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The increasing involvement of wild waterfowl in H5 Highly Pathogenic Avian Influenza Virus (HPAIV) circulation continues to pose a threat to animal and public health worldwide. In winter 2020–2021, two field surveillance activities were carried out on a weekly basis, through virological and serological analyses, in 823 hunted and 521 trapped migratory aquatic birds in northeast Italy. Sixty Eurasian teals were recaptured several times, which allowed us to follow the progression of the HPAI H5 infection in naturally infected wild waterfowl. Oropharyngeal, cloacal, and feather swabs (OS, CS and FS) were collected from each duck and tested by real time rRT-PCR Type A influenza. The identified viruses were characterized and pathotyped by sequencing. Several viruses belonging to three different HPAI H5 subtypes were detected: H5N8, H5N5, and H5N1. High prevalence of infection with HPAI H5 clade 2.3.4.4b during November–December 2020 (up to 27.1%) was observed in captured Eurasian teals, while infection rates in hunted dabbling ducks, mainly Eurasian wigeons, showed the highest prevalence of infection in November 2020 (8.9%) and January 2021 (10.2%). All HPAI positive birds were also clinically healthy when recaptured weeks apart. The OS and FS showed the highest detection efficiency of HPAIV. Our results highlight that HPAI passive surveillance should be complemented by a targeted active surveillance to more efficiently detect novel HPAI viruses

Simple Summary Most of the newly emerging infections arise from animal reservoirs, frequently represented by wildlife species. Western European hedgehogs (Erinaceus europaeus) are mammalian hibernators, mainly nocturnal and insectivorous, living in natural open and green spaces as well as artificial, rural and urban, areas. They are generalist predators of macro-invertebrates, but they may also eat meat, bird eggs and on occasion pet food. These ecological and feeding habits, along with their high population densities, notable synanthropic attitudes, frequent contacts with sympatric wild and domestic species, including humans, implicate the possibility of intra- and interspecies interactions accounting for the possible involvement of E. europaeus in the ecology of several potentially emerging pathogens, including coronaviruses. Using PCR-based and virus isolation methods, we found that 58.3% of 24 hedgehogs’ fecal samples were PCR-positive for Erinaceus coronaviruses (EriCoVs). We did not observe any clinical disease related to the EriCoV infection in hedgehogs. However, the high mutation rates characterizing members of the Coronaviridae family and their potential successful interspecies host jumps—as that likely occurred in the Novel coronavirus (2019-nCoV) emergence—should be considered in the management of hedgehogs admitted to multi-species wildlife rehabilitation centers, recommending their return back to the original recovery areas. Abstract The Western European Hedgehog (Erinaceus europaeus) is one of the four hedgehog species belonging to the genus Erinaceus. Among them, E. amurensis is extant in East Asia’s areas only, whereas E. europaeus, E. roumanicus and E. concolor are mainly found in Europe. E. europaeus is endemically distributed from western to central and southern Europe, including Italy. Western European hedgehogs’ ecological and feeding habits, along with their high population densities, notable synanthropic attitudes, frequent contacts with sympatric wild and domestic species, including humans, implicate the possible involvement of E. europaeus in the ecology of potentially emerging viruses, such as coronaviruses, influenza A and influenza D viruses, canine distemper virus, pestiviruses and Aujeszky’s disease virus. We examined 24 E. europaeus individuals found injured in urban and rural areas of Northern Italy. Of the 24 fecal samples collected and tested for the above-mentioned pathogens by both PCR-based and virus isolation methods, 14 were found PCR-positive for betacoronaviruses belonging to lineage C and related to the known Erinaceus coronaviruses (EriCoVs), as determined by partial sequencing of the virus genome. Our findings suggest that hedgehogs could be considered natural reservoirs of CoVs, and also act as chronic shedding carriers of these potentially emerging RNA viruses.

