during a first step ( november 2009february 2010 ) , seroprevalence rates for a(h1n1)pdm09 virus were assessed in 120 breeding pigs ( > 4 years old ) from 57 farms .
blood was obtained from randomly selected pigs at the only slaughterhouse on the island , where pigs are held for <3 hours .
we screened the samples for antibodies to influenza a viruses by using the i d screen antibody influenza a kit ( id.vet , montpellier , france ) , and titers were determined by using hemagglutination - inhibition ( hi ) assays ( 5 ) against all classical swine influenza viruses and a(h1n1)pdm09 virus ( table 1 ) .
ninety - eight ( 81.7% ; 95% ci 74.7%88.5% ) of the 120 serum samples were positive for a(h1n1)pdm09 virus ( hi titers > 20 ) ; the range of positive titers was 40640 , and 54.2% of the samples expressed high hi titers ( 160640 ) .
of the 98 serum samples , 5 reacted at low titer and with only 1 european a ( h1n1 ) swine virus ( titer < 20 ) , i.e. , > 4 dilutions lower than for a(h1n1)pdm09 virus , indicating cross reactivity ( 6 ) .
thus , pigs from 47 ( 82.4% ) of 57 tested farms had been infected by a(h1n1)pdm09 virus ; the seroprevalence rate was 81%100% for pigs on 79.0% of the farms .
farms with affected pigs were located throughout the island ( figure ) . * hi , hemagglutination inhibition . 
hi tests were performed according to standard procedure ( 5).titers are expressed as the reciprocal of the highest dilution of serum that inhibits 4 hemagglutination units of virus .  a(h1n1)pdm09 lineage .  
location of farms tested for antibodies against influenza a(h1n1)pdm09 virus in serologic surveys , runion island , 20092011 .
blue dots , seronegative farms ; red dots , seropositive farms . in a second step ( june 2010 , when a(h1n1)pdm09 infection was no longer detected among humans ) , we tested whether the virus was still circulating among pigs born that year . to obtain nasal swab and blood samples for testing
, we randomly selected 390 fattening pigs ( 2527 weeks old ) at the slaughterhouse ; the pigs originated from 45 farms . at the time of sampling ,
the veterinary surveillance system did not report any clinical signs suggesting virus circulation among herds .
however , 3.5% of the serum samples ( 9% of tested farms ) contained antibodies to a(h1n1)pdm09 virus ( hi titers 20160 ) . nasal swab specimens from 6.7% ( 26/390 ) of pigs were positive for a(h1n1)pdm09 virus as determined by using a specific real - time reverse transcription pcr ( rrt - pcr ) ; the pigs originated from 13 ( 28.8% ) farms ( 7 ) . two strains , a / sw / la reunion/0164/10 and a / sw / lareunion/110348/10 , were isolated onto mdck cell cultures ( 5 ) . during july  december 2010 ,
11 farms reported influenza - like clinical signs in pigs , and proof of a(h1n1)pdm09 virus infection was established on 3 farms ( farms a 
fattening pigs on farm a were seronegative for a(h1n1)pdm09 virus . in july , when acute respiratory disease was reported among pigs , 12 of 39 fattening pigs ( 1821 weeks old ) sampled on farm a were still seronegative for a(h1n1)pdm09 virus ; however , rrt - pcr results were positive for a(h1n1)pdm09 virus .
four weeks later , when pigs had recovered from influenza , only 7.7% ( 3/39 ) of sampled pigs on farm a had rrt - pcr results positive for a(h1n1)pdm09 virus , and all 39 were seropositive for the virus .
high rates of rrt - pcr positivity were also noted for pigs on farms b ( 17/30 pigs ) and c ( 6/15 pigs ) .
two a(h1n1)pdm09 strains ( a / sw / lareunion/0167/10 and a / sw / la reunion/110194/10 ) were isolated from pigs on farms a and b , respectively .
