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Evidence of the concurrent circulation of H1N2, H1N1 and H3N2 influenza A viruses in densely populated pig areas in Spain
The Veterinary Journal The Veterinary Journal 172 (2006) 377–381 www.elsevier.com/locate/tvjl
Short communication
Evidence of the concurrent circulation of H1N2, H1N1 and H3N2 influenza A viruses in densely populated pig areas in Spain Jaime Maldonado a,*, Kristien Van Reeth b, Pere Riera a, Marta Sitja` a, Narcı´s Saubi a, Enric Espun˜a a, Carlos Artigas a b
a Laboratorios HIPRA, S. A., Av. La Selva No. 135, Amer, 17170 Girona, Spain Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
Abstract This paper reports on a serological and virological survey for swine influenza virus (SIV) in densely populated pig areas in Spain. The survey was undertaken to examine whether the H1N2 SIV subtype circulates in pigs in these areas, as in other European regions. Six hundred sow sera from 100 unvaccinated breeding herds across Northern and Eastern Spain were examined using haemagglutination inhibition (HI) tests against H1N1, H3N2 and H1N2 SIV subtypes. Additionally, 225 lung samples from pigs with respiratory problems were examined for the presence of SIV by virus isolation in embryonated chicken eggs and by a commercial membrane immunoassay. The virus isolates were further identified by HI and RT-PCR followed by partial cDNA sequencing. The HI test on sera revealed the presence of antibodies against at least one of the SIV subtypes in 83% of the herds and in 76.3% of the animals studied. Of the 600 sow sera tested, 109 (18.2%), 60 (10%) and 41 (6.8%) had SIV antibodies to subtype H1N2 alone, H3N2 alone and H1N1 alone, respectively. Twelve H3N2 viruses, 9 H1N1 viruses and 1 H1N2 virus were isolated from the lungs of pigs with respiratory problems. The analysis of a 436 nucleotide sequence of the neuraminidase gene from the H1N2 strain isolated further confirmed its identity. Demonstrably, swine influenza is still endemic in the studied swine population and a new subtype, the H1N2, may be becoming established and involved in clinical outbreaks of the disease in Spain. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Influenza A viruses; H1N2; Swine; Influenza; Flu; Spain
Surveillance of swine influenza (SI) viruses is important because new influenza viruses and influenza virus reassortants regularly emerge in pig populations, and because there are considerable antigenic and genetic differences between swine influenza viruses (SIVs) in different geographic regions. Monitoring and characterization of SIV in pig producing regions is needed for the adequate control and diagnosis of infection. Furthermore, there are concerns that pigs may serve as intermediate hosts for the introduction of new influenza viruses into the human population (Brown et al., 1998). *
Corresponding author. Tel.: +34 972 43 06 55; fax: +34 972 43 06
61. E-mail address: [email protected] (J. Maldonado). 1090-0233/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2005.04.014
The H1N1 and H3N2 SIV subtypes have been enzootic in several swine producing countries in Europe for more than 20 years (Brown, 2000). A third subtype, H1N2, was first isolated in Great Britain in 1994 (Brown et al., 1995). Thereafter, the H1N2 virus subtype has also been found in France, Belgium, Italy and Germany, where it appears to co-circulate with H1N1 and H3N2 viruses (Gourreau et al., 1994; Van Reeth et al., 2000; Marozin et al., 2002; Schrader and Suss, 2003). In Spain, SIV subtypes H1N1 and H3N2 have been circulating since the mid 1980s. Both H1N1 (Plana Duran et al., 1984) and H3N2 (Castro et al., 1988) SIV strains have been isolated from pigs with acute respiratory problems. Serological examinations of fattening pigs between 1987 and 1989 revealed high
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seroprevalences for both H1N1 (between 72.8% and 78.5% seropositive pigs) and H3N2 (between 61.6% and 62.5% seropositive pigs) (Yus et al., 1989, 1992). Recent reports point towards a continuous circulation during the 1990s of H1N1 and H3N2 SIV in the Spanish pig population (Oliveira et al., 1999; Gutierrez-Martin et al., 2000). However, there is little information on the current seroprevalences of H1N1 and H3N2 subtype influenza viruses. Moreover, it is unknown whether H1N2 SIV viruses are circulating in Spain, as neither virus isolation nor prevalence of SIV subtype specific antibodies have been recently reported. In this paper, we report on a recent virological and serological survey for different SIV subtypes in a representative part of the Spanish swine population. It was the purpose of this survey to determine the prevalence of H1N2 virus antibodies in the studied population, and to compare it with that