Due to their need for living cells, viruses have developed adaptive evolutionary strategies to survive and perpetuate in reservoir hosts that play a crucial role in the ecology of emerging pathogens. Pathogenic and potentially pandemic betacoronaviruses arose in humans in 2002 (SARS-CoV, disappeared in July 2003), 2012 (MERS-CoV, still circulating in Middle East areas), and 2019 (SARS-CoV-2, causing the current global pandemic). As universally recognized, bats host ancestors of the above-mentioned zoonotic viruses. However, hedgehogs have been recently identified in Europe and Asia as possible reservoirs of MERS-CoV-like strains classified as Erinaceus coronavirus (EriCoV). To elucidate the evolution and genetics of EriCoVs, NGS (next generation sequencing) and Sanger sequencing were used to examine fecal samples collected in Northern Italy in 2018/2019 from 12 hedgehogs previously found EriCoV-positive by RT-PCR. By sequence analysis, eight complete EriCoV genomes, obtained by NGS, showed a high phylogenetic correlation with EriCoV strains previously reported in Eurasia. Interestingly, eight viral strains presented an additional ORF encoding for the CD200 ortholog located between the genes encoding for the Spike and the ORF3a proteins. The CD200 ortholog sequences were closely similar to the host CD200 protein but varying among EriCoVs. The result, confirmed by Sanger sequencing, demonstrates for the first time that CoVs can acquire host genes potentially involved in the immune-modulatory cascade and possibly enabling the virus to escape the host defence.

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.

Swine influenza viruses (SIVs) have been repeatedly demonstrated to circulate in wild boar (Sus scrofa) populations, whereas no evidence of exposure to avian influenza viruses (AIVs) has been described in wild boar. To better understand how different environments may influence the ecology of influenza A viruses (IAVs) in wild suid populations, we examined biologic samples of wild boars from two study areas represented by an upland (UL) and a wetland (WL) in northern and central Italy, respectively. Serum samples were collected from 388 wild boars sampled in the UL, whereas both a serum sample and a nasal swab were obtained from each of 35 wild boars sampled in the WL. Twenty of 388 (5.2%) sera from the UL were positive by enzyme-linked immunosorbent assay for the presence of antibodies against influenza A nucleoprotein and some of these samples showed antibodies by hemagglutination inhibition to SIVs of H1N1 (1/20), H1N2 (10/20), and H3N2 (1/20) antigenic subtypes. No IAV-seropositive wild boar was detected in the WL, although one of 35 animals was found to be IAV-positive by both a reverse transcriptase PCR and a real-time reverse transcriptase PCR. We hypothesize an SIV exposure for IAV-seropositive wild boars occupying the UL, whereas a possible AIV spillover from aquatic bird species—natural reservoirs of IAVs— to wild boars in the WL cannot be ruled out. Further research is needed to better understand the role played by wild boars in IAV ecology in Mediterranean habitats.

Sardinia is a Mediterranean island with a long geological history, leading to a separation process from continental Europe during the Miocene. As a consequence, in this insular habitat some wild bird species developed endemic forms, some of which are currently threatened. The aim of this study is to evaluate the possible animal health risk associated with a potential avian influenza virus (AIV) circulation in Sardinian wild bird populations. Overall, 147 cloacal swabs were sampled in the Sardinia region from June 2009 to September 2011. Samples were obtained from 12 taxonomic orders, including 16 families and 40 species of birds. Based on the endangered host status or on the ecology of the host-virus interaction, samples were categorized into three groups of species: 1) endemic, endangered, or both (17 species); 2) potential reservoir (21 species); and 3) potential spillover (two species). Cloacal swabs were tested by reverse transcription (RT)-PCR for influenza A virus matrix gene amplification. Forty-one serum samples were tested by nucleoprotein-enzyme-linked immunosorbent assay (NP-ELISA) for antibodies against influenza A virus nucleoprotein and by hemagglutination inhibition assay for detection of seropositivity against H5 and H7 AIV subtypes. No cloacal swabs tested RT-PCR positive for AIV, whereas two weak seropositive results were detected by NP-ELISA in a mallard (Anas platyrhynchos) and in a yellow-legged gull (Larus michahellis). The low or absent AIV circulation detected in Sardinia's wild birds during the study suggests a naïve status in these avian populations. These data provide new information on AIV circulation in Sardinia's wild birds that could be applied to implement conservation strategies for threatened species.

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....

