Coronaviruses (CoVs) comprise a family under the order Nidovirales (family Coronaviridae) and infect a wide variety of mammals and birds. The course of infection varies greatly from asymptomatic to severe disease, depending on the host and virus species in question. The genome of CoVs is one of the largest (25–32 kb) viral RNA-genomes. Based on phylogenetic analysis, the CoVs are divided into four different genera: Alpha-, Beta-, Gamma-, and Deltacoronavirus. The alpha- and betacoronaviruses are carried by mammals, whereas the gamma- and deltacoronaviruses mainly infect birds, with few exceptions. The large genomes, infidelity of the RNA-dependent RNA polymerase, and high frequency of homologous RNA recombination are the main factors contributing to the high genetic diversity of CoVs [4–6].

The first CoV, Infectious bronchitis virus (IBV), was identified in 1937. IBV mainly infects chickens, but may infect other bird species as well. IBV is highly contagious and affects the respiratory tract, gut, kidney, and reproductive systems, causing substantial economic losses in the poultry industry. Despite the global distribution of IBV, poultry in Finland remained free of clinical cases until April 2011 after which outbreaks involving several CoV genotypes have occurred in Southern Finland.

The first human CoVs were identified in 1960s [10–12]. The human CoVs cause generally mild to moderate upper respiratory tract infections [13–15]. In 2003, a novel highly pathogenic betacoronavirus emerged in China, causing severe disease characterized by acute respiratory distress and it became known as severe acute respiratory syndrome (SARS)-CoV. The emergence of SARS-CoV inspired virologists to more explore the highly divergent group of coronaviruses and their hosts, leading to the identification of a rapidly growing number of CoV species particularly in bats. More recently, another highly pathogenic betaCoV infecting humans, the Middle East Respiratory Syndrome (MERS) CoV emerged in 2012 with a case fatality rate of over 40%.

Migratory birds have the ability to facilitate the dispersion of microorganisms with zoonotic potential. Wild birds have been associated with the ecology and dispersal of at least West Nile virus, tick-borne encephalitis virus, influenza A virus (IAV) and Newcastle disease virus (NDV) [19–21]. Since the discovery of IBV in 1937, it remained the only known Gammacoronavirus for over 50 years, but the number has increased dramatically during the last 10 years. Thereafter, representatives of the genera Gamma- and Deltacoronavirus have been isolated from both wild and domestic birds including species from the order Anseriformes, Pelecaniformes, Ciconiiformes, Galliformes, Columbiformes, and Charadriiformes [22–24]. In this report we provide a description of CoV species circulating in wild birds in Finland. Altogether 939 samples representing 61 different bird species were collected during 2010–2013 and examined for the presence of CoV RNA.

About 70% of microbial agents causing outbreaks of emerging infectious diseases in humans originate directly from animals. Among respiratory virus infections, the influenza A viruses H5N1 and H7N9 from avian species, and the severe acute respiratory syndrome coronavirus from bats have caused large epidemics–[3]. Atypical bacterial pathogens causing community-acquired pneumonia include Chlamydophila psittaci from psittacine birds and Coxiella burnetti from livestock and other animals. However, human outbreaks due to zoonotic bacteria associated with the emergence of a novel animal virus in the animal host were not previously documented.

In November 2012, an outbreak of human psittacosis affecting six staff members occurred at the New Territories North Animal Management Centre (NTNAMC) in Hong Kong. The human outbreak was preceded by an outbreak of avian chlamydiosis among the detained Mealy Parrots (Amazona farinose). Although birds in the tropical and sub-tropical areas are commonly infected with C. psittaci, most infected birds are asymptomatic,. Large human outbreaks are rare even among bird handlers. Although co-infection of C. psittaci and viruses has been reported in outbreaks of avian species–[12], no virus-bacterium co-infection of implicated avian species has ever been reported in outbreaks of human psittacosis. In this study, we sought to investigate viruses that cause avian co-infection, which may have led to this outbreak of psittacosis.

A case was defined as a staff member working at the NTNAMC who was hospitalized for respiratory tract infection between November 1 and November 30, 2012, and confirmed to have C. psittaci infection by polymerase chain reaction (PCR) and/or a four-fold rise in serum microimmunofluorescent antibody titer against C. psittaci (Focus Diagnostics, Cypress, California, USA).

Besides the strong innate immune responses, waterfowl are generally considered long-term carrier of APMV-1 and disease outbreaks have been reported since 1997 [12–14], and were confirmed by follow up experimental studies. Clinically and naturally infected ducks and geese with APMV-1 show clinical signs such as elevated body temperature, excessively excreted oral mucus, dried cloaca, watery, greenish-white diarrhea, vain attempts of eating and drinking, listlessness, anorexia, crouch, eyelid edema and emaciation [14, 23, 46, 47]. Ducks may show up to 70% decrease in egg production, 80% morbidity and 67% mortality [15, 48] however the mortality in ducks varies with the different breeds, virus strain and dose of virus. Some birds also show weakness of legs and wing along with unilateral or bilateral incomplete paralysis and the effects of this paralysis increases with progression of the disease. Duck and geese also show the neurological signs such as muscular trembles, muscular dis-coordination, circling, and twisting of head and neck [23, 46, 48]. These clinical signs disappear according to infection status; mildly affected recover sooner and severely affected birds may recover after 15 days of infection [22, 46].

APMV-1 infected ducks and geese show the gross lesions on the immune organs such as bursa, spleen, thymus, mild to severe tracheitis, kidney enlargement, necrosis of pancreas, congestion on the meninx and in the brain and diffuse brain edema, focal hemorrhages in the mucosa of the proventriculus and intestine (especially duodenum and upper part of jejunum) [14, 22, 23, 46]. Bursal atrophy, hemorrhagic thymus and splenomegaly with white necrotic spots were found in the APMV-1 infected geese and duck [23, 46]. These lesions and histopathological changes may be due to higher viral loads, multi-systemic distribution of the virus in these immune organs. As these immune organs are the reservoir of immune cells, and their destruction may lead to low antibody titer and other infections.

Astroviruses (AstVs) are non-enveloped, positive-sense, single-stranded RNA viruses belonging to the Astroviridae family. Currently, two genera: namely Mamastrovirus and Avastrovirus are distinguished within this family. The genus Mamastrovirus includes astrovirus species isolated from humans and a number of mammals. Isolates originated from avian species, such as turkey, chickens, ducks, and other birds are classified into the genus Avastrovirus1, 2. AstVs have been detected in humans and a variety of animal species, including non-human primates, other mammals and avian species3–5. Their genomes are 6.8–7.9 kb in length, consisting of a 5′-untranslated region (UTR), three open reading frames (ORFs), a 3′-UTR and a poly (A) tail6. The high degree of genetic diversity among AstVs and their recombination potential signify their capacity to cause a broad spectrum of diseases in multiple host species3, 7, 8. Human classical AstVs are a frequent cause of acute gastroenteritis in young children and the elderly, occasionally with encephalitis8.

