinces of southern china, where novel h7n <dig> aiv are currently circulating or were frequently detected in  <dig>  for inclusion in the study, we selected swine farms by location , and we studied only pigs on large-scale farms that denied having a swine influenza vaccine program.
pigs were selected using a stratified random sampling method.
three groups of pigs were selected from each farm: weaning pigs, finishing pigs, and sows.
no anesthesia was used during phlebotomy.
all the owners of the swine farms gave permission for their animals to be sampled in this study.
pigs were restrained by rope or wire loop while a health professional performed the  <dig> - <dig>  ml serum draw.
our sampling processes were assisted by local authorities and licensed veterinarians.
the animal research in this study protocol was reviewed and approved by the guangdong center for disease control and prevention.
as reported in previous studies  <cit> , we employed a hemagglutination inhibition  assay according to the who’s protocol.
an h <dig> antigen ) derived from the emergent h7n <dig> aiv was obtained from the college of veterinary medicine, south china agricultural university.
negative control serum  and positive control  were also prepared by the college of veterinary medicine, south china agricultural university.
all serum samples were treated with a receptor-destroying enzyme  and absorbed with erythrocytes to remove nonspecific inhibitors before use in the assays.
briefly, two-fold serial dilutions of serum samples were added to v-shaped micro titer plates, and  <dig> ha units of virus were added to each well.
the mixture was incubated at room temperature for 35 minutes.
then, 1%  horse erythrocytes were added in each well.
the plates were left at room temperature for 40 minutes.
the hi titers were expressed as the highest dilution of serum producing complete inhibition of agglutination.
all positive control serum specimens had hi titers of 1: <dig> or more.
we isolated the other aiv antigens used in this study: low pathogenic avian influenza viruses  a/chicken/guangdong/v/ <dig> , at the college of veterinary medicine, south china agricultural university.
the lpaiv a/chicken/guangdong/v/ <dig> , beijing/1/94-like, was selected for a control as it is the most prevalent subtype of influenza virus in poultry in china  <cit> .
the antigens a/california/7/2009pandemic   <dig> virus, recently circulating among pigs in china, was also used for hi testing.
negative control serum and positive control serum specimens from different species were included in each plate to provide a full range of controls.
sera from pigs that were hi titer ≥  <dig> by horse-rbc hi assay were confirmed with a microneutralization assay  procedure recommended by the who  <cit> .
both the hi titer ≥ 1: <dig> and mn titer ≥ 1: <dig> were considered as positive evidence of previous h7n <dig> infection.
RESULTS
during the period between january  <dig> and november  <dig>  we drew blood from a total of  <dig> pigs from  <dig> farms in jiangsu, zhejiang, guangdong and fujian provinces of southern china.
these  <dig> provinces have had numerous recent detections of h7n <dig>  a total of  <dig> of the  <dig> pigs had clinical signs of influenza-like infection .
the results from the  <dig> pig blood samples were obtained by hi assay and mn assay.
hi titers of ≥1: <dig> were detected in  <dig> of the  <dig> serum samples using the h9n <dig> aiv antigens.
only  <dig> of the  <dig> samples had hi titers of ≥1: <dig>  using the h7n <dig> aiv antigens.
although  <dig> of the  <dig> h9n <dig> aiv hi-positive samples were also positive by the mn assay , none of the  <dig> h7n <dig> aiv hi-positive samples were positive by the mn assay , revealing that no samples were positive for h7n <dig>  as a control,  <dig> %  of sera were found positive by hi assay  for h1n <dig> pdm <dig>  and  <dig> %of these hi-positive samples were also positive by the mn assay  .
ili = influenza-like-illness; hi = horse rbc hemagglutination inhibition assay; mn = microneutralization assay; h9n2 = a/chicken/guangdong/v/2008; h1n1 = a/california/7/2009pandemic  ; h7n9 = a/guangdong/gh074/ <dig>
none of the four provinces studied had statistically significant different rates of swine infection with avian influenza virus.
however, by hi assay, samples from  <dig> showed an increased risk of elevated antibodies against avian influenza virus h7n <dig> as compared to samples from  <dig>  sows had a significant increased risk of elevated antibodies against avian influenza virus h7n <dig>  and finishing pigs had increased risk , compared to weaning pigs.
also, pigs without signs of influenza-like-illness had a significant increased risk of elevated antibodies to avian h9n <dig> and pdm <dig> h1n <dig> as compared to pigs with signs of influenza-like-illness.
influenza-like-illness had no significant effect on antibodies against h7n <dig> virus .