La tubercolosi, importante patologia riemergente sostenuta da micobatteri, causa nell’uomo e negli animali morbilità, mortalità e danni economici. In particolare Mycobacterium bovis rappresenta un importante problema di sanità pubblica per la sua capacità di infettare animali domestici, selvatici e uomo, mentre M. avium è patogeno per il suino ed è segnalato come patogeno umano in soggetti immunodepressi. L’infezione da M.bovis, M. avium e altri micobatteri è stata diagnosticata nel cinghiale (Sus scrofa) in molti Paesi europei, compresa l’Italia. Erysipelothrix rhusiopathiae è l’agente causale del mal rosso, malattia zoonosica diffusa in tutto il mondo con importante impatto economico sull’allevamento suino. Sebbene descritta nel cinghiale, esistono attualmente pochi studi sulla suscettibilità all’infezione e sul ruolo epidemiologico di tale suide selvatico. Nel presente lavoro sono state svolte indagini microbiologiche volte alla ricerca di tali agenti zoonosici in una popolazione di cinghiali a vita libera in Provincia di Bologna, per valutare l’implicazione di questo ungulato, in continua espansione demografica, nell’epidemiologia di tali infezioni.

Il maiale domestico (Sus scrofa domestica), condividendo con gli uccelli e i mammiferi i recettori cellulari per i virus influenzali, svolge un ruolo epidemiologico chiave nell’ecologia del virus dell’influenza di tipo A ed in particolare nell’emergenza di nuovi ceppi, come confermato dall’origine suina del virus pandemico H1N1 del 2009. Il cinghiale (Sus scrofa) è il progenitore selvatico del maiale domestico, con cui condivide sia una stretta affinità genetica sia la suscettibilità verso numerosi agenti patogeni, tra cui i virus influenzali. La sottospecie nominale (Sus scrofa scrofa) è la più comune e diffusa in Eurasia, dove può arrecare gravi danni economici e ambientali. Se da un lato l’epidemiologia delle infezioni da virus influenzali di tipo A ed il relativo impatto economico negli allevamenti suini sono noti, vi sono ancora scarse informazioni sulla dinamica di circolazione del virus nelle popolazioni di cinghiali a vita libera. Nel presente lavoro è stata condotta un’indagine sierologica per valutare l’esposizione a virus influenzali di tipo A di una popolazione di cinghiali a vita libera distribuiti in un'area protetta della Provincia di Bologna. Metodi. Durante i periodi maggio 2002-luglio 2003 e aprile 2010-aprile 2011, sono stati raccolti 741 campioni di sangue prelevati da cinghiali catturati o abbattuti selettivamente nel Parco Regionale dei Gessi Bolognesi e Calanchi dell’Abbadessa. In questa area protetta di 4.844 ettari situata nella fascia collinare preappenninica bolognese, dal maggio 2002 è applicato un modello di gestione demografica densità-dipendente basato sulla selezione ed il mantenimento di una popolazione a densità nota strutturata in base ad una piramide di popolazione sesso-età dipendente. In funzione dell’eruzione dentaria, gli animali campionati sono stati suddivisi in tre classi di età: 1a classe, 14 mesi. Per differenziare le positività sierologiche dovute ad immunità passiva materna (IPM) da quelle indotte da una risposta sierologica attiva (RSA) post-infezione, la 1a classe è stata ulteriormente suddivisa nelle sottoclassi 1a-IPM e 1a-RSA che comprendono rispettivamente i cinghiali di età

Nelle prime due settimane di gennaio 2011, circa 3.000 carcasse di tortore dal collare (Streptopelia decaocto) a vita libera sono state rinvenute nei pressi di un sito industriale, in provincia di Ravenna, dedito alla lavorazione di partite di semi o loro residui. L’esame autoptico di 322 soggetti evidenziava prevalentemente un grave danno renale, epatomegalia e ipoplasia splenica. Gli esami istologici a livello del rene mostravano nefrosi associata a necrosi tubulari e una nefrite interstiziale linfoplasmacellulare. Inoltre, si osservavano deplezione linfocitaria nella milza unitamente ad emosiderosi splenica ed epatica. La stima dell’età su 46 soggetti identificava il 78% di adulti e il 22% di giovani (entro il primo anno di vita). Le analisi chimiche sulle ingesta e su pool di fegati non evidenziavano tossicità da pesticidi clorurati e fosforati, carbammati, triazine, piretroidi, stricnina, neonicotinoidi, Pb, Cd, Cr, Hg, Zn, Cu, micotossine, perossidi. Indagini molecolari (RT-PCR Real-Time) escludevano la presenza di virus influenzali, mentre veniva evidenziato RNA di Paramyxovirus aviario tipo 1 (APMV-1) tramite RTPCR in 176 su 193 animali analizzati. L’isolamento di 27 ceppi di APMV-1 è stato ottenuto inoculando pool di organi su uova embrionate di pollo SPF. Tali ceppi risultavano patogeni in base alla sequenza aminoacidica del sito di clivaggio della proteina di fusione. Il sequenziamento parziale del gene F ha permesso di identificare 2 differenti genotipi cocircolanti: il lineaggio 4b (APMV-1 ceppo piccione) e un distinto cluster genetico del lineaggio 4.

