Hydropericardium Syndrome In Chickens Research Articles

Recombinant fiber-1 protein of fowl adenovirus serotype 4 induces high levels of neutralizing antibodies in immunized chickens.

Virulent fowl adenovirus serotype 4 (FAdV-4) causes hydropericardium syndrome (HPS) with high mortality in chickens, leading to significant economic losses to the poultry industry. The development of an effective vaccine is essential for successful disease control. Here, we produced recombinant fiber-1 protein of FAdV-4, isolated from a Japanese HPS outbreak strain, JP/LVP-1/96, using a baculovirus expression system and evaluated its immunogenicity and protective efficacy. Recombinant fiber-1 protein induced high levels of neutralizing antibodies in immunized chickens, which were maintained for a minimum of 10 weeks. After being challenged with the virulent FAdV-4 strain JP/LVP-1/96, the immunized chickens did not exhibit clinical signs of infection or histopathological changes, there was a significant reduction in the viral load in various organs and total serum proteins, and albumin levels did not decline. These results suggest that the recombinant fiber-1 protein produced in this study can serve as a subunit vaccine to control HPS in chickens.

Characterization of a hypervirulent fowl adenovirus 4 with the novel genotype newly prevalent in China and establishment of reproduction infection model of hydropericardium syndrome in chickens

Severe hydropericardium syndrome (HPS) has been present in layers in the northeast of China since June 2015, with mortality rates varying from 30 to 90%. Dead layers had severe hydropericardium with pericardial volumes of 5 to 20 mL, as well as inclusion body hepatitis. Laboratory investigations led to the isolation of a fowl adenovirus strain, HLJFAd15, from the liver tissue of dead layers. Natural deletions of ORF19 and ORF27 were found in this clinical strain by complete genome sequencing, which was identified with the novel genotype recently prevalent in China. The pathogenicity characterization was conducted in 35-day-old SPF chickens using HLJFAd15 with novel genotype of fowl adenovirus serotype 4 (FAdV-4). The reproduction disease cases of HPS with mortality rates of 76.9% by oral administration and 100% by intramuscular injection were induced successfully by challenging SPF chickens, respectively. Non-enveloped viral particles with a mean diameter of approximately 80 nm were found in the livers of virus-infected SPF chickens. Our study revealed that HLJFAd15 was identified with the novel genotype strains recently emerging in China by complete genome sequencing, and the strain was capable of causing HPS by the pathogenicity analysis. However, although there is currently no commercial vaccine against the novel genotype FAdV-4, the animal infection model established in this study was valuable for vaccine evaluation and development.

Hydropericardium syndrome outbreak caused by fowl adenovirus serotype 4 in China in 2015.

There have been many outbreaks of hydropericardium syndrome (HPS), which is characterized by pericardial effusion and hepatitis, in Chinese chicken farms since June 2015. HPS was mainly found in miscellaneous meat-type chickens, Ma chickens, layer chicks and Three-yellow chickens, while it was occasionally found in white broilers. To determine the specific causative pathogen and pathogenicity of HPS in chickens, we collected 25 suspected cases and performed clinical pathology and aetiology analyses. The results showed that the 25 cases exhibited multifocal hepatitis with intra-nuclear inclusion bodies and 70 nm-latticed viral particles in the cell nuclei. All samples were positive for fowl adenovirus (FAdV), and sequencing results showed that the hexon gene shared the highest nucleotide similarities with the hexon gene of group 1 serotype 4 (FAdV-4). FAdV-4 was highly pathogenic to embryos and specific pathogen-free chickens, causing 100 and 70 % mortality rates, respectively. Thus, FAdV-4 is associated with HPS outbreaks in China.

Molecular cloning, expression and characterization of 100K gene of fowl adenovirus-4 for prevention and control of hydropericardium syndrome

Fowl adenovirus-4 is an infectious agent causing Hydropericardium syndrome in chickens. Adenovirus are non-enveloped virions having linear, double stranded DNA. Viral genome codes for few structural and non structural proteins. 100K is an important non-structural viral protein. Open reading frame for coding sequence of 100K protein was cloned with oligo histidine tag and expressed in Escherichia coli as a fusion protein. Nucleotide sequence of the gene revealed that 100K gene of FAdV-4 has high homology (98%) with the respective gene of FAdV-10. Recombinant 100K protein was expressed in E. coli and purified by nickel affinity chromatography. Immunization of chickens with recombinant 100K protein elicited significant serum antibody titers. However challenge protection test revealed that 100K protein conferred little protection (40%) to the immunized chicken against pathogenic viral challenge. So it was concluded that 100K gene has 2397 bp length and recombinant 100K protein has molecular weight of 95 kDa. It was also found that the recombinant protein has little capacity to affect the immune response because in-spite of having an important role in intracellular transport & folding of viral capsid proteins during viral replication, it is not exposed on the surface of the virus at any stage.

Pathogenicity and Complete Genome Characterization of Fowl Adenoviruses Isolated from Chickens Associated with Inclusion Body Hepatitis and Hydropericardium Syndrome in China.