of H1N1 and H3N2 antibodies. We also took the opportunity to subtype SIV isolates from acute respiratory disease outbreaks. Farm selection followed the criteria established by the European Surveillance Network for Influenza in Pigs (ESNIP group). Breeding herds were included in the study when they met the inclusion criteria of being located in the most densely populated pig areas in Spain and not having had vaccination programme against SIV (confirmed by telephone interviews with the farmers). Vaccination against SIV in Spain is neither compulsory nor common practice. The number of samples was calculated from an estimated population of 875,000 females, with 4% precision at a 95% confidence level (Epi Info software package, version 5.01). Serological testing was performed on 600 sow sera from 100 conventional two-site operations (six samples per unit including sows of all parities) located in the four major Spanish swine producing autonomous regions (Arago´n, Castilla and Leon, Catalonia and Murcia) and distributed around 22 provinces in Northern and Eastern Spain (accounting for 65% of the national pig inventory). Sera were collected during the period February–June 2003. On arrival at the laboratory, sera were separated by centrifugation, split and stored in vials at 20 °C until analysed. Haemagglutination inhibition (HI) tests against H1N1, H3N2 and H1N2 subtypes were performed using standard methods (Webster and Krauss, 2002). The SIV strains Sw/Spain/45304/2003(H1N1), Sw/Spain/46356/ 2003(H3N2) and Sw/Scotland/410440/94(H1N2) were used as antigens. Two-fold serum dilutions were tested starting at a dilution of 1/20. The results were recorded as the log2 of the HI titre, and the geometric mean titre (GMT) for each virus subtype was calculated. A farm was classified as positive and assigned to a SIV subtype when at least one of the six sows had HI titres above the cut-off value against 1 of the SIV subtypes alone, regardless of the presence of individuals showing mixed infections.
In the virological surveillance, diagnostic specimens were examined. Between November 2001 and April 2004, 225 pig lungs from unrelated growing-finishing pig units (housing an average of 800 pigs per unit) were submitted to the Veterinary Diagnostic Centre, Laboratorios HIPRA, S.A. (Girona, Spain) for the aetiological diagnosis of pneumonia. The herds of origin (located all around Spain) were experiencing acute episodes of respiratory disease, mainly at the end of the finishing period. Affected animals showed clinical signs and macroscopic lesions suggestive of acute viral pneumonia. Bacteriological and virological analyses were performed to detect the most relevant respiratory pathogens for swine (data not shown). Samples (lung and bronchial tissues) for SIV investigation were systematically collected. Ten per cent lung tissue homogenates were examined for the presence of SIV antigen by a commercial membrane enzyme immunoassay (EI) (BD Directigen Flu A; Becton Dickinson Microbiology Systems) and for virus isolation by inoculation in 9- to 11-day old specific-pathogen-free embryonated chicken eggs (ECE), as described by Swenson et al. (2001). Allantoic fluids were harvested after 3–4 days of incubation at 37 °C. The presence of SIV was determined in a haemagglutination (HA) assay with 0.5% chicken red blood cells. To determine the subtype of SIV isolates, allantoic fluids were examined in a HI assay with swine hyperimmune sera using standard methods (Webster and Krauss, 2002) and by single-step reverse transcription-polymerase chain reaction (RT-PCR). RNA was extracted from 200 lL of allantoic fluids, by using the RNeasy Protect Mini Kit (QIAGEN). PCR amplification conditions and primers designed to amplify partial sequences of the H1, H3, N1 and N2 SIV genes were adapted from Chiapponi et al. (2003). Briefly, 0.6 lM of each primer and 5 lL of the extracted RNA were added to four separate RT-PCR mixtures (one for each target) (OneStep RT-PCR Kit, QIAGEN) to obtain final volumes of 50 lL. The following conditions were set up in a Px2 Thermal Cycler (Thermo Electronic Corporation): the first strand cDNA synthesis was conducted for 30 min at 50 °C. After denaturation for 15 min at 95 °C, the samples were submitted to 40 cycles of PCR amplification as follows: 1 min at 95 °C, 1 min at 50 °C and 1 min at 72 °C. The final extension step was for 7 min at 72 °C. In all PCR reactions, predefined SIV-positive, -negative and nontemplate controls were tested simultaneously with the field isolates. PCR products were analysed by agarose gel electrophoresis. To further confirm the identity of some of the SIV isolates, selected PCR products (N2 fragments) were sequenced using an ABI 373 DNA sequencer, together with the Taq dye-Deoxy terminator cycle sequencing kit (Applied Biosystems). Comparative nucleotide and amino acid sequence analysis were conducted using
