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Background: infectious low pathogenic avian influenza viruses (LPaIVs) have been recently detected on feathers of wild ducks. Laboratory trial results suggested that the preen oil gland secretion, covering waterbirds’ feathers, may attract and concentrate virus particles from aIV-contaminated waters to birds’ bodies. We evaluated whether ducks can become infected by the ingestion of preen oil-associated viral particles, experimentally smeared on their plumage. In addition, we compared virologic and serologic results obtained from mallards whose feathers were experimentally infected, with those from wild mallards naturally carrying aIVs on feathers.Methods: we experimentally coated 7 mallards (anas plathyrynchos) using preen oil mixed with a LPaIV (h10n7 subtype), and housed them for 45 days with a control, uncoated duck. cloacal, oropharyngeal and feather swabs were collected from all birds and examined for aIV molecular detection and isolation. Blood samples were also taken to detect influenza specific antibodies. In addition, sera from 10 wild mallards, carrying on feathers infectious LPaIV h10n7, were examined.Results: virologic and serologic results indicated that through self- and allopreening all the birds experimentally coated with the preen oil/aIV mix and the control duck ingested viruses covering feathers and became infected. Virus isolation from feathers was up to 32 days post-coating treatment. one out of 8 wild mallards showing antibodies against type a influenza virus was seropositive for h10 subtype too.Conclusions: our experimental and field results show evidences suggesting that uninfected birds carrying viruses on their feathers, including immune ones, might play an active role in spreading aIV infection in nature. For this reason, routine aIV surveillance programs, aimed at detecting intestinal and/or respiratory viruses, should include the collection of samples, such as feather swabs, enabling the detection of viruses sticky to preened birds’ bodies....

Wild aquatic birds are reservoir hosts perpetuating the genetic pool of all influenza A viruses, including pandemic ones. High viral loads in feces of infected birds allow fecal-oral transmission. However, this route does not fully account for the efficiency of avian influenza virus (AIV) spread since dilution of infectious feces in water progressively decreases the chances of virus/host interaction. We investigated whether preen oil gland secretion, by which all aquatic birds make their feathers waterproof, could support a natural concentration mechanism of AIVs from water to birds’ bodies, thus favouring virus spread and persistence in the aquatic environment. First, we detected consistently both viral genome and infectious AIVs on swabs taken by rubbing preened feathers of 345 wild mallards and examined by reverse transcription (RT)-PCR and virus isolation (VI) assays. Second, in two laboratory experiments using a quantitative real-time (qR) RT-PCR, we demonstrated that feather samples (n=5) and cotton swabs (n = 24) experimentally impregnated with preen oil, when soaked in AIV-contaminated waters, attracted and concentrated AIVs on their surfaces, as shown by statistical analysis. Third, we experimentally coated 7 mallards with a preen oil-AIV mix, and housed them with a control, uncoated, duck. Through self- and/or allopreening behaviour, all birds ingested the virus, as shown by virus detection in both oro-pharyngeal and cloacal samples. Virus isolation from cloacal swabs and virus-specific antibody response confirmed the occurrence of mallards’ infection. Infectious AIVs were isolated from ducks’ body surface until 32 days after the experimental coating.Our field and experimental results indicate that AIVs can be naturally concentrated and carried on the feather surface of infected ducks (i.e., those VI-positive from both cloacal and feathers swabs) and uninfected ones (i.e., those VI-positive from feathers only). In such a context, the natural preening behaviour, by which waterbirds spread preen oil all over their plumage (self-preening) or other birds’ plumage (allo-preening), could facilitate the ingesion of AIV particles stuck on birds’ feathers, thus promoting a preening-mediated infection route. Our findings also suggest that during the time period between the virus adhesion to the bird’s body and the infection (possibly due to self- and/or allopreening), the virus could move in nature with the host by an undescribed circulation mechanism. We demonstrate here a novel viral transmission route that adds to, and possibly contributes to explain the knowledge of longdistance movements and long-term infectivity of lowly and highly pathogenic AIVs in nature.