In this study, we determined and genetically characterized three fowl adenoviruses isolated from chickens with inclusion body hepatitis (IBH) and hydropericardium syndrome (HPS) in China and assessed their pathogenicity. The full genome of HBQ12, BJH13 and JSJ13 was found to be 44,081, 43,966 and 43,756 nucleotides long, respectively. Sequence alignment and phylogenetic analysis revealed that strain HBQ12 and BJH13 were clustered together belonging to fowl adenoviruses D species and serotyped as FAdV-11, whereas strain JSJ13 was classified into fowl adenoviruses C species and serotyped as FAdV-4. To our knowledge, this is the first report of FAdV-4 strain circulating in China. The pathogenicity test showed that mortality for chickens infected with HBQ12 and JSJ13 within 21 days post infection (dpi) was 8.6% and 28.6%, respectively. Necropsy displayed mild or severe hepatitis and hydropericardium at 3 and 5 dpi as well as dead chickens. Viral DNA was detected in almost all tissues sampled from dead chickens. These results revealed that fowl adenovirus strains HBQ12 and JSJ13 are capable of causing IBH and HPS in chickens, indicating that preventive measures against FAdV infection on poultry farms should be implemented in China.

Recombinant hexon antigen based single serum dilution ELISA for rapid serological profiling against fowl adenovirus-4 causing hydropericardium syndrome in chickens

A recombinant hexon antigen based single serum dilution enzyme linked immunosorbent assay (ELISA) was developed to measure the specific antibody in sera of chickens against Fowl adenovirus-4 (FAdV) causing Hydropericardium syndrome (HPS). An immunodominant partial hexon gene of 737bp was cloned into pRSET vector and expressed in Escherichia coli strain BL21 DE3 pLys S. Expression was tested by Western Blot test. The purified recombinant protein antigen was used in coating ELISA plate for FAdV-4 serology. A linear relationship was found between the predicted antibody titres at a single working dilution of 1:100 and the corresponding observed serum titres as determined by the standard serial dilution method. Regression analysis was used to determine a standard curve from which an equation was derived that allowed the demonstration of this correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay proved to be sensitive, specific and accurate as compared to Q-AGID test. Recombinant antigen was also used in Dot ELISA. In an experimental vaccination of broiler chicken at 10 days old age, the geometric mean (GM) antibody titres as measured by ELISA ranged from 5.006±0.11log10 to 4.526±0.04log10 and by Dot ELISA titre were from 2.240±0.08log10 to 0.180±0.04log10 during 5th–8th weeks of age, results were compared with Q-AGID results. Field samples were collected randomly from breeder flocks, found to have antibody titre by both ELISA and Dot ELISA at 10th and only 75% samples were positive at 14th weeks of age. After revaccination at 16th weeks of age, all sera samples were found have considerably high antibody titre on 24th week but all samples were negative at 32nd weeks. Advantages of recombinant hexon antigen based ELISA and Dot ELISA in FAdV-4 serology have been discussed.

Recombinant FAdV-4 fiber-2 protein protects chickens against hepatitis–hydropericardium syndrome (HHS)

Virulent fowl adenovirus (FAdV) serotype 4 strains are the etiological agents of hepatitis–hydropericardium syndrome (HHS), a highly infectious disease in chickens with severe economic impact.In the present study, three different FAdV-4 derived capsid proteins, fiber-1, fiber-2, and hexon loop-1, were expressed in a baculovirus system and tested for their capacity to induce protection in chickens. Purified recombinant proteins were administered to day-old specific pathogen-free (SPF) chickens allocated in three separate groups and challenged with virulent FAdV-4 at 21 days of life. Two additional groups served as controls, a challenge control group with mock-vaccinated but infected birds and a negative control group with PBS injection substituting both vaccination and challenge.The fiber-2 vaccinated group displayed high resistance against the adverse effects of the challenge with only one dead bird out of 28, as compared to the challenge control group where the infection caused 78% mortality. A moderate protective effect resulting in 38% mortality was observed for fiber-1, whereas the hexon loop-1 vaccinated group was not effectively protected as manifested by 73% mortality.While a fiber-2 specific ELISA showed a gradual antibody increase after immunization of birds with the homologous protein, a commercial ELISA did not detect vaccination-induced antibodies in any of the groups but displayed a difference in challenge virus-directed response in protected and non-protected birds. Although immunoblotting confirmed the presence of specific antibodies in all vaccinated groups, the anti-protein sera did not exhibit neutralizing activity.Fecal excretion of challenge virus DNA was detected with a real-time PCR in the majority of tested birds until termination of the study independent of protection, indicating the prevention of clinical symptoms, but not infection, by vaccination.In conclusion, recombinant fiber-2 was identified as a protective immunogen and is proposed as an attractive candidate for a subunit vaccine to prevent hepatitis–hydropericardium syndrome in chickens.

Some strains of serotype 4 fowl adenoviruses cause inclusion body hepatitis and hydropericardium syndrome in chickens

Fowl adenoviruses were isolated and characterized from severe cases of hydropericardium syndrome in Ecuador and Pakistan. All were neutralized by antibodies against serotypes 4 and 11. Cross‐neutralization tests and restriction enzyme analysis strengthen the classification as serotype 4 strains. The restriction endonucleases used allowed the differentiation among field isolates and reference strains. All field isolates tested induced high embryo mortality. One‐day‐old specific pathogen free (SPF) chicks were infected with 105 plaque‐forming units by natural routes to reproduce the disease with plaque purified virus. Whereas no mortality was seen with the reference strain, the mortality with the field isolates was 100%. A reduced mortality occurred with a lower infectious dose. Field isolate K1013 from Ecuador was also highly pathogenic for 1‐ to 3‐week‐old SPF chicks after intramuscular inoculation. The main pathological signs were swollen livers, severe hydropericardium and nephritis, reflecting the field situation and underlining the role of FAV4 strains in the aetiology of infectious hydropericardium.