Presence of antibodies against H5, H7 and H9 influenza A virus in wild birds in the State of São Paulo, Brazil

Presence of antibodies against H5, H7 and H9 influenza A virus in wild birds in the State of São Paulo, Brazil

Sousa E de^I; Costa T P^I,*; Werther K^I; Durigon EL^II; Araujo J de^II; Ferreira CS^II; Pinto AA^I

^IDepartamento de Patologia Veterinária, Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista, Jaboticabal, São Paulo, Brazil

^IIDepartamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil

^{Correspondence}

ABSTRACT

Although the natural reservoirs of the avian influenza (AI) virus have been extensively studied in many countries, there is a clear lack of information on this subject in South America, particularly in Brazil. The objective of this study was to conduct a serological survey for H5, H7 and H9 antibodies to AI-subtype viruses in wild birds in the state of São Paulo , Brazil. Serum samples were tested using the hemagglutination-inhibition assay. Out of the 31 wild birds sampled between January and December of 2006, seven (22.58%), were seropositive for H5, H7 and H9; four (12.90%) were seropositive for H5 and H7; 13 (41.94%), were seropositive only for H7; three (9.7%), were seropositive only for H9; and four (12.90%) were negative for all three hemagglutinin subtypes. These results indicate that AI viruses belonging to H5, H7 and H9 subtypes circulate among wild birds in the state of São Paulo in the form of either concurrent or consecutive infections. This study contributes to the knowledge of AI epidemiology in Brazil, and stresses the need of further detailed and long-term epidemiological and ecological investigation to determine the current status of this virus.

Keywords: Avian influenza virus, Brazil, hemagglutination inhibition, reservoir, wild bird.

INTRODUCTION

Influenza virus type A of bird origin, also called avian influenza (AI) virus, has been implicated in endemic infections and outbreaks in poultry and wild birds and in human infection and fatalities, as well as in important economic losses (Martins, 2001; Mcleod, 2008; Moraes et al., 2009; Malik, 2009; Lupiani & Reddy, 2009; Kalthoff et al., 2010). AI viruses belong to the Orthomyxoviridae family, and are classified into subtypes based on two surface glycoproteins used for host-cell entry: hemagglutinin (H) and neuraminidase (N) (Moraes et al., 2009). Different 16 H and 9 N subtypes are currently identified (Halvorson, 2002; Suarez, 2008). AI viruses are further classified into low pathogenic AI (LPAI) or highly pathogenic AI (HPAI) viruses according to their ability to cause illness and death of 4- to 6-week-old chickens infected intravenously and/or the presence of multiple basic amino acids at the cleavage site of the H molecule (World Organisation For Animal Health Avian Influenza, 2009). So far, only H5 and H7 AI viruses have been identified as HPAI, and these viruses have been responsible for outbreaks in many countries, causing the death of many thousands of domestic poultry and wild birds (Capua & Alexander, 2004; Kalthoff et al., 2010). In 1999, an H9N2 AI virus was implicated in human infections in Asia, suggesting a pandemic threat related to this virus (Lin et al., 2000; Peiris et al., 1999).

AI viruses have been isolated in more than 100 wild avian species worldwide (Stallknecht & Shane, 1988; Alexander, 2000; Olsen et al., 2006; Stallknecht & Brown, 2008). Most have been isolated from species present in wetlands and aquatic habitats, particularly Anseriformes and Charadriiformes, which are considered as natural reservoirs of AI viruses (Stallknecht & Brown, 2007; Webster et al., 1992). AI infections in these birds are usually subclinical, and can be produced by a single or multiple AI viruses simultaneously (concurrent infections) or subsequently (consecutive infections) (Sinnecker et al., 1982; Suss et al., 1994). Although the diagnosis of AI is usually based on virus isolation and identification (Martins, 2001; Swayne & Halvorson, 2008), serological tests, such as agar gel immunodiffusion, enzyme-linked immunosorbent assay, and hemagglutination inhibition (HI) test, have been widely used for AI surveillance (Allwinn et al., 2002; Brown et al., 2010; Moraes et al., 2009; Swayne & Halvorson, 2008).