J. Maldonado et al. / The Veterinary Journal 172 (2006) 377–381
software tools from the National Centre for Biotechnology Information (NCBI, Maryland, USA [Available from: http://www.ncbi.nlm.nih.gov/]). The HI test revealed the presence of antibodies against at least one of the SIV subtypes in 83% of the herds and 76.3% of the animals studied. Of the 600 sow sera tested, 109 (18.2%), 60 (10%) and 41 (6.8%) had SIV antibody to subtype H1N2 alone, H3N2 alone and H1N1 alone, respectively. Mixed infections were detected in 41.3% of the animals tested. Most were housed in farms in which the identification of the dominant subtype of circulating virus was not possible. Table 1 summarizes the HI results at the sow and farm levels. Twenty-two SIV lung samples (9.8%) were found to be positive for SIV by isolation. All tested positive in the EI, HI and PCR tests, and total agreement between HI and PCR allowed the classification of the isolates as being of the H3N2 (12 isolates), H1N1 (9 isolates) and H1N2 (1 isolate) subtypes. The H1N2 virus strain A/sw/Polen˜ino/40564/ 02(H1N2) was isolated from the lungs of a 5-month-old fattening pig, during a typical outbreak of acute respiratory disease. No other relevant pathogen from that particular sample was detected or isolated. The affected farm had 1600 pigs and morbidity and mortality rates during the respiratory disease outbreak were 50% and 1%, respectively. Alignment of the N2 gene partial sequences obtained from isolate A/sw/Polen˜ino/40564/02(H1N2) with those of the most closely related Italian and French H1N2 strains available in electronic data bases (GenBank, EMBL, DDBJ and PDB) (Accession Nos.: AJ412696, AJ412705, AJ412701, AJ412693, AJ412698, AJ412695, AJ412694 and AJ412704) revealed 95–97% homology. Deduced protein sequences from both the Italian A/swine/Italy/1081/00(H1N2) and the French A/ swine/Cotes dÕArmor/790/97(H1N2) strains (first and second places in the nucleotide–nucleotide matching list of the BLAST analysis) showed 97% homology with the A/ sw/Polen˜ino/40564/02(H1N2) isolate (Fig. 1).
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Our data show that SIVs of H1N1 and H3N2 subtype have remained endemic in the Spanish swine population. A new finding is that the recently emerged H1N2 SIV is widespread in Spain and that it is involved in respiratory outbreaks, as is the case in other European countries (Van Reeth et al., 2000; Marozin et al., 2002; Schrader and Suss, 2003). Our serology results may raise the question of whether the positive reactions to the H1N2 virus in the HI test may in part be due to serological crossreaction with the H1N1 subtype or vice versa. However, in experimental infection studies with both a single subtype and a combination of two SIV subtypes it has been clearly demonstrated that there is no serological crossreaction between H1N1, H1N2 and H3N2 subtypes in the HI test (Van Reeth et al., 2003). It is likely therefore that those animals in this study with HI antibodies against H1N1 or H1N2 have been previously infected with the respective subtypes. In our study, the serological determination of the dominant serotype infecting a high proportion of the tested sera was not feasible. It has recently been demonstrated that when field serum contains significant amounts of anti-SIV antibody to more than one subtype after natural infections, it cannot be classified in a reliable way by using current HI methods (Long et al., 2004). This could be the situation in this study, as the seroprevalence rates for H1N1, H3N2 and H1N2 SIV observed suggest that the three subtypes are circulating concurrently and cause mixed infections. SIV was isolated from 9.8% of the 225 lung samples tested. This cannot be considered as a prevalence rate as it is the result of a diagnostic exercise rather than a prevalence survey. In other surveys of acute respiratory disease outbreaks in the Netherlands, SIVs were isolated from 45% of the acute respiratory disease outbreaks examined (Loeffen et al., 1999) and similar SIV isolation rates have been reported in Belgium (K. Van Reeth, personal communication) and in the US (Choi et al., 2003). It must be mentioned, therefore, that some of the
Table 1 Numbers of individual animals (n = 600) and herds (n = 100) with HI antibodies to one single SIV subtype or to combinations of 2 or 3 subtypes (Spain, 2003) Virus subtype(s)
Number and percentage of positivesa Individual sows
None (negative to all 3 subtypes) H1N1 only H3N2 only H1N2 only H1N1 + H3N2 H3N2 + H1N2 H1N1 + H1N2 H1N1 + H3N2 + H1N2 a b
b
142 41 60 109 40 58 80 70
The cut-off value for HI was 1/40. Cumulative number of negative sows housed in positive and negative herds.