In poultry, AstV infections have been found to be associated with multiple diseases, such as poult enteritis mortality syndrome, runting-stunting syndrome of broilers, white chick syndrome, kidney and visceral gout in broilers and fatal hepatitis of ducklings, leading to substantial economic losses9–16. Increasing evidence indicates that there is a high degree of cross species transmission of AstVs between domestic birds, and even the potential to infect humans17. By comparison, fewer AstV infection cases have been described in domestic goose flocks. Bidin et al.18 reported the detection of avian nephritis virus infection in Croatian goose flocks and provided evidence that this AstV was associated with stunting and pre-hatching mortality of goose embryos. Studies to detect AstV genomes from the clinical samples of geese suggested that these viruses might distribute widely among goose flocks, as seen in other poultry flocks19, 20. In February 2017, an outbreak of disease was reported in a goose farm in Weifang, Shandong Province, China. Affected flocks (containing 2000–3000 goslings) experienced continuous mortality rates ranging from 20 to 30% during the first 2 weeks of the outbreak despite antibiotic and supportive treatment. We conducted a systematic investigation to identify the causative agent of this disease and report here the isolation and characterization of a genetically distinct avian AstV. The pathogenicity of this virus was evaluated by experimental infection of goslings.

Increasing morbidity from zoonoses is a significant problem in terms of global health, and they are currently considered exotic in Europe. Zoonoses are infectious or parasitic diseases transmitted by animals to humans (Spahr et al. 2018). These infections as aetiological factors can develop in humans in several ways:

by the digestive tract, where the microorganism gets into the body via infected feed (meat) or water, which is quite often in large-scale farming. Therefore, Escherichia and Salmonella transmission to people occurs mainly through ingestion or frequent contact with infected birds. The above microorganisms can live in the external environment for a long period of time without damage to the pathogenic properties.by skin laceration; a break in the continuity of the skin promotes infection by pathogens of the staphylococci and streptococci families. In addition, the infection is intensified by the release of toxins into the host system.by the respiratory system (by inhalation of dust in which the pathogens are raised); infection occurs through direct contact of a healthy individual with contaminated excretion or through indirect contact (low hygiene at the slaughterhouse) with contaminated faeces and secretion on bird feathers (Hugh-Jones and de Vos 2002).

Humans are more susceptible to zoonoses carried by mammals than birds, and they share more diseases with them. This sharing is due to a higher degree of similarity between the intracellular environment of mammals (to which they belong), rather than birds. Many microorganisms need proper conditions and parameters to determine their host, for example, the presence of its receptor on the surface of the cells. These receptors can serve as a site for attachment and penetration into cells, which provides a pathway for the development of infection. The above situation determines the susceptibility of one animal species to a pathogen and resistance of another species. Vectors, such as insects, are also involved in the transmission of various pathogens, which may have an effect on the immune system. In the case of infecting a human with an avian zoonosis, the course of the disease is usually severe with general life-threatening symptoms. People who have been infected with an avian zoonosis require in-hospital treatment in isolation. Avian zoonoses in humans may end in death or a chronic disease requiring prolonged administration of antibiotics (Hugh-Jones and de Vos 2002).

Hence, the aim of this work is to present the aetiological factors of bird zoonoses, which are currently the most threatening to the European population. In addition, the epidemiological and economic analysis of the above infections in humans is presented. In general, zoonoses from birds can be divided by the type of the infectious agent: bacterial, viral and fungal.

Worldwide, the use of vaccines is seen as critical for the prevention and control of many economically damaging outbreaks of poultry diseases. In Caribbean countries, the use of vaccination to prevent poultry diseases is variable and is mainly influenced by the size of the poultry industry in the various countries. Many of the larger poultry-producing CARICOM countries (for example, T & T, Jamaica, Belize, Barbados, and Guyana) with large poultry (broiler and layer) operations have structured and rigid regimes of vaccination in place, while smaller island states (for example, Grenada and St. Lucia), with smaller poultry and egg production operations, often do not vaccinate their poultry. The larger intensive broiler and layer production units in the Caribbean routinely vaccinate their birds against IBV, NDV, and IBDV, whereas some smaller semi-intensive and backyard operations often do not carry out vaccination against these three viral pathogens. Routine vaccination is not carried out in the region against other viral pathogens (AIV, ILTV, APV, FADV Gp1, and EDSV) included in this review, although occasional vaccination against FADV Gp1 has been carried out in the face of disease outbreaks. All reports/publications describing the detection of viruses and outbreaks of disease in both vaccinated and unvaccinated poultry were reviewed. When information pertaining to vaccination history was given in the relevant report/publication, this information was included.

Recent outbreaks of novel diseases in humans and domestic animals underscore the critical need to elucidate the factors that enable pathogens to become established in new host species (1–5). Host shifts require not only that pathogens come in direct contact with the novel host but also that they have the capacity to infect and be transmitted by the new host (6, 7). Contact depends on opportunities for the pathogen to leave the original host and gain access to a novel host and, because of this, is mitigated by the geographic ranges and ecologies of both the hosts and the pathogen (8). For instance, exposure of European rabbits (Oryctolagus cuniculus) to the myxoma virus during an eradication attempt in Australia in the mid-20th century was sufficient to allow for pathogen emergence, even though the virus' natural host is a South American leporid rabbit (Sylvagus brasiliensis) (9, 10). Infectiousness and transmission, in contrast, will be determined primarily by pathogen and host genotypes (11, 12). For example, humans have long been in contact with pathogens of Himalayan palm civets (Paguma larvata), which are traditional food items in China. Despite this, there had been no known host shifts from civets to humans until the emergence of the severe acute respiratory syndrome (SARS) virus in 2002, then made possible by adaptive genetic changes in the virus' receptor binding domain (13, 14). The extent to which hosts shifts are limited by opportunities for contact with novel hosts versus by fortuitous mutations that predispose the pathogen to infect the novel host remains, however, understudied despite the potential impacts on humans and livestock of host shifts by pathogens.

In recent decades, molecular analyses have revealed that host shifting by bacterial pathogens may occur more frequently than previously thought (15–18). For example, phylogenetic analyses suggest that Wolbachia bacteria independently colonized multiple species of arthropods via horizontal transmission (15, 19, 20). Staphylococcus aureus similarly exhibits a diverse host range, including poultry, ruminants, and other mammals, likely the result of host shifting from humans (17, 18). Indeed, the jump of S. aureus from humans to rabbits required only a single mutation in a gene encoding an integral membrane protein (1). Yet, for other bacteria, there is evidence of more restrictive host ranges despite regular contact with other potential host species. For example, wood mice (Apodemus sylvaticus) and bank voles (Myoedes glareolus) in the United Kingdom harbor unique variants of Bartonella despite the collection of fleas carrying bank vole-specific variants from wood mice and vice versa (21). Experimental studies on cotton rats (Sigmodon hispidus) and white-footed mice (Peromyscus leucopus) similarly found that Bartonella infections were successful only when bacteria originated from the same host species or from their close phylogenetic relatives (22). An important limitation to understanding the role of contact versus host suitability in bacterial host shifts is that a majority of studies have focused on viral pathogens (11). Yet bacterial host shifts may be subject to different constraints than viral host shifts; unlike viruses, bacteria must also be able to extract essential metabolic substrates, nutrients, and enzymatic cofactors, such as iron, from their host and may face a suite of different host immune defenses (23). As a result, further studies are required to better understand the role of ecological versus evolutionary factors in bacterial host shifts.

One notable host shift by a bacterial pathogen occurred when Mycoplasma gallisepticum emerged in eastern North American house finches (Haemorhous mexicanus) in 1994. Comparative genomic analyses confirmed that this epizootic, which caused measurable declines in eastern U.S. house finch populations, resulted from a single host shift event of M. gallisepticum from poultry that occurred in the mid-1990s (2, 24–28). The subsequent spread of M. gallisepticum throughout North American house finches was uniquely well documented thanks to externally visible symptoms of conjunctivitis, quick identification of M. gallisepticum as the causative agent, and active disease monitoring (2, 24). Since then, spillover infections have been documented in numerous other wild bird species (29, 30), although none have led to an epizootic-scale outbreak, the reason for which remains unclear.