Il passaggio del Virus dell'Influenza Aviaria (AIV) dal serbatoio naturale rappresentato dalle specie aviarie selvatiche alle specie aviarie domestiche, può indurre cambiamenti aminoacidici a livello delle glicoproteine di superficie e delle proteine interne strutturali. Lo studio, di cui si presentano alcuni dati preliminari, ha l'obiettivo di identificare le modificazioni nucleotidiche a livello dei geni virali interni PA, PB1 e PB2 a seguito di trasmissione sperimentale di un virus isolato da anatra selvatica in due diverse specie aviarie domestiche, tacchino e quaglia. A tale scopo è stato utilizzato il virus A/Mall/It/33/01 (A/H7N3), identificato quale precursore diretto dei virus A/H7N3 a bassa patogenicità circolati negli allevamenti italiani dal 2002 al 2004. Le principali modificazioni amminoacidiche tra virus A/H7N3 isolati nel corso dell'epizoozia nel pollame ed il virus isolato dall'anatra, sono state identificate come sostituzioni di due amminoacidi nell'emoagglutinina, delezione di 23 amminoacidi nello stalk della neuraminidasi ed un numero limitato di cambiamenti a carico delle proteine strutturali interne. Metodi. Per lo studio molecolare sono stati selezionati 4 isolati virali da 10 passaggi (ps.) seriali del virus A/H7N3 effettuati rispettivamente in tacchino e quaglia ed indicati Tk-p.4 (4° ps. in tacchino), Tk-p.10 (10° ps. in tacchino), Qu-p.6 (6° ps. in quaglia), Qup. 10 (10° ps. in quaglia). Per ciascuno dei passaggi indicati sono state ottenute le sequenze dei geni interni PA (bp.1-2233), PB1 (bp.1-2341) e PB2 (bp.1-2341). L'assemblaggio e l'allineamento con sequenze di virus aviari, sono stati effettuati col software DNASTAR-Lasergene. Risultati e conclusioni. L'analisi molecolare dei geni interni PA, PB1 e PB2, relativi ad alcuni passaggi in vivo del virus A/Mall/It/33/01 ed il confronto di analoghe sequenze ottenute dal precursore selvatico, hanno evidenziato mutazioni sinonime e non sinonime nei geni in studio rispetto al ceppo originale; inoltre alcune sostituzioni amminoacidiche distinguono gli isolati ottenuti in quaglia rispetto a quelli replicati in tacchino. I risultati 55 dello studio contribuiscono alla identificazione dei cambiamenti molecolari che possono avere un ruolo importante nella trasmissione interspecie degli AIV dalle specie aviarie selvatiche a quelle domestiche.

Tra gli uccelli acquatici gli Anseriformi rappresentano il principale serbatoio dei virus influenzali di tipo A e possono albergare la maggior parte dei sottotipi virali caratterizzati sino ad oggi da 16 emoagglutinine e 9 neuroaminidasi diverse. Occasionalmente virus influenzali aviari possono essere trasmessi ad altre specie, compresi i volatili domestici ed i mammiferi. Scopo del lavoro è stato quello di valutare sia la circolazione di virus influenzali tipo A in Anatidi selvatici sia i patterns di prevalenza correlati a stagionalità, sesso e classi di età degli animali campionati all’interno dell’Oasi WWF “Laguna di Orbetello” (GR) nel periodo ottobre 2005/settembre 2006. L’RNA virale è stato estratto da 146 tamponi cloacali prelevati da anatre di superficie catturate con gabbie ad invito. Mediante one-step RT-PCR diretta ad amplificare un tratto specifico del gene M, sono stati identificati i campioni contenenti virus influenzali. Tali campioni sono stati quindi inoculati, per l’isolamento virale, nella cavità allantoidea di uova embrionate di pollo all’undicesimo giorno di incubazione. I liquidi allantoidei ottenuti sono stati analizzati in prove di emoagglutinazione ed ELISA diretta verso la nucleoproteina virale e sono stati considerati positivi i liquidi allantoidei emoagglutinanti positivi ad entrambe queste metodiche. I test del Chi-quadrato ed Esatto di Fisher sono stati utilizzati al fine di evidenziare differenze statisticamente significative in funzione dell’età e sesso dei soggetti campionati e di tre periodi di campionamento (sovrapponibili rispettivamente alla migrazione di discesa, allo svernamento ed al periodo post-riproduttivo). La prevalenza totale è risultata del 26% in RT-PCR e del 9,6% nelle prove di isolamento su uova embrionate. Durante l’intero anno di campionamento nessuna differenza significativa è stata riscontrata tra le classi di età, di sesso e tra i diversi periodi di campionamento mentre dal confronto degli isolamenti ottenuti in adulti e giovani, durante la stagione di svernamento (gennaio/marzo 2006) emerge una differenza significativa (P