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.

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.

Wild aquatic birds are the main reservoir hosts harbouring the genetic pool of all influenza A viruses, including pandemic ones. Among waterbirds, wild ducks can contribute to perpetuate these viruses in a well-adapted host/parasite relationship [1]. The goal of this work was to determine how population parameters such as sex and age classes could affect the isolation prevalences of influenza A viruses in ducks trapped in a Mediterranean area during three crucial period of ducks’ biology. Viral RNA was extracted from cloacal swabs taken from 146 dabbling ducks trapped from October 2005 to September 2006 in Laguna di Orbetello WWF Oasis (Central Italy). Avian influenza viruses (AIVs) were detected by a one-step RT-PCR targeting the matrix gene [2] and RT-PCR positive samples were inoculated into specific-pathogen-free embryonated chicken eggs [3]. Harvested allantoic fluids were tested by hemagglutination assay [3] and ELISA test directed to viral nucleoprotein [4]. Chi-square test and Fisher's exact test were used to highlight significant differences in viral isolation prevalences related to ducks age, ducks sex, during an yearly sampling periods (fall migration, wintering period, post-reproductive period). RT-PCR and isolation prevalences for AIVs were 26% (38/146) and 9.6% (14/146), respectively. During the overall sampling period no age- and sex-related differences in virus isolation prevalences were detected. Considering each sampling period, significant differences were only found during the wintering season January–March 2006, when the juvenile ducks showed higher isolation prevalence than the adult ones. The particularly low temperatures recorded in north-eastern Europe during winter 2005-2006 [5] could have caused erratic movements of juvenile ducks, increasing the interaction among different bird subpopulations and the opportunity for virus exchanges. Our results confirm that the juvenile ducks play a key role in the movement of AIVs. Moreover, climatic variations may induce changes in the host species behavior affecting viral populations ecology.

Il Circovirus suino tipo 2 (Porcine Circovirus type 2, PCV2) riveste un ruolo primario nel determinismo della Sindrome multisistemica del deperimento post-svezzamento del suino (Post-weaning Multisystemic Wasting Syndrome, PMWS), patologia a diffusione mondiale e responsabile di gravi ripercussioni zoo-economiche. L'infezione da PCV2 è stata descritta in Europa anche nel cinghiale (Sus scrofa), talvolta associata alla PMWS. Dal 2002 al 2006, al fine di verificare la presenza dell'infezione da PCV2 e di studiarne la dinamica all'interno di una popolazione selvatica di cinghiale sottoposta a gestione demografica densità dipendente, è stata effettuata un'indagine siero-epidemiologica. Gli anticorpi anti PCV2 sono stati ricercati, mediante l'impiego di un test ELISA competitivo, in 1499 sieri di cinghiale raccolti nell'ambito di catture o abbattimenti selettivi effettuati nel Parco Regionale dei Gessi Bolognesi e Calanchi della Abbadessa, provincia di Bologna (4.844 ettari). In tale area la popolazione del suide selvatico viene mantenuta annualmente su valori noti di densità e struttura. Il numero medio di cinghiali stimati/anno dal 2002 al 2006 e le corrispondenti intensità percentuali di campionamento sono state: 700 e 21,9%, 600 e 66,2%, 450 e 82,7%, 500 e 64%, 550 e 47,5%. In funzione dell'età, i soggetti campionati sono stati suddivisi in classe 1 (0-6 mesi: n. 742), classe 2 (7-12 mesi: n. 376), classe 3 (13-24 mesi: n. 263), classe 4 (>24 mesi: n. 118). Nei 5 anni di studio è stata riscontrata una prevalenza complessiva pari al 39,8% (596/1.499). La prevalenza totale nelle femmine (42,8%) è risultata significativamente superiore (p=0,02) a quella dei maschi (36,9%). Le prevalenza più elevate sono state registrate nella classe 1 e nella classe 4 (48,2% e 49,2% rispettivamente). Nelle rimanenti classi di età la prevalenza era pari al 22,6% e 36,1%. Analizzando i valori di prevalenza per sesso e classe di età, solo nella classe 3 i maschi presentavano valori significativamente inferiori (p=0,005) rispetto alle femmine. Tale osservazione è probabilmente spiegabile con la parziale segregazione che i maschi presentano in questa classe di età. Per quanto riguarda i titoli anticorpali, è stata osservata una differenza statisticamente significativa in funzione dell'anno di campionamento, con titoli progressivamente crescenti fino al 2004. Gli animali adulti (classi 3 e 4) presentano titoli anticorpali superiori a quelli degli animali giovani (p=0,028). Le femmine evidenziano valori anticorpali superiori a quelli dei maschi (p=0,003): questo probabilmente è dovuto ad un effetto booster indotto dall'infezione dei giovani al calo dell'immunità materna mentre sono ancora in branco con femmine adulte.