To investigate whether direct contact was sufficient for M. gallisepticum to jump into house finches, we experimentally inoculated house finches either with an M. gallisepticum strain obtained from the original poultry host (Rlow) or with a strain collected during the epizootic outbreak in the novel house finch host (HF1995) (26, 30–32). Whole-genome comparisons have revealed that HF1995 exhibits widespread genomic changes compared to Rlow (26), but the functional significance of these genomic changes for colonizing the novel host remains unknown. We predicted that if contact with the novel host alone was sufficient for M. gallisepticum to infect house finches, then Rlow and HF1995 should display similar abilities to establish an infection and cause clinical disease in house finches. Conversely, if Rlow showed low or no capacity to infect house finches, then this would support the hypothesis that mutations arising in the original poultry host would have been necessary for successful pathogen emergence in the novel finch host.

Colibacillos is suspected based on the clinical features and the typical macroscopic lesions. The diagnosis is obtained by E. coli isolation from cardiac blood and affected tissues, like liver, spleen, pericard or bone marrow. Experimentally it was shown that in acute cases, isolation is possible from six hours to three days after infection; in subacute cases, isolation is only possible until seven days after infection. Contamination from the intestines is rarely a problem, if fresh material is used and standard bacteriological procedures are applied. Selective media like McConkey, eosin-methylene blue or drigalki agar are used for isolation. Further identification of the isolated colonies is based on biochemical reactions (indol production, fermentation of glucose with gas production, presence of ß-galactosidase, absence of hydrogen sulphite production and urease, and the inability to use citrate as a carbon source). O-serotyping is a frequently used typing method. An ELISA, based on sonicated E. coli, has been developed for detection of antibodies against two important pathogenic serotypes of E. coli: O78:K80 and O2:K1. Another ELISA was based on fimbrial antigen. Both have limited value because they can only detect homologous APEC types. All currently known virulence-associated factors, detected in strains isolated from colibacillosis lesions, can also be detected in faecal isolates from clinically healthy chickens. For this reason, none of these traits can be used for APEC identification.

Diagnosis of avian salmonellosis should be confirmed by isolation, identification, and serotyping of Salmonella strains. Infections in mature birds can be identified by serologic tests, followed by necropsy evaluation complemented by microbiologic culture and typing for confirmation. A serological ELISA test for the diagnosis of avian salmonellosis either with S. typhimurium or S. enteritidis has been established. Szmolka et al. established a diagnostic and a real-time PCR system for rapid and reliable genus- and serovar- (S. enteritidis and S. typhimurium) specific detection of Salmonella for monitoring purposes in the poultry food chain.

Botulism, caused by neurotoxins produced by the bacterium Clostridium botulinum, affects a wide range of birds and mammals, including humans. The symptoms typically include paralysis. The disease is found globally and is usually acquired via oral intake of the toxin, or – especially in birds – intestinal growth of the bacteria, which then produce toxins in the guts. Different strains of C. botulinum produce different toxins, usually referred to as types A through G. Avian botulism is considered the most significant disease of migratory waterfowl in North America,[130] and is caused by other strains than human botulism.

Avian botulism has been known for more than a century. For example, Hay and co-workers already in 1973 published a paper on botulism type C in wild spur-winged geese Plectropterus gambensis in South Africa, where they traced descriptions of this disease back to 1893. There are also indications of botulism being present in North America as early as in 1890.[130] In Spain, outbreaks in waterfowl are mainly seen in the warmer seasons, i.e. summer and autumn, due to the combination of high temperatures, large amounts of biomass, and anaerobic conditions in wetlands.[131] To our knowledge, botulism has not been reported as a significant problem in swans and geese (albeit in other types of wild birds, such as gulls) in Northern Europe, and no link between waterfowl and outbreaks in domestic poultry has been established here. For mammals including humans, botulism is linked to consumption of feed or food containing the neurotoxin, i.e. not to contact with birds, which are affected by a different strain. Hence, the risk of wild geese and swans transmitting botulism to humans is negligible, and the risk of transfer to poultry appears limited, and should not warrant any specific action.

Live animal markets (LAMs) represent a traditional place for congregation and commerce, particularly in developing countries. Owing to their role as a source of affordable, live or freshly slaughtered animals, LAMs act as a source for transmission of pathogens, especially viruses.1, 2, 3 Spread of a virus within the market is often enhanced due to high density, close contact animal housing, increasing the risk of zoonotic and anthroponotic transmission.4 Animals remain in the LAM for extended periods of time until sold and can consequently transform these markets into viral reservoirs. As new animals are introduced to the LAM, Infected animals easily transmit viruses to these naïve hosts, thus perpetuating and amplifying viral circulation.5 In addition, LAMs are often part of a larger marketplace ecosystem, potentially exposing people to zoonotic pathogens with little to no direct contact with infected animals.1 This is especially true with avian influenza viruses (AIV).

LAMs in many parts of the world harbor highly pathogenic as well as low pathogenic AIV, which can spread asymptomatically through poultry and are difficult to detect without routine surveillance.6, 7, 8, 9 Although AIV has been detected in North American and Caribbean LAMs, there is no information about South America LAMs,10, 11 likely due to minimal surveillance.12 During active surveillance at the largest LAM in Medellin, Colombia, we isolated two H11N2 viruses from asymptomatic birds. At the peak of the occurrence, 17.0% of the birds in the market tested positive. Genetically, the circulating virus was most similar to viruses isolated from North American migratory birds and to viruses isolated in 2013 from Chilean shorebirds. H11 viruses are distributed worldwide10, 13, 14, 15 primarily in wild ducks and shorebirds16, 17 but rarely are found in domestic poultry.9, 10, 18, 19, 20 Given this unique occurrence and the fact that H11 have been reported to cause human infections21, 22 we characterized the viruses in vitro and in vivo. The Colombian H11 viruses displayed no molecular markers associated with increased virulence in birds or mammals and had an α2,3-sialic acid binding preference. They replicated and transmitted effectively in chickens, explaining the spread throughout the market, but caused little morbidity in Balb/c mice. The genetic similarity to H11 viruses isolated from South American shorebirds suggests that the LAM occurrence may have resulted from a wild bird to domestic poultry spillover. These findings highlight the need for enhanced AIV surveillance in South America, especially in areas of high-risk, such as LAMs.

Toxoplasmosis is one of the most common parasitic zoonoses, caused by the obligate intracellular protozoan Toxoplasma gondii, which can infect almost all warm-blooded animals, including birds. It has been estimated that approximately one third of the world population and 7.88% of population in China have been infected. Toxoplasmosis is generally benign or associated with mild nonspecific clinical symptoms in most patients. However, blindness and mental retardation can be caused in congenitally infected children, and T. gondii infection is ranked as a leading cause of death in immuno-compromised individuals, especially in acquired immuno-deficiency syndrome patients.

Wild and domestic felids are the definitive hosts of this protozoan parasite, being able to excrete sporulated oocysts into the environment. Intermediate hosts such as humans or birds can become infected post-natally by ingesting tissue cysts from undercooked meat, consuming food or drink contaminated with oocysts, or ingesting oocysts from the environment accidentally. Birds are important intermediate hosts of T. gondii and infection of birds with T. gondii is considered important epidemiologically because infection of ground-foraging birds with T. gondii can indicate soil contamination with oocysts, which also represent a source of infection for cats. Surveys of T. gondii infection in wild birds have been reported extensively in the world, clinical cases have been reported and T. gondii is considered to be one of the causes of mortality in birds of different species.

