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Background and Aim: Live-attenuated vaccines are the most successful type of vaccine and could be useful in controlling fowl adenovirus (FAdV) 8b infection. This study aimed to attenuate, molecularly characterize, and determine the immunogenicity, efficacy, and challenge virus shedding in broiler chickens. Materials and Methods: The FAdV 8b isolate (UPM08136) was passaged onto chicken embryo liver (CEL) cells until attenuation. We sequenced and analyzed the hexon and fiber genes of the passage isolates. The attenuated bioreactor-passage isolate was inoculated into 1-day-old broiler chickens with (attenuated and inactivated) and without booster groups and challenged. Body weight (BW), liver weight (LW), liver: body weight ratio (LBR), FAdV antibody titers, T-lymphocyte subpopulation in the liver, spleen, and thymus, and challenge virus load and shedding were measured. Results: Typical cytopathic effects with novel genetic changes on CEL cells were observed. The uninoculated control-challenged (UCC) group had significantly lower BW and higher LW and LBR than the inoculated groups. A significantly higher FAdV antibody titer was observed in the challenged non-booster and attenuated booster groups than in the UCC group. T cells in the spleen and thymus of the liver of inoculated chickens were higher than uninoculated control group levels at all-time points and at different times. A significantly higher FAdV challenge virus load was observed in the liver and shedding in the cloaca of UCC chickens than in non-booster chickens. Conclusion: The FAdV 8b isolate was successfully attenuated, safe, and immunogenic. It reduces virus shedding and is effective and recommended as a vaccine against FAdV infection in broiler chickens.

Inclusion body hepatitis (IBH) is a devastating disease of chickens caused by Fowl adenovirus (FAdV) and molecular studies of hexon gene is important for classification of FAdV isolates, epidemiology of IBH infection and development of effective control strategy. The objective of the study was to molecularly characterize FAdV isolates obtained from the field outbreaks of IBH in broiler chicken from 2017 to 2019. Liver and gizzard samples named UPM1701, UPM1801, UPM1802, UPM1901 and UPM1902 were collected and processed for FAdV detection and characterization by PCR. Swollen, necrotic and haemorrhagic liver; erosion of koilin layer of gizzard and enlargement of the proventriculus were observed. The samples were positive for FAdV with 98% to 100% identical to serotype 8b reference strains based on NCBI Blast. Thirty-one nucleotide changes that produced 10 amino acid substitutions were observed in L1 loop region of UPM1701, UPM1901 and UPM1902 isolates. All isolates clustered together with FAdV 8b reference strains and shared common ancestor with UPM1137E2 and UPM04217. Gizzard erosion in FAdV 8b infection is not common and conciding with mutations indicate evolving novel pathogenicity. These mutations could have effect on the epidemiology of IBH and be useful in designing effective prevention and control strategy.

Fowl adenovirus (FAdV) infection is a major threat in commercial poultry farms which exerts serious economic impacts on the poultry industry. At the end of 2018, it was reported that a decrease of 9.0% in revenue to RM692.9 million was due to high mortality and low broiler production volume as a result of inclusion body hepatitis (IBH) outbreaks in Malaysia. Fowl adenovirus is a double-stranded DNA virus made up of 5 genotypes and 12 serotypes. The potential danger posed by this virus to the Malaysian poultry industry is hereby discussed. Fowl adenovirus serotype 8b has been reported to be predominant in Malaysian chicken where it causes IBH. It predominantly affects 3 to 7 weeks old broiler chickens as well as layer chickens. Inclusion body hepatitis has been reported in farms in the states of Perak, Johore, and Malacca in Malaysia with a mortality range of 9.6-30%. Morbidity is low and infected chickens may present crouching position with ruffled feathers and die within 48 hours or may recover. Recovered chickens usually indicate low feed intake, feed conversion, and weight gain. Typical IBH lesions include friable, and inflamed liver, petechial hemorrhages on the musculature, and microscopic basophilic/eosinophilic inclusion bodies in the hepatocytes. Fowl adenovirus can be transmitted vertically from hen to offspring through the eggs and cause disease conditions to chicks especially those with no or low maternal antibodies. It is also transmitted horizontally through contact with feces and fluids from infected birds or humans as well as contaminated fomites. Although adequate biosecurity measures could reduce the incidences of this infection, some strains are resistant to disinfectants. Therefore, the major form of control is vaccination which makes the development of live attenuated and potent inactivated vaccines imperative. To avoid a crisis in broiler meat production in the country, regional cooperations among major stakeholders in the Malaysian poultry indu