The authors reported some occurrences of the coronavirus infection in pheasants and red-partridges reared in the Emilia-Romagna Region of Northern Italy. During May 1996 a pathological condition was seen in a pheasant farm of approximately 8,000 breeders with up to 10% mortality observed in younger birds. Kidney lesions were constantly seen and urolithiasis and visceral gout were sometimes observed. Ultrastructural exams have been made on the kidney lesions. Coronavirus particles were isolated after three chicken embryo passages of cecal tonsil and kidney suspensions. In order to characterise the virus, serum neutralisation tests in chick embryo tracheal organ cultures were carried out using 12 infectious bronchitis virus (IBV) strains. The isolate designed Pheasant/Italy(Ra)/1754-13/1996 only showed low serum neutralisation titres with the French IBV type CR-84221. A second isolate (Pheasant/Italy(Ra)/5700/2000) was obtained from a game bird farm where about 1,000 pheasant breeders were reared. In July 2000, mortality, kidney lesions and visceral gout were seen in young birds (2-7-week-old). Coronavirus particles, isolated after two chicken embryo blind passages of kidney suspension, were detected by electron microscopy. Finally, a third strain (Red-legged Partridge/Italy(Ra)/191390/2004) was isolated in August 2004 from kidney lesions of one out 13 scanty red-legged partridges chosen from 10,000 birds recently imported from France (Region of Western Loire) for hunting purposes. Coronavirus infection was diagnosed by RT-PCR carried out on allantoic fluid (third passage) of chicken embrionated eggs. Genomic characterization of both S1 and M proteins of these strains and their phylogenetic correlation with other avian coronavirus is presented. The first pheasant outbreak described above, induced us to carry out a serological survey in the Emilia-Romagna Region in order to establish the occurrence and diffusion of coronavirus infection in farm-bred pheasants. Seven hundred and four sera were obtained during 1998 from 16 game farms. Moreover, 275 sera were collected from 1995 to 2002 from free-living pheasants, belonging to a natural population: these were classified as “wild” and “restocked” (a few reared birds released in the study area). A commercial blocking ELISA (Svanovir) test was employed for the detection of cross reactive antibodies anti-fowl coronavirus (Infectious Bronchitis virus) in sera. Seropositive animals were detected in 5 out of 16 game farms examined, while only two free-living pheasants (a restocked bird and an unclassified one) were seropositive. Serological data confirm that the infection was present in Italian-reared pheasants, but the free-living sampled population appeared to be free from the infection. The authors emphasise the risk of spreading the infection to wild bird populations by game restocking activities.

Pheasant coronavirus (PhCoV) has been isolated in the UK and Italy in the last few decades. A serological survey was carried out in the Emilia-Romagna Region (Northern Italy) in order to establish the occurrence and diffusion of coronavirus infection in farm-bred and free-living pheasants. Seven hundred and four sera were obtained during 1998 from 16 game farms. 275 sera were collected from 1995 to 2002 from free-living pheasants, belonging to a natural population: these were classified as “wild” and “restocked” (a few reared birds released in the study area). A blocking ELISA (Svanovir) test was employed for the detection of cross reactive antibodies anti-fowl coronavirus (Infectious Bronchitis Virus) in sera. Seropositive animals were detected in 5 out 16 game farms examined, while only two free-living pheasants (a restocked bird and an unclassified one) were seropositive. These data confirm that the infection was present in Italian-reared pheasants, but the free-living sampled population appeared to be free from the infection. The authors emphasise the risk of spreading the infection to wild bird populations by game restocking activities.