The peafowls (Pavo) include two Asiatic species of flying birds in the genus Pavo of the pheasant family Phasianidae, best known for the male's extravagant display feathers. The blue peafowl (Pavo cristatus) is widely distributed naturally in the tropical forests of Southeast Asia, but the green peafowl (P. muticus) is only naturally distributed in Yunnan Province and Tibetan areas in China. Due to hunting and a reduction in extent and quality of habitat, the green peafowl is considered endangered on the International Union for Conservation of Nature (IUCN) Red List of Threatened Species, and is listed as Category I in the list of Key Protected Wildlife in China. In addition to its ornamental value, blue peafowl is a rare breeding bird domesticated for meat in some areas of China. Recent studies have identified a number of pathogens (such as avian influenza, avian pox) of potential conservation concern for this species, but such information still remains relatively limited.

Data on peafowl infection with T. gondii is limited in the world, to date only one survey has been conducted in Shanghai Zoological Garden in China in 2000. The objective of the present investigation was to determine the seroprevalence of T. gondii infection in peafowls in Yunnan Province, southwestern China, and the results obtained will provide base-line information on potential risk factors associated with infection and potential implications for public health.

Some bacteria of the genus Yersinia, namely Y. enterocolitica and Y. pseudotuberculosis, are considered pathogenic for animals and humans, symptoms mainly being gastrointestinal illness.[109] Geese have been shown to carry the strain of Y. enterocolitica that causes disease in humans, but also several non-pathogenic species and strains. For example, Niskanen and co-workers found Yersina spp. in 42 out of 105 faecal samples from barnacle geese, but none in brent geese, Canada geese, greylag geese, or mute swans (seven, one, one and one samples respectively). Many of these were, however, non-pathogenic species of Yersinia, or non-pathogenic strains of Y. enterocolitica. The authors commented that as these barnacle geese were sampled on migration they had most likely become infected at a previous location, and then acted as long-distance dispersers. Furthermore, they concluded that, because of the low prevalence of pathogenic strains isolated, birds – including geese – are not likely to be a direct source of Yersinia infections in humans.[129]

The most pest-prone species of poultry are primarily hens, quail and turkeys. The condition is caused by a virus belonging to the Herpesvirus genus. The way they penetrate the organism is through the respiratory system or gastrointestinal tract. Infection usually occurs immediately after hatching. The virus contained in the exfoliated warts of the pen still retains its virulence for more than 12 months after initiation. Infected birds show weight loss and paroxysmal symptoms. However, it often occurs that the course of this disease is very violent and no clinical symptoms were observed in humans (Ryan and Ray 2004; Koelle and Corey 2008; Johnston et al. 2011; Schiffer et al. 2014).

Avian species serve as hosts for a diversity of viruses that pose both biosecurity and zoonotic risks. Numerous outbreaks of disease in collections of caged birds and commercial domestic poultry have been attributed to viruses introduced by direct or indirect contact with wild birds [1–7]. Some avian viruses can also cause disease in humans [8–14]. As such, understanding the viruses present in wild birds is important for managing potential risks to human and animal health, and for understanding the biology of these viruses in their natural hosts. Studying viruses in wild birds can be challenging as gaining access to these birds for the purpose of sample collection can be difficult, and their capture may be a cause of stress or injury. Collecting and testing samples from avian patients presenting to veterinary hospitals can be a convenient and useful approach to detecting and studying avian viruses. Importantly this approach does not involve additional capturing or handling of birds, and allows the risks to staff and other avian patients/collections within the facility to be more accurately assessed, since the species from which the samples are collected reflects the species being handled at the participating facility. This approach has been successful in previous studies utilizing veterinary hospitals and wildlife rescue centres in several countries including Australia, the United Kingdom, France and Belgium [15–18].

In Australia, the Australian Wildlife Health Centre (AWHC) at Zoos Victoria’s Healesville Sanctuary functions as a large primary care and referral centre for sick, injured and orphaned native wildlife, including providing veterinary care for approximately 700–900 wild birds each year, as well as Healesville Sanctuary’s collection of captive animals. Patients may be presented to the AWHC by members of the public, wildlife carers, keepers, government wildlife officers or by referral from other veterinary clinics for veterinary diagnosis and treatment. During this period in captivity there is an elevated risk of stress-induced shedding of pathogens, presenting a risk for both the veterinary staff and other animals in care, including the Sanctuary’s collection animals and captive breeding programs. Close contact between infected and susceptible animals can facilitate the transmission of pathogens between species, particularly when the hosts are taxonomically related. The large number of avian patients passing through the AWHC provides an opportunity to study the viruses present in Australian birds and better understand the potential risks these viruses present.

Of particular importance and conservation value at Healesville Sanctuary are the captive breeding and insurance programs for threatened species. This includes a captive breeding program for the critically endangered orange-bellied parrot (Neophema chrysogaster), which is facing imminent extinction in the wild. The orange-bellied parrot is arguably the most threatened parrot species in the world, with the wild-bred population declining to only three females and 13 male birds in the 2016/2017 breeding season. Infectious diseases, including viral diseases, are a recognized threat to this and other threatened bird species both within Australia and around the world. Beak and feather disease virus (BFDV), a circovirus, is of particular concern. Our past work has shown that BFDV is prevalent in both psittacine and non-psittacine birds presenting to the AWHC, and has revealed that non-psittacine birds may represent a previously unrecognized risk for spreading BFDV infection. Other viruses are also a potential threat to Psittaciformes in Australia. Psittacid alphaherpesvirus 1 (PsHV1) is the aetiologic agent of both Pacheco’s disease and mucosal papillomas in Psittaciformes. PsHV1 has not been reported in wild Psittaciformes in Australia, but has been detected in Australia in captive green-winged macaws (Ara chloropterus) imported from the United Kingdom in the 1990s [23, 24]. It is not known how far the virus has been transmitted from these birds but it is likely to be present in at least some avicultural collections in Australia. The inadvertent introduction of such a virus into the remaining population of a threatened avian species, such as through the Neophema chrysogaster recovery program, would be potentially devastating. Avian paramyxoviruses (APMVs) also pose a threat to Australian Psittaciformes. Avian avulavirus 3 (previously avian paryamyxovirus serotype 3, APMV-3) has been documented causing mortality rates of up to 70% in captive collections of Neophema species and other Australian psittacines overseas [25–28]. APMV-3 strains have been isolated from a diversity of avian species in different parts of the world, but has not yet been reported in Australia [25–31]. However we do know that the Australian parrot genus Neophema is highly susceptible to APMV-3, with infection frequently resulting in fatal neurological disease [26, 28]. Another avian paramyxovirus, Avian avulavirus 5 (avian paramyxovirus 5, APMV-5) has caused outbreaks of severe disease with up to 100% mortality in captive budgerigars (Melopsittacus undulatus), including an outbreak in a captive budgerigar flock in Queensland in 1972 [25, 32–34].

