Prevalence of Fowl Adenovirus Serotype 4 and Co-Infection by Immunosuppressive Viruses in Fowl with Hydropericardium Hepatitis Syndrome in Shandong Province, China.

Fowl adenovirus serotype 4 (FAdV-4) is the pathogenic agent of hydropericardium hepatitis syndrome (HHS) in chickens and ducks, which has caused huge economic losses for the Chinese poultry industry since 2015. In order to objectively determine the prevalence and co-infection status of the virus in Shandong province in China, we analyzed a total of 679 clinical cases of chickens and ducks from 36 farms in the province. The results showed that the FAdV-4 infection rate was 65.2% (443/679), and the rate in breeder ducks was almost two-fold higher than that in breeder chickens (68.57% vs. 34.30%). Notably, co-infection by H9N2 avian influenza virus, infectious bursal disease virus, and/or chicken infectious anemia virus was very common in the 443 FAdV-4-positive cases. Furthermore, phylogenetic analysis of the hexon genes of four Shandong FAdV-4 isolates revealed that these strains clustered into Indian reference strains, indicating that the Shandong FAdV-4 strains might have originated in India. These findings provide the first data on the prevalence and co-infection status of FAdV-4 in Shandong province, which may serve as a foundation for the prevention of FAdV-4 in the field.

Development of Colloidal Gold-Based Immunochromatographic Assay for Rapid Detection of Goose Parvovirus.

Goose parvovirus (GPV) remains as a worldwide problem in goose industry. For this reason, it is necessary to develop a new diagnostic approach that is easier and faster than conventional tests. A rapid immunochromatographic assay based on antibody colloidal gold nanoparticles specific to GPV was developed for the detection of GPV in goose allantoic fluid and supernatant of tissue homogenate. The monoclonal antibodies (Mab) was produced by immunizing the BALB/c mice with purified GPV suspension, and the polyclonal antibody (pAb) was produced by immunizing the rabbits with recombinant VP3 protein. The colloidal gold was prepared by the reduction of gold salt with sodium citrate coupled with Mab against GPV. The optimal concentrations of the coating antibody and capture antibody were determined to be 1.6 mg/ml and 9 µg/ml. With visual observation, the lower limit was found to be around 1.2 µg/ml. Common diseases of goose were tested to evaluate the specificity of the immune colloidal gold (ICG) strip, and no cross-reaction was observed. The clinical detection was examined by carrying out the ICG strip test with 92 samples and comparing the results of these tests with those obtained via agar diffusion test and polymerase chain reaction (PCR) test. Therefore, the ICG strip test was a sufficiently sensitive and accurate detection method for a rapid screening of GPV.

Serological and Pathogenic Analyses of Fowl Adenovirus Serotype 4 (FAdV-4) Strain in Muscovy Ducks.

Hydropericardium hepatitis syndrome (HHS) is a lethal disease caused by Fowl adenovirus serotype 4(FAdV-4) that mainly infects 3- to 6-week-old broiler chicks. In 2015, an infectious disease characterized similar symptom to HHS in broilers outbroke in commercial duck flocks in Shandong province. FAdV-4 was isolated from naturally infected ducks and determined by polymerase chain reaction (PCR) amplification and DNA sequence analysis. In order to investigate the effect of FAdV-4 infection on muscovy ducks, we determined and characterized the FAdV-4 Isolate, and assessed its pathogenicity. In this study, HHS was respectively reproduced in 5-week-old muscovy duck by intramuscular injection and intranasal inoculation of allantoic fluid containing FAdV-4, ducks in the negative control group were inoculated with allantoic fluids of healthy duck embryos in the same manner. Clinical symptoms, gross and microscopic lesions, cytokines and antibodies, blood biochemical indices were detected and recorded for 12 days after infection. Typical hydropericardium and hepatitis was observed in experimental muscovy duck in the 3rd day post-inoculation (dpi). FAdV-4 can be replicated in tissues and cause pathological damage, especially in the liver and immune organs. Most of the immune-related cytokines and antibodies levels are up-regulated and then decreased, which may be caused by the initial infection and the normal immune response, later the virus caused the immunosuppression and led to the decrease of levels. To the best of our knowledge, this is the first systematic trial of the pathogenicity of FAdV-4 in muscovy ducks mainly based on the serological test, which will provide new insights into the study of the disease.

Preparation and evaluation of goose reovirus inactivated vaccine.

BACKGROUND: Infection with Goose Reovirus (GRV) can cause serious economic losses in the goose breeding industry. In this study, the GRV allantoic fluid was concentrated and used as an antigen in a formalin-inactivated oil-emulsion vaccine. RESULTS: When 6 day-old geese were inoculated, antibodies against GRV became detectable at 6 days post-vaccination, their concentration peaked at 3 weeks. These antibodies were maintained for longer than 2 weeks. As the most susceptible age for GRV infection is birds under 2 weeks of age this vaccine should provide adequate cover for the most at risk birds. When geese were exposed to reovirus at different time intervals after immunization, the data revealed that the vaccine can provide a protection rate of 80%. The developed vaccine has good stability and could be stored at 4 °C for at least 12 months. CONCLUSION: These results indicate that the developed GRV vaccine is safe, effectively absorbed, efficacious in inducing a rapid immune response, and effective in controlling GRV infection. Our results should be useful for the application of vaccines for controlling GRV in different goose flocks.

