Pathogenicity of fowl adenovirus isolates in specific pathogen-free embryonated chicken eggs

Fowl adenovirus (FAdV) is the primary pathogen in inclusion body hepatitis(IBH) in chickens and causes sudden onset of mortality in broiler and layer chickens. Inclusion body hepatitis outbreak has a worldwide distribution and has recently been reported in the region. The clinical signs of the infection were weakness, dehydration, ruffled feather and paleness of comb in the affected chickens. Upon necropsy, the infected chickens showed swollen liver with petechial to focal haemorrhages, hydropericardium, and gizzard erosion and haemorrhages. Isolation of the virus from clinical cases is needed to determine the pathogenicity of the avian adenovirus. Thus the objective of this study was to determine the pathogenicity of recent FAdV isolates in specific pathogen-free (SPF) embryonated chicken eggs. Two isolates of FAdV were obtained from recent outbreaks of the disease in layer (Group A) and broiler (Group B) chicken farms. Isolate from each liver sample was processed and inoculated in SPF eggs. The eggs were harvested and liver of the embryo collected for preparation of FAdV inocula. Sixty 9-day-old SPF embryonated chicken eggs were used in the study. They were divided into three major groups, namely, Group A [A1 (sacrifice) and A2 (mortality)], B [B1 (sacrifice) and B2 (mortality)] and C [C1 (sacrifice) and C2 (mortality)]. Five eggs each from Groups A2, B2 and C2 were labeled as mortality groups and observed for mortality throughout the trial. Twenty eggs from Groups A and B were inoculated with 0.1 mL FAdV isolates A and B, respectively. Twenty eggs from Group C were not inoculated and served as the control group. The eggs were candled twice daily and mortality recorded. Three eggs each from Groups A1, B1 and C1 were sacrificed at days 1, 3, 6, 9 and 12 post-inoculation (pi). At necropsy, the gross lesions were recorded and liver, gizzard and chorioallantoic membrane (CAM) samples were fixed in 10% buffered formalin for histological examination. The study showed 100% mortalities in Groups A and B within 1 to 9 days pi for the mortality group. The control group had no mortality throughout the trials. The number of dead embryo from the sacrifice group was 7 and 11 in the groups A and B, respectively. Control group did not show mortality. Gross lesions in the sacrifice group of Group A were mainly observed in the CAM, liver and gizzard. The CAM became thickened and cloudy beginning day 3 pi. Lesions in the liver revealed enlarged, pale, petechial haemorrhages with multifocal area of necrosis, which were first observed on day 6 pi. The gizzard was congested at day 9 pi. The gross lesions observed in Group B were mainly in the CAM and liver. The lesions were observed as early as day 3 pi with thickening and cloudiness of the CAM as well as enlargement, pale to yellowish liver. The control group remained normal throughout the trial. Histologically, typical intranuclear inclusion bodies were observed in the CAM, liver and gizzard in Group A. The lesions were confined to the CAM and liver in Group B. It was concluded that FAdV is highly pathogenic to SPF embryonated chicken eggs and the embryonic liver should be used for isolation and propagation of the virus

Pathogenicity of fowl adenovirus seroype 8B isolates of Malaysia in specific pathogen free chicken

Highly pathogenic Fowl Adenovirus (FAdV) is a causative agent of Inclusion Body Hepatitis (IBH) in poultry. It causes mortality and poor performance of the affected chickens. It was objectives of the study to determine pathogenicity of FAdV serotype 8b isolates of Malaysia in Specific Pathogen Free (SPF) chickens. The virus isolates namely, UPM11134 and UPM11142 were obtained from IBH outbreaks in broiler chicken farms and characterized as FAdV serotype 8b. The liver samples of affected chickens were prepared and inoculated into SPF embryonated chicken eggs via. Chorioallantoic Membrane (CAM) route. The liver of embryos was subsequently harvested for preparation of virus inoculum. The 36 days old SPF chicks were divided into 3 groups namely groups A-C. Each group was further divided into sacrificed and mortality groups. Chickens in groups A and B were inoculated with UPM11134 and UPM11142 FAdV isolates, respectively, via. intraperitoneal route at day old. All chicken in group C remained uninoculated and acted as the control group. The chicken were monitored for any clinical abnormalities throughout the trial. Gross lesions were recorded on necropsy and samples of liver and gizzard were collected and fixed in 10% buffered formalin for histological examination. The study showed 100 and 91% mortality of SPF chickens in groups A and B, respectively, at day 4 post inoculation (pi). Clinical signs of IBH such as depression, weakness, ruffled feathers and diarrhea were recorded within 12-24 h prior to death. On necropsy, hydropericadium with pale, friable and petechial haemorrhages of liver were recorded. Hepatitis with areas of hepatic necrosis and haemorrhages and presence of numerous basophilic Intranuclear Inclusion Bodies (INIB) in degenerated hepatocytes were recorded in all chickens in groups A and B, whilst INIB were also observed in glandular epithelium of gizzard in group A. All chicks in group C remained normal throughout the trial. It was concluded that Malaysian FAdV serotype 8b isolates are highly pathogenic in SPF chickens and acted as the primary agent of IBH