Few avian viruses represent a risk to veterinary staff and others handling birds, but avian influenza viruses (AIVs) can threaten human health [8, 9, 11–13]. Wild birds, particularly waterfowl, waders and sea birds, are the natural reservoirs of low pathogenicity avian influenza (LPAI) viruses and play important roles in the circulation of AIVs [35–38], with potential for transmission of AIVs both to and from poultry and the potential for mutation to generate highly pathogenic avian influenza (HPAI) viruses [6, 7, 39]. Some AIVs can be transmitted from poultry to humans, and some have caused severe disease or even death in humans [8, 9, 11–13, 40, 41]. Transmission of AIVs from wild birds to commercial poultry has occurred recently in Australia in Victoria, New South Wales and Queensland [10, 42–45]. The Australian National Avian Influenza Wild Bird Surveillance Program has been in place since 2006 but primarily targets Anseriformes (ducks, swans, geese) and Charadriiformes (gulls, terns and shorebirds) [46, 47]. The prevalence of AIV infection in birds presenting to veterinary care facilities, such as the AWHC, has not been as comprehensively studied. In Australia, avian coronaviruses (AvCoVs) are also of particular interest in wild birds because of the presence of variant forms of the poultry pathogen infectious bronchitis virus (IBV) in Australian commercial poultry [48–50]. It is suspected that the variant IBV strains contain genetic material derived from AvCoVs hosted by wild birds, but this requires further investigation.

This study aimed to collect samples from birds presenting to the AWHC and use them for the molecular detection and characterization of viruses with potential significance to biosecurity, human health or animal health. Four virus families were targeted in this study: avian influenza viruses, herpesviruses, paramyxoviruses and coronaviruses. Samples were screened using a combination of genus- or family-wide polymerase chain reaction methods coupled with sequencing and phylogenetic analyses for detection and identification of both known and novel viruses. Two novel viruses were detected in this study, hence contributing to our knowledge of avian virodiversity, while the overall low level of virus detection adds to our understanding about the presence or absence of viruses of birds in Australia and their potential risk to avian and human health.

The mechanisms by which avian pathogenic E. coli cause infection are largely unknown. The virulence factors contributing to the pathogenesis of avian colibacillosis are summarized in Table 3.

Recently, Hughes et al. described a cross-sectional study of wild birds in northern England to determine the prevalence of E. coli-containing genes that encoded Shiga toxins (stx1 and stx2) and intimin (eae), important virulence determinants of STEC associated with human disease and they stated that while wild birds were unlikely to be direct sources of STEC infections, they did represent a potential reservoir of virulence genes.

APEC are responsible for a considerable number of various diseases at different ages. Neonatal infection of chicks can occur horizontally, from the environment, or vertically, from the hen. A laying hen suffering from E. coli-induced oophoritis or salpingitis may infect the internal egg before shell formation. Faecal contamination of the eggshell is possible during the passage of the egg through the cloaca and after laying. The latter possibility is considered as the main route of infection for the egg. Before hatching, APEC causes yolk sac infections and embryo mortality. The chick can also be infected during or shortly after hatching. In these cases, retained infected yolk, omphalitis, septicemia and mortality of the young chicks up to an age of three weeks is seen. Broilers may be affected by necrotic dermatitis, also known as cellulitis, characterized by a chronic inflammation of the subcutis on abdomen and thighs.

Swollen head syndrome (SHS), mainly a problem in broilers, causes oedema of the cranial and periorbital skin. SHS can cause a reduction in egg production of 2 to 3%, and a mortality of 3 to 4%. Data on this disease are contradictory. Picault et al. and Hafez & Löhren considered SHS as a disease caused by avian pneumovirus (APV), usually followed by an opportunistic E. coli infection. Nakamura et al. however reported that APEC were probably playing a significant part in the disease, but that the role of APV was not at all clear. This had been confirmed by Georgiades et al., who did not detect APV in any of the flocks affected by SHS during a field study, but instead detected infectious bronchitis virus (IBV), avian adenovirus, avian reovirus, and Newcastle disease virus (NDV), as well as Mycoplasma synoviae and M. gallisepticum (MG).

APEC probably do not cause intestinal diseases. Nevertheless, enterotoxigenic E. coli (ETEC) are occasionally isolated from poultry suffering from diarrhoea [161–163] and diarrhoea was experimentally induced after intramuscular inoculation of APEC. On the other hand, enteropathogenic E. coli (EPEC) were isolated from clinically healthy chickens. In turkeys, experimentally inoculated EPEC can only cause enteritis in combination with coronavirus.

Layers as well as broilers may suffer from acute or chronic salpingitis. Salpingitis can be the result of an ascending infection from the cloaca or an infection of the left abdominal airsac, although Bisgaard and Dam considered the latter possibility less likely than an ascending infection. Salpingitis can lead to the loss of egg-laying capacity. In the case of chronic salpingitis, the oviduct has a yellowish-gray, cheese-like content, with a concentric structure. In layers, salpingitis can cause egg peritonitis if yolk material has been deposited in the peritoneal cavity, characterised by a sero-fibrinous inflammation of the surrounding tissues.

Airsacculitis is observed at all ages. The bird is infected by inhalation of dust contaminated with faecal material, which may contain 106 CFU of E. coli per gram. This aerogenic route of infection is considered as the main origin of systemic colibacillosis or colisepticemia.

Septicemia also affects chickens of all ages, and is mainly described in broilers. It is the most prevalent form of colibacillosis, characterised by polyserositis. It causes depression, fever and often high mortality. Although its pathogenesis has not been elucidated, several routes of infection are possible: neonatal infections, infections through skin lesions, infection of the reproductive organs, of the respiratory tract and even infection per os. When E. coli reaches the vascular system, the internal organs and the heart are infected. The infection of the myocard causes heart failure. Septicemia occasionally also leads to synovitis and osteomyelitis and on rare occasions to panophthalmia. Coligranuloma or Hjarre’s disease is characterised by granulomas in liver, caeca, duodenum and mesenterium, but not in the spleen. It is a rare form of colibacillosis, but in affected flocks it may cause up to 75% mortality.

Further studies are needed to determine the role of newly identified putative virulence genes and genes with unknown functions as virulence markers of APEC to strengthen the current understanding of mechanisms underlying the pathogenesis of avian colibacillosis.

Infectious bronchitis virus (IBV) is, by definition, the coronavirus of the domestic fowl. Although it does indeed cause respiratory disease, it also replicates at many nonrespiratory epithelial surfaces, where it may cause pathology, for example, kidney and gonads [1, 2]. Strains of the virus vary in the extent to which they cause pathology in nonrespiratory organs. Replication at enteric surfaces is considered to not normally result in clinical disease, although it does result in faecal excretion of the virus. Infectious bronchitis (IB) is one of the most important diseases of chickens and continues to cause substantial economic losses to the industry. Infectious bronchitis is caused by IB virus (IBV), which is one of the primary agents of respiratory disease in chickens worldwide. All chickens are susceptible to IBV infection, and the respiratory signs include gasping, coughing, rales, and nasal discharge. Sick chicks usually huddle together and appear depressed. The severity of the symptoms in chickens is related to their age and immune status. Other signs of IB, such as wet droppings, are due to increased water consumption. The type of virus strain infecting a flock determines the pathogenesis of the disease, in other words, the degree and duration of lesions in different organs. The upper respiratory tract is the primary site of infection, but the virus can also replicate in the reproductive, renal, and digestive systems. The conventional diagnosis of the IBV is based on virus isolation in embryonated eggs, followed by immunological identification of isolates. Since two or three blind passages are often required for successful primary isolation of IBV, this procedure could be tedious and time consuming. Alternatively, IBV may be isolated by inoculation in chicken tracheal organ cultures. Furthermore, IBV may be detected directly in tissues of infected birds by means of immunohistochemistry [6, 7] or in situ hybridization. The reverse transcription-polymerase chain reaction (RT-PCR) has proved useful in the detection of several RNA viruses [9, 10]. Outbreaks of the disease can occur even in vaccinated flocks because there is little or no cross-protection between serotypes [2, 11]. The necessity of IB prevention in chicken regarding the nature of the virus with a high mutation rate in the S1 gene dictates the necessity to develop effective vaccines. The first step is to study the virus strains distributed in the geographical region and determine their antigenicity and pathogenicity in order to choose a suitable virus strain for vaccination. This virus was isolated from a flock suspected of IB suffering from severe respiratory distress and experiencing high mortality. The objective of the present study was to clarify some aspects of pathogenesis of the disease caused by IRFIBV32 (793/B serotype) in experimentally infected broilers. RT-PCR test was performed to detect the presence of the virus in body tissues and samples. The clinical signs, gross lesions, and antibody response of the affected chicks were also monitored.