With regard to the role of wild ducks and coots as a source of viruses potentially pathogenic for domestic poultry we observed some differences: in coots, no evidence of H5 and H7 virus circulation was found either at serological or virological level whereas ducks serologically positive to H5 virus were detected every year. In the present study, the highest H5 antibody frequencies that were observed in DWMA during the first sampling period overlapped with the H5N2 Italian poultry epidemic (Donatelli et al., 2001). Moreover, even if no H5 viruses were isolated during the present study period, seroconversion observed in a mallard indicated that circulation of H5 subtype viruses occurred in the sampling area during the first sampling period (Table 2). Unlike H5 AIVs, no evidence of H7 virus circulation was found either at serological or virological level in ducks trapped in the area under study (Table 1). Thus it appears that no H7 viruses circulated among ducks sampled in the study area since 1992 (De Marco et al., 2004). In particular, duck sera resulted negative for H7 subtype both before and after the beginning of the catastrophic Italian poultry epidemic due to the H7N1 serovar (Capua & Alexander, 2004), suggesting a possible implication of other bird reservoirs. With regard to virus circulation, the H1N1 subtype appears to be endemic in Italian wetlands since 1992 (De Marco et al., 2004). H2N3 and H11N6 strains were isolated for the first time, even though ducks serologically positive to both subtypes had been found in previous years in the same study area (De Marco et al., 2004). The present results, together with data obtained before and after this study period in the same study area (De Marco et al., 2004, Campitelli et al., 2004b), suggest a perpetuation of H5 AIVs in the wild duck reservoir in Italy. This evidence is all the more relevant when we consider that the duck populations we analysed are representative of waterfowl populations that very likely make use of wetlands of Northern Italian regions (where H5N2, H7N1 and H7N3 poultry epidemics occurred in recent years) during their migrations northward in spring and southward in autumn (De Marco et al., 2000). The identification in 2001, in these wetlands, of H7N3 duck viruses almost identical to H7N3 strains affecting domestic poultry in Northern Italy in 2002-03 (Campitelli et al., 2004a) supports the existence of an epidemiological connection between these two areas and confirms the importance of maintaining an active influenza surveillance program in wild waterfowl reservoirs.

La diffusione dei focolai di influenza aviaria A/H5N1 e gli episodi di mortalità nell’uomo registrati nei paesi del Sud-Est asiatico, hanno determinato un crescente allarme nella comunità scientifica e nell’opinione pubblica a causa del potenziale pandemico rappresentato dalla evoluzione del virus. La tempestiva identificazione del virus nelle specie aviarie domestiche e selvatiche, attraverso l’impiego di strumenti diagnostici altamente sensibili, specifici e di rapida esecuzione, è essenziale per l’adozione rapida ed efficace delle misure sanitarie di controllo. La real-time RT-PCR, basata sull’impiego di sonde fluorescenti, rappresenta lo strumento diagnostico in grado di garantire l’identificazione dei virus influenzali, sia umani che aviari, presentando notevoli vantaggi in termini di rapidità, sensibilità e specificità rispetto alle metodiche classiche di isolamento del virus e alle altre metodiche molecolari. Nell’ottica di un continuo miglioramento dell’iter diagnostico per l’identificazione del virus dell’influenza aviaria, è stato sviluppato un saggio di real-time RT-PCR, impiegando una sonda fluorescente Minor Groove Binder, che presenta una serie di innovazioni rispetto alle piattaforme per real-time sinora disponibili per la diagnostica virologica. Il sistema diagnostico è stato calibrato e ottimizzato in termini di specificità, utilizzando un panel di virus influenzali aviari, di origine suina, equina, oltre che umana, dimostrando inoltre una sensibilità tale da renderlo applicabile per lo screening di campioni biologici prelevati dalle specie aviarie domestiche e selvatiche.

