In June-July 2009, an outbreak of pandemic (H1N1) 2009 infection occurred on a pig farm in Argentina. Molecular analysis indicated that the virus was genetically related to the pandemic (H1N1) 2009 influenza virus strain. The outbreak presumably resulted from direct human-to-pig transmission.Facultad de Ciencias Veterinaria

Balzano, Rodrigo

Barral, Leonardo

Baumeister, Elsa

Cannilla, María L.

Capalbo, Santiago

Cappuccio, Javier A.

Craig, María I.

Debenedetti, Rosa T.

Escobar Cabrera, Oscar E.

Franzese, Débora

Ibar, Mariela Paula

Insarralde, Lucas

Machuca, Mariana Alejandra

Pereda, Ariel

Perfumo, Carlos Juan

Quiroga, María Alejandra

Rimondi, Agustina

Risso, Adriana

Sanguinetti, Ramón

Zenobi, Carlos

English

SEDICI - Repositorio de la UNLP

DISPATCHES
Pandemic (H1N1) 
2009 Outbreak 
on Pig Farm, 
Argentina
Ariel Pereda,1 Javier Cappuccio,1 
Maria A. Quiroga, Elsa Baumeister, 
Lucas Insarralde, Mariela Ibar, 
Ramón Sanguinetti, Maria L. Cannilla, 
Dé bora Franzese, Oscar E. Escobar Cabrera, 
Maria I. Craig, Agustina Rimondi, 
Mariana Machuca, Rosa T. Debenedetti, 
Carlos Zenobi, Leonardo Barral, 
Rodrigo Balzano, Santiago Capalbo, 
Adriana Risso, and Carlos J. Perfumo
In June-July 2009, an outbreak of pandemic (H1N1) 
2009 infection occurred on a pig farm in Argentina. Molecu­
lar analysis indicated that the virus was genetically related 
to the pandemic (H1N1) 2009 influenza virus strain. The 
outbreak presumably resulted from direct human-to-pig 
transmission.
Swine influenza viruses (SIVs) occur worldwide; they usually cause asymptomatic infection but occasion­
ally cause outbreaks of varying intensity (7,2). In North 
America, the landscape of swine influenza lias changed 
substantially since the late 1990s. when human (H3N2) vi­
ruses were first isolated from swine and triple reassortant 
viruses carrying influenza genes (TRIG) of avian, swine, 
and human origin and showing great reassortment flex­
ibility emerged concurrently. TRIGs of influenza subtypes 
H3N2, H1N1, H3N1, and H1N2 appear to be circulating in 
swine in North America. Coincidentally. 1 of these TRIG 
viruses led to emergence of the pandemic (H1N1) 2009 vi­
rus (3). Testing by the Canadian Food Inspection Agency 
identified pandemic (H1N1) 2009 virus in a swine herd in
Author affiliations: Instituto Nacional de Tecnologia Agropecuaria, 
Castelar, Buenos Aires, Argentina (A. Pereda, M.l. Craig, A. Rimon­
di); Universidad Nacional de La Plata, La Plata, Buenos Aires (J. 
Cappuccio, M.A. Quiroga, L. Insarralde, M. Ibar, M. Machuca, C.J. 
Perfumo); Institute Nacional de Enfermedades Infecciosas-Admin- 
istraciôn Nacional de Laboratorios e Institutes de Salud, Buenos 
Aires (E. Baumeister); Servicio Nacional de Sanidad Animal, Mar­
tinez, Buenos Aires (R. Sanguinetti, M.L. Cannilla, D. Franzese, 
O.E. Escobar Cabrera, R.T. Debenedetti, C. Zenobi, L. Barral, R. 
Balzano); and private veterinary practice, Buenos Aires (S. Capal­
bo, A. Risso)
DOI: 10.3201/eid1602.091230
Alberta (-/). The pigs may have been exposed to the virus 
by a farm worker who returned from Mexico with influen­
za-like symptoms (5). Affected pigs showed clinical signs 
of infection with SIV (5).
During the past influenza season (April-September 
2009) in the Southern Hemisphere, pandemic (H1N1) 2009 
virus predominated in humans and accounted for >90% of 
influenza cases in humans in Argentina. In Argentina, se- 
roepidemiologic analyses of pigs on 17 farms showed that 
»41% of pigs had antibodies against Hl and H3 (6). Vac­
cines against SIV are not licensed for use in Argentina.
We describe the epidemiology, clinical outcome, pa­
thology. and persistence of pandemic (H1N1) 2009 infec­
tion on a pig farm 103 km from Buenos Aires. Molecular 
analysis indicated that the virus was genetically related 
to the pandemic (H1N1) 2009 vims strain and indicated 
no evidence of further reassortment with any other influ­
enza strains. The outbreak presumably resulted from direct 
human-to-pig transmission at the peak of the pandemic in 
Argentina.
The Study
The pig farm, a closed farrow-to-finish 1-site opera­
tion in Buenos Aires province, had 519 sows. Pig death rate 
from farrowing to slaughter remained at 9.5% before and 
after the outbreak. Ten days before the outbreak, the farm 
manager and his wife reported clinical signs of influenza in 
themselves.
On June 15, 2009, nursery bam pigs >40 days of age 
showed clinical signs, e.g.. cough, dyspnea, fever, nasal 
discharge, and inappetence. Illness affected 30% of nurs­
