Introduction
Haemorrhagic nephritis enteritis (HNEG) is a major disease of geese in Europe (Guerin, Reference GUERIN2008). HNEG was first described in 1969 in Hungary in association with the passive immunisation of goslings with convalescent goose serum used to treat Derzsy's disease. HNEG was described a few years later in Germany (Schettler, Reference SCHETTLER1977), France (Schettler, Reference SCHETTLER1977) and Hungary (Palya et al., Reference PALYA, IVANICS, GLÁVITS, DÁN, MATÓ and ZARKA2004). In a recent study in Poland, most goose flocks were negative for HNEG, as identified by end-point PCR. Only three flocks were HNEG positive out of 122 sampled (2.46%) (Kozdruń et al., Reference KOZDRUŃ, WOŹNIAKOWSKI, SAMOREK-SALAMONOWICZ and CZEKAJ2012).
HNEG is commonly known as ‘young geese disease’ or ‘late form Derzy's disease’ due to confusion with infections with parvovirus, however, based on its aetiology a more accurate name could be ‘goose polyomavirosis’. Although this disease has been known for more than 30-years, isolation and identification of its causative agent were not carried out until 2000 by Guerin et al. (Reference GUERIN, GELFI, DUBOIS, VUILLAUME, BOUCRAUT-BARALON and PINGRET2000). Further analyses demonstrated that this virus was closely related to other polyomarivuses and identified as the novel virus goose haemorrhagic polyomavirus (GHPV) (Guerin et al., Reference GUERIN, GELFI, DUBOIS, VUILLAUME, BOUCRAUT-BARALON and PINGRET2000).
Polyomaviruses are widely known as tumour-inducing agents, however, this property is observed only after experimental infection of newborn laboratory rodents with mammalian polyomaviruses (Johne and Müller, Reference JOHNE and MÜLLER2007). In contrast to the well-documented ability of mammalian polyomaviruses to induce neoplastic transformation of cultured cells and neoplasias in vivo, none of the avian polyomaviruses display these abilities (Johne et al., Reference JOHNE, BUCK, ALLANDER, ATWOOD, GARCEA, IMPERIALE, MAJOR, RAMQVIST and NORKIN2011).
The Polyomaviridae Study Group of the International Committee on Taxonomy of Viruses (ICTV) has recommended several taxonomical revisions as follows: the family Polyomaviridae which currently represents the single genus Polyomavirus is comprised of three genera: two containing mammalian viruses and one containing avian viruses. The two mammalian genera were designated Orthopolyomavirus and Wukipolyomavirus, and the avian genus consisting of four species was designated Avipolyomavirus (Johne et al., Reference JOHNE, BUCK, ALLANDER, ATWOOD, GARCEA, IMPERIALE, MAJOR, RAMQVIST and NORKIN2011). Well-known polyomaviruses isolated from birds include Avian Polyomaviruses (APV) or Goose Haemorrhagic Polyomaviruses (GHPV), which are highly pathogenic and cause acute and chronic inflammatory diseases, especially in young birds (Johne et al., Reference JOHNE, BUCK, ALLANDER, ATWOOD, GARCEA, IMPERIALE, MAJOR, RAMQVIST and NORKIN2011). Two other bird polyomaviruses, the finch polyomavirus (FPyV) and the crow polyomavirus (CPyV) were detected in 2006 by a screening approach designed to identify novel polyomaviruses using broad spectrum PCR (Johne and Müller, Reference JOHNE and MÜLLER2007; Wittig et al., Reference WITTIG, HOFFMANN, MÜLLER and JOHNE2007).
The GHPV genome is circular and comprised of double-stranded DNA (5256 bp). Genome organisation of all polyomaviruses share common features in the form of a set of early genes encoding polymerases (t and T antigens) and late genes that encode structural proteins such as VP1 (the main capsid protein) VP2 and VP3. However, avian polyomaviruses possess an additional capsid protein VP4 with yet to be defined functions (Johne and Müller, Reference JOHNE and MÜLLER2003).
Replication of GHPV occurs in the nucleus based on observations that infected cells contained significant amounts of viral material in the nucleus regardless of whether the cells were cultured or harvested from infected goslings. GHPV viruses are easily detected in the nuclei by immunofluorescence using serum isolated from convalescent geese. Release of virions is suggestive of cell membrane disruption. GHPV is highly resistant to heating and freezing, that is, the virus is still virulent after 2 h incubation at 55°C or following freeze-thaw cycles, in addition to being resistant to lipid solvents (1% phenol). By contrast, other avian polyomaviruses such as FPyV are sensitive to chloride-derived products, and so can be controlled by sanitising agents (Kisary, Reference KISARY1993; Ritchie et al., Reference RITCHIE, PRITCHARD, PEST, NIAGRO, LATIMER and LUKERT1993).
Sources and transmission routes
Growing goslings are particularly susceptible to GHPV infections, although infections in wild geese and ducklings have been reported. Until recently, it was thought that GHPV infectious were the result of horizontal transmission since infected birds excrete large amounts of virus, resulting in spread through the environment. However, vertical transmission is thought to occur based on the high mortality rates of four day old goslings (Palya et al., Reference PALYA, IVANICS, GLÁVITS, DÁN, MATÓ and ZARKA2004) and data from earlier studies demonstrating that the shortest incubating period was six days after subcutaneous infection of one day old goslings (Bernath and Szalai, Reference BERNATH and SZALAI1970).
Recently Farsang et al. (Reference FARSANG, BERNATH and DOBOS-KOVACS2011) reported a case of goose haemorrhagic polyomavirus in four day old goslings, providing further support for vertical transmission of GHPV. Vertical transmission of GHPV affects goslings when they are most vulnerable; therefore preventing vertical transmission of GHPV is critical to controlling the spread of infection and understanding its epizootiology. Farsang et al. (Reference FARSANG, BERNATH and DOBOS-KOVACS2011) speculated that introduction of an infectious agent into a ‘clean’ farm usually occurred via vertical transmission or as a consequence of persistently infected adult carriers with the majority of infections resulting in death due to horizontal transmission. Corrand et al. (Reference CORRAND, GELFI, ALBARIC, ETIEVANT and PINGRET2011) recently demonstrated that ducks are asymptomatic carriers of GHPV but show no pathological evidence of infection and therefore should be considered potential reservoirs. For this reason, mixed goose and duck farming should be carefully reconsidered.
Epizootiology and clinical observations
The majority of the outbreaks in Hungary have occurred in three to six week old birds, but some outbreaks were observed in much younger goslings (four days old) or in older flocks (16-20 weeks old) with mortality rates ranging between 4-67% (Palya et al., Reference PALYA, IVANICS, GLÁVITS, DÁN, MATÓ and ZARKA2004). Other studies indicated that infections were characterised by sudden death in goslings at two to 10 weeks of age, resulting in cumulative mortality rates ranging between 4-64% (Miksch et al., Reference MIKSCH, GROSSMANN, KÖHLER and JOHNE2002) and sometimes up to 80% (Guerin, Reference GUERIN2008). Clinical signs present only a few hours before death and are preceded by behaviours that include birds sitting alone followed by coma and death. During acute disease presentation the most common symptoms were oedema of subcutaneous tissues, subcutaneous haemorrhages, ataxia, tremors of the head and neck, and blood-stained faeces.
Chronic disease progression results in urate deposits on the viscera and joints resulting in lameness. Mortality rates are less in animals presenting with these late manifestations of disease until birds reach 12 weeks of age. However, mortality in several flocks was associated with chronic disease that lasted for up to one to two months or was interrupted i.e., two separate mortality peaks were observed (Palya et al., Reference PALYA, IVANICS, GLÁVITS, DÁN, MATÓ and ZARKA2004; Guerin, Reference GUERIN2008). In France, Lacroux et al. (Reference LACROUX, ANDREOLETTI, PAYRE, PINGRET, DISSAIS and GUERIN2004) reported 30-80% morbidity among geese in a field study, and in animals aged four to 10 weeks old, mortality was 100%. In these animals death was preceded by coma and, in a few cases, nervous system-related symptoms.
The anatomopathological changes observed in dead geese include systemic oedema, mostly in the subcutaneous areolar tissue of the neck and inner thigh. Oedema was observed in the abdominal cavity, pericardium, lungs, trachea, septa of the Bursa of Fabricius, and in the mesentery. Petechiea were seen in the kidneys and occasionally in the Bursa of Fabricius. A few suffuse haemorrhages were observed primarily in the lumen or as blood-stained faeces. Apart from the lesions mentioned above, acute degeneration of the kidneys and sometimes dystrophy of the liver were reported. Pathologic changes to the spleen were not observed. Lesions presented in various combinations and with differing degrees of severity between geese, and some pathological changes were absent in some cases (Lacroux et al., Reference LACROUX, ANDREOLETTI, PAYRE, PINGRET, DISSAIS and GUERIN2004; Dobos-Kovács et al., Reference DOBOS-KOVÁCS, HORVÁTH, FARSANG, NAGY, KOVÁCS, SZALAI and BERNÁTH2005).
Histopathologically, the most common findings were zonal necrosis of proximal tubular epithelial cells with small haemorrhages in acute cases, or the presence of urate crystal deposits and intertubular fibroblast proliferation in sub-acute cases. Other suggestive histological lesions were serous hepatitis with fatty infiltration of hepatocytes, haemorrhagic enteritis (frequently with necrosis in the crypt epithelium), and haemorrhages in various organs. In the lymphoid organs (Bursa of Fabricius, thymus, spleen) lymphocyte depletion was generally observed (Palya, 2004).
During histological examinations of experimentally infected goslings, Lacroux et al. (Reference LACROUX, ANDREOLETTI, PAYRE, PINGRET, DISSAIS and GUERIN2004) observed that the majority of lesions developing eight days after infection were tubular epithelial necrosis in kidneys and moderate lymphocytic depletion of the cloacal bursa and spleen. No lymphocyte depletion was observed in the thymus. Despite systemic arteriolitis, only a few haemorrhages and thrombotic lesions were observed during the clinical stage of experimentally infected animals. Vascular lesions consisted of a multifocal, moderate endothelial cell nuclear enlargement sometimes associated with light lymphocytic infiltration of the vessel wall. The central nervous system was affected with mild to moderate vascular lesions, but no major degenerative lesions were observed.
Disease pathogenesis
Disease pathology was consistent with viral replication in blood vessel endothelial cells including arteries and veins throughout the body, resulting in endothelial cell necrosis associated with significantly increased blood vessel permeability. This manifestation likely explains the oedema and diapedetic haemorrhages observed throughout the body, especially in the kidney and the brain. It is possible that the degenerative changes observed in the epithelial cells of the renal tubules were not due to primary effects of the virus, but occurred as a secondary consequence of blood circulation disorders caused by damage and dysfunction of capillary endothelial cells (Dobos-Kovécs et al., 2005).
Based on these observations it is likely that the oedema and blood haemorrhages presenting in the subcutaneous tissues and kidneys of infected geese resulted from the destruction of blood vessel endothelia cells via undefined mechanisms, although induction of apoptosis may contribute to pathogenicity of the virus (Johne and Müller, Reference JOHNE and MÜLLER2007). Induction of apoptosis may facilitate the efficient release of virus progeny from infected cells in the absence of a primary inflammatory response leading to efficient spread of the virus systemically.
Diagnosis
GHPV infections of geese are confirmed during autopsy when characteristic lesions are observed and confirmed by histopathological examinations. However, PCR amplification of polyomavirus DNA (primarily of the gene encoding VP1) is the only practical and rapid method of confirming diagnosis. GHPV DNA can be amplified from various tissues including kidney, liver, spleen, lungs, Bursa of Fabricius, and intestinal contents (Palya, 2004). Furthermore, PCR-based tests may prove valuable to the success of epizootiological investigations as well as in demonstrating subclinical shedders and/or carriers. The virus can be isolated by means of kidney cell culture or by inoculating goose embryos with material harvested from infected birds (Bernath and Szalai, Reference BERNATH and SZALAI1970), but these methods are time-consuming and seldom applied to the routine diagnosis of HNEG. Serologic based tests do not appear to be an option for the diagnosis of polyomavirus infections since serologic responses are greatly variable due to the ability of the virus to persist in infected birds for months or even years (Guerin et al., 2004).
Prevention and treatment
Considering factors such as vertical and horizontal transmission routes, reservoir duck populations (serving as asymptomatic carriers), and excretion of the virus by clinically infected geese (and other carriers), it is necessary to carry out non-specific prophylaxis (bio-security) and specific prophylaxis. Non-specific prophylaxis should include raising birds according to the rule ‘all in – all out’, maintaining consistent disinfection practices after each rearing cycle and avoiding the simultaneous rearing of ducks and geese. However, management procedures are unlikely to be sufficient for controlling GHPV infections. Vaccination of breeders will confer maternal immunity to goslings at an age when they are highly susceptible to infection (Guerin et al., Reference GUÉRIN, GELFI, LEON, CLAVERYS and PAPPALARDO2004).
Taking the above into consideration, Gelfi et al. (Reference GELFI, PAPPALARDO, CLAVERYS, PERALTA and GUERIN2010) developed an inactivated vaccine comprised of viral particles produced in goose kidney cells. A carbopol-adjuvanted vaccine induced a high serological response. Moreover, goslings hatched from vaccinated breeders were protected against viral challenge, indicating that maternally-derived neutralising antibodies (MDA) were efficiently transferred. MDA were still detectable 15 days post-hatching. Mato et al. (Reference MATO, PENZES, RUEDA, VELA, KARDI, ZOLNAI, MISAK and PALYA2009) showed that immunisation with a recombinant subunit vaccine containing the GHPV VP1 capsid protein (the most important antigenic epitope) provided excellent protection against challenge with a field virus. A single vaccine dose induced 95% protection and 100% protection was achieved following booster vaccination. To date, no effective treatments for HNEG infections exist; however, immunoprophylaxis appears to be effective.
Conclusions
HNEG is a fatal disease of young geese caused by infections with GHPV that has been reported in all major goose-producing countries in Europe. Horizontal and vertical transmission of GHPV has been confirmed. Disease is diagnosed at necropsy following the identification of lesions consistent with oedema of subcutaneous tissues, gelatinous ascites, inflammation of the kidneys and often, haemorrhagic enteritis. PCR can be used to amplify polyomavirus DNA and represents the only practical and rapid diagnostic method. To date, no treatment for HNEG exists but immunisation of breeders with an inactivated vaccine formulation protected offspring against viral challenge. Day-old goslings immunised with a subunit vaccine were also protected against challenge with virulent GHPV strains.
