the siv h1n1 and h3n2 subtypes have been circulating for more than 30 years , and the siv subtype h1n2 has been circulating since it was first isolated in great britain in 1994 [ 2 , 3 ] . in april 2009 , a new influenza a virus of subtype h1n1 emerged in the human population in mexico and the united states .
this was a multiple reassortant virus containing genes from north american and eurasian lineages [ 4 , 5 ] .
human - to - pig transmission was first reported in canada in late april 2009 , then in european pig population in early september 2009 , and has since been reported in several countries all over the world [ 69 ] . in october 2009 ,
siv was reported for the first time in norway when an integrated pig herd tested positive for pandemic ( h1n1 ) 2009 virus after showing mild clinical signs of respiratory disease .   the clinical picture of pandemic ( h1n1 ) 2009 infections in experimentally and naturally infected pigs was described as mild respiratory illness , increased temperature , and decreased appetite [ 6 , 11 , 12 ] . in some infected herds , clinical signs were not detected .
studies have shown that immunological nave pigs experimentally infected with pandemic ( h1n1 ) 2009 virus could transmit the virus efficiently to other nave pigs [ 11 , 14 , 15 ] .
although pandemic ( h1n1 ) 2009 virus contains gene segment genetically related to other swine influenza virus strains circulating in europe and north america , it shows antigenetic differences in the major glycoproteins of the virus .
however , it has been shown that pigs infected or vaccinated with h1n1 european avian - like swine influenza virus could produce cross - reactive antibodies against pandemic ( h1n1 ) 2009 virus which protected pigs against the infection of pandemic ( h1n1 ) 2009 virus [ 13 , 16 ] .  
the pig production in norway consists of approximately 2500 herds , mainly small family farms , with about 1.5 million slaughtered pigs in 2010 .
national surveillance and control program shows that the pig population is free for aujeszky 's disease ( ad ) , transmissible gastroenteritis ( tge ) , porcine respiratory corona virus ( prcv ) , porcine reproductive and respiratory syndrome ( prrs ) , and mycoplasma hyopneumoniae [ 17 , 18 ] .
the norwegian food safety authority has been running annual surveillance programs to document the status since 1994 .
blood samples from about 500 randomly selected herds are collected annually for specific disease surveillance .
the surveillance has never detected siv infection in norway until october 2009 , except for pigs in one herd that tested seropositive for subtype h3n2 in 1998 without showing clinical signs or further spread of the infection .  
this paper consists of studies that describe the initial spread of pandemic ( h1n1 ) 2009 virus amongst norwegian pig herds , the control measures initiated , and the infection status 20 months after the introduction .
the paper also discusses the manifestation of the infection in the norwegian pigs given that they had no prior immunity to any influenza a viruses and are free from most other viral respiratory diseases .
following the primary outbreak in october 2009 , an intensified surveillance was immediately initiated to investigate the extent of siv infection in the pig population in norway , referred to as study 1 in the results section .
the study targeted the following categories of herds : ( i ) herds where pigs with influenza - like signs were observed , ( ii ) herds where the pigs had been exposed to humans with verified pandemic ( h1n1 ) 2009 virus infection or influenza - like illness ( ili ) , and ( iii ) herds with a history of contact with or in close proximity to infected herds through transfer of animals , usage of common equipment , for example , transport vehicles to slaughterhouse , and visitations from farmers of infected farms . in herds suspected to have an active infection , nasal swabs were collected from 20 pigs at different ages , with priority of sampling pigs with clinical symptoms . in herds without clinical signs , blood samples were collected from at least 20 pigs ( sows or slaughtered pigs ) for serological testing .
calculation of sample size was done assuming 100% test sensitivity and a within - herd prevalence > 15% .
the sample size implies that the risk of having 20 nonreactors from a positive farm is less than 4% .   during the follow - up study
, there was a special focus on the prevalence in nucleus and multiplier herds , hereafter referred to as study 2 .
the nucleus herds are closed herds meaning that they have no contact with other herds except via semen from the norsvin ai station .
the multiplying herds are either closed or obtain replacement sows from a single nucleus herd .