AI is considered an exotic disease in Brazil, and has not been reported in commercial poultry (Martins, 2001; Moraes et al., 2009). Few studies have been conducted in Brazil aiming at either isolating (Couceiro, 1986; Kawamoto et al., 2005) or identifying (Soares, 2002) AI viruses, or at investigating the production of antibodies (Oliveira Junior et al., 2001; Viegas, 2006) in wild or ornamental birds and domestic poultry. The present study reports the results of a serological surveillance of H5, H7 and H9 AI viruses in wild birds in the state of São Paulo, Brazil, and aims at contributing to the current knowledge on AI in South America.

MATERIAL AND METHODS

Birds

Thirty-one wild birds (Table 1) were included in this study. Birds were admitted to the Exotic and Wild Animal Service of Hospital Veterinário Governador Laudo Natel (Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista, Jaboticabal, SP, Brazil) between January and December of 2006. Birds had been found injured and had been taken to the hospital by firefighters, forest rangers, or employees of the highway concessionaires in the area. Access to birds and collection of biological samples were authorized by the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA, process # 02027.000775/2005-25; license # 500/2005).

Blood collection and Serology

Blood was collected from the right jugular vein. Plasma was separated by centrifugation, inactivated at 56 °C for 30 minutes, and stored at -20°C.

HI test was used for the detection of influenza antibodies. In order to remove nonspecific hemagglutination inhibitors, 0.2 mL of plasma were added to 0.8 mL of 25% kaolin and 1.0 mL of phosphate buffered saline solution (PBS) at pH 7.2. The mixture was homogenized, incubated overnight at 37°C, and centrifuged at 600g for 30 minutes. The supernatant transferred to clean tubes. In order to remove nonspecific hemagglutinating agents, plasma was then incubated in 0.2mL suspension of chicken erythrocytes at 50% in PBS for one hour at room temperature (25°C). After incubation, erythrocytes were removed by centrifugation at 600g for 10 minutes at 8°C and the sample was stored at -20°C. The influenza virus strains A/RT/DE/244/94 (H5N2); A/Equine/Prague/56 (H7N7) and A/Hong Kong/1073/99 (H9N2) were inactivated in β-propiolactone and used as viral antigens.

HI was performed in 96-well U-shaped microtiter plates. Treated plasma was diluted in 50µL aliquots in PBS, and mixed with four hemagglutination units of viral antigen. Plates were incubated at room temperaturefor 30 minutes, and 50µL of a suspension of washed SPF chicken red blood cells (1% in PBS) was added to each well. After 30 minutes incubation at room temperature, the microtiter plates were evaluated for the presence of hemagglutination inhibition. Samples with HI titers < 40 for H5, < 64 for H9, and < 256 for H7 were considered negative. Analyses were performed at the Laboratory of Clinical and Molecular Virology of the Institute of Biomedical Sciences II, University of São Paulo (São Paulo, Brazil).

RESULTS AND DISCUSSION

Between January and December of 2006, a total of 31 wild birds (11 species and 7 orders) were tested (Table 1). Twenty-seven (87.0%) of the 31 samples analyzed were positive for AI by the HI test (Table 1). Antibodies against one, two or all three AI subtypes tested were present in a single bird, indicating previous concurrent or consecutive infections with different AI virus subtypes. Overall, 24 birds (77.4%) had detectable antibody titers against H7, 11 birds (35.5%) against H5, and ten (32.3%) against H9. Seven birds (22.6%) were positive for the presence of antibodies against H5, H7 and H9; four (12.9%) for the presence of antibodies against H5 and H7; 13 (41.9%) for the presence of antibodies against H7 only; three (9.7%) for the presence of antibodies only against H9; and four (12.9%) were negative for all three H subtypes analyzed. The lack of data on the prevalence of type-specific antibodies against AI in wild birds in Brazil limits the interpretation and comparison of the results of the present study .