(%)
Herds
(%)
23.7 6.8 10.0 18.2 6.6 9.7 13.3 11.7
17 9 20 21 6 6 5 16
17.0 9.0 20.0 21.0 6.0 6.0 5.0 16.0
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(a)
(b)
Fig. 1. Partial nucleotide and deduced amino acid sequences for the neuraminidase gene of the virus strain A/sw/Polen˜ino/40564/02(H1N2). The nucleotide sequence (436 nucleotides) is shown in the top lines (a). Alignment with the predicted amino acid sequences for A/swine/Italy/1081/ 00(H1N2) (Accession No. AJ412696) and A/swine/Cotes dÕArmor/790/97(H1N2) (Accession No. AJ412705) was performed by ClustalW programme (http://www.ebi.ac.uk/clustalw/): Shaded areas denote sequence differences.
diagnostic submissions in the present study were accompanied by rather vague descriptions of the clinical symptoms, and only the known acute cases of respiratory disease were examined in the studies mentioned above. Other factors that may account for the rather low SIV isolation rates in the present study are suboptimal conservation and autolysis of part of the lung tissue samples. Although direct PCR could give a better prevalence estimation of SIV infection on autolytic samples, this was not the objective of our survey. With the limited clinical information available, we cannot assume that the SIVs isolated played a central role in the course of the studied outbreaks. Furthermore, other bacteria and viruses were also present in some of the SIV-positive lungs (data not shown). It should be mentioned nevertheless, that SIV is recognized as an important contributor in the aetiology of the porcine respiratory disease complex, infecting alone or in combination with other pathogens (Thacker et al., 2001). The origin of the strain A/sw/Polen˜ino/40564/ 02(H1N2) remains elusive, but genetic analyses of the N2 suggest that this strain is related to H1N2 viruses isolated in France and in Italy since the late 1990s. Based on the antigenic and genetic relationships of viruses isolated in France, Italy and the UK, Marozin et al. (2002) stated that H1N2 viruses were introduced into continental Europe from the UK. However, sequencing of the internal genes is required to further determine the origin of the Spanish H1N2 strain, as recent studies have demonstrated extensive antigenic and genetic heterogeneity among H1N2 viruses (Marozin et al., 2002).
Results obtained in the present study highlight the need for continuous surveillance for SIVs in pig producing areas, as new and potentially pathogenic SIV subtypes readily emerge. Also, this study will contribute significantly to the task of the ESNIP group in monitoring the evolution of SI in Europe during the next years. All the data collected and viruses isolated until now, and the obtained ones in a future, will be deposited in the virus bank and the electronic database for further studies on molecular epidemiology, immunology and genetics of SI in Europe (http://www.esnip.wur.nl/).
Acknowledgements The authors thank Romney Jackson for helpful language revision. Technical assistance of Corona Vin˜als is gratefully acknowledged. This research was supported by Laboratorios HIPRA S. A., Amer, Gerona, Spain and ESNIP (EC concerted action, 6th Framework Programme, QLK2-CT-2000-01636).
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J. Maldonado et al. / The Veterinary Journal 172 (2006) 377–381 Brown, I.H., 2000. The epidemiology and evolution of Influenza viruses in pigs. Veterinary Microbiology 74, 29–46. Castro, J.M., del Pozo, M., Simarro, I., 1988. Identification of H3N2 Influenza virus isolated from pigs with respiratory problems in Spain. The Veterinary Record 122, 418–419. Chiapponi, C., Fallacara, F., Foni, E., 2003. Subtyping of H1N1, H1N2 and H3N2 swine Influenza viruses by two multiplex RTPCR. In: Proceedings of the 4th International Symposium on Emerging and Re-emerging Pig Diseases, Rome, Italy, p. 257. Choi, Y.K., Goyal, S.M., Joo, H.S., 2003. Retrospective analysis of etiologic agents associated with respiratory diseases in pigs. Canadian Veterinary Journal 44, 735–737. Gourreau, J.M., Kaiser, C., Valette, M., Douglas, A.R., Labie, J., Aymard, M., 1994. Isolation of two H1N2 Influenza viruses from swine in France. Archives of Virology 35, 365–382. Gutierrez-Martin, C.B., Rodriguez-Delgado, O., Alvarez-Nistal, D., De La Puente-Redondo, V., Garcia-Rioja, A.F., Martin-Vicente, J., Rodriguez Ferri, E.F., 2000. Simultaneous serological evidence of Actinobacillus pleuropneumoniae, PRRS, AujeszkyÕs disease and Influenza viruses in Spanish finishing pigs. Research in Veterinary Science 68, 9–13. Loeffen, W.L., Kamp, E.M., Stockhofe-Zurwieden, N., van Nieuwstadt, A.P., Bongers, J.H., Hunneman, W.A., Elbers, A.R., Baars, J., Nell, T., van Zijderveld, F.G., 1999. Survey of infectious agents involved in acute respiratory disease in finishing pigs. The Veterinary Record 31, 123–129. Long, B.C., Goldberg, T.L., Swenson, S.L., Erickson, G., Scherba, G., 2004. Adaptation and limitations of established hemagglutination inhibition assays for the detection of porcine anti-swine influenza virus H1N2 antibodies. Journal of Veterinary Diagnostic Investigation 16, 264–270. Marozin, S., Gregory, V., Cameron, K., Bennett, M., Valette, M., Aymard, M., Foni, E., Barigazzi, G., Lin, Y., Hay, A., 2002. Antigenic and genetic diversity among swine Influenza A H1N1 and H1N2 viruses in Europe. Journal of General Virology 83, 735– 745.