serological surveillance was carried out to investigate the possible spread of pandemic ( h1n1 ) 2009 to these herds in the early phase of the outbreak . between 1st of october 2009 and 31st of january 2010
, blood samples were collected from 20 slaughtered pigs from 112 nucleus and multiplier herds .  
a third study was initiated to follow the course of the pandemic ( h1n1 ) 2009 infection . from the herds that tested positive for pandemic ( h1n1 ) 2009 in 2009
, 38 herds consisting of 13 nucleus herds , 20 multiplier herds , and five conventional integrated herds were selected for the study . from each herd , blood samples from 20 slaughtered pigs were tested for antibodies against pandemic ( h1n1 ) 2009 .
blood samples were taken on the condition that 6 to 12 months had elapsed since the herd first tested positive and that the pigs tested were born at least 2 months after that time .
an additional testing was performed in 9 herds about 18 months after the pandemic ( h1n1 ) 2009 introduction .  
study 4 consists of data from the national surveillance programme for si in norwegian pigs in 2009 and 2010 .
the program includes all nucleus and multiplier breeding herds and the nucleus unit of all sow pools .
in addition , a random selection of conventional pig herds , 300 integrated and piglet - producing herds and 60 fattening herds , was selected from a population of approximately 2500 herds .
ten pigs in each herd were tested . with the exception of pigs from finishing herds , which are sampled at the abattoirs ,
clinical and epidemiological information recorded by the food safety authority was available from 43 pig herds suspected to be infected .
the information was collected , while the herd was being sampled during the early phase of the outbreak between 10th of october 2009 and 31st of december 2009 .
the size of these herds ranged in numbers from 5 to 2000 pigs ( average 550 pigs ) .
the investigation used a structured questionnaire consisting of questions on epidemiological information including clinical picture within the herd , likely source of infection , contact with people diagnosed with pandemic influenza or had ili symptoms and being in contact with pigs while sick , movement of animals in or out of the farm , and handling and biosecurity measures .
initially , serum samples were analyzed for specific antibodies against a / sw / belgium/1/98(h1n1 ) and a / sw / flanders/1/98(h3n2 ) using hemagglutination - inhibition ( hi ) assays according to the method described in the oie manual of diagnostic tests and vaccines for terrestrial animals .
following introduction of pandemic ( h1n1 ) 2009 virus in the norwegian pig population , serum samples were tested for influenza - a - specific np antibodies using an elisa kit ( i d screen influenza a antibody competition test , i d vet , montpellier , france ) .
a herd was considered seropositive if three or more blood samples from the herd were positive for influenza - a - specific np antibodies . in case one or two of the 20 blood samples from a herd
were elisa positive , the herd was retested with samples from another 20 pigs , and the herd concluded positive if at least one of these samples was seropositive .
positive and/or inconclusive samples were retested for hemagglutinin - specific antibodies using hi assay with a / sw / belgium/1/98(h1n1 ) , a / sw / flanders/1/98(h3n2 ) , and a / california/07/2009 ( pandemic ( h1n1 ) 2009 ) as antigens .
the antigens for the hi tests were produced in chicken eggs at the norwegian veterinary institute in oslo .
nasal swabs ( copan innovation ltd , brescia , italy ) , individual or in pools of two , were placed in 1  ml of transport medium ( emem 2% ibfs / tris ) before they were shipped to the laboratory . total rna was extracted from 200  l of the sample material using the automatic extraction instrument nuclisens easymag ( biomrieux , norge as , oslo , norway ) according to the manufacturer 's instruction .
detection of influenza a virus was performed as described by the world health organization , the who collaborating centre for influenza at cdc atlanta .
specific detection of the ha gene of the pandemic ( h1n1 ) 2009 subtype was carried out on influenza - a - positive samples as described by robert koch institute .
amplification was performed on a stratagene mx3500p ( lajolla , ca , usa ) with superscript iii platinum one - step quantitative rt - pcr system ( invitrogen , paisley , uk ) .
in addition , some samples were analyzed at dtu veterinary ( national veterinary institute , technical university of denmark ) using an inhouse real - time rt - pcr for detection of pandemic ( h1n1 ) 2009 virus ( ref statens serum institut , denmark , unpublished ) .
one rt - pcr - positive sample was sufficient to confirm a herd as positive for pandemic ( h1n1 ) 2009 virus .  