Pheasant coronavirus (PhCoV) has been isolated in the UK and Italy in the last few decades. A serological survey was carried out in the Emilia-Romagna Region (Northern Italy) in order to establish the occurrence and spread of coronavirus infection in farm-bred and free-living pheasants. Seven hundred and four sera were obtained during 1998 from 16 game farms. 275 sera were collected from 1995 to 2002 from free-living pheasants, belonging to a natural population: these were classified as “wild” and “restocked” (a few reared birds released in the study area). A blocking ELISA (Svanovir) test was employed for the detection of antibodies cross reactive to Infectious Bronchitis Virus in sera. Seropositive animals were detected in 5 out 16 game farms examined, while only two free-living pheasants (a restocked bird and an unclassified one) were seropositive. These data confirm that the infection was present in Italian-reared pheasants, but the free-living sampled population appeared to be free from the infection. The authors emphasise the risk of spreading the infection to wild bird populations by game restocking activities.

This report describes a pandemic A/H1N1 (H1N1 pdm) virus outbreak occurred in December, 2009 in a swine farm used as research facility (Istituto Mediterraneo Trapianti e Terapie ad Alta Specializzazione) for preclinical studies, located in Sicily, Italy. All the 13 pigs of the farm, showed cough, fever, inappetence and weakness. At the same time, an unvaccinated worker of the stabling showed influenza-like symptoms. RNAv extracted from two swabs collected from infected pigs resulted positive by Real Time RT-PCR for Influenza A virus. Furthermore, after growth on embryonated eggs, viral isolates were identified by Real Time RT-PCR specific for H1N1 pdm virus and characterized antigenically. Sequencing of the whole genome was also performed. All sera taken from animals and from the worker were tested by a competitive Influenza A ELISA and by the haemoagglutination inhibition test. Serological findings confirmed the circulation of influenza virus H1N1 pdm in pigs and the presence of specific antibodies against H1N1 pdm in human serum. The results of this study seem to support a H1N1 pdm transmission from man to animals showing the importance of serological and virological investigation to control the pig farms and the importance of close cooperation between the different authorities like veterinarian and human public. © 2012 Triveni Enterprises.

Ecological interactions between wild aquatic birds and outdoor-housed poultry can enhance spillover events of avian influenza viruses (AIVs) from wild reservoirs to domestic birds, thus increasing the related zoonotic risk to occupationally exposed workers. To assess serological evidence of AIV infection in workers operating in Northern Italy at the wildfowl/poultry interface or directly exposed to wildfowl, serum samples were collected between April 2005 and November 2006 from 57 bird-exposed workers (BEWs) and from 7 unexposed controls (Cs), planning three sample collections from each individual. Concurrently, AIV surveillance of 3587 reared birds identified 4 AIVs belonging to H10N7, H4N6 and H2N2 subtypes while serological analysis by hemagglutination inhibition (HI) assay showed recent infections caused by H1, H2, H4, H6, H10, H11, H12, and H13 subtypes. Human sera were analyzed for specific antibodies against AIVs belonging to antigenic subtypes from H1 to H14 by using HI and virus microneutralization (MN) assays as a screening and a confirmatory test, respectively. Overall, antibodies specific to AIV-H3, AIV-H6, AIV-H8, and AIV-H9 were found in three poultry workers (PWs) and seropositivity to AIV-11, AIV-H13—still detectable in October 2017—in one wildlife professional (WP). Furthermore, seropositivity to AIV-H2, accounting for previous exposure to the “extinct” H2N2 human influenza viruses, was found in both BEWs and Cs groups. These data further emphasize the occupational risk posed by zoonotic AIV strains and show the possible occurrence of long-lived antibody-based immunity following AIV infections in humans.

During an influenza A(H7N7) virus outbreak among poultry in Italy during August–September 2013, infection with a highly pathogenic A(H7N7) avian influenza virus was diagnosed for 3 poultry workers with conjunctivitis. Genetic analyses revealed that the viruses from the humans were closely related to those from chickens on affected farms.