Virus isolation was not attempted in any of the birds in this study. Therefore, it is not known whether these birds were actively shedding AI virus at the time of admittance, and it is not possible to make any statements relating the type of injury birds presented with their AI status. Due to the relatively small number of samples analyzed each month, it is also not possible to make any statistical correlations relative to seasonal variation with AI status.

The results of this study evidenciate that AI viruses belonging to H5, H7 and H9 subtypes circulate among wild birds in the state of São Paulo. Moreover, 11 birds included in this study were previously infected with more than one subtype of AI virus, either concurrently or consecutively. This is an evidence of the complex natural history of AI in wild avian populations (Dugan et al., 2008). Avian influenza, particularly in wild birds, has received little attention in Brazil. Although some studies report the isolation and identification of this agent in Brazil (Couceiro, 1986; Kawamoto et al., 2005), the AI virus is still considered exotic in this country. More detailed and long-term AI serosurveillance studies in wild birds, associated with attempts to isolate the virus, are needed to determine the status and the epidemiological characteristics of this disease in Brazil.

ACKNOWLEDGMENTS

We are grateful to the personnel of the Laboratory of Clinical and Molecular Virology of the Institute of Biomedical Sciences II, University of São Paulo, for their help with sample analysis.

Submitted: January/2012

Approved: February/2013

• Alexander DJ. A review of avian influenza in different bird species. Veterinary Micobiology 2000; 74:3-13
• Allwinn R, Preiser W, Rabenau H, Buxbaum S, Sturmer M, Doerr HW. Laboratory diagnosis of avian influenza - virology or serology? Medical Microbiolology Immunolology 2002;191:157-160.
• Brown JD, Luttrell MP, Uhart MM, Del Valle Ferreyra H, Romano MM, Rago MV, Stallknecht DE. Antibodies to type A influenza virus in wild waterbirds from Argentina. Journal Wildlife Diseases 2010;46:1040-1045.
• Capua I, Alexander DJ. Avian influenza: recent developments. Avian Pathology 2004;33:393-404.
• Couceiro JNSS. Estudo da ocorrência de infecções por vírus influenza A e vírus da doença de Newcastle numa comunidade de aves ornamentais em cativeiro [thesis]. Rio de Janeiro (RJ): Universidade Federal do Rio de Janeiro; 1986.
• Dugan VG, Chen R, Spiro DJ, Sengamalay N, Zaborsky J, Ghedin E, Nolting J, Swayne DE, Runstadler JA, Happ GM, Senne DA, Wang R, Slemons RD, Holmes EC, Taubenberger JK. The evolutionary genetics and emergence of avian influenza viruses in wild birds. PLoS Pathogens 2008;4: e1000076.
• Halvorson DA. The control of H5 or H7 mildly pathogenic avian influenza: a role for inactivated vaccine. Avian Pathology 2002;31:5-12.
• Kalthoff D, Globig A, Beer M. (Highly pathogenic) avian influenza as a zoonotic agent. Veterinary Micobiolology 2010;140:237-245.
• Kawamoto AHN, Mancini DAP, Pereira LE, Cianciarullo AM, Cruz AS, Dias ALF, Mendonça RMZ, Pinto JR, Durigon EL. Investigation of influenza in migrating birds, the primordial reservoir and transmitters of influenza in Brazil. Brazilian Journal Microbiology 2005;36:88-93.
• Lin YP, Shaw M, Gregory V, Cameron K, Lim W, Klimov A, Subbarao K, Guan Y, Krauss S, Shortridge K, Webster R, Cox N, Hay A. Avian-to-human transmission of H9N2 subtype influenza A viruses: relationship between H9N2 and H5N1 human isolates.  Proceedings of the National Academy of Science USA 2000;97:9654-9658.