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Oliveira, J., Guitia´n, J., Yus, E., Sanjua´n, M.L., 1999. Description of serological results against several strains of Influenza virus in a population of slaughter pigs in NW Spain. In: Proceedings of the Symposium on Animal Influenza Viruses, Gent, Belgium, pp. 38– 39. Plana Duran, J., Vayreda, M., Vila, X., Marrull, L., 1984. Isolation for the first time in Spain of the swine Influenza virus. In: Proceedings of the 8th International Pig Veterinary Society Congress, Gent, Belgium, p. 62. Schrader, C., Suss, J., 2003. Genetic characterization of a porcine H1N2 Influenza virus strain isolated in Germany. Intervirology 46, 66–70. Swenson, S.L., Vincent, L.L., Lute, B.M., Janke, B.H., Lechtenberg, K.E., Landgraf, J.G., Schmitt, B.J., Kinker, D.R., McMillen, J.K., 2001. A comparison of diagnostic assays for the detection of type A swine Influenza virus from nasal swabs and lungs. Journal of Veterinary Diagnostic Investigation 13, 36–42. Thacker, E.L., Thacker, B.J., Janke, B.H., 2001. Interaction between Mycoplasma hyopneumoniae and swine influenza virus. Journal of Clinical Microbiology 39, 2525–2530. Van Reeth, K., Brown, I.H., Pensaert, M., 2000. Isolations of H1N2 Influenza A virus from pigs in Belgium. The Veterinary Record 146, 588–589. Van Reeth, K., Gregory, V., Hay, A., Pensaert, M., 2003. Protection against a European H1N2 swine influenza virus in pigs previously infected with H1N1 and/or H3N2 subtypes. Vaccine 21, 1375–1381. Webster, R.G., Krauss, S. (Eds.), 2002. World Health Organization Manual on Animal Influenza Diagnosis and Surveillance. World Health Organization, Geneva, Switzerland. Yus, E., Laviada, M.D., Moreno, L., Castro, J.M., Escribano, J.M., Simarro, I., 1989. The prevalence of antibodies to Influenza virus and respiratory corona virus in fattening pigs in Spain. Zentralblatt fur Veterina¨rmedizin Reihe B 36, 551–556. Yus, E., Sanjuan, M.L., Garcia, F., Castro, J.M., Simarro, I., 1992. Influenza A viruses: epidemiologic study in fatteners in Spain (1987–89). Zentralblatt fur Veterina¨rmedizin Reihe B 39, 113–118.
Short communication
Evidence of the concurrent circulation of H1N2, H1N1 and H3N2 influenza A viruses in densely populated pig areas in Spain Jaime Maldonado a,*, Kristien Van Reeth b, Pere Riera a, Marta Sitja` a, Narcı´s Saubi a, Enric Espun˜a a, Carlos Artigas a b
a Laboratorios HIPRA, S. A., Av. La Selva No. 135, Amer, 17170 Girona, Spain Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
Abstract This paper reports on a serological and virological survey for swine influenza virus (SIV) in densely populated pig areas in Spain. The survey was undertaken to examine whether the H1N2 SIV subtype circulates in pigs in these areas, as in other European regions. Six hundred sow sera from 100 unvaccinated breeding herds across Northern and Eastern Spain were examined using haemagglutination inhibition (HI) tests against H1N1, H3N2 and H1N2 SIV subtypes. Additionally, 225 lung samples from pigs with respiratory problems were examined for the presence of SIV by virus isolation in embryonated chicken eggs and by a commercial membrane immunoassay. The virus isolates were further identified by HI and RT-PCR followed by partial cDNA sequencing. The HI test on sera revealed the presence of antibodies against at least one of the SIV subtypes in 83% of the herds and in 76.3% of the animals studied. Of the 600 sow sera tested, 109 (18.2%), 60 (10%) and 41 (6.8%) had SIV antibodies to subtype H1N2 alone, H3N2 alone and H1N1 alone, respectively. Twelve H3N2 viruses, 9 H1N1 viruses and 1 H1N2 virus were isolated from the lungs of pigs with respiratory problems. The analysis of a 436 nucleotide sequence of the neuraminidase gene from the H1N2 strain isolated further confirmed its identity. Demonstrably, swine influenza is still endemic in the studied swine population and a new subtype, the H1N2, may be becoming established and involved in clinical outbreaks of the disease in Spain. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Influenza A viruses; H1N2; Swine; Influenza; Flu; Spain