The effect of Tembusu virus infection in different week-old Cherry Valley breeding ducks.

To study the effect of Tembusu virus (TMUV) infection on Cherry Valley Breeding ducks of different ages, 350 five-week-old ducks were divided into 14 groups. Ducks in seven experimental group were respectively infected with 1.265×10(5) mean embryo lethal dose (ELD50) of TMUV-AHQY strain (in 4.2mL) by intravenous route. Ducks in control groups were inoculated with Phosphate-buffered Saline (PBS) in the same way. Clinical symptoms, gross and microscopic lesions, viral loads and serum antibodies were detected and recorded for 20days after infection. Some ducks infected at 7 and 21 week s of age showed severe clinical symptoms including depression and inappetence, and no obvious clinical symptoms were seen in other week-old infected ducks. Severe gross lesions including hepatomegaly, meningeal congestion, myocardial hemorrhage, intestinal, myocardial and pulmonary edema were observed in ducks infected at 7, 18 and 21 weeks of age. No or mild gross lesions were observed in ducks infected at 14 and 16 weeks of age. The main microscopic lesions including hyperaemia, degeneration and necrosis of different cells and inflammatory cellular infiltration mainly consisting of mononuclear cells or lymphocytes were observed in ducks infected at 7 and 21 week of age. But relatively intact structures and rare lymphocytic infiltration were presented in ducks infected at 14 and 16 weeks of age. Viral antigen was more frequently observed in organ slices collected from 7 week-old infected ducks and few positive staining was found in 14 and 16 week-old infected ducks. Less viral loads in different tissues and swabs were detected by a quantitative real-time PCR assay. The level of viral loads in the tissues of ducks infected at 14 and 16 weeks of age was very lower than that of ducks infected at 7 and 21 weeks of age. Meanwhile, less viral copy numbers were detected in swab samples collected from 14 and 16 week-old infected ducks. Ducks infected at 14-week-old developed significantly higher serum neutralizing antibody titers than those infected at other week of age. These results indicated that the effect of TMUV infection on Cherry Valley ducks is partly related to weeks of age. 7-10 week-old and 18-21 week-old ducks were more susceptible to TMUV infection, but 14-16 week-old ducks were more resistant to this disease.

Experimental reproduction of beak atrophy and dwarfism syndrome by infection in cherry valley ducklings with a novel goose parvovirus-related parvovirus.

Infection of clinically susceptible ducks, including cherry valley and Muscovy ducks, with a novel goose parvovirus (GPV)-related virus (N-GPV) can result in beak atrophy and dwarfism syndrome (BADS). To obtain new insights into the host range and pathogenic potential of this novel waterfowl parvovirus, cherry valley ducklings (n=20) were experimentally infected with N-GPV strain SDLC01. An equal number of ducklings served as uninfected controls. The appearance of clinical signs, histopathological changes, viral shedding, and seroconversion was monitored for 20 days post-infection. Infection status of all ducks was monitored using indirect ELISA, virus neutralization test, nested PCR, clinical indicators, and microscopic examination. Three ducks developed the typical clinical, gross, and histological changes of BADS. By study day 6, the infected ducks had seroconverted to N-GPV. The antibodies raised were neutralizing against the SDLC01 strain in vitro. Here we successfully developed an experimental infection model for studying the pathogenicity and role of N-GPV in BADS.

Isolation and Genomic Characterization of a Duck-Origin GPV-Related Parvovirus from Cherry Valley Ducklings in China.

A newly emerged duck parvovirus, which causes beak atrophy and dwarfism syndrome (BADS) in Cherry Valley ducks, has appeared in Northern China since March 2015. To explore the genetic diversity among waterfowl parvovirus isolates, the complete genome of an identified isolate designated SDLC01 was sequenced and analyzed in the present study. Genomic sequence analysis showed that SDLC01 shared 90.8%-94.6% of nucleotide identity with goose parvovirus (GPV) isolates and 78.6%-81.6% of nucleotide identity with classical Muscovy duck parvovirus (MDPV) isolates. Phylogenetic analysis of 443 nucleotides (nt) of the fragment A showed that SDLC01 was highly similar to a mule duck isolate (strain D146/02) and close to European GPV isolates but separate from Asian GPV isolates. Analysis of the left inverted terminal repeat regions revealed that SDLC01 had two major segments deleted between positions 160-176 and 306-322 nt compared with field GPV and MDPV isolates. Phylogenetic analysis of Rep and VP1 encoded by two major open reading frames of parvoviruses revealed that SDLC01 was distinct from all GPV and MDPV isolates. The viral pathogenicity and genome characterization of SDLC01 suggest that the novel GPV (N-GPV) is the causative agent of BADS and belongs to a distinct GPV-related subgroup. Furthermore, N-GPV sequences were detected in diseased ducks by polymerase chain reaction and viral proliferation was demonstrated in duck embryos and duck embryo fibroblast cells.