• Lupiani B, Reddy SM. The history of avian influenza. Comparative Immunology Microbiology Infectious and Diseases 2009;32:311-323.
• Malik Peiris JS. Avian influenza viruses in humans. Revue Scientifique et Technique 2009;28:161-173.
• Martins N. Avian influenza: a review of the last ten years. Revista Brasileira Ciencia Avicícola 2001; 3: 97-140.
• Mcleod A. The economics of avian influenza. In: Swayne DE, editor. Avian influenza. Ames, IA: Blackwell Publishing; 2008. p.537-560.
• Moraes HSL, Salle CTP, Caron LF. Influenza aviária. In: Berchieri Junior A, Silva EN, Di Fabio J, Sesti L, Zuanaze MAF, editors. Doença das aves. Campinas: FACTA; 2009. p.611-627.
• Oliveira Junior JG, Belluci MSP, Vianna JSM, Mazur C, Andrade CM, Fedullo LPL, Portz C, Loureiro BO. Avaliação soroepidemiológica do vírus influenza em aves domésticas e silvestres no Estado do Rio de Janeiro. Arquivo Brasileiro de Medicina Veterinaria e Zootecnia 2001;53:299-302.
• Olsen B, Munster VJ, Wallensten A, Waldenstrom J, Osterhaus ADME, Fouchier RAM. Global patterns of influenza a virus in wild birds. Science 2006;312:384-388.
• Peiris M, Yuen KY, Leung CW, Chan KH, Ip PL, Lai RW, Orr WK, Shortridge KF. Human infection with influenza H9N2. Lancet 1999;354:916-917.
• Sinnecker H, Sinnecker R, Zilske E, Koehler D. Detection of influenza A viruses and influenza epidemics in wild pelagic birds by sentinels and population studies. Zentralblatt Bakteriologie fur Mikrobiologie und Hygiene A 1982;253:297-304.
• Soares PBM. Padronização da RT-PCR dúplex para detecção dos vírus da influenza A e doença de Newcastle em aves migratórias [dissertation]. São Paulo (SP): Universidade de São Paulo; 2002.
• Stallknecht DE, Brown JD. Ecology of avian influenza in wild birds. In: Swayne DE, editor. Avian influenza. Ames, IA: Blackwell Publishing; 2008. p. 43-58.
• Stallknecht DE, Brown JD. Wild birds and the epidemiology of avian influenza. Journal of  Wildlife of Diseases 2007;43:S15-S20.
• Stallknecht DE, Shane SM. Host range of avian influenza virus in free-living birds. Veterinary Research Communications 1988;12:125-141.
• Suarez DL. Influenza A virus. In: Swayne DE, editor. Avian influenza. Ames: Blackwell Publishing; 2008. p. 3-22.
• Suss J, Schafer J, Sinnecker H, Webster RG. Influenza virus subtypes in aquatic birds of eastern Germany. Archives of Virolology 1994;135:101-114.
• Swayne DE, Halvorson DA. Influenza. In: Saif YM, Glisson JR, Fadly AM, Mcdougald LR, Nolan L. editors. Diseases of poultry. Ames, IA: Blackwell Publishing; 2008. p. 153-184.
• Viegas RE. Levantamento sorológico para o virus da influenza A (subtipos H6, H7 e H9) em pinguin-de-Magalhães do Aquário Municipal de Santos e Acqua Mundo-Guarujá [monograph]. Santos (SP): Centro Universitário Monte Serrat; 2006.
• Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y. Evolution and ecology of influenza A viruses. Microbiological  Reviews 1992;56:152-179.
• World Organisation For Animal Health. Avian influenza. Terrestrial Animal Health Code. Paris; 2009. p. 1-20.

*

Current address: Centre de Recerca en Sanitat Animal. Edifici CReSA, Campus UAB, Bellaterra, 08193, Barcelona, Spain

Publication Dates

• Publication in this collection
  01 Oct 2013
• Date of issue
  Sept 2013

History

• Received
  Jan 2012
• Accepted
  Feb 2013