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

All'interno di una sperimentazione gestionale densità-dipendente applicata ad una popolazione di cinghiale (Sus scrofa) in area protetta, si è investigata la presenza e la distribuzione di anticorpi per la malattia di Aujeszky al fine di valutare nel tempo il rapporto tra densità della specie ospite e prevalenza sierologica della malattia. Lo studio si è svolto all'interno dei 4.844 ettari del Parco regionale dei Gessi Bolognesi e Calanchi dell'Abbadessa. Durante il triennio 2002/2004 sono stati campionati settimanalmente, mediante catture o abbattimenti selettivi, 805 cinghiali. Il campione risultava costituito da: 444 animali di classe 1 (1-5 mesi), 180 di classe 2 (6-13 mesi), 104 di classe 3 (14-23 mesi) e 65 di classe 4 (>24 mesi) (12 soggetti indeterminati). La sex-ratio femmine:maschi era pari a 1:1,1. Gli emosieri stoccati a -20°C sono stati processati per la ricerca di anticorpi anti glicoproteine gB e gE del SHV-1 impiegando un test ELISA blocking. L'elaborazione statistica è stata effettuata mediante il test del Chi-quadrato di Pearson. Le prevalenze sierologiche totali sono state del 18% (gB) e del 16,6% (gE). Sieroprevalenze diverse sono state riscontrate sia tra le quattro classi di età sia nella stessa classe nel tempo. Globalmente la sieroprevalenza anti gB in classe 1 è stata del 21,8%; in classe 2 del 3,3%; in classe 3 del 13,5% mentre in classe 4 del 40%. La diminuzione progressiva della stessa, nella classe 1 può essere correlata all'esaurimento dell'immunità materna. La minor prevalenza nella classe 2 trova probabili giustificazioni da un lato nella non riattivazione di infezioni latenti, dall'altro nella mancata esposizione all'agente eziologico. La cessazione di tali condizioni potrebbe invece aver incrementato la sieroprevalenza nelle classi 3 e 4. Dallo studio emerge come al diminuire della densità di popolazione corrisponda una incrementata circolazione virale. Il confronto statistico degli anni 2003/2004 dimostra un rapporto inversamente proporzionale tra densità di popolazione e prevalenza anticorpale. Tale dato deve far riflettere qualora si pensi di contenere questo agente eziologico mediante piani di controllo della specie serbatoio. Infine le diverse prevalenze per classi di età indicano come piani di sorveglianza che prevedono campionamenti non stratificati per età possano portare ad una erronea valutazione dello stato sanitario della popolazione.

Cloacal swabs collected from mallard ducks (Anas platyrhynchos) experimentally infected with a H7N1 avian influenza strain were examined by Reverse Transcription Polymerase Chain Reaction to detect the influenza A virus. Reverse Transcription Polymerase Chain Reaction was compared with standard methods: inoculation of embryonated chicken eggs and inoculation of three established cell lines: Newborn Swine Kidney cells, Newborn Pig Trachea cells and Madine Darby Canine Kidney cells. Reverse Transcription Polymerase Chain Reaction was performed using a set of primers based on conserved regions of the matrix and nucleoprotein genes.

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.