ery pigs. On June 17, pigs in 8 growing and fattening bams 
showed similar clinical signs; 4,000 (15%) pigs were af­
fected). Clinical signs lasted for 1 week; on June 22, resolu­
tion of clinical signs was evident, with cough index <2% in 
growing and fattening.
At the onset of the outbreak, 5 clinically affected pigs 
were submitted for postmortem examination. Tissue sam­
ples from nose, trachea, and lungs were fixed in 10% buff­
ered formalin and processed for routine histopathologic 
examination. We conducted immunohistochemical (IHC) 
analysis using antinucleoprotein influenza A monoclonal 
antibody, isotype MSIg2a (Chemicon International Inc.. 
Temecula. CA, USA) according to Vincent et al. (7). In ad­
dition, we obtained 30 serum samples from nursery, grow­
ing, and fattening pigs.
All pigs had cranioventral lung consolidation on 5%- 
60% of the total surface. Two of 5 pigs had distinctive, 
scattered, dark-red foci of lobular consolidation (chess- 
board-like) (Figure, panel A). In 3 pigs, we observed sero­
fibrinous polyserositis.
’These authors contributed equally to this article.
304 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 2, February 2010
Pandemie (H1N1) 2009 on Pig Farm
Figure. Postmortem samples from clinically affected pigs, Argentina, 2009. A) Macroscopic lung lesions with distinctive scattered dark-red 
foci of lobular consolidation (chessboard-like) in all lobes. B) Severe necrotizing bronchiolitis with partially denuded epithelia. Hematoxylin- 
eosin stain; original magnification »400. C) General view of the alveolar walls showing moderate interstitial thickening by leukocytes. 
Hematoxylin-eosin stain; original magnification »200. D) Bronchioli. Positive-labeled nuclei and cytoplasm of bronchial epithelial cellsand 
mononuclear cells in the alveoli. 3,3'-diaminobenzidine counterstain with hematoxylin stain; original magnification »200.
Four pigs had severe necrotizing bronchiolitis. Small 
and medium bronchioles were plugged with neutrophils 
cellular debris and exudates. Affected airways were de­
nuded or lined with flat epithelium (Figure, panel B). The 
adjacent alveolar walls were infiltrated and thickened in a 
lobular fashion by mononuclear cells (Figure, panel C). In 
other pigs, the alveolar lumen was filled with macrophages, 
neutrophils, and fibrinous exudates. We observed a positive 
IHC reaction in bronchiolar epithelia only, which showed 
severe inflammatory changes (Figure, panel D).
We processed 5 bronchial swabs and lung samples 
from postmortem-examined pigs for real-time reverse 
transcription-PCR (rRT-PCR) and for virus isolation in 
MDCK cells. Viral RNA was extracted from swab suspen­
sion and from culture supernatant (Total RNA Isolation 
Kit; Applied Biosystems. Foster City. CA, USA), and viral 
cDNA was synthesized. We tested the cDNA for influenza 
type A by rRT-PCR directed to the matrix (M) gene, as pre­
viously reported (8), and tested for pandemic (H1N1) 2009 
virus by rRT-PCR using the protocol released by the Cen­
ters for Disease Control and Prevention (CDC) on April 
30, 2009 (revision 1) (www.who.int/csr/resources/publica- 
tions/swineflu/realtimeptpcr/en/index.html).
Next, we conducted a cross-sectional study of 60 na­
sal swabs from pigs of 3 categories (i.e.. nursery, grow­
ing, fattening) on June 25, July 2, July 10, and July 31. All 
these samples were negative. Also. 120 bronchial swabs 
were sampled at slaughter on June 30, July 3, and July 7; 1 
sample from June 30 and 5 from July 3 were positive, but 
thereafter, all bronchial swabs were negative. A serologic 
survey was conducted of 214 fattening pigs on June 30 and 
every week thereafter by using ELISA (HerdChek Swine 
Influenza H1N1 Antibody Kit; IDEXX Laboratories. West­
brook. ME. USA).
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 2, February 2010 305
DISPATCHES
All postmortem samples were positive for subtype 
(H1N1) according to both rRT-PCR protocols, and cycle 
threshold values ranged from 16 to 27 cycles according to 
the CDC protocol. Nasal samples from pigs in the cross- 
sectional study were negative. However, 5% of the bronchi­
al swabs sampled from clinically healthy pigs at slaughter 
were positive according to both rRT-PCR protocols. At the 
onset of the outbreak, all pig serum samples were negative 
for swine (H1N1) influenza virus. However, 98% of serum 
samples from slaughter pigs were positive 15 days later.
Cytopathic effects in MDCK cells were noted on first 
passage at 72 h postinoculation, and this virus was charac­
