Veterinary Research Communications, 27 Suppl. 1 (2003) 107–114© 2003 Kluwer Academic Publishers. Printed in the Netherlands

Long-term Monitoring for Avian Influenza Viruses in Wild Bird

Species in Italy

M.A. De Marco1*, E. Foni2, L. Campitelli3, E. Raffini4, M. Delogu5 and I. Donatelli31Istituto Nazionale per la Fauna Selvatica, Ozzano Emilia (BO); 2IstitutoZooprofilattico Sperimentale della L ombardia e dell’Emilia Romagna, Parma;3Department of V irology, Istituto Superiore di Sanita, Rome; 4Istituto ZooprofilatticoSperimentale della L ombardia e dell’Emilia Romagna, L ugo (RA); 5Department ofPublic Health and Animal Pathology, Faculty of Veterinary Medicine, University ofBologna, Italy*Correspondence: Istituto Nazionale per la Fauna Selvatica ‘A. Ghigi ’, 9 V ia Ca’Fornacetta, 40064 Ozzano Emilia (BO), ItalyE-mail: infs.adem@iperbole.bologna.it

Keywords: avian influenza, Italy, serological surveys, virological surveys, wild birds

Abbreviations: HA, hemagglutinin; HI, haemagglutination inhibition; HPAI, highly pathogenic avianinfluenza; IVPI, intravenous pathogenicity index; LPAI, low-pathogenicity avian influenza; N, neura-mindase; NP-ELISA, nucleoprotein ELISA

INTRODUCTION

The first reported isolation of an influenza virus from wild birds was obtained in 1961from common terns (Sterna hirundo) in South Africa, where a highly pathogenic avianinfluenza (HPAI) virus (H5N3 subtype) caused the death of about 1300 birds(Stallknecht and Shane, 1988). Although evidence for the infection of wild birds withinfluenza viruses existed prior to the 1970s, it was not until then that systematicsurveillance studies revealed the widespread distribution of influenza viruses in wildavian populations (Webster et al., 1992). To date a vast number of low pathogenicityavian influenza (LPAI) viruses have been isolated from a wide variety of birds. TheHPAI viruses have rarely been isolated from feral birds, and when they have it hasusually been from wild birds found dead in the vicinity of farms infected by HPAIviruses (Capua et al., 2000). The above-mentioned mass mortality among commonterns represents the only case reported to date of influenza infection in free-livingbirds that is associated with obvious signs of disease and is unrelated to outbreaks ofHPAI viruses in domestic birds (Alexander, 2000).Aquatic birds are considered to be the main reservoir of influenza A viruses innature, harbouring in their populations viruses of all 15 (H1–H15) haemagglutinin(HA) and all 9 (N1–N9) neuraminidase (NA) subtypes, with almost every possiblecombination of antigenic subtypes. In particular, among wild waterfowl, ducks are ofparamount importance for the ecology of this infection, providing, by various mecha-nisms, most of the gene pool that is the source of all influenza viruses both in human

107

108

and in animal populations (Webster et al., 1992). Wild waterfowl are often consideredto be the primary source of poultry outbreaks – H5 or H7 LPAI viruses circulating inwild populations could infect poultry reared in semi-wild conditions. The kind ofbreeding, such as that where poultry species are kept in bird-proof confinement,provides strategies for the prevention of the spread of infection by wild birds(Alexander, 2000).

LONG-TERMMONITORING IN FREE-LIVINGWATERFOWL

Numerous studies suggest the important role of waterfowl in the ecology of avianinfluenza viruses; therefore, in 1992 we initiated a long-term monitoring programmeto evaluate the presence and diffusion of influenza viruses in waterfowl caught forringing purposes in two neighbouring wildlife refuges (‘Laguna di Orbetello’ and‘Lago di Burano’ WWFOases) located on the west coast of Italy, about 140 km northof Rome. Both represent important wintering sites for migratory and resident water-fowl; the main breeding sites of these migrant populations are wetlands in central andnorth-eastern Europe. Serum samples and cloacal swabs were collected from ducksand coots during consecutive wintering periods, when numerous flocks of these birdswere concentrated in a few clustered sites. These studies were performed to evaluatethe circulation of avian influenza viruses in wild reservoirs in the Mediterranean area,and to investigate possible epidemiological relationships with influenza in poultry.The main serological and virological results are described below.