full genome sequencing of isolate a / sw / norway/02_11342/2009(h1n1 ) was carried out through a modified version of the who sequencing primers and protocol provided by the who collaborating centre for influenza in cdc , atlanta , usa ( 28 april 2009 ) .
briefly , 1  l extracted rna was amplified using 46 primer pairs and a one - step rt - pcr kit ( qiagen , oslo , norway ) .
the resulting overlapping amplicons , carrying primer - derived m13 terminal sequences , were sequenced with m13 primers using the abi bigdye terminator v1.1 cycle sequencing kit and the abi 3130 genetic analyzer ( applied biosystems , warrington , uk ) .
sequences were assembled using sequencher v.4.9 ( genecodes corporation , ann arbor , mi , usa ) and compared to related h1n1 virus sequences using bioedit software .
descriptive statistics and calculation of true population prevalence using confidence intervals were used . to measure the association between county prevalence and the pig density by county
after the introduction of pandemic ( h1n1 ) 2009 , the intensified surveillance ( study 1 ) shows that 54 of the 114 herds tested were positive for pandemic ( h1n1 ) 2009 virus by rt - pcr , and 55 of the 140 herds tested by serology were positive for antibodies against pandemic ( h1n1 ) 2009 . altogether
91 herds tested positive from 10th of october to 31st of december 2009 . in the 55 seropositive herds , 59% ( range 5% to 100% ) of the individual samples from each herd were positive . from the 54 rt - pcr - positive herds ,
an average of 65% ( range 5% to 100% ) of the individual samples from each herd were positive .
geographically , the infected herds were found throughout norway ( figure 1 ) . in 2010 , two out of nine herds tested were positive for pandemic ( h1n1 ) 2009 by rt - pcr . during the first six months of 2011 ,
these herds were sampled on suspicion of infection with siv based on clinical signs in the pigs . in study 2 , a total of 2349 blood samples from 112 nucleus and multiplier herds were tested for antibodies against influenza a virus .
testing of the nucleus herds was completed by the 18th of december 2009 , and the multiplier herds were finished on the 31st of january 2010 .
the surveillance shows that 27.1% of the nucleus herds and 34.4% of the multiplier herds were seropositive for pandemic ( h1n1 ) 2009 .   between six and 12 months after a herd
was seropositive for antibodies against pandemic ( h1n1 ) 2009 virus , blood samples were collected from slaughtered pigs born at least two months after the initial detection ( study 3 ) . in total , 1127 pigs from 38 herds were tested .
three of the 38 herds tested positive for antibodies against pandemic ( h1n1 ) 2009 virus .
the seropositive herds were multiplier herds located in the south west region where pig farming density is highest ( figure 1 ) .
pigs from all the 13 previously positive nucleus herds selected for the study were found negative at this time . however , the intensified surveillance is still ongoing in the nucleus herds . by the end of july 2011 , nine of the 13 nucleus herds
pigs from five of these herds were positive for antibodies against pandemic ( h1n1 ) 2009 virus , between 12 to 20 months of time after the primary infection in the herds .   within the 2009 routine surveillance of study 4 ,
4724 samples from 452 herds were tested for antibodies against siv ( table 1 ) . in total , 16 herds were found seropositive for pandemic ( h1n1 ) 2009 . retrospectively , the earliest seropositive samples were collected from a herd in the county of rogaland on the 30th of september 2009 , suggesting that pandemic ( h1n1 ) 2009 has been circulating in the norwegian pig population since september 2009 .
the results from the surveillance program in 2010 show that 41% of 459 tested herds were seropositive ( table 1 ) .
the proportion of seropositive herds in different counties varied from 21% in the low pig density counties of vestlandet to 57% in the high pig density county of rogaland and agder .
simple regression analysis shows that herd prevalence is positively correlated ( r = 0.013 , p = 0.023 ) with the pig density by county ( figure 1 ) .
moreover , the surveillance program in 2010 shows that 42% ( 19 positive/45 tested herds ) of nucleus and 44% ( 30 positive/68 tested herds ) of multiplier herds were seropositive .  