hi geometric mean antibody titers  of aiv h9n <dig> and h1n <dig> pdm <dig> were  <dig>  and  <dig>  respectively .
the gmt of h7n <dig> was  <dig> , indicating again that all serum samples were negative.
pigs were studied during the period january  <dig> to november  <dig>
discussion
china has been identified as a hot spot for the generation of novel influenza viruses.
in this report, we identified large farms that seemed to have a high probability of exposure to the novel h7n <dig> virus and found no evidence of infection in pigs.
the surveillance of pigs has focused on the subtype of h <dig>  h <dig>  h <dig> and h <dig>  <cit> - <cit> , while prior to the recent zoonosis of h7n <dig>  <cit> , <cit> - <cit> , <cit> , <cit> , there were very few studies on the seroprevalence of the h <dig> subtype.
our results matched the results reported previously, with an  <dig> % prevalence of h <dig> infection.
although we found no definitive serological evidence of avian h <dig> influenza virus in swine in southern china,  <dig> % of the specimens had an hi titer ≥1: <dig>  this suggests that pig herds of southern china may have some potential risks of subtype h <dig> infection.
while we agree that pigs have potential to serve as mixing vessels for the novel influenza virus generation  <cit> , <cit> , this study found no evidence that pigs were involved in the ecology of the h7n <dig> virus.
our study had a number of limitations.
first, it is possible that results may have been confounded due to serologic cross-reactivity between different viral subtypes.
in our study, the possibility of cross-reaction between subtypes was not evaluated.
however, there were no positive serum against h7n <dig> via two serological methods , which indicates that no cross-reaction occurred between h7n <dig> and other avian viruses of this study.
second, we did not study pigs from small farms or slaughterhouses, and thus our study population may not be representative of all pigs in these provinces.
CONCLUSIONS
the unique environment on swine farms and live animal markets  in southern china provides many opportunities for wild aquatic birds, domestic poultry, and pigs to come in close contact with one another, thereby increasing the likelihood of interspecies transmission and generation of novel influenza viruses through reassortment  <cit> , <cit> .
as novel viruses may change rapidly, it seems prudent to continue surveillance for the h7n <dig> virus among non-poultry domestic animal species such as pigs and dogs  <cit> , <cit> .
zhu et al.
reported that pigs were infected productively by human-isolated h7n <dig> influenza virus and shed virus for 6 days  <cit> .
xu and colleagues reported rapid adaptation of avian h7n <dig> virus in pigs; the avian h7n <dig> virus replicated to a high titer after only one passage  <cit> .
p. horby suggests that a hidden epidemic of h7n <dig> in other animals may be well under way and that it will provide opportunities for further adaptation of h7n <dig> to mammals and for re-assortment with human- or pig-adapted viruses if h7n <dig> is circulating in the large populations of pigs  <cit> .
therefore, such studies are crucial in understanding and reducing risk factors for human h7n <dig> infection in china.
ethical approval
this study protocol was reviewed and approved by the institutional review board of south china agricultural university.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
pz and gz designed the experiments.
mh, lw, hh carried out the tests.
pz, mmm and ls drafted the manuscript.
all authors have read and approved the final manuscript.

 among the hemagglutinin subtype viruses detected among domestic birds, wild birds and bats, h subtypes have occasionally infected humans and generated significant public health concerns. in a novel h7n avian influenza virus was isolated from ill humans in shanghai and anhui province, china.since then, the virus has spread quickly throughout the country. as of august a total of human h7n infections in china had been reported to the world health organization. recent research has shown that the novel h7n aiv may be transmissible between mammals,. the h7n aiv has also been isolated from one retail pork worker in shanghai. h7n zoonosis was not preceded by overt epizootics in domestic poultry or other avian species in the wild, and the source of human infection remains to be definitively established. in response to the reported human infections with h7n virus, the ministry of agriculture of the people’s republic of china expanded and enhanced surveillance in live bird markets and poultry farms, as well as in swine farms and slaughterhouses, across the whole country, particularly in the affected region and surrounding provinces of southern china. within six weeks of the initial case report, testing of tens of thousands of samples from poultry and their environment resulted in the identification of h7n virus isolates from the provinces of anhui, guangdong, zhejiang, fujian, and jiangsu, as well as the shanghai municipality, mostly from live poultry markets. for multiple reasons, it seems biologically plausible that pigs could be involved in the ecology of this emergent h7n virus in southern china. some large-scale swine farms are adjacent to lakes that are home to multiple species of wild birds. in southern china, many farms raise pigs and poultry in close proximity. recently, avian-origin h7n influenza viruses have been isolated