Serological results

In order to detect antibodies against type A influenza virus, serum samples weretested using a double antibody sandwich ELISA, nucleoprotein-ELISA (NP-ELISA).The haemagglutination-inhibition (HI) assay was performed to assess the presence ofantibodies against different H subtypes.The NP-ELISA (De Marco et al., 1996) was used to test 1040 serum samples

obtained from 591 ducks and 449 coots caught during six wintering periods, from1992 through 1998. The bird age was identified whenever possible. According to themain biological features of the avian species, birds were divided into four groups:(1) mallards (Anas platyrhynchos), 407 specimens (this species is employed in heavyrestocking activities; at least 71 400 birds are reared and released in hunting groundseach year in Italy); (2) other dabbling ducks (including Tadorna tadorna, Anas penel-ope, Anas crecca, Anas acuta), 70 specimens; (3) diving ducks (including Aythya ferina,Aythya nyroca, Aythya fuligula), 114 specimens; (4) coots (Fulica atra), 449 specimens.The overall seroprevalences detected for the four bird groups were 63.9% (mallards);31.4% (other dabbling ducks); 22.8% (diving ducks); 7.1% (coots). Figure 1 shows the

109

Figure 1. Seroprevalences of influenza A viruses detected in free-living waterfowl (1040 speci-mens) caught in Italy between 1992 and 1998

seroprevalences observed during the study period. Statistical analysis (De Marcoet al., 2000) showed that mallards were more positive than the two other duck groupsand that ducks in general were more positive than coots. No age group differenceswere noted among ducks; however, the seroprevalence was higher for adult cootsthan for the juvenile ones. The size of the coot wintering population is similar to thatof the duck population; nevertheless, the seroprevalence is lower and more adultsthan juveniles are positive. These results indicate a very slow spread of influenzainfection in the sampled coots.Using the NP-ELISA as a screening assay, the type A-positive serum samples,when available, were tested by HI (Anonymous, 1982; Alexander, 1989). The HI testwas performed to evaluate the influenza virus subtypes circulating in duck and cootpopulations and, in particular, the occurrence of H5 and H7 subtypes that couldrepresent risk factors for poultry. Fourteen reference strains of avian influenza viruseswere used in the HI test (from H1 to H14 subtypes). Serum samples were also testedusing the LPAI virus A/Turkey/Italy/6423-1/99 (H7N1). Figure 2 shows the percen-tage of HI-seropositive birds. These percentage values were calculated on NP-ELISA-seropositive ducks (278 specimens) and coots (24 specimens). From 1992 through1998, the ducks sampled showed HI antibodies against most of the H subtypes, exceptfor the H3, H4, H7 and H12 antigenic subtypes. In contrast, the coots were seroposi-tive against the H10 subtype only.

V irological results

In order to evaluate the circulation of avian influenza viruses in wild reservoirs,detailed virological studies were carried out in North America (Hinshaw et al., 1985)and northern Europe (Suss et al., 1994).

110

Figure 2. Percentage of HI-seropositive birds. The percentage values were calculated onNP-ELISA-seropositive ducks (278 specimens) and coots (24 specimens) caught in Italybetween 1992 and 1998. Fourteen reference strains (from H1 to H14) were used in the HI test.Serum samples were also tested using the H7N1 LPAI virus A/Turkey/Italy/6423–1/99 (H7*)

To study the epidemiology of avian influenza in a Mediterranean area, between1993 and 1998, a total of 802 cloacal swabs were collected, concomitantly with bloodsamples, from 469 ducks and 333 coots. Virus isolation and characterization wereperformed as described (Anonymous, 1982; De Marco et al., 2000). A total of 22viruses were isolated, as shown in Table I. The results obtained showed the importantrole in these Italian wetlands of ducks in the influenza ecology. The low overallisolation prevalence observed in ducks (18/469) showed only a few individuals har-bouring the virus during the winter period, whereas the high seroprevalences observedby the NP-ELISA’ in the absence of age group differences, indicated that the presentstudies were performed after a very rapid spread of the infection, occurring towards

TABLE IAvian influenza viruses isolated from waterfowl wintering in Italybetween 1993 and 1998

Bird species Influenza virus subtype No. isolates

Fulica atra H3N8 1H10N8 2H1N1 1

Anas platyrhynchos H5N2 1H1N1 16

Tadorna tadorna H1N1 1

111

the end of the breeding season, when the susceptible population is mainly representedby newly hatched ducks.

INFLUENZA IN GALLIFORMES, PASSERIFORMES, RAPTORS

In addition to the main research activities performed on waterfowl, we carried outepidemiological surveys on other wild bird species.