During the last decade, awareness concerning the intimate links between human and animal health has rapidly increased in the context of disease emergence. Indeed, approximately 80% of the infectious diseases that recently emerged were zoonotic. The role of wildlife in emerging pathogen transmission to humans and domestic animals has in many cases been pointed out–[5]. Conversely, pathogen transmission from domestic animals to wildlife has received far less attention, although the importance of this issue was often mentioned,. Indeed, contacts between wildlife and livestock or their environment sometimes result in wildlife diseases with conservation issues,. Besides, hand-reared animal releases into the wild for either conservation or exploitation purposes represent a particular case in which hand-reared individuals eventually share natural habitats with their wild congeners. In both cases such releases can dramatically influence disease dynamics in the surrounding wild animal populations–[14].

In the present study we focused on the case of game restocking, which implies the release of millions of individuals worldwide each year. Birds are the most frequently involved, with millions of individuals being released annually in Europe only. For example more than 3 millions red-legged partridges are released annually in Spain, and ca. 1.4 million Mallards are being so in France. The Camargue region, a complex network of wetlands situated in the Rhone delta, is a major duck winter quarter and a central place for wildfowl hunting in France. Hunting is also among the most important economic activities in the area, which is one of the reasons for the massive Mallard releases in the Camargue. At least 30 000 hand-reared individuals are released annually in the region. Maximum Mallard numbers in the wild are reached in September after the beginning of the hunting season, with 56 500 individuals on average over the last seven years (Gauthier-Clerc, unpubl. data), these numbers certainly include a mixing of wild and released Mallards. Given its central position on the flyway of many European migratory species, the Camargue is also a potential hotspot for the introduction and transmission of bird-borne pathogens.

For this reason, avian influenza viruses (AIV) have been studied since 2004 in the area. These negative-sense single stranded RNA viruses belonging to the Orthomyxoviridae family are commonly characterized by the combination of their surface proteins: hemagglutinin (HA) and neuraminidase (NA),. AIVs are highly variable and undergo continuous genetic evolution via two mechanisms: i) accumulation of point mutations at each replication cycle, ii) reassortment involving gene segment exchanges that occur when a cell is co-infected by different viruses. These mechanisms contribute to the emergence of new variants with the ability to transmit to new hosts and/or with epidemic or even pandemic potential. Aquatic birds, particularly Anseriforms (ducks, geese and swans) and Charadriiforms (gulls, terns and shorebirds) constitute their major natural reservoir,. The AIV circulating in wild birds are usually low pathogenic ones (LPAIV). LPAIV generally have little impact on their host,, although some studies have reported a possible influence on migration capacities. Besides, when LPAIV of H5 or H7 subtypes are transmitted from wild birds to domestic ones reared in artificial environments, their virulence can evolve to high pathogenicity,. Highly pathogenic avian influenza viruses (HPAIV), such as HP H5N1 strains currently circulating in Asia and Africa, are still of great economic concern, notably due to the cost of preventive actions including vaccination and massive birds culling. Moreover, HPAIV infections represent a threat for human health since 603 HPAIV H5N1 human infections including 356 fatal cases have been reported worldwide since 2003.

Relatively high prevalence of AIV was regularly detected in the wintering Mallard population of the Camargue (e.g. 5.4% prevalence during the 2006–2007 hunting season). Moreover a seasonal infection pattern was identified in Mallards during autumn and winter, with higher infection rates in early fall. Mallards hence represent a focal study species in AIV research. Indeed wild Mallards are one of the main low pathogenic AIV natural reservoir host, and have proven to be healthy carriers of some of the H5N1 HPAIV strains. However, to our knowledge no study ever aimed at investigating the potential role of hand-reared Mallards released for hunting in the epidemiology of AIV, despite the very large number of ducks being released in the wild annually.

To clear this gap we conducted a 2-year study in the Camargue to investigate the potential influence of hand-reared Mallard releases on AIV dynamics in surrounding wildlife. We first hypothesized that, owing to high density rearing conditions and to their genetic uniformity, hand-reared Mallards should be highly susceptible to AIV infections and could play an amplification role in AIV dynamics. This phenomenon has already been pointed out in red-legged partridge (Alectoris rufa) reared for hunting in Spain, where Escherichia coli prevalence was much higher in hand-reared populations before their release than in the wild ones. To test this assumption we collected cloacal swabs from Mallards reared for hunting in several game bird facilities (GBF) in 2009 and 2010, and analysed these samples to measure AIV prevalence. Second, we hypothesized that AIV exchange occurs between wild and hand-reared Mallards, potentially leading either to the circulation of new strains in wild populations or to the amplification and dispersal of wild strains. Indeed, no barrier prevents AIV exchange between wild birds and hand-reared ducks in the GBF since water flows exist between pens and ponds used by wild birds. Moreover, as the GBF roofs are made of nets wild birds can deposit feces in the pens. Finally, hand-reared ducks are in direct contact with wild ones after their release. To investigate these issues, we tested shot waterfowl before and after hand-reared Mallards were sampled. Noteworthy, genetic analyses suggest that 76% of hunted Mallards in the Camargue have a captive origin. Considering the low annual survival of released Mallards (0.8–15.9% depending on the release site), the individuals we tested from the Camargue hunting bags certainly included a large proportion of ducks released some months before being shot. These released ducks cannot be differentiated morphologically from the wild ones. Here we hence analyzed shot ducks as a whole since they are a representative sample of the Mallard population wintering in the Camargue, which is composed of individuals of both wild and captive origin that share habitats and can thus be considered as a single epidemiological unit.

Our third hypothesis was that any AIV strain potentially found in captive reared Mallards might present genetic characteristics linked to its circulation in domestic populations. We therefore performed a molecular study of the identified strains in order to look for such characteristics, and in particular to test for the presence of mutations known to be associated with increased virulence, since it has been highlighted that artificial environments are favorable to the appearance of such mutations in birds. We also searched for mutations linked with transmission to other species including humans, since some studies proved that they can be acquired during their transmission among birds. Thirdly, we tested for the presence of mutations conferring resistance to common antiviral drugs, since such resistance has recently been recorded in wild birds, notably in Sweden. Lastly, full sequence analysis of some strains was performed, so as to get insight into their geographic origin through a phylogenetic study, and to determine their relatedness with strains, which have caused human infections in the past.

