Presence of antibodies against H 5 , H 7 and H 9 influenza A virus in wild birds in the State of São Paulo , Brazil

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 hemagglutinationinhibition 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.


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 hemagglutinationinhibition 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.

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 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.

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 Durigon EL, Araujo J de, Ferreira CS, Pinto AA

Presence of antibodies against H5, H7 and H9 influenza A virus in wild birds in the State of São Paulo , Brazil
analyzed.The lack of data on the prevalence of typespecific 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.