Wild aquatic birds in the Orders Anseriformes and Charadriiformes are the main reservoir hosts perpetuating the genetic pool of all influenza A viruses, including pandemic viruses. High viral loads in feces of infected birds permit a fecal-oral route of transmission. Numerous studies have reported the isolation of avian influenza viruses (AIVs) from surface water at aquatic bird habitats. These isolations indicate aquatic environments have an important role in the transmission of AIV among wild aquatic birds. However, the progressive dilution of infectious feces in water could decrease the likelihood of virus/host interactions. To evaluate whether alternate mechanisms facilitate AIV transmission in aquatic bird populations, we investigated whether the preen oil gland secretions by which all aquatic birds make their feathers waterproof could support a natural mechanism that concentrates AIVs from water onto birds' bodies, thus, representing a possible source of infection by preening activity. We consistently detected both viral RNA and infectious AIVs on swabs of preened feathers of 345 wild mallards by using reverse transcription-polymerase chain reaction (RT-PCR) and virus-isolation (VI) assays. Additionally, in two laboratory experiments using a quantitative real-time (qR) RT-PCR assay, we demonstrated that feather samples (n = 5) and cotton swabs (n = 24) experimentally impregnated with preen oil, when soaked in AIV-contaminated waters, attracted and concentrated AIVs on their surfaces. The data presented herein provide information that expands our understanding of AIV ecology in the wild bird reservoir system.

We report the results of a 6-year serological and virological monitoring performed in ducks and coots in Italy, in order to assess the degree of influenza A virus circulation in these birds during wintering. A total of 1039 sera collected from 1992 to 1998 was screened by a double antibody sandwich blocking ELISA (NP-ELISA): seroprevalence of antibodies to influenza A viruses was significantly higher in ducks compared to coots (52.2% vs. 7.1%, respectively). The hemagglutination-inhibition (HI) assay, performed on NP-ELISA positive sera, showed that 16.9% of these duck sera and 33.3% of these coot sera had antibodies to at least one influenza virus HA subtype: ducks showed HI antibodies against most of the HA subtypes, except for the H3, H4, H7, and H12; coots were seropositive to the H3 and H10 subtypes, only. From 1993 to 1998, 22 virus strains were obtained from 802 cloacal swabs, with an overall virus isolation frequency of 2.7%. Viruses belonging to the H1N1 subtype were by far the most commonly circulating strains (18/22) and were isolated mainly from ducks (17/18). The remaining viruses were representative of the H10N8, H5N2 and H3N8 subtypes. Our data indicate some differences between influenza A virus circulation in sympatric ducks and coots and a significant antigenic diversity between some reference strains and viruses recently isolated in Italy.

From October 1997 to January 1998, highly pathogenic H5N2 avian influenza viruses caused eight outbreaks of avian influenza in northern Italy. A nonpathogenic H5N9 influenza virus was also isolated during the outbreaks as a result of virological and epidemiological surveillance to control the spread of avian influenza to neighbouring regions. Antigenic analysis showed that the Italian H5N2 isolates were antigenically similar to, although distinguishable from, A/HK/156/97, a human influenza H5N1 virus isolated in Hong Kong in 1997. Phylogenetic analysis of the haemagglutinin (HA) genes showed that the highly pathogenic Italian viruses clustered with the Hong Kong strains, whereas the nonpathogenic H5N9 virus, despite its epidemiological association with the highly pathogenic Italian isolates, was most closely related to the highly pathogenic A/Turkey/England/91 (H5N1) strain. Like the HA phylogenetic tree, the nonstructural (NS) phylogenetic tree showed that the H5N2 Italian virus genes are clearly separate from those of the H5N9 strain. In contrast, results of the phylogenetic analysis of nucleoprotein (NP) genes indicated a closer genetic relationship between the two Italian virus groups, a finding suggesting a common progenitor. Comparison of the HA, NS and NP genes of the Italian H5 strains with those of the H5N1 viruses simultaneously circulating in Hong Kong revealed that the two groups of viruses do not share a recent common ancestor. No virological and serological evidence of bird-to-human transmission of the Italian H5N2 influenza viruses was found.