four influenza virus strains were isolated from pigs , and all induced a cytopathic effect and displayed hemagglutinating activity on chicken erythrocytes ; all 4 were confirmed as a(h1n1)pdm09 virus by specific rrt - pcrs . in addition cross - hi assays ( 5 ) revealed that these strains exhibit antigenic relationships with swine influenza a(h1n1 ) viruses from classical and avian - like lineages , although they reacted most strongly with a(h1n1)pdm09 virus ( table 1 ) .
genome sequencing of these strains showed high ( > 98% ) nucleotide sequence homology to the corresponding genes of a / california/04/09 and 2009 human strains from runion island , suggesting human - to - swine transmission ( h. pascalis , unpub .
data ) . in a third step ( march , july  august , and october 2011 ) ,
3 other sampling campaigns were conducted at the slaughterhouse , including 831 fattening pigs from 104 farms .
nasal swab samples for 7 ( 8.4% ) pigs from 3 ( 2.9% ) farms still had rrt - pcr  positive results .
however , serologic analyses revealed that pigs on 40% of the farms ( distributed throughout the island ) were seropositive for a(h1n1)pdm09 virus , indicating continuing circulation of the virus in swine herds ( table 2 ) .
seroprevalence rates are % farms positive at the herd level among all farms ( n ) and % pigs positive at the animal level among all animals ( n ) .
consistent with findings elsewhere ( 8) , our results show that a(h1n1)pdm09 virus has substantially affected swine herds in runion island .
results of our long - term ( 2 years ) investigation show that a(h1n1)pdm09 virus has circulated in pigs beyond the 5-week epidemic among humans during the austral winter 2009 ( 3 ) and has become a novel enzootic pathogen in runion island .
several facts may account for the heavy human - to - swine transmission of a(h1n1)pdm09 virus .
first , the reassortant pandemic virus contains genomic segments originating from swine influenza viruses established in pigs ( 1 ) .
second , pigs are highly susceptible to experimental inoculations with a(h1n1)pdm09 virus and support high intraspecies transmissibility ( 9 ) .
third , the pressure of infection caused by a(h1n1)pdm09 virus among humans in runion island was high but most infections were mild or asymptomatic ( 3 ) ; therefore , people pursued their professional activities , acting as silent spreaders of the virus .
last , pigs on runion island had no history of previous passages of swine influenza viruses ; thus , the lack of specific immunity to influenza a viruses would have contributed to the high sensitivity of the pigs to infection , as described ( 10,11 ) . despite serologic proof of large numbers of infected pigs during late 2009early 2010 in runion island ,
influenza - like signs were not exhibited and reported until july 2010 ; this finding was similar to that in new caledonia ( 8) . in july 2010 , several herds showed symptomatic changes in infection that could indicate either a change in virulence of the circulating strain or the intervention of co - infecting pathogens or some other environmental factor(s ) .
mycoplasma hyopneumoniae and pasteurella multocida were co - detected on farms with pigs with signs of infection ( data not shown ) .
co - infection with swine influenza virus and these bacteria is known to contribute to severe respiratory disorders among pigs ; thus , these bacteria may have enhanced pathogenicity of a(h1n1)pdm09 virus on affected farms ( 12 ) .
because specific immunity to a(h1n1)pdm09 virus will decline over time when the virus is no longer circulating among humans , persistence of the virus in an animal reservoir raises concerns about the risk for genetic evolution of the virus and retransmission back to humans of variants with potentially increased virulence . as an example , during the 2011 austral winter , only influenza a(h3n2 ) and b viruses were recorded ( 13 ) .
novel reassortant viruses containing genomic segments from a(h1n1)pdm09 and enzootic swine influenza viruses have been isolated in pigs ( 14 ) .
such a reassortant was responsible for several cases of influenza among humans in 2011 ( 15 ) ; these cases were mild , but other , more virulent pathogenic viruses could emerge . hence continuous surveillance of a(h1n1)pdm09 infection in pigs
 during 2009 , pandemic influenza a(h1n1)pdm09 virus affected humans on runion island . 
 since then , the virus has sustained circulation among local swine herds , raising concerns about the potential for genetic evolution of the virus and possible retransmission back to humans of variants with increased virulence . 
 continuous surveillance of a(h1n1)pdm09 infection in pigs is recommended . 
The Study
Conclusions