Surveillance of swine influenza (SI) viruses is important because new influenza viruses and influenza virus reassortants regularly emerge in pig populations, and because there are considerable antigenic and genetic differences between swine influenza viruses (SIVs) in different geographic regions. Monitoring and characterization of SIV in pig producing regions is needed for the adequate control and diagnosis of infection. Furthermore, there are concerns that pigs may serve as intermediate hosts for the introduction of new influenza viruses into the human population (Brown et al., 1998). *
Corresponding author. Tel.: +34 972 43 06 55; fax: +34 972 43 06
61. E-mail address: [email protected] (J. Maldonado). 1090-0233/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2005.04.014
The H1N1 and H3N2 SIV subtypes have been enzootic in several swine producing countries in Europe for more than 20 years (Brown, 2000). A third subtype, H1N2, was first isolated in Great Britain in 1994 (Brown et al., 1995). Thereafter, the H1N2 virus subtype has also been found in France, Belgium, Italy and Germany, where it appears to co-circulate with H1N1 and H3N2 viruses (Gourreau et al., 1994; Van Reeth et al., 2000; Marozin et al., 2002; Schrader and Suss, 2003). In Spain, SIV subtypes H1N1 and H3N2 have been circulating since the mid 1980s. Both H1N1 (Plana Duran et al., 1984) and H3N2 (Castro et al., 1988) SIV strains have been isolated from pigs with acute respiratory problems. Serological examinations of fattening pigs between 1987 and 1989 revealed high
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seroprevalences for both H1N1 (between 72.8% and 78.5% seropositive pigs) and H3N2 (between 61.6% and 62.5% seropositive pigs) (Yus et al., 1989, 1992). Recent reports point towards a continuous circulation during the 1990s of H1N1 and H3N2 SIV in the Spanish pig population (Oliveira et al., 1999; Gutierrez-Martin et al., 2000). However, there is little information on the current seroprevalences of H1N1 and H3N2 subtype influenza viruses. Moreover, it is unknown whether H1N2 SIV viruses are circulating in Spain, as neither virus isolation nor prevalence of SIV subtype specific antibodies have been recently reported. In this paper, we report on a recent virological and serological survey for different SIV subtypes in a representative part of the Spanish swine population. It was the purpose of this survey to determine the prevalence of H1N2 virus antibodies in the studied population, and to compare it with that of H1N1 and H3N2 antibodies. We also took the opportunity to subtype SIV isolates from acute respiratory disease outbreaks. Farm selection followed the criteria established by the European Surveillance Network for Influenza in Pigs (ESNIP group). Breeding herds were included in the study when they met the inclusion criteria of being located in the most densely populated pig areas in Spain and not having had vaccination programme against SIV (confirmed by telephone interviews with the farmers). Vaccination against SIV in Spain is neither compulsory nor common practice. The number of samples was calculated from an estimated population of 875,000 females, with 4% precision at a 95% confidence level (Epi Info software package, version 5.01). Serological testing was performed on 600 sow sera from 100 conventional two-site operations (six samples per unit including sows of all parities) located in the four major Spanish swine producing autonomous regions (Arago´n, Castilla and Leon, Catalonia and Murcia) and distributed around 22 provinces in Northern and Eastern Spain (accounting for 65% of the national pig inventory). Sera were collected during the period February–June 2003. On arrival at the laboratory, sera were separated by centrifugation, split and stored in vials at 20 °C until analysed. Haemagglutination inhibition (HI) tests against H1N1, H3N2 and H1N2 subtypes were performed using standard methods (Webster and Krauss, 2002). The SIV strains Sw/Spain/45304/2003(H1N1), Sw/Spain/46356/ 2003(H3N2) and Sw/Scotland/410440/94(H1N2) were used as antigens. Two-fold serum dilutions were tested starting at a dilution of 1/20. The results were recorded as the log2 of the HI titre, and the geometric mean titre (GMT) for each virus subtype was calculated. A farm was classified as positive and assigned to a SIV subtype when at least one of the six sows had HI titres above the cut-off value against 1 of the SIV subtypes alone, regardless of the presence of individuals showing mixed infections.