questionnaires with epidemiological information were available from 35 pig herds confirmed positive by laboratory testing ( serology or pcr ) . in 60% ( 21/35 ) of these infected herds
, there was evidence to suggest that humans were the most likely source of infections . in these herds ,
the farmer , farm worker , or the farmer 's family members were diagnosed with pandemic ( h1n1 ) 2009 ( 33% or 7/21 ) or had ili just before or during the outbreak ( 66% or 14/21 ) .
twenty percent ( 7/35 ) reported contact with another infected farm through humans or movement of infected animals as source of infection .
one farmer of a positive herd had bought pigs from a positive herd and also had one farm worker with ili during this period .
forty - nine percent ( 17/35 ) had reported that clinical signs were observed in the pigs .
the most common clinical signs reported were coughing , fever , and loss of appetite , lasting for less than two weeks .
nineteen percent ( 8/43 ) of the herds from which epidemiological information was gathered tested negative although 3 of these herds had histories of contact with humans with ili .
full genome sequence analysis was carried out on nasal swab rna from an infected pig in the index herd , as well as from the virus isolated from a sick staff member at the same farm .
the sequences of these two full viral genomes were virtually identical ( 99.9985% identity ) ( acc .
no jq253790-jq253797 ) . moreover , the ha sequence showed at least 99.8% identity to other pandemic ( h1n1 ) 2009 circulating in humans in norway at the same time .
the complete sequence further indicated that the virus had not acquired known resistance mutations in the neuraminidase gene .
pandemic ( h1n1 ) 2009 virus was first detected in norwegian pigs on the 10th of october 2009 .
as it was reported that the pandemic ( h1n1 ) 2009 virus was transmitted rapidly between humans and also from humans to pigs , the norwegian food safety authority and the pig industry initiated biosecurity measures such as use of gloves and gauze masks to protect the norwegian pig population from this virus . following the detection of the index case , an intense screening based on detection of virus and antibodies against the virus
, a total of 91 herds had tested positive for pandemic ( h1n1 ) 2009 virus and/or antibodies against the virus .
our investigations showed that despite rapid implementation of control measures , the virus had in a short period of time been spread to pig herds throughout norway , including herds that had high biosecurity and to closed herds .  
the first confirmed human case of pandemic ( h1n1 ) 2009 virus in norway was in may 2009 .
it was followed by a first minor wave of infected people during the summer and a second major wave in the autumn . shortly after the onset of the autumn wave , the first pig herd positive for pandemic ( h1n1 ) 2009 virus was detected .
hence , the farmer and family members must care for the pigs even when being ill .
many of the first positive pig herds had been in contact with people that were confirmed with pandemic ( h1n1 ) 2009 virus infection or people with ili .
herd - to - herd transmission was ruled out because there was no history of contact between herds that were positive for the virus or were seropositive .
it is also known that many people could be infected with pandemic ( h1n1 ) 2009 without showing clinical symptoms .
this suggests that infected humans , with or without clinical symptoms , were an important factor in the introduction and transmission of pandemic ( h1n1 ) 2009 virus to the norwegian pig population .
sequencing analysis of virus isolated from humans and from pigs indicated that almost identical viruses had infected both species .
furthermore , the high prevalence in nucleus and multiplying herds , that normally have no or few contacts with other herds , indicates that humans , and not trade with pigs , were responsible for introduction of pandemic ( h1n1 ) 2009 virus to norwegian pigs .  
the national surveillance program for siv in norway shows that the first herd positive for antibodies against pandemic ( h1n1 ) 2009 virus was sampled on the 30th of september 2009 . as shown in table 1 ,
almost four percent of the herds that had been tested in this program in 2009 were seropositive .
the results also show that in areas with high pig density , a high proportion of the herds were seropositive .
this suggests virus transmission between pig herds , either by movement of pigs and people visiting infected herds .
however , air transmission can not be excluded . the farm size and density of pigs in the area around the farm
experimental studies have shown that pandemic ( h1n1 ) 2009 virus is efficiently transmitted between pigs [ 11 , 14 , 15 ] .
it is also known that other siv subtypes are efficiently spread between pigs and herds .  
norway has previously been free of siv as documented by a national surveillance program for swine .
although one multiplier herd was tested seropositive in 1998 for antibodies against influenza a h3n2 virus , follow - up studies for many years in this herd did not reveal new seropositive pigs , nor any spread of the virus in contact herds .