Despite a lack of empirical studies of the transmission of influenza in rLBMs, well-established ideas from epidemic theory enable us to make mechanistic predictions about prevalence patterns within them. Incubation periods for AIVs in poultry can be up to 2 days when birds are inoculated with doses less than or equal to 103, and around 1 day when doses are higher. Making the worst-case assumption that susceptible and infectious hosts are in constant contact, this means that the minimum time infectious individuals can create other infectious individuals is 1 day, and higher on average. Thus, if the average stay-time of birds in rLBMs is ≤ 2 days, there is not time for exponential growth of prevalence due to direct transmission within rLBMs (e.g., “outbreaks”). In addition, direct transmission alone may not cause significant amplification of prevalence within rLBMs because birds that entered the market uninfected have a high probability of being slaughtered before they begin shedding AIV. A similar principle has been identified in other animal-disease systems. For example, epizootics of plague in prairie dog populations have been shown not to occur by blocked-flea or pneumonic transmission alone, because both blocked vectors and hosts capable of direct transmission are removed from the population by death before they reliably create large chains of transmission required for outbreaks. Essentially, direct transmission in rLBMs should be limited by the interplay of stay-times and incubation periods.

Retail LBMs are also thought to foster persistence of AIVs in the environment, creating another source of transmission. The importance of an environmental factor in viral persistence has been shown in an experiment that monitored AIV isolation rates before and after days that the market was disinfected. However, whether this environmental persistence adds to transmission has not been determined empirically. Theoretically, indirect transmission via an environmental reservoir could contribute to the overall force of infection within rLBMs by providing a sustained source of AIV (i.e., by providing a transmission link between birds even if they do not occupy the market at the same time). Indirect transmission can occur through a variety of routes, including viruses in drinking water, in feces on the ground or on surfaces in cages. All of these routes rely on three main processes: shedding rates into/on a particular environmental feature, decay rates of the virus in it, and contact of susceptible birds with it. Intuitively, one would predict that indirect transmission would be most significant when shedding rates are high, decay rates are low and contact rates are high.

Clinical monitoring of infected chicks reveals at first, apparent respiratory symptoms beginning at day 2 post-inoculation (dpi). Respiratory clinical signs were predominant in all of the inoculated groups and were intense and more severe until 7 dpi, with no clear differences in the pathogenicity of the three strains. The most prominent clinical signs were characterized by gasping, depression, sneezing, difficulty in breathing, cough, pulmonary and tracheal rales, with high scores were reported for all the tested strains with clinical score of 108, 126 and 140 for IBV/RA, IBV/MN and IBV/TU strain respectively, (Tables 1, 2 and 3).

Nasal discharge and watery eyes were also observed but were transient in some of infected by IBV/RA and IBV/MN chicks. These clinical symptoms were persisted in all groups until 12dpi. The birds infected by IBV/MN and IBV/TU appeared lethargic, reluctant to move (Fig. 1), whereas, chicks infected with by IBV/RA were not as apathetic. At autopsy, all infected chicks that were killed at 5 dpi to prevent their suffering were examined for the macroscopic lesions in the trachea, lung and kidney. These gross lesions consisted in hemorrhagic tracheitis, mucosal congestion and catarrhal exudates that mainly progressed. For the three strains used, the signs were observed in the most infected chicks with dominance in the lungs that were hemorrhagic and sometimes cyanotic at one lobe. Therefore, samples were taken at 14 dpi just for a macroscopic examination of organs to confirm whether the absence of clinical signs is correlative with the gross lesions. Whereas, gross lesions of kidney, were not observed in all inoculated chicks. During the experiment, the non-infected control group stayed normally without clinical signs or gross lesions. The statistical analysis was not performed as the three tested strains are phylogentically related to each other and the difference in clinical and tissues scores is not very significant.

Approximately one month after confirmation of the first three cases of human infection with the new reassortant avian influenza virus (H7N9), the disease had extended to 42 cities in 12 provinces, infecting 134 people and killing 45 people in China as of September 10, 2013.1 The viral isolates from patients were similar to the isolate from an epidemiologically linked market chicken.2 Approximately 77% of patients had a history of exposure to live poultry.3 The virus was clearly of avian origin, and the patients were infected in farmers' markets with live birds (FMLB) through an unknown mechanism. Suspending the FMLB can prevent new human infections in cities with a high population density.4 The sporadic human infection patterns were distributed in 39 cities and occurred at various times, indicating that the virus is silently spreading in live birds over a far larger geographical area and at a far greater speed than originally thought.2,5,6 The movement of live birds is a well-known risk factor for the geographic dissemination of the virus among poultry flocks. The daily incidence of H5N1 virus outbreaks in Vietnam peaks around the annual holiday festivities in February coinciding with poultry movement increases.7,8 Therefore, determining the location of live birds carrying the virus and the FMLB that is contaminated by the live-bird trade is critical in stopping the surge of human infection and the geographical dissemination of the disease. The traditional investigation of animals carrying the virus is based on the virological diagnosis of animals with a possible epidemiological link.9,10 This task can be difficult because China has approximately six billion domestic birds. Until the source of infection has been identified and controlled, more cases of human infection by the virus are expected.6,11 Crowd-powered expansion and the distributed focused crawler are methods that use large-scale online data to reveal human behavior accessible to research.12 The method as used in this study to reveal live-bird-trading information to predict the potential nationwide geographic spreading of H7N9 virus, which could not be identified by traditional epidemiological investigation methods.

Enteric viruses are the etiological agents for a series of health disturbances for commercial chickens around the world. They cause severe economic losses for the poultry industry because they negatively affect productive parameters, causing growth retardation, low feed consumption, high mortality, poor egg and meat production, and Runting-Stunting Syndrome (RSS). These kinds of infections affect mostly young birds, but it is common to find viral infections in birds of all ages, including broilers, layers, and breeders. The main enteric viruses reported to cause enteric diseases are found in single and multiple infections and include the Fowl Adenovirus of group I (FAdV-I); Chicken Parvovirus (ChPV); two viruses from the Astroviridae family: Chicken Astrovirus (CAstV) and Avian Nephritis Virus (ANV); two viruses from the Reoviridae family: Avian Reovirus (AReo) and Avian Rotavirus (ARtV); and a member of the Coronaviridae family, Infectious Bronchitis Virus (IBV). Several laboratory analytical methods have been used to detect enteric viruses in organic tissues from sick and healthy birds. Conventional polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR) are two of the most commonly used methods for diagnosis and characterization of viruses in the poultry industry. The objective of this study was to determine the prevalence of enteric viruses affecting commercial chicken flocks in Brazil, encompassing an analysis of the relationships between single and multiple infections, the age of birds, clinical signs, and geographical distribution in the Brazilian states of Mato Grosso, Goias, Piaui, Ceara, Paraiba, Pernambuco, Bahia, Minas Gerais, Espirito Santo, São Paulo, and Santa Catarina.

Avian influenza viruses (AIV) of the H5, H7 and H9 subtypes represent an enormous commercial burden for the poultry sector, at a global level. Commercial, as well as political and sanitary issues related to the circulation of AIV, prompted policymakers and intergovernmental health organizations to set subtype and pathotype-specific international standards to better control H5 and H7 outbreaks, as these subtypes might cause epidemics of huge proportions, in different poultry species. On the other hand, AIV of the H9N2 subtype are not subject to specific international control measures, as they are considered of low-pathogenicity for poultry and their subtype has never mutated to the highly pathogenic form.

Genetically, distinct H9N2 lineages have been described, the G1 being probably the most widespread. In fact, H9N2 viruses of the G1 lineage emerged in Hong Kong in 1997, and steadily spread to the rest of Asia, reaching the Middle East and North Africa [4–7], by the year 2000 and 2006, respectively. In 2016, the virus severely affected Morocco and, for the first time, was reported in sub-Sahara Africa, being reported in Burkina Faso, in West Africa. In many of these regions, the virus has become an endemic presence notwithstanding the implementation of vaccination, increased biosafety and surveillance measures, and is frequently associated with moderate-to-high mortality in broilers and long-lasting drops in egg production in layers and breeders [4, 5, 9].