Newcastle disease (ND), together with highly pathogenic avian influenza, is one of the most devastating diseases of poultry and other birds and vaccination is one method of control.. Susceptibility to Newcastle disease virus (NDV) of both free-living and reared pheasants is well known and was confirmed during the epidemic of ND that affected Italy during 2000. On that occasion the advisability of releasing ND vaccinated, semi-intensive reared pheasants into the wild was questioned. There was the possibility that vaccinated pheasants might develop an infection with a velogenic NDV which would then spread.. In this paper an experimental infection of eight pheasants vaccinated with live and killed NDV vaccines, is reported. The birds were infected by the oronasal route with a velogenic strain of NDV which was isolated in 2000 from a wild pheasant. Birds were observed daily for clinical signs and mortality throughout the experiment. Tracheal and cloacal swabs were collected for virus isolation on tracheal organ cultures.and recovery was confirmed by RT nested PCR using extracted RNA. Blood samples were collected and tested for NDV antibodies by hemagglutination-inhibition assay. Severe nervous signs were observed in one bird 6 days post infection. NDV was isolated from the trachea of 5 pheasants from day 2 up to day 5 post infection. It was never recovered from the cloaca. Seroconversion was observed in all the birds. It was shown that that vaccinated pheasants can become infected with a velogenic NDV and shed it by the respiratory route, though not themselves show any disease. Therefore they pose a potential threat to wild and domestic birds with which they will come into contact. This experiment highlights important health implications which may arise from administration of avifauna and which should be given due consideration.

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.

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.

0:74 Escherichia coli epidemic in a Roe deer metapopulation of the Northern Apennines, Italy. An epidemic of enteropathogenic (EPEC) E. coli in a in a free living metapopulation of Roe deer Capreolus capreolus of the Northern Apennines (Italy) is described. 40 out of the 132 censued animals were radiocollared. In the radiocollared animals the infection resulted in a morbility of 12.5% (SD 5.2%), with a lethality of 80% (SD 18%) and the whole mortality reached 10%.(SD 4.7%). The E. coli responsible of the infection was classified as 0:74. The pathological lesions and some clinical features of the infection are described.

Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS) in pigs. Serological and pathological studies have shown that European wild boars (Sus scrofa) are also susceptible to both PCV2 infection and PMWS (2, 3, 4). The aims of this study were: i) to evaluate the prevalence of PCV2 antibodies in a wild boar population of a protected area in Northern Italy during a 3 years period; ii) to evaluate possible changes of PCV2 seroprevalence after the application of a demographic control program aimed at reducing the wild boars density in the area.

Since the “bird flu” incident in Hong Kong SAR in 1997, several studies have highlighted the substantial role of domestic birds, such as turkeys and chickens, in the ecology of influenza A viruses. Even if recent evidence suggests that chickens can maintain several influenza serotypes, avian influenza viruses (AIVs) circulating in domestic species are believed to be introduced each time from the wild bird reservoir. However, so far the direct precursor of influenza viruses from domestic birds has never been identified. In this report, we describe the antigenic and genetic characterization of the surface proteins of H7N3 viruses isolated from wild ducks in Italy in 2001 in comparison to H7N3 strains that circulated in Italian turkeys in 2002–2003. The wild and domestic avian strains appeared strictly related at both phenotypic and genetic level: homology percentages in seven of their genes were comprised between 99.8% (for PB2) and 99.1% (for M), and their NA genes differed mainly because of a 23-aminoacid deletion in the NA stalk. Outside this region of the molecule, the NAs of the two virus groups showed 99% similarity. These findings indicate that turkey H7N3 viruses were derived “in toto” from avian influenza strains circulating in wild waterfowl 1 year earlier, and represent an important step towards the comprehension of the mechanisms leading to interspecies transmission and emergence of potentially pandemic influenza viruses.

A molecular and serological survey was performed in order to investigate the role of some wild avian species in the epidemiology of Avian Pneumovirus (APV) infection. During 2001, 440 aquatic birds, including sea gulls, terns, herons, flamingos and shorebirds, were sampled in north-eastern wetlands of Italy. According to the taxonomic group, the sample sizes were: 179 Laridae, 161 Sternidae, 46 Ardeidae, 35 Phoenicopteridae, 19 Scolopacidae. Swabs from the choanal cleft were collected from 394 of these birds and tested by a subtype-specific RT-nested PCR, based on oligonucleotides specific for G gene sequence and able to differentiate A and B subtypes. At the same time, whenever possible, blood samples were collected (n. 368) and analysed using a blocking ELISA serological assay. All molecular and serological tests produced negative results.

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.

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.