Fowl adenovirus (FAdV) 8b causes inclusion bodies hepatitis (IBH) in chicken with 10- 30%. mortality worldwide and was first reported in Malaysia in 2005, but control has been difficult due to unavailability of vaccines. The objectives of this study were to propagate Malaysian isolates of FAdV 8b in chicken embryo liver (CEL) cells, attenuate, molecularly characterize and inactivate the virus and determine the pathogenicity, immunogenicity and efficacy of the attenuated and inactivated isolates in commercial broiler chickens. UPM11142, UPM11134 and UPM08136 FAdV isolates from IBH outbreaks in Malaysia were each inoculated into specific pathogen free (SPF) chicken embryonated eggs (CEE). Liver from dead embryo were processed to obtain FAdV inocula which were each propagated and attenuated onto CEL cells till passage 20 (P1-P20). The P5 and P20 of UPM11142 and UPM08136 FAdV were propagated in Cytodex™ 1 microcarrier adapted CEL cells in a stirred tank bioreactor (P5B1 and P20B1). TCID50 of all isolates were determined. Virus infectivity and localization of the virus in CEL cells were determined by immunocytochemical and HE stainning assays. PCR amplification of hexon and fibre genes were carried out using established and novel primers. Amplified genes were sequenced, analysed and phylogenetic trees constructed. UPM11142P5B1 and UPM08136P5B1 isolates were inactivated using binary ethylene imine (BEI) method, adjuvanted with Montanide 71VG and tested for immunogenicity and efficacy (with or without booster) on commercial broiler chickens. UPM11142P20B1 and UPM08136P20B1 attenuated vaccine candidates either with or without booster were tested for pathogenicity, immunogenicity and efficacy in commercial broiler chickens. Challenge group chickens of all groups were challenged with pathogenic FAdV at 28- day post inoculation (dpi). Clinical signs, gross and histopathological changes of organs were recorded. Body weight, liver weight and liver to body weight ratio were recor

Large volume production of vaccine virus is essential for prevention and control of viral diseases. The objectives of this study were to propagate Fowl adenovirus (FAdV) isolate (UPM08136) in chicken embryo liver (CEL) cells adapted to Cytodex™ 1 microcarriers using stirred tank bioreactor (STB) and molecularly characterize the virus. CEL cells were prepared and seeded onto prepared Cytodex™ 1 microcarriers and incubated first in stationary phase for 3 h and in STB at 37 °C, 5% CO2, and 20 rpm for 24 h. The CEL cells were infected with FAdV isolate (UPM08136) passage 5 (UPM08136CELP5) or passage 20 (UPM08136CELP20) and monitored until cell detachment. Immunofluorescence, TCID50, sequencing, alignment of hexon and fiber genes, and phylogenetic analysis were carried out. CEL cells were adapted well to Cytodex™ 1 microcarriers and successfully propagated the FAdV isolates in STB with virus titer of 107.5 (UPM08136CELP5B1) and 106.5 (UPM08136CELP20B1) TCID50/mL. These isolates clustered with the reference FAdV serotype 8b in the same evolutionary clade. The molecular characteristics remained unchanged, except for a point substitution at position 4 of the hexon gene of UPM08136CELP20B1, suggesting that propagation of the FAdV isolate in STB is stable and suitable for large volume production and could be a breakthrough in the scale-up process.

The objectives of this study were to characterize fowl adenovirus (FAdV) isolates of Malaysia propagated in chicken embryo liver (CEL) cells. Three isolates of FAdV namely; UPM11142, UPM11134 and UPM08136 were used. They were first propagated in specific pathogen free (SPF) chicken embryonated eggs (CEE) once (E1) and further propagated in CEL cells for 5 consecutive passages (CELP1 to CELP5). The E1, CELP1 and CELP5 of all of the isolates were characterized by PCR technique. The PCR products were then sequenced and analyzed. The study showed that all the isolates caused mortality in SPF CEE eggs and adapted well in CEL cells. Eosinophilic intranuclear inclusion bodies were observed in the cells. The UPM11142 and UPM11134 exhibited more pathogenic in CEE and CEL cells than UPM08136. All of the FADV isolates were characterized as FAdV group E serotype 8b. Nucleotide and amino acid changes were recorded in CELP1 and CELP5 when compared to E1. These isolates have potential to be used for vaccine development against the disease.