Several experimental studies have tried to reproduce the clinical presentations observed in the field, but mortality and severe respiratory signs were rarely observed unless the infection was artificially coupled with secondary pathogens such as Escherichia coli, Mycoplasma gallisepticum, Staphylococcus aureus, Haemophilus paragallinarum, Chlamydia psittaci, Ornithobacterium rhinotracheale and live infectious bronchitis virus vaccine [10–15] or in the presence of an immunocompromised flock. In our opinion, these data together with the lack of ad hoc restrictive control measures and in-depth diagnostic analyses during outbreaks, have somehow mitigated the perception of H9N2 viruses as primary pathogens of poultry.

Although evidence from the field clearly indicates the ability of H9N2 viruses to induce pathology at the level of the oviduct, only few studies have investigated the tropism of these agents for this organ, in a controlled experimental setting [18–21].

Wang et al. proved in fact, that a H9N2 virus of the Y280 lineage isolated in the Shangxii province in 2011, could replicate at high titers in the oviduct, trigger apoptosis and cause a decrease in the number of laid eggs in the first week post infection and a reduction in egg-shell thickness [18, 20].

Inspired by this work, we decided to study the acute and chronic impact of a G1-lineage H9N2 virus on the performance of Hy-Line Brown hens at the peak of lay, to fully elucidate the etiology and pathogenesis of the disease in commercial layers.

Enteric diseases are an important health problem for the intensive poultry industry and can result in considerable economic losses. The most characteristic of these conditions are diarrhea, stunting, huddling, increased feed conversion and extended time-to-market. In the more severe forms, immune dysfunction and increased mortality may occur. Different terms have been used to describe enteric disease syndrome in turkey: poult enteritis complex (PEC) and poult enteritis mortality syndrome (PEMS) or light turkey syndrome (LTS) [1–3]. However, none of these descriptions relate to any specific agents, and numerous factors have been associated with them, including environmental, such as housing, ventilation, temperature and humidity; management, such as biosecurity programmes; and microbiological agents (viruses, bacteria and parasites). In recent years there has been a lot of research regarding the viral origin of enteric diseases, and different viruses, such as astrovirus, coronavirus, reovirus, rotavirus, parvovirus, and adenovirus have been identified in intestinal tract of not only diseased but also healthy poultry. The role of some viruses in enteritis is well-defined, i.e. coronavirus is a known factor of mud fever/bluecomb disease of turkeys. However, the clinical and epidemiological significance of turkey infection with most of them still remains unclear. It seems that viruses are present in large amounts and different combinations in bird intestines, but in optimal nutritional and environmental conditions they do not cause clinical disease. In favorable conditions, such as feed change, sudden temperature shift or other stress factors, the viruses present in the gut might damage its walls making birds more susceptible to secondary bacterial infection. Furthermore, it is also possible that enteric viruses could contribute to the development of symptoms only by co-infections with multiple viruses. For a better understanding and control of enteric disease in turkeys, more studies on enteric viruses are needed.

In Polish commercial turkey flocks, despite the high level of hygiene/biosecurity implemented, the clinical signs of enteritis are often observed. In such situations, bacteria and parasites are most often investigated microbiological agents. The aim of the study was to examine the prevalence of four enteric viruses, namely astrovirus (AstV), coronavirus (CoV), rotavirus (RoV) and parvovirus (PV) in Polish meat-type turkey flocks. Recently statistical methods have been used more often to explain which factors contribute to disease characteristics, thus we also attempted to determine whether a statistical correlation occurs between the presence of the viruses studied and the health condition and age of turkeys.

Bornaviruses are enveloped, 80 to 100 nm in diameter with a non-segmented genome of single-stranded negative sense RNA of around 8900 nucleotides in length, belonging to the order Mononegavirales. Bornaviruses replicate in the nucleus of the nerve cells of various organs and establish persistent, non-cytolytic infections by exploiting the cellular splicing mechanisms to efficiently use its genome, organized into six open reading frames (ORFs) (Figure 1). Alternative transcription start and stop sites and splicing produce mRNAs that are translated to produce the viral-encoded proteins (Figure 1). The first transcription unit contains an ORF for the nucleoprotein (N), the second transcription unit contains two overlapping ORFs for the phosphoprotein (P), and the X protein (X) (Figure 1). The third transcription unit is spliced differently, and also has different transcription initiation and termination signals, enabling polymerase read-through during transcription, which results in expression of the matrix protein (M), the glycoprotein (G), and the RNA-dependent RNA-polymerase (L) (Figure 1). The P and X ORFs overlap; as well as, the M and G ORFs (Figure 1). The immunogenicity of phosphoprotein, as well as, the degree of conservation of the phosphoprotein and its gene, within and between bornavirus species, make them good candidates as universal targets in laboratory diagnosis. Once the family Bornaviridae is expanding speedily, producing knowledge about highly conserved regions within phosphoprotein will be useful for the development of sensitive laboratory diagnostic tools. So far, genus Carbovirus, Cultervirus and Orthobornavirus have been identified, which comprise 11 species. From those, 10 species are associated with the development of severe neurological and/or gastrointestinal disease and death of its hosts [5–15] (Figure 2). The disease has been reported in humans, several species of pets, production and wild animals [5–15]. Namely, two species infect mammals (Mammalian 1 to 2 orthobornavirus), five infect birds (Passeriform 1 to 2 orthobornavirus, Psittaciform 1 to 2 orthobornavirus and Waterbird 1 orthobornavirus) and three infect reptiles (Queensland carbovirus, Southwest carbovirus and Elapid 1 orthobornavirus) (Figure 2). However, some Mammalian 1 orthobornavirus showed the ability to also infect farmed ostriches and wild birds (mallards and jackdaws). Wild birds are hosts of avian bornaviruses (e.g. strains of Waterbird 1 orthobornavirus and Psittaciform 1 orthobornavirus) [13–15,17] and mammalian bornaviruses (e.g. genotypes of Mammalian 1 orthobornavirus). Therefore, co-infection may play a role in the emergence of new pathogenic and zoonotic bornavirus species; once X and P proteins of PaBV-4 look like to have different ancestors.

In Psittaciformes, parrot bornavirus 1 to 8 (PaBV-1 to 8) can cause proventricular dilatation disease (PDD), characterized by a flaccid and distended proventriculus impacted with feed, as a result of the inability of seeds’ digestion (however throughout the gastrointestinal tract can occur variable distention). Psittaciformes can also show uncoordinated movements, postural disorders, apathy, blindness, and behavioural disorders, such as loss of appetite and self-mutilation (resulting from lesions in the central nervous system). Within captive birds, the virus has become relevant for Psittaciformes housed in reserves, in breeding projects of rare species, in private collections and zoos, because of the severe effects it may cause for bird welfare, economy, and biodiversity levels. Analyses of molecular epidemiology suggested that a world trade of psittacines without biosafety measures has been carried out. There is, to the best of our knowledge, no publications identifying and characterizing the avian bornaviruses infecting pet parrots in Portugal. Moreover, there are still unresolved questions on the epidemiology of bornaviruses, such as the role of waterbirds in the emergence and dissemination of new pathogenic and zoonotic species, and the localization of highly conserved regions within P gene inter- and intra-species of bornaviruses.

The aim of this study was to identify and phylogenetically characterize the etiologic agent associated with clinical signs and necropsy findings consistent with avian bornavirus infection in two pet parrots in Portugal, as well as to produce molecular epidemiologic knowledge on bornaviruses.