based on this , it was concluded that the pigs in this herd had probably been infected by a human - adapted h3n2 virus that did not have the capacity to be transmitted further between pigs .
since the norwegian pig population has been free of siv , there are no cross - reactive antibodies to pandemic ( h1n1 ) 2009 virus or any of the other siv subtypes .
this has probably contributed to the high prevalence of pandemic ( h1n1 ) 2009 virus in the norwegian pig population .
it has been shown that european pigs naturally infected or vaccinated with european sivs have cross - reactive hemagglutinating antibodies against pandemic ( h1n1 ) 2009 virus [ 15 , 16 ] . moreover ,
natural infection with influenza virus stimulates mucosal immunity and cellular immune responses , responses that give partial protection against infection with an antigenetically unrelated strain [ 27 , 28 ] .
surveillance of siv in europe prior to the emergence of pandemic ( h1n1 ) 2009 virus showed that the three siv subtypes   avian - like 
 human - like  h1n2 were most often found in animals between 3- and 6 months old .
it should be noted that norway is carrying out surveillance for siv , including 45005000 samples from between 450 and 500 herds per year .
consistent with other studies , there were mild or no clinical signs observed in herds that tested positive for pandemic ( h1n1 ) 2009 virus .
for example , in australia , pigs naturally infected with pandemic ( h1n1 ) 2009 virus only showed mild clinical signs .
it was suggested that a lower infection pressure in natural infection as compared to experimental infection , as well as absence of other respiratory pathogens , could result in milder clinical signs . in norway ,
only a few pigs showed clinical signs like coughing , fever , and loss of appetite which are typical for siv .
as norway is free from most other viral respiratory diseases , presence of clinical signs like coughing would alert farmers who would notify their veterinarian or the authorities .  
herds positive for pandemic ( h1n1 ) 2009 virus in 2009 were tested again in 2010 .
the results show that there was no seroconversion in pigs born at least two months after the virus was first detected , indicating that there was no ongoing infection in the herds .
a possible explanation may be that the norwegian pig herds are small , and many herds practice batch farrowing of three , five and a half , or seven weeks intervals , with strict sectioning of the herds with all - in all - out system .
however , results from the surveillance in 2011 show that some pigs born in 2010/2011 are seropositive , indicating an ongoing spread of the infection .
it is not clear to what extent this is caused by new introduction from humans or continued virus circulation between susceptible pigs since 2009 .
continuing surveillance is important to show whether the virus has become enzootic in the norwegian pig population .  
there have been several reports of reassortant virus that have genes from pandemic ( h1n1 ) 2009 virus and other siv subtypes [ 3033 ] . as long as norway is free of other siv subtypes ,
however , reassortant viruses evolving in humans might be a risk for the norwegian pig population .
it is therefore important to continue the surveillance of siv and genetic characterization of positive samples . given the potential long - term presence of influenza virus in norwegian pigs , it is also interesting to evaluate the economic impact of this disease and assess the cost effectiveness of new biosecurity measures or change in husbandry practices .
pandemic ( h1n1 ) 2009 virus was introduced to the norwegian pig population in september / october 2009 .
the virus spread rapidly to a large proportion of all categories of herds all over the country , including closed , high - biosecurity nucleus herds .
the clinical impact was mild , and in many infected herds , no clinical signs were observed .
the ongoing national surveillance program will reveal whether the virus will become enzootic in the norwegian pig population .
 pandemic ( h1n1 ) 2009 influenza a virus was detected in norwegian pigs in october 2009 . until then , norway was regarded free of swine influenza . intensified screening revealed 91 positive herds within three months . 
 the virus was rapidly transmitted to the susceptible population , including closed breeding herds with high biosecurity . 
 humans were important for the introduction as well as spread of the virus to pigs . 
 mild or no clinical signs were observed in infected pigs . 
 surveillance of siv in 2010 revealed that 41% of all the norwegian pig herds had antibodies to pandemic ( h1n1 ) 2009 virus . 
 furthermore , this surveillance indicated that pigs born in positive herds after the active phase did not seroconvert , suggesting no ongoing infection in the herds . 
 however , results from surveillance in 2011 show a continuing spread of the infection in many herds , either caused by new introduction or by virus circulation since 2009 . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions