Visual detection of goose haemorrhagic polyomavirus in geese and ducks by loop-mediated isothermal amplification

Goose haemorrhagic polyomavirus (GHPV) is an aetiological agent of haemorrhagic nephritis and enteritis of geese occurring in geese (Anser anser). GHPV may also infect Muscovy ducks (Carina mochata) and mule ducks. Early detection of GHPV is important to isolate the infected birds from the rest of the flock thus limiting infection transmission. The current diagnosis of haemorrhagic nephritis and enteritis of geese is based on virus isolation, histopathological examination, haemagglutination inhibition assay, ELISA and polymerase chain reaction (PCR). Recently, real-time PCR assay was developed which considerably improved detection of GHPV. In spite of many advantages, these methods are still time-consuming and inaccessible for laboratories with limited access to ELISA plate readers or PCR thermocyclers. The aim of our study was to develop loop-mediated isothermal amplification (LAMP) that may be conducted in a water bath. Two pairs of specific primers complementary to VP1 gene of GHPV were designed. The results of GHPV LAMP were recorded under ultraviolet light. Our study showed LAMP was able to specifically amplify VP1 fragment of a GHPV without cross-reactivity with other pathogens of geese and ducks. LAMP detected as little as 1.5 pg of DNA extracted from a GHPV standard strain (150 pg/µl). The optimized LAMP was used to examine 18 field specimens collected from dead and clinically diseased geese and ducks aged from 1 to 12 weeks. The positive signal for GHPV was detected in three out of 18 (16.6%) specimens. These results were reproducible and consistent with those of four real-time PCR. To the best of our knowledge this is the first report on LAMP application for the GHPV detection.     

Pathology of goose haemorrhagic polyomavirus infection in goose embryos.

Goose embryos were infected with goose haemorrhagic polyomavirus (GHPV) onto the chorioallantoic membrane (CAM) in order to examine the effect of GHPV on the embryos and to obtain data on whether embryos could develop into infected, virus-shedding goslings, as well as to present an accurate biological method for virus titration. The reported method of infection could offer a possibility to express the virus titre as the median embryo infective dose (EID(50)). As a special pathological feature of the disease, extensive cerebral haemorrhages were observed, which protruded the skullcap in many cases. Some embryos infected with 10(1.25) or 10(0.25) EID(50)/0.2 ml were able to hatch; however, they were in poor physical condition and died by post-hatching day 4 showing haemorrhagic nephritis and enteritis of geese. Virus shedding was revealed by polymerase chain reaction. The ability of some of the infected goose embryos to hatch may indicate the potency of GHPV to spread vertically, although this needs further study for confirmation.     

Pathology of spontaneous and experimental infections by Goose haemorrhagic polyomavirus.

Haemorrhagic nephritis enteritis of geese (HNEG) is a fatal disease of geese aged from 3 to 12 weeks. The causative virus, Goose haemorrhagic polyomavirus (GHPV), is a member of the Polyomaviridae family We examined goslings either spontaneously or experimentally infected with GHPV. Tissues were sampled for histology, GHPV DNA detection and electron microscopy. Clinical signs and gross lesions observed in experimentally infected goslings were largely consistent with those noticed in field cases. Histological examination showed that, in the acute phase of HNEG, GHPV replicates in almost all the tissues with a particular tropism for endothelial and lymphoid cells. Haemorrhagic foci were widespread in many tissues, including brain. Ultrastructural features were largely consistent with other polyomavirus infections, with accumulation of virions in the nucleus. Non-typical, double-membraned organelles were observed in the cytoplasm. GHPV DNA distribution was widespread in tissues of infected birds, from day 5 post-infection. GHPV therefore induces a systemic disease in its host, leading to severe vascular dysfunction and immunosuppressive B-cell depletion.     

Observations on host range and control of goose virus hepatitis

A number of experiments were done with a strain of goose hepatitis virus (isolated from sick goslings in the Netherlands in 1969. This virus was easily grown in embryonating eggs of the Muscovy duck (Cairina moschata) as well as in embryonating goose eggs. It also proved possible to adapt it to White Pekin duck eggs. The susceptibility of embryonating eggs obtained by crossing Muscovy drakes with Pekin ducks was intermediate between that of eggs from the two parental breeds. It was not possible to adapt goose hepatitis virus to growth in chicken embryos. The sensitivity of goslings to the goose hepatitis virus was found to vary considerably according to the farms from which they came. These differences were caused by variations 'in the quantity of parentally derived antibodies. One day-old Muscovy ducklings appeared to be at least as sensitive to the virus as goslings. However, disease symptoms could not be produced with goose hepatitis virus in Pekin ducklings nor in ducklings obtained by crossing Muscovy drakes and Pekin ducks. Treatment with homologous immune serum protected susceptible goslings and Muscovy ducklings against the disease. Under farm conditions, mortality was reduced from 30 to 3%. A breeding flock of adult Muscovy ducks, which had by serum therapy survived a goose hepatitis infection at an early age, produced ducklings resistant to the goose hepatitis virus. Over 100 flocks of geese, totalling more than 6,000 birds, were actively immunized with goose hepatitis Virus. The progeny produced in the following breeding season resisted, almost without exception, a challenge at one day of age with virulent goose hepatitis virus.     

Safety and efficacy of an inactivated Carbopol-adjuvanted goose haemorrhagic polyomavirus vaccine for domestic geese

Haemorrhagic nephritis enteritis of the goose (HNEG) is an epizootic viral disease in domestic geese. The causal agent is a polyomavirus, namely goose haemorrhagic polyomavirus. To help control the disease, an inactivated vaccine was developed, based on viral particles produced in goose kidney cells. Viral material was quantified using real-time quantitative polymerase chain reaction, inactivated with β-propiolactone and adjuvanted with Carbopol, an acrylic acid polymer. Carbopol proved to be more immunogenic than aluminium hydroxide and was totally safe when administered to young goslings and breeders alike. Carbopol-adjuvanted vaccine induced a high serological response. Moreover, goslings hatched from vaccinated breeders were protected against viral challenge, indicating that maternally-derived neutralizing antibodies (MDA) were efficiently transferred. MDA were still detectable 15 days post-hatch. Clinical trials will be necessary to accurately evaluate a vaccine-based HNEG control strategy under field conditions.     

The role of egg drop syndrome virus in acute respiratory disease of goslings

An outbreak of severe acute respiratory disease characterized by tracheitis and bronchitis was observed in young goslings on a large-scale goose farm in Hungary. Histological examination revealed amphophilic intranuclear inclusion bodies in the superficial epithelial cells of the trachea and bronchi. Adenovirus-like particles were detected by electron microscopy, and the virus isolated from the trachea and the lungs was identified as egg drop syndrome (EDS) virus by serological and genomic examination. The clinical and pathological signs were reproduced by intratracheal administration of the virus isolate to 1-day-old goslings free of EDS antibodies. The presence of EDS virus DNA in different organs of the naturally and experimentally infected goslings was detected by polymerase chain reaction. This is the first report on the involvement of EDS virus in severe respiratory disease of geese.     

Experimental infection of young and laying geese with egg drop syndrome 1976 adenovirus strain B8/78

Of 19 large flocks of geese in Hungary 15 were serologically positive to the egg drop syndrome 1976 (EDS-76) avian adenovirus strain B8/78. Sixty-five to 100% of the samples taken from each flock were positive with haemagglutination inhibition (HI) titres between 1:8 and 1:256. All the progeny of geese with HI antibodies had maternally derived antibodies. After infection per os of 8-, 19- and 29-day-old goslings with the B8/78 strain of EDS-76 adenovirus clinical signs of disease and mortality were not seen although the birds developed antibodies and shed the virus in the faeces. No lesions were noted using light microscopy. Egg production was unaffected in geese experimentally infected with B8/78 virus strain and there was no change in egg quality. The layers developed antibodies to EDS-76 adenovirus and shed the virus in the faeces. Contact-controls responded in the same way. The results of these experiments suggest that when investigating the origin of EDS-76 avian adenovirus infection not only ducks but also geese should be considered as a potential source of infection.     

Epizootic occurrence of haemorrhagic nephritis enteritis virus infection of geese.

Recent outbreaks of haemorrhagic nephritis enteritis in geese flocks of 3 to 10 weeks in age in Hungary were investigated. Mortality varied between 4% and 67%. Affected birds generally died suddenly. Occasional clinical signs included tremors of the head and neck, subcutaneous haemorrhages and excretion of faeces containing partly digested blood. At necropsy the most frequent findings were a turgid wall and reddish mucosa of the intestines and reddish discolouration of the swollen kidneys, but oedema and haemorrhages of the subcutaneous connective tissue, hydropericardium and ascites were also seen. In subacute cases, visceral gout was frequently observed. Histological examination revealed zonal necrosis of the tubular epithelial cells with haemorrhages in the kidney. Other histological findings were serous hepatitis with fatty infiltration, necrotizing haemorrhagic enteritis and haemorrhages in the different organs including the brain. Experimental geese infected parenterally with crude liver and spleen homogenates prepared from diseased birds died after 8 to 20 days without premonitory signs, and had typical gross and histological lesions. Attempts to isolate cytopathic virus on different tissue cultures failed. The presence of polyomavirus was proven by polymerase chain reaction. Five isolates were further investigated by analysing their complete VP1 gene sequence. All tested strains were very closely related to each other on the basis of the nucleotide sequence, and they were identical at the deduced amino acid level.     

Characterization of a herpesvirus isolated from domestic geese in Australia

A herpesvirus (GHV 552/89) associated with high mortality in a flock of domestic geese in Australia was compared with duck virus enteritis (DVE) herpesvirus by cross-protection studies in domestic geese, Muscovy ducks and commercial Pekin ducks. In DVE-vaccinated geese, Muscovy ducks and Pekin ducks, mortality levels of 100, 50 and 0%, respectively, were recorded following challenge with GHV 552/89. Conversely, in geese, Muscovy ducks and Pekin ducks immunized with inactivated GHV 552/89, 100% mortality was observed in the geese and Muscovy ducks, and 80% in the Pekin ducks following challenge with DVE virus. The isolate was also compared with six other avian herpesviruses using cross-neutralization tests in cell cultures. No detectable cross-neutralization occurred with any of the avian herpesviruses tested. Further characterization of GHV 552/89 was undertaken by comparing its genome with strains of DVE herpesvirus using restriction endonuclease analysis of the viral DNA and a polymerase chain reaction (PCR) test. Following digestion with HindIII, the DNA fragment pattern of GHV 552/89 was found to be completely different from the DVE viruses. Similarities were found between the digestion patterns of a UK and a US DVE isolate, but both were distinguishable from a UK vaccine strain. The results of the PCR analysis and comparison using two DVE-specific primer sets did not produce specific amplification products of expected molecular weights (603 and 446 base pairs) from the GHV 552/89 genome. The PCR products derived from the DVE strains were similar to those derived from the DVE control DNA. From the results of this study, it is concluded that the goose herpesvirus GHV 552/89 is antigenically and genomically distinct from DVE herpesvirus.     

Mycoplasma infection of geese. II. Studies on pathogenicity of mycoplasmas in goslings and goose and chicken embryos

Of various mycoplasma strains of goose original, axanthum strains (609 and 612) caused the death both of goose and chicken embryos, A. laid-lawii strain (606) killed only goose embryos, whereas M. gallinarum (598) failed to kill either. Infection of 3-day-old goslings with these mycoplasmas resulted in no mortality but lesions were produced with A. axanthum in 9 of 10 birds. Less severe lesions were seen in fewer birds infected with other strains. Dual infection of 3-day-old goslings, with maternal antibody to goose parvovirus, with M. gallinarum (598) or A. axanthum (612) and a virulent parvovirus resulted in some death and all birds showed lesions.     

The genome of goose hemorrhagic polyomavirus,a new member of the proposed subgenus Avipolyomavirus

The full-length genome of goose hemorrhagic polyomavirus (GHPV), the ethiologic agent of hemorrhagic nephritis and enteritis of geese, was cloned and sequenced. Transfection of the circular ds DNA with a size of 5256 bp and an organisation typical of polyomaviruses produced viral progeny in cultured goose cells. According to the splicing sites determined by RT-PCR, five open reading frames (ORFs) were found to encode putative proteins with significant similarities to large T antigen and small t antigen as well as VP1, VP2, and VP3 of other polyomaviruses. An additional ORF located in the 5' region of late mRNA, with a coding capacity for 169 amino acids, shows a low degree of homology to VP4 of avian polyomavirus (APV). The alignment of nucleotide sequences and amino acid sequences revealed a relatively close relationship between GHPV and APV. Therefore, grouping of this new polyomavirus into the proposed subgenus Avipolyomavirus is suggested.     

Geese not susceptible to virulent subgroup J avian leukosis virus isolated from chickens

ABSTRACT

To determine whether geese are susceptible to infection by avian leukosis virus (ALV), 702 serum samples from domestic and foreign goose breeds were screened for p27 antigen as well as being inoculated into DF-1 cell cultures to isolate ALV. Although 5.7% of samples were positive for p27 antigen, reactivity appeared to be non-specific because no ALV was detected in the corresponding DF-1 cultures. To further determine whether geese are susceptible to ALV-J isolated from chickens, ALV-J strain JS09GY7 was artificially inoculated into 10-day-old goose embryos, with one-day-old hatched goslings then screened for p27 antigen and the presence of ALV. In all cases, the results of both tests were negative. Liver tissues from the 1-day-old goslings were screened using a polymerase chain reaction-based assay, which failed to amplify ALV-J gene fragments from any of the samples. Further, no histopathological damage was observed in the liver tissues. ALV-J was further inoculated intraperitoneally into one-day-old goslings, with cloacal swabs samples and plasma samples then collected every 5 days for 30 days. All samples were again negative for the presence of p27 antigen and ALV, and liver tissues from the challenged geese showed no histopathological damage and were negative for the presence of ALV-J gene fragments. Furthermore, p27 antigen detection, PCR-based screening, and indirect immunofluorescence assays were all negative following the infection of goose embryo fibroblasts with ALV-J. Together, these results confirm that virulent chicken-derived ALV-J strains cannot infect geese, and that p27 antigen detection in goose serum is susceptible to non-specific interference.     

Reovirus identified as cause of disease in young geese

The pathology, epizootiology and aetiology of a specific disease of young geese, which has been seen in Hungary for more than three decades, were investigated. The disease was characterised by splenitis and hepatitis with miliary necrotic foci during the acute phase, and epicarditis, arthritis and tenosynovitis during the subacute/chronic phase. Clinical signs usually appeared at 2 to 3 weeks of age and persisted for 3 to 6 weeks. From different organs of the affected birds, a reovirus was isolated in embryonated eggs and tissue cultures ofMuscovy duck or goose origin, as well as in Vero cells. In experimental infections, the dominant features of thedisease were reproduced in day-old and young goslings. The biological and partial molecular characterisation ofone of the isolated strains (D15/99) showed that it was related to the reovirus described as the cause of a similardisease of Muscovy ducks. An RT-PCR method suitable for the detection of reoviruses was also elaborated andtested. This is the first report on the involvement of reovirus in arthritis of geese.     

Astroviruses associated with stunting and pre-hatching mortality in duck and goose embryos

The first detection of avian nephritis virus (ANV) in goose embryos and of turkey astrovirus-1 (TAstV-1) in duck embryos is described. Intestinal samples from duck and goose embryos from five duck and four goose flocks in Croatia were tested by polymerase chain reaction for the presence of ANV, TAstV-1, turkey astrovirus-2, chicken astrovirus, duck astrovirus and also for the presence of avian reovirus, Derzsy's disease virus and duck enteritis virus. The kidneys from duck and goose embryos were also tested for ANV, while liver samples were tested for duck astrovirus. Duck embryos were also tested to detect duck circovirus and goose embryos for the presence of goose circovirus and goose haemorrhagic polyomavirus. All embryos were in the final stage of incubation and were characterized by moderate to markedly retarded growth. ANV was confirmed in the intestines and kidneys of embryos from two duck and two goose flocks and TAstV-1 was found in embryos from two duck flocks. One duck flock was positive for both ANV and TAstV-1. No other viruses were found in tested flocks. Phylogenetic analysis based on the ANV polymerase gene fragment of ANV sequences detected in duck and goose embryos revealed greatest similarity (88.1 to 97.2%) with ANV isolates from chickens. Further, the existence of at least two types of ANV circulating in Croatian duck and goose flocks was confirmed. Based on the phylogenetic analysis of the portion of TAstV-1 polymerase gene, two detected TAstV-1 nucleotide sequences were 99.5% similar. Compared with six TAstV-1 sequences, Croatian sequences showed one unique nucleotide change. In addition to other possible causes of stunted growth and late embryonic death, these findings suggest that ANV and/or TAstV-1 infection may be a contributing factor in the pre-hatching mortality of ducklings and goslings.     

Infectivity of Histomonas meleagridis in ducks.

The susceptibility of mule and muscovy ducks to "blackhead" disease caused by Histomonas meleagridis was studied, using an experimental intracloacal inoculation. Turkeys were used as controls. Morbidity, mortality and body weight gain were recorded regularly during the experiments. A direct examination of the caecal content was made to determine the absence or presence of the parasite. Gross and microscopic lesions were observed on days 7, 14, 21, 28 and 35 post infection to evaluate any clinical histomoniosis in ducks and to appraise the histomonad's carriage. A scoring system was developed both for gross and histological lesions of the caecum and liver. Infected mule and muscovy ducks (n = 83) never developed any clinical signs of histomoniasis. Weight gains of infected mule and muscovy ducks were similar to those of uninfected ducks. In 67% of the ducks (56/83), it was possible to demonstrate the parasite in the caecal content throughout the experiment. Typical macroscopic caecal lesions were observed in five of the ducks between days 7 and 21 post infection, with a caecal necropsy main lesion score (MLS = 1.6) less severe than that in turkeys (MLS = 2.9). Only caecal histological lesions occurred in six of the cases. Therefore, ducks do not seem to be a susceptible host for "blackhead" but may act as carrier animals for H. meleagridis. The virulence was apparently not changed, since 67% of turkeys (10/15) infected with the caecal content of positive ducks displayed classical signs of blackhead disease. Even if H. meleagridis alone does not represent a substantial danger in the duck production, its infectivity should to be taken into account in the transmission to more susceptible species.     

Pathogenicity of a variant goose parvovirus,from short beak and dwarfism syndrome of Pekin ducks,in goose embryos and goslings

The pathogenicity of a variant goose parvovirus (GPV), isolated from short beak and dwarfism syndrome of Pekin ducks (strain Cherry Valley), was investigated in embryonating goose eggs and goslings. The virus was easily grown in GPV antibody-free goose embryos and caused high mortality and severe lesions of goose embryos, indicating that the variant GPV has good adaptation and high pathogenicity to embryonated goose eggs similar to the classical GPV. Like the third egg-passage virus (strain H) of a classical GPV, the third egg-passage virus (strain JS1) of the variant GPV caused Derzsy’s disease in 2-day-old goslings with high mortality. The findings suggest that the variant GPV strain, which had specifically adapted to Pekin ducks, still retained high pathogenicity for its original host. The mortality (73.3–80%) caused by the first and third egg-passages of the variant GPV was somewhat lower than that (93.3%) caused by the third passage virus of the classical GPV, reflecting the higher pathogenicity of the classical GPV for its original host. These findings are likely to reinforce the importance of surveillance for parvoviruses in different waterfowl species and stimulate further study to elucidate the impact of mutations in the GPV genome on its pathogenicity to goslings and ducks.     

Generation of virus-like particles consisting of the major capsid protein VP1 of goose hemorrhagic polyomavirus and their application in serological tests

Goose hemorrhagic polyomavirus (GHPV) is the causative agent of hemorrhagic nephritis and enteritis of geese (HNEG), a fatal disease of young geese with high mortality rates. GHPV cannot be efficiently propagated in tissue culture. To provide antigens for diagnostic tests and vaccines, its major structural protein VP1 was recombinantly expressed in Sf9 insect cells and in the yeast Saccharomyces cerevisiae. As demonstrated by density gradient centrifugation and electron microscopy, GHPV-VP1 expressed in insect cells formed virus-like particles (VLPs) with a diameter of 45 nm indistinguishable from infectious polyomavirus particles. However, efficiency of VLP formation was low as compared to the monkey polyomavirus SV-40-VP1. In yeast cells, GHPV-VP1 alone formed smaller VLPs, 20 nm in diameter. Remarkably, co-expression of GHPV-VP2 resulted in VLPs with a diameter of 45 nm. All three types of GHPV-VLPs were shown to hemagglutinate chicken erythrocytes. ELISA and hemagglutination inhibition tests using the VLPs as antigen detected GHPV-specific antibodies in up to 85.7% of sera derived from flocks with HNEG but in none of the sera of a clinically healthy flock. However, GHPV-specific antibodies were also detected in sera from two other flocks without HNEG indicating a broad distribution of GHPV due to subclinical or unrecognised infections.     

Isolation and molecular characterization of a new Muscovy duck parvovirus from Muscovy ducks in the USA.

Between 1997 and 1999 several cases of a new disease in Muscovy ducks were reported in Pennsylvania, USA. The cases were characterized by locomotor dysfunction, weakness, recumbency, 40 to 60% morbidity and 10 to 40% mortality. The most characteristic microscopic lesions were moderate to severe degenerative rhabodomyopathy. In order to characterize the aetiological agent, virus isolation was attempted from the spleen, liver, heart, skeletal muscle and intestine by inoculation of 14-day-old Muscovy duck embryos with tissue homogenates. Deaths occurred on the second egg passage and parvoviruses were isolated by serial passage of allantoic fluid from dead embryos and then in Muscovy duck embryo fibroblast (MDEF) cultures. Parvovirus particles were observed in allantoic fluids and supernatants of MDEF cultures by transmission electron microscopy. Two genomic fragments, comprising 1108 nucleotides of the right open reading frame that codes for the structural viral proteins 1, 2 and 3, were amplified by polymerase chain reaction from one of the isolates, Muscovy duck parvovirus (MDPV)/PSU-31010. Comparison of this fragment with available sequences of other MDPV and related goose parvovirus (GPV) isolates showed that it had only 84.5% sequence identity with other MDPV isolates and 84.6% identity with the GPV isolates. This region shares over 99% identity among previously sequenced MDPV isolates and 95% identity among the related GPV isolates. This suggests that MDPV/PSU-31010 is divergent from all other sequenced MDPV and GPV isolates, and may represent a new group of avian parvoviruses.     

Mortality in Muscovy ducks (Cairina moschata) and domestic geese (Anser anser var. domestica) associated with natural infection with a highly pathogenic avian influenza virus of H7N1 subtype

Among the 413 outbreaks of highly pathogenic avian influenza (HPAI) caused by a virus of the H7N1 subtype, which occurred in Italy during 1999 and 2000, an outbreak diagnosed in a backyard flock was characterized by mortality and nervous signs in ducks and geese. Dead geese (Anser anser var. domestica) and Muscovy ducks (Cairina moschata) were submitted to the laboratory for bacteriological, virological, histological and immunohistochemical investigations. Routine bacteriological tests resulted negative, while a HPAI virus of the H7N1 subtype was isolated from the geese. Pancreatic damage was observed in both the geese and the ducks, and the pancreas was also positive by immunohistochemistry for avian influenza in the geese. Histopathological lesions were observed in the central nervous system of both species, and this result was supported by positive immunohistochemical findings for the presence of the virus.     

Attenuation of the goose parvovirus strain B. Laboratory and field trials of the attenuated mutant for vaccination against Derzsy's disease

Serial transfer of the goose parvovirus strain B, causal agent of Derzsy's gosling disease, in cultured goose-embryo fibroblast (GEF) resulted in a mutant (designated as Bav) apathogenic for both goose embryos and susceptible goslings. Goose embryos inoculated with the 38th or higher passages of strain B survived the infection, although the virus replicated in their organs. Susceptible goslings survived challenge with the Bav strain without showing symptoms, and developed normally. Only 4.2% of gosling progeny of parents vaccinated twice with strain Bav died after challenge with the virulent strain B goose parvovirus compared with 95% of gosling progeny of unvaccinated parents. Progeny of vaccinated and unvaccinated geese were placed on a farm on which Derzsy's disease was present. During the first month of life mortality was 7.7% in the progeny of vaccinated geese compared with 59.8% in the progeny of the unvaccinated geese. At 8 weeks of age the mean weight of the vaccinated goslings was 20% greater than for the unvaccinated goslings. These results indicate that the attenuated apathogenic Bav mutant is suitable for the immunisation of layers to protect their progeny by passive immunisation against Derzsy's disease.