In the virological surveillance, diagnostic specimens were examined. Between November 2001 and April 2004, 225 pig lungs from unrelated growing-finishing pig units (housing an average of 800 pigs per unit) were submitted to the Veterinary Diagnostic Centre, Laboratorios HIPRA, S.A. (Girona, Spain) for the aetiological diagnosis of pneumonia. The herds of origin (located all around Spain) were experiencing acute episodes of respiratory disease, mainly at the end of the finishing period. Affected animals showed clinical signs and macroscopic lesions suggestive of acute viral pneumonia. Bacteriological and virological analyses were performed to detect the most relevant respiratory pathogens for swine (data not shown). Samples (lung and bronchial tissues) for SIV investigation were systematically collected. Ten per cent lung tissue homogenates were examined for the presence of SIV antigen by a commercial membrane enzyme immunoassay (EI) (BD Directigen Flu A; Becton Dickinson Microbiology Systems) and for virus isolation by inoculation in 9- to 11-day old specific-pathogen-free embryonated chicken eggs (ECE), as described by Swenson et al. (2001). Allantoic fluids were harvested after 3–4 days of incubation at 37 °C. The presence of SIV was determined in a haemagglutination (HA) assay with 0.5% chicken red blood cells. To determine the subtype of SIV isolates, allantoic fluids were examined in a HI assay with swine hyperimmune sera using standard methods (Webster and Krauss, 2002) and by single-step reverse transcription-polymerase chain reaction (RT-PCR). RNA was extracted from 200 lL of allantoic fluids, by using the RNeasy Protect Mini Kit (QIAGEN). PCR amplification conditions and primers designed to amplify partial sequences of the H1, H3, N1 and N2 SIV genes were adapted from Chiapponi et al. (2003). Briefly, 0.6 lM of each primer and 5 lL of the extracted RNA were added to four separate RT-PCR mixtures (one for each target) (OneStep RT-PCR Kit, QIAGEN) to obtain final volumes of 50 lL. The following conditions were set up in a Px2 Thermal Cycler (Thermo Electronic Corporation): the first strand cDNA synthesis was conducted for 30 min at 50 °C. After denaturation for 15 min at 95 °C, the samples were submitted to 40 cycles of PCR amplification as follows: 1 min at 95 °C, 1 min at 50 °C and 1 min at 72 °C. The final extension step was for 7 min at 72 °C. In all PCR reactions, predefined SIV-positive, -negative and nontemplate controls were tested simultaneously with the field isolates. PCR products were analysed by agarose gel electrophoresis. To further confirm the identity of some of the SIV isolates, selected PCR products (N2 fragments) were sequenced using an ABI 373 DNA sequencer, together with the Taq dye-Deoxy terminator cycle sequencing kit (Applied Biosystems). Comparative nucleotide and amino acid sequence analysis were conducted using
J. Maldonado et al. / The Veterinary Journal 172 (2006) 377–381
software tools from the National Centre for Biotechnology Information (NCBI, Maryland, USA [Available from: http://www.ncbi.nlm.nih.gov/]). The HI test revealed the presence of antibodies against at least one of the SIV subtypes in 83% of the herds and 76.3% of the animals studied. Of the 600 sow sera tested, 109 (18.2%), 60 (10%) and 41 (6.8%) had SIV antibody to subtype H1N2 alone, H3N2 alone and H1N1 alone, respectively. Mixed infections were detected in 41.3% of the animals tested. Most were housed in farms in which the identification of the dominant subtype of circulating virus was not possible. Table 1 summarizes the HI results at the sow and farm levels. Twenty-two SIV lung samples (9.8%) were found to be positive for SIV by isolation. All tested positive in the EI, HI and PCR tests, and total agreement between HI and PCR allowed the classification of the isolates as being of the H3N2 (12 isolates), H1N1 (9 isolates) and H1N2 (1 isolate) subtypes. The H1N2 virus strain A/sw/Polen˜ino/40564/ 02(H1N2) was isolated from the lungs of a 5-month-old fattening pig, during a typical outbreak of acute respiratory disease. No other relevant pathogen from that particular sample was detected or isolated. The affected farm had 1600 pigs and morbidity and mortality rates during the respiratory disease outbreak were 50% and 1%, respectively. Alignment of the N2 gene partial sequences obtained from isolate A/sw/Polen˜ino/40564/02(H1N2) with those of the most closely related Italian and French H1N2 strains available in electronic data bases (GenBank, EMBL, DDBJ and PDB) (Accession Nos.: AJ412696, AJ412705, AJ412701, AJ412693, AJ412698, AJ412695, AJ412694 and AJ412704) revealed 95–97% homology. Deduced protein sequences from both the Italian A/swine/Italy/1081/00(H1N2) and the French A/ swine/Cotes dÕArmor/790/97(H1N2) strains (first and second places in the nucleotide–nucleotide matching list of the BLAST analysis) showed 97% homology with the A/ sw/Polen˜ino/40564/02(H1N2) isolate (Fig. 1).