Efficacy of live attenuated fowl adenovirus serotype 8B isolate of Malaysia in spesific pathogen-free chickens

Fowl adenovirus (FAdV) is the primary agent of inclusion body hepatitis (IBH) in poultry and caused serious economic impact due to high mortality and poor productivity. To date, clinical cases of IBH in chickens increases over the years in Malaysia, thus, an effective local vaccine to control the disease outbreak is in dire need. The objective of the study was to determine the efficacy of live attenuated FAdV serotype 8b isolate, UPM1137CEL35, in specific-pathogen-free chickens. Attenuated isolate UPM1137CEL35 conferred full protection against the virulent FAdV isolate, UPM11134 in the vaccinated chickens. Neither clinical signs nor mortality were recorded in all vaccinated groups. However, in the challenged unvaccinated group, chickens showed clinical signs of weakness, reduced feed consumption, and lateral recumbency at day 4 to 7 postchallenge. Additionally, the body weight was low significantly (p<0.05) compared to the challenged vaccinated groups. Upon necropsy, the liver from the challenged unvaccinated group was pale at peripheral bilateral lobes with the presence of focal lymphoid aggregation microscopically, while, challenged vaccinated groups were normal without significant changes. All vaccinated chickens were protected from disease manifestations with antibody response compared to the challenged unvaccinated group. It was concluded that the attenuated FAdV isolate, UPM1137CEL35, has a high potential to be used as a vaccine against the FAdV serotype 8b of Malaysian isolates

Fowl adenovirus in chickens: diseases, epidemiology, impact, and control strategies to the Malaysian poultry industry – a review

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 industry are advised to eradicate this disease. Inclusion body hepatitis in Malaysia could cause a significant reduction in broiler meat production and therefore is a potential danger to the Malaysian poultry industry

Pathogenicity and immunogenicity of live attenuated and inactivated fowl adenovirus in commercial broiler chickens

Fowl Adenovirus (FAdV) is a non-enveloped DNA virus which is the primary pathogen of Inclusion Body Hepatitis (IBH) in chickens. IBH outbreaks were reported worldwide and was first reported in Malaysia in 2005 due to FAdV strain of serotype 8b infection. It was objective of the study to determine pathogenicity and immunogenicity of live attenuated and/or inactivated FAdV strain of serotype 8b (UPM1137) of Malaysian isolate in commercial broiler chickens. The 54, 1-day-old Cobb 500 broiler chicks were divided into four groups, namely groups A-D. Feed and water were provided ad-libitum. The chicks in groups A-C were inoculated with inactivated FAdV (0.2 mL) with virus titer of 106.5 TCID50 /0.2 mL, live attenuated FAdV (0.1 mL) with virus titer of 105.2 TCID50 /0.1 mL and the combination of the inactivated (0.2 mL) and live attenuated (0.1 mL) FAdV, respectively at day old and day 14 post-inoculation (pi). Body weight and blood samples were collected prior to necropsy at days 14 and 28 pi, except sampling was also conducted at day 0 pi in the group D (control). On necropsy, the gross lesions and liver weight were recorded and samples of liver were collected for histological examination. The study showed that neither clinical signs nor gross and histological lesions were recorded in all group of chickens throughout the trial. The body weight of chickens at days 14 and 28 pi were not significantly different (p>0.05) among all the groups. The liver to body weight ratio of group C was significantly higher (p<0.05) than groups A and D at day 28 pi. The FAdV antibody titer in group D (control) was 938±1596 on day old and was not detected at days 14 and 28 pi. However, the FAdV antibody was induced at high titer in all the inoculated groups at days 14 and 28 pi. The FAdV antibody titer of group C was significantly (p<0.05) higher than groups A and B at day 28 pi. It was concluded that the live attenuated and inactivated FAdV are safe and able to induce FAdV antibody titer in broiler chickens with moderate level of maternally derived antibody at day old of age. The combination of live attenuated and inactivated FAdV was able to induce higher antibody titer when compared to sole use of live attenuated or inactivated FAdV. It has high potential to be used as vaccination strategy against IBH outbreaks