Galliformes

The limited number of cases of influenza in free-living Galliformes suggests thedynamics of a self-limiting infection within these populations (Stallknecht and Shane,1988). Nevertheless, the fact that the pheasant is resistant to some influenza virusstrains that are pathogenic for poultry (Wood et al., 1985), makes this species a riskfactor for the spread of infection. During February 1992 and February 1994, 194 and185 serum samples, respectively, were collected from free-living pheasants (Phasianuscolchicus) caught in an area of the Po Valley. Furthermore, since epidemiological dataon free-living quails (Coturnix coturnix coturnix) are not present in the literature,during May 1998, 260 serum samples were collected from 226 juvenile and 34 adultmigrating quails caught for ringing purposes in the Pesaro province. All the pheasantsand the quails sampled were seronegative to influenza A by agar gel precipitation (DeMarco et al., 1999) and by NP-ELISA (De Marco et al., 1996), respectively, so thesebirds did not seem to play a relevant role in the spread of infection.

Passeriformes

Although passerine birds are not currently considered important in the epidemiologyof avian influenza, this order includes over 5000 individual species; thus any conclu-sion regarding the entire order may be premature (Stallknecht and Shane, 1988).During August and September 1993, 83 cloacal swabs were collected from 79 juvenileand 4 adult reed warblers (Acrocephalus scirpaceus). The sampled species was associ-ated with aquatic habitats, where the species known to be epidemiological reservoirslive. The passerine birds were caught in the reed thicket of Lago di Massaciuccoli(PI). Virus isolation was performed as described (DeMarco et al., 2000). No influenzavirus isolation was achieved and this passerine species did not seem to be of impor-tance in influenza epidemiology during this seasonal sampling period.

Raptors

Influenza virus isolations have been reported for numerous free-living bird species(Stallknecht and Shane, 1988) and, considering that raptors are at the top of the food

112

pyramid, epidemiological surveys carried out in birds of prey could indicate thecirculation of influenza viruses in nature. Between July 1993 and December 1996, 192serum samples were collected from raptors (137 owls and 55 diurnal raptors) recentlyhospitalized in two rehabilitation centres in the Emilia Romagna region. TheNP-ELISA (De Marco et al., 1996) showed antibodies against the influenza A virusonly in two birds, a buzzard (Buteo buteo) and a peregrine (Falco peregrinus), indicat-ing the occurrence of infection in the wild. Seronegative results observed for the owlsseem to highlight their very low susceptibility to the infection, as has been previouslydescribed for these birds (Butterfield et al., 1973).

CONCLUSIONS

The results reported in this paper demonstrate the central role of waterfowl, particu-larly ducks, in the ecology of avian influenza in the Mediterranean area. The followingconclusions can be drawn.(a) The low virus isolation prevalences observed during the winter period arein agreement with data obtained in the northern hemisphere, in North America(Stallknecht and Shane, 1988).(b) The management of bird species could influence the dynamics of this infection:mallards (species possibly including birds reared and released in the wild) have sig-nificantly higher seroprevalences to influenza viruses. Previous studies showed sig-nificantly higher values in waterfowl species employed in restocking operations, bothfor hunting and for conservation purposes, whereas lower seroprevalences and meanantibody titres were observed in the species belonging to wild populations only (DeMarco et al., 1999).(c) Epidemiological data regarding the circulation in the wild of H5 and H7 influ-enza virus subtypes were obtained. Occurrence of the H5 subtype was demonstratedby both virus isolation and serological results; sequencing of the genome regioncoding for the cleavage site of the haemagglutinin molecule, allowed classification ofthe isolated A/Mallard/Italy.80.93 (H5N2) as a LPAI virus (L. Campitelli, personalcommunication, 1996). There is a notable lack of evidence, both virological andserological, regarding the circulation of the H7 subtype in the sampled waterfowlspecies. Both the long-term monitoring study (the data were obtained from six consec-utive wintering periods) and the large number of sampled birds corroborate theresults obtained. Additional research, which is still in progress in the same study area,has produced similar results in ducks and coots. No H7 subtypes of influenza viruseswere isolated, either from 145 cloacal swabs collected during the period betweenDecember 1998 and January 1999 or from 143 cloacal swabs collected duringDecember, 1999 (E. Foni, personal observation, 2000). Moreover, the HI assay wasperformed in order to test, for the H7 serotype, the NP-ELISA-positive sera ofwaterfowl; this was done using the LPAI virus A/Turkey/Italy/6423-1/99 (H7N1) asan antigen. Neither the 91 sera sampled during the period between December 1998

113

and January 1999 nor the 48 sera collected during December 1999 showed HI anti-bodies against this H7 serotype (De Marco, personal observation, 2000). The latterresults further support the absence, during the period considered in this report, of H7subtype virus circulation in the sampled avian species, which represent the principalreservoir of influenza viruses in nature. The data obtained include samples collectedat the end of 1999, when the H7N1 influenza virus involved in the last catastrophicItalian epidemic was already circulating in intensively reared poultry (Capua andMarangon, 2000).