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Our data show that SIVs of H1N1 and H3N2 subtype have remained endemic in the Spanish swine population. A new finding is that the recently emerged H1N2 SIV is widespread in Spain and that it is involved in respiratory outbreaks, as is the case in other European countries (Van Reeth et al., 2000; Marozin et al., 2002; Schrader and Suss, 2003). Our serology results may raise the question of whether the positive reactions to the H1N2 virus in the HI test may in part be due to serological crossreaction with the H1N1 subtype or vice versa. However, in experimental infection studies with both a single subtype and a combination of two SIV subtypes it has been clearly demonstrated that there is no serological crossreaction between H1N1, H1N2 and H3N2 subtypes in the HI test (Van Reeth et al., 2003). It is likely therefore that those animals in this study with HI antibodies against H1N1 or H1N2 have been previously infected with the respective subtypes. In our study, the serological determination of the dominant serotype infecting a high proportion of the tested sera was not feasible. It has recently been demonstrated that when field serum contains significant amounts of anti-SIV antibody to more than one subtype after natural infections, it cannot be classified in a reliable way by using current HI methods (Long et al., 2004). This could be the situation in this study, as the seroprevalence rates for H1N1, H3N2 and H1N2 SIV observed suggest that the three subtypes are circulating concurrently and cause mixed infections. SIV was isolated from 9.8% of the 225 lung samples tested. This cannot be considered as a prevalence rate as it is the result of a diagnostic exercise rather than a prevalence survey. In other surveys of acute respiratory disease outbreaks in the Netherlands, SIVs were isolated from 45% of the acute respiratory disease outbreaks examined (Loeffen et al., 1999) and similar SIV isolation rates have been reported in Belgium (K. Van Reeth, personal communication) and in the US (Choi et al., 2003). It must be mentioned, therefore, that some of the
Table 1 Numbers of individual animals (n = 600) and herds (n = 100) with HI antibodies to one single SIV subtype or to combinations of 2 or 3 subtypes (Spain, 2003) Virus subtype(s)
Number and percentage of positivesa Individual sows
None (negative to all 3 subtypes) H1N1 only H3N2 only H1N2 only H1N1 + H3N2 H3N2 + H1N2 H1N1 + H1N2 H1N1 + H3N2 + H1N2 a b
b
142 41 60 109 40 58 80 70
The cut-off value for HI was 1/40. Cumulative number of negative sows housed in positive and negative herds.
(%)
Herds
(%)
23.7 6.8 10.0 18.2 6.6 9.7 13.3 11.7
17 9 20 21 6 6 5 16
17.0 9.0 20.0 21.0 6.0 6.0 5.0 16.0
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(a)
(b)
Fig. 1. Partial nucleotide and deduced amino acid sequences for the neuraminidase gene of the virus strain A/sw/Polen˜ino/40564/02(H1N2). The nucleotide sequence (436 nucleotides) is shown in the top lines (a). Alignment with the predicted amino acid sequences for A/swine/Italy/1081/ 00(H1N2) (Accession No. AJ412696) and A/swine/Cotes dÕArmor/790/97(H1N2) (Accession No. AJ412705) was performed by ClustalW programme (http://www.ebi.ac.uk/clustalw/): Shaded areas denote sequence differences.
diagnostic submissions in the present study were accompanied by rather vague descriptions of the clinical symptoms, and only the known acute cases of respiratory disease were examined in the studies mentioned above. Other factors that may account for the rather low SIV isolation rates in the present study are suboptimal conservation and autolysis of part of the lung tissue samples. Although direct PCR could give a better prevalence estimation of SIV infection on autolytic samples, this was not the objective of our survey. With the limited clinical information available, we cannot assume that the SIVs isolated played a central role in the course of the studied outbreaks. Furthermore, other bacteria and viruses were also present in some of the SIV-positive lungs (data not shown). It should be mentioned nevertheless, that SIV is recognized as an important contributor in the aetiology of the porcine respiratory disease complex, infecting alone or in combination with other pathogens (Thacker et al., 2001). The origin of the strain A/sw/Polen˜ino/40564/ 02(H1N2) remains elusive, but genetic analyses of the N2 suggest that this strain is related to H1N2 viruses isolated in France and in Italy since the late 1990s. Based on the antigenic and genetic relationships of viruses isolated in France, Italy and the UK, Marozin et al. (2002) stated that H1N2 viruses were introduced into continental Europe from the UK. However, sequencing of the internal genes is required to further determine the origin of the Spanish H1N2 strain, as recent studies have demonstrated extensive antigenic and genetic heterogeneity among H1N2 viruses (Marozin et al., 2002).
Results obtained in the present study highlight the need for continuous surveillance for SIVs in pig producing areas, as new and potentially pathogenic SIV subtypes readily emerge. Also, this study will contribute significantly to the task of the ESNIP group in monitoring the evolution of SI in Europe during the next years. All the data collected and viruses isolated until now, and the obtained ones in a future, will be deposited in the virus bank and the electronic database for further studies on molecular epidemiology, immunology and genetics of SI in Europe (http://www.esnip.wur.nl/).
Acknowledgements The authors thank Romney Jackson for helpful language revision. Technical assistance of Corona Vin˜als is gratefully acknowledged. This research was supported by Laboratorios HIPRA S. A., Amer, Gerona, Spain and ESNIP (EC concerted action, 6th Framework Programme, QLK2-CT-2000-01636).
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