terized by sequencing as pandemic (H1N1) 2009. All ge­
nome segments were fully amplified by RT-PCR ( 9) and 
directly sequenced from the isolate in MDCK. Regions cor­
responding to the entire open reading frame of hemagglu­
tinin (HA), nucleoprotein, neuraminidase (NA), Ml, and 
nonstructural and partial polymerase base (PB) 2 (nt 580- 
1140), PB1 (nt 390-990), and polymerase acid (PA) (nt 
720-1230) segments were obtained by using appropriate 
set of primers and submitted to GenBank (accession nos. 
CY044256-CY044260 and CY047749-CY047751). High 
(>99.99%) nucleotide identity was found between these se­
quences and pandemic strain A/Califomia/04/2009 (H1N1) 
and A/swine/Alberta/OTH-33-8/2009 (H1N1) (Table).
Conclusions
Young pigs are susceptible to pandemic (H1N1) 2009 
as reported in Canada (4,5). Recently, experimental infec­
tions showed that the pandemic (H1N1) 2009 virus induced 
mild clinical signs, virus shedding, and gross and histo­
pathologic lesions similar to those caused by SIV (10,11). 
Successful cross-species transmission usually requires a 
period of adaptation to the new host (72). However, phylo­
genetic analyses of pandemic (H1N1) 2009 vims showed 
that the HA and NA glycoproteins arose from the classical 
swine (H1N1) and avian-like Eurasian swine (H1N1) lin­
eages, respectively (5), which might explain the high sus­
ceptibility and transmissibility among pigs.
Table. Nucleotide identity percentage of swine influenza viruses 
in a study of a pandemic (H1N1) 2009 outbreak at a pig farm, 
Argentina, June-July 2009
Genome 
segment*
A/California/04/
2009 (H1N1)
A/swine/AI be rta/OTH -33-8/
2009 (H1N1)
1 (PB2) 100 99.994
2 (PB1) 100 99.998
3 (PA) 100 99.998
4 (HA) 99.997 99.994
5 (NP) 99.996 99.993
6 (NA) 99.998 99.995
7 (M1) 99.997 99.996
8 (NS) 99.999 99.994
*PB, polymerase base; PA, polymerase acid; HA, hemagglutinin; NP, 
nucleoprotein; NA, neuraminidase; M, matrix; NS, nonstructural protein.
From an epidemiologic standpoint, the 25%-30% 
morbidity rate with few deaths agreed with the clinical pre­
sentation of SIV (H1N1) (1,13) and was similar to the rates 
found in the outbreak in Canada (4.5). Clinical signs lasted 
1 week. However, 5% of the bronchial swabs taken 15-18 
days after onset of the outbreak from slaughter-weight pigs 
showed positive results. Virus shedding, detected by rRT- 
PCR, from nasopharyngeal samples peaked at 4 days posti­
noculation and ceased at ~ 11 days postinoculation.
Epithelial cell necrosis, sloughing, and neutrophil in­
filtration with total or partial obstruction of bronchioles 
is the hallmark of SIV infection (14) and was distinctive 
for influenza A virus (H1N1). However, IHC results were 
strongly positive for virus antigen in only those bronchioles 
that showed severe bronchiolitis. Our findings from field 
observation confirm findings from experimental studies 
with pandemic (H1N1) 2009 virus (11).
The National Veterinary Services Laboratory has 
established a farm biosecurity program to survey the 
infection. The program includes movement restrictions 
and clinical surveillance of farms within 3-km of the in­
dex case. No other farm had clinically ill pigs. The farm 
was allowed to move animals to slaughter after the nasal 
swabs of those animals tested negative by rRT-PCR with 
the CDC protocol.
The suspected human source of infection was not con­
firmed. This outbreak supports the belief that influenza 
cross-species transmission may occur between pigs and 
humans and vice versa.
Acknowledgments
We thank Carolina Aralda and Lorena Diaz for their valuable 
help making histopathologic and immunohistochemical slides and 
Yibin Cai for sequencing the PB1, PB2, and PA genes.
This work was supported by grants from Agenda Nacional 
de Promoción Científica y Tecnológica Préstamo BID 1728 OC/ 
AR PICT 2005-33987, P.E. Otras Exóticas AESA1573-INTA, 
National Institute for Allergy and Infectious Diseases, National 
Institutes of Health contract no. HHSN266200700010C; Center 
of Excellence for Influenza Research and Surveillance; and Secre­
taría de Ciencia y Técnica, Universidad Nacional de La Plata.
Dr Pereda is a veterinarian at the University of Buenos Aires. 
His main research interests are emerging porcine and avian viral 
diseases, including influenza.
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Pandemic (H1N1) 2009 outbreak on pig farm, Argentina

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Pandemic (H1N1) 2009 outbreak on pig farm, Argentina

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