from pigs in south korea. the isolation of swine h7n virus suggests zoonotic significance, highlighting the probable transmission of h subtype aivs through pigs and prompting us to perform surveillance in this species. prior to the recent zoonosis of h7n there were very few studies on the seroprevalence of the h subtypes in pigs. serologic surveys are urgently needed to help us understand outbreaks of influenza a virus. thus, we conducted surveillance from january to november in the jiangsu, zhejiang, guangdong and fujian provinces in eastern china to evaluate whether pigs were infected with the novel h7n virus. methods swine farms in southern china vary in size from large-scale farms to backyard farms. farms often raise pigs indoors, have little biosafety and seldom use swine influenza vaccines. many pigs on swine farms have contact with wild birds or domestic poultry. we worked with local veterinary department leaders who regularly inspect swine farms in jiangsu, zhejiang, guangdong and fujian provinces of southern china, where novel h7n aiv are currently circulating or were frequently detected in for inclusion in the study, we selected swine farms by location, and we studied only pigs on large-scale farms that denied having a swine influenza vaccine program. pigs were selected using a stratified random sampling method. three groups of pigs were selected from each farm: weaning pigs, finishing pigs, and sows. no anesthesia was used during phlebotomy. all the owners of the swine farms gave permission for their animals to be sampled in this study. pigs were restrained by rope or wire loop while a health professional performed the -  ml serum draw. our sampling processes were assisted by local authorities and licensed veterinarians. the animal research in this study protocol was reviewed and approved by the guangdong center for disease control and prevention. as reported in previous studies, we employed a hemagglutination inhibition assay according to the who’s protocol. an h antigen ) derived from the emergent h7n aiv was obtained from the college of veterinary medicine, south china agricultural university. negative control serum and positive control were also prepared by the college of veterinary medicine, south china agricultural university. all serum samples were treated with a receptor-destroying enzyme and absorbed with erythrocytes to remove nonspecific inhibitors before use in the assays. briefly, two-fold serial dilutions of serum samples were added to v-shaped micro titer plates, and ha units of virus were added to each well. the mixture was incubated at room temperature for 35 minutes. then, 1% horse erythrocytes were added in each well. the plates were left at room temperature for 40 minutes. the hi titers were expressed as the highest dilution of serum producing complete inhibition of agglutination. all positive control serum specimens had hi titers of 1: or more. we isolated the other aiv antigens used in this study: low pathogenic avian influenza viruses a/chicken/guangdong/v/, at the college of veterinary medicine, south china agricultural university. the lpaiv a/chicken/guangdong/v/, beijing/1/94-like, was selected for a control as it is the most prevalent subtype of influenza virus in poultry in china. the antigens a/california/7/2009pandemic virus, recently circulating among pigs in china, was also used for hi testing. negative control serum and positive control serum specimens from different species were included in each plate to provide a full range of controls. sera from pigs that were hi titer ≥  by horse-rbc hi assay were confirmed with a microneutralization assay procedure recommended by the who. both the hi titer ≥ 1: and mn titer ≥ 1: were considered as positive evidence of previous h7n infection.  during the period between january and november we drew blood from a total of pigs from farms in jiangsu, zhejiang, guangdong and fujian provinces of southern china. these provinces have had numerous recent detections of h7n a total of of the pigs had clinical signs of influenza-like infection. the results from the pig blood samples were obtained by hi assay and mn assay. hi titers of ≥1: were detected in of the serum samples using the h9n aiv antigens. only of the samples had hi titers of ≥1: using the h7n aiv antigens. although of the h9n aiv hi-positive samples were also positive by the mn assay, none of the h7n aiv hi-positive samples were positive by the mn assay, revealing that no samples were positive for h7n as a control, % of sera were found positive by hi assay for h1n pdm and %of these hi-positive samples were also positive by the mn assay. ili = influenza-like-illness; hi = horse rbc hemagglutination inhibition assay; mn = microneutralization assay; h9n2 = a/chicken/guangdong/v/2008; h1n1 = a/california/7/2009pandemic; h7n9 = a/guangdong/gh074/ none of the four provinces studied had statistically significant different rates of swine infection with avian influenza virus. however, by hi assay, samples from showed an increased risk of elevated antibodies against avian influenza virus h7n as compared to samples from sows had a significant increased risk of elevated antibodies against avian influenza virus h7n and finishing pigs had increased risk, compared to weaning pigs. also, pigs without signs of influenza-like-illness had a significant increased risk of elevated antibodies to avian h9n and pdm h1n as compared to pigs with signs of