ACKNOWLEDGEMENTS

The authors acknowledge Antonio Canu, Marco Carsughi, Luigi Calchetti and FabioCianchi (WWF Italian Delegation); Paolo Cordioli, Fausto Marzadori andGiampaolo Tozzoli (Istituto Zooprofilattico Sperimentale della Lombardia edell’Emilia Romagna); Amministrazioni Provinciali di Ferrara e Pesaro; and theLIPU Centres of Bologna and Ferrara. This work was partly funded by the grantsnos. 98JI and IZSLER 09/99-PRC99007 from the Ministero per la Salute (Italy).

REFERENCES

Alexander, D.J., 1989. Avian influenza (fowl plague) (A/015). In: Standards Commission of the OIE (ed.),OYce International des Epizooties Manual of Recommended Diagnostic T echniques and Requirements forBiological Products for L ist A & B Diseases, vol. I, (Office International des Epizooties, Paris), 1/6–6/6

Alexander, D.J., 2000. A review of avian influenza in different bird species. VeterinaryMicrobiology, 74, 3–13Anonymous, 1982. WHO Collaborating Centers for Reference and Research on Influenza. Concepts andProcedures for L aboratory-based Influenza-Surveillance, part B, Procedures, B1–B67

Butterfield, W.K., Yedloutschnig, R.J. and Dardiri, A.H., 1973. Isolation and identification of myxovirusesfrom domestic and imported avian species. Avian Diseases, 17, 155–159

Capua, I. and Marangon, S., 2000. The avian influenza epidemic in Italy, 1999–2000: a review. AvianPathology, 29, 289–294

Capua, I., Grossele, B., Bertoli, E. and Cordioli, P., 2000. Monitoring for highly pathogenic avian influenzain wild birds in Italy. Veterinary Record, 147 (22), 640

DeMarco, M.A., Guberti, V., Raffini, Delogu, M. and Govoni, S., 1996. Virus influenzali aviari e virus dellamalattia di Newcastle: indagine sierologica in uccelli acquatici svernanti in Toscana. In: M. Spagnesi,V. Guberti and M. A. De Marco (eds), Atti del Convegno Nazionale: Ecopatologia della Fauna Selvatica,Supplemento alle Ricerche di Biologia della Selvaggina, XXIV, 653–661

De Marco, M.A., Guberti, V., Raffini, E., Foni, E:, Delogu, M. and Donatelli, I., 1999. Influenza aviaria:indagini epidemiologiche in specie selvatiche. L a Selezione Veterinaria (12), 897–907

De Marco, M.A., Guberti, V., Foni, E., Raffini, E., Campitelli, L., Barigazzi, G., Delogu, M. and Donatelli, I.,2000. Waterfowl wintering in Italy: a serological and virological survey for type A influenza viruses. In:E. Brocchi and A. Lavazza (eds in chief ), Proceedings of the 5th International Congress of the EuropeanSociety for Veterinary V irology, Brescia, (Istituto Zooprofilattico Sperimentale della Lombardia edell’Emilia Romagna, Brescia and Fondazione Iniziative Zooprofilattiche e Zootecniche, Brescia),303–304

Hinshaw, V.S., Wood, J.M., Webster, R.G., Deibel, R. and Turner, B., 1985. Circulation of influenza virusesand paramyxoviruses in waterfowl originating from two different areas of North America. Bulletin ofthe World Health Organization, 63(4), 711–719

Stallknecht, D.E., and Shane, S.M., 1988. Host range of avian influenza virus in free-living birds. VeterinaryResearch Communications, 12, 125–141

114

Suss, J., Schafer, J., Sinnecker, H. and Webster, R.G., 1994. Influenza virus subtypes in aquatic birds ofeastern Germany. Archives of V irology, 135, 101–114

Webster, R.G., Bean, W.J., Gorman, O.T., Chambers, T.M. and Kawaoka, Y., 1992. Evolution and ecologyof influenza A viruses.Microbiological Reviews, 56 (1), 152–179

Wood, J.M., Webster, R.G. and Nettles, V.F., 1985. Host range of A/Chicken/Pennsylvania/83 (H5N2)influenza virus. Avian Diseases, 29(1), 198–207.