influenza-like-illness. influenza-like-illness had no significant effect on antibodies against h7n virus. hi geometric mean antibody titers of aiv h9n and h1n pdm were and respectively. the gmt of h7n was, indicating again that all serum samples were negative. pigs were studied during the period january to november discussion china has been identified as a hot spot for the generation of novel influenza viruses. in this report, we identified large farms that seemed to have a high probability of exposure to the novel h7n virus and found no evidence of infection in pigs. the surveillance of pigs has focused on the subtype of h h h and h -, while prior to the recent zoonosis of h7n, -,,, there were very few studies on the seroprevalence of the h subtype. our results matched the results reported previously, with an % prevalence of h infection. although we found no definitive serological evidence of avian h influenza virus in swine in southern china, % of the specimens had an hi titer ≥1: this suggests that pig herds of southern china may have some potential risks of subtype h infection. while we agree that pigs have potential to serve as mixing vessels for the novel influenza virus generation,, this study found no evidence that pigs were involved in the ecology of the h7n virus. our study had a number of limitations. first, it is possible that results may have been confounded due to serologic cross-reactivity between different viral subtypes. in our study, the possibility of cross-reaction between subtypes was not evaluated. however, there were no positive serum against h7n via two serological methods, which indicates that no cross-reaction occurred between h7n and other avian viruses of this study. second, we did not study pigs from small farms or slaughterhouses, and thus our study population may not be representative of all pigs in these provinces.  the unique environment on swine farms and live animal markets in southern china provides many opportunities for wild aquatic birds, domestic poultry, and pigs to come in close contact with one another, thereby increasing the likelihood of interspecies transmission and generation of novel influenza viruses through reassortment,. as novel viruses may change rapidly, it seems prudent to continue surveillance for the h7n virus among non-poultry domestic animal species such as pigs and dogs,. zhu et al. reported that pigs were infected productively by human-isolated h7n influenza virus and shed virus for 6 days. xu and colleagues reported rapid adaptation of avian h7n virus in pigs; the avian h7n virus replicated to a high titer after only one passage. p. horby suggests that a hidden epidemic of h7n in other animals may be well under way and that it will provide opportunities for further adaptation of h7n to mammals and for re-assortment with human- or pig-adapted viruses if h7n is circulating in the large populations of pigs. therefore, such studies are crucial in understanding and reducing risk factors for human h7n infection in china. ethical approval this study protocol was reviewed and approved by the institutional review board of south china agricultural university. competing interests the authors declare that they have no competing interests. authors’ contributions pz and gz designed the experiments. mh, lw, hh carried out the tests. pz, mmm and ls drafted the manuscript. all authors have read and approved the final manuscript. 
however, prior to the recent zoonosis of h7n there were very few studies on the seroprevalence of the h subtypes in this species. in a novel h7n avian influenza virus was isolated from ill humans in shanghai and anhui province, china. pigs in southern china have been shown to be infected with multiple avian influenza viruses. only % of the pig sera had antibodies with an hi titer ≥1: and none had an mn titer ≥1: against the h antigen. thus, no samples were found to be positive against h7n however, % of the pig sera had antibodies with hi titer ≥1: and % by mn titer ≥1: against h antigen. thirty-seven percent of the pig sera had antibodies with hi titer ≥1: and % had mn titer ≥1: against pandemic this surveillance may help us understand risk factors for outbreaks of influenza a virus.
 in a novel h7n avian influenza virus was isolated from ill humans in shanghai and anhui province, china. since then, the virus has spread quickly throughout china. previous isolation of h7n virus from swine suggests that additional h subtype aivs may be transmitted through pigs. however, prior to the recent zoonosis of h7n there were very few studies on the seroprevalence of the h subtypes in this species. thus, there is a need to perform serological surveys for novel h7n as well as other h subtype aivs in swine. this surveillance may help us understand risk factors for outbreaks of influenza a virus.  only % of the pig sera had antibodies with an hi titer ≥1: and none had an mn titer ≥1: against the h antigen. thus, no samples were found to be positive against h7n however, % of the pig sera had antibodies with hi titer ≥1: and % by mn titer ≥1: against h antigen. thirty-seven percent of the pig sera had antibodies with hi titer ≥1: and % had mn titer ≥1: against pandemic  pigs in southern china have been shown to be infected with multiple avian influenza viruses. as the prevalence of novel influenza a viruses may be increasing among poultry in china, similar seroepidemiological studies of pigs should be conducted in the future. h7n9aivpig 