Genetic diversity of fowl adenovirus serotype 8b isolated from cases of inclusion body hepatitis in commercial 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

Hexon and fiber gene changes in an attenuated fowl adenovirus isolate from Malaysia in embryonated chicken eggs and its infectivity in chickens

Fowl adenovirus (FAdV) is distributed worldwide and causes economic losses in the poultry industry. The objectives of this study were to determine the hexon and fiber gene changes in an attenuated FAdV isolate from Malaysia in specific pathogen-free chicken embryonated eggs (SPF CEE) and its infectivity in commercial broiler chickens. SPF CEE were inoculated with 0.1 mL FAdV inoculum via the chorioallantoic membrane (CAM) for 20 consecutive passages. The isolate at passage 20 (E20), with a virus titer of 108.7TCID50/mL (TCID50, 50% tissue culture infective dose), was inoculated (0.5 mL) into one-day-old commercial broiler chicks either via oral or intraperitoneal routes. The study demonstrated that 100% embryonic mortality was recorded from E2 to E20 with a delayed pattern at E17 onwards. The lesions were confined to the liver and CAM. Substitutions of amino acids in the L1 loop of hexon at positions 49 and 66, and in the knob of fiber at positions 318 and 322 were recorded in the E20 isolate. The isolate belongs to serotype 8b and is non-pathogenic to broiler chickens, but it is able to induce a FAdV antibody titer. It appears that molecular changes in the L1 loop of hexon and the knob of fiber are markers for FAdV infectivity

Molecular characterization of fowl adenovirus isolate of Malaysia attenuated in chicken embryo liver cells and its pathogenicity and immunogenicity in chickens

Fowl adenovirus (FAdV) is the causative agent of inclusion body hepatitis (IBH) in chickens with significant economic losses due to high mortality and poor production. It was objectives of the study to attenuate and determine the molecular characteristic of FAdV isolate (UPM1137) of Malaysia passages in primary chicken embryo liver (CEL) cells. The cytopathic effect (CPE) was recorded and the present of the virus was detected by polymerase chain reaction (PCR). Nucleotide and amino acid changes were determined and a phylogenetic tree was constructed. The pathogenicity and immunogenicity of the virus at passage 35 (CEL35) with virus titre of 106.7TCID50/mL was determined in day old specific pathogen free (SPF) chicks via oral or subcutaneous route of inoculation. The study demonstrated that the FAdV isolate was successfully propagated and attenuated in CEL cells up to 35th consecutive passages (CEL35) with delayed of CPE formation within 48 to 72 post inoculation (pi) from CEL20 onwards. The virus caused typical CPE with basophilic intranuclear inclusion bodies, refractile and clumping of cells. The virus is belong to serotype 8b with substitution of amino acid at position 44, 133 and 185 in L1 loop of hexon gene and in knob of fiber gene at position 348 and 360 at CEL35. It is non-pathogenic, but immunogenic in SPF chickens. It was concluded that the FAdV isolate was successfully attenuated in CEL cells with molecular changes in major capsid proteins which affect its infectivity in cell culture and SPF chickens

Discerning the antimicrobial resistance, virulence, and phylogenetic relatedness of Salmonella isolates across the human, poultry and food materials sources in Malaysia

Salmonella enterica subspecies enterica serovar Enteritidis is one of the major foodborne zoonotic pathogens globally. It has significantly impacted human health and global trade. In this investigation, whole-genome sequencing was employed to determine the antimicrobial resistance (AMR) pattern of a collection of Salmonella Enteritidis isolated from humans, poultry, and food sources. The study also investigated the virulence genes profile of the isolates as well as the phylogenetic relationships among strains. Illumina NextSeq technology was used to sequence the genome of 82 Salmonella Enteritidis strains isolated over 3 years (2016-2018) in Peninsular Malaysia. The pattern of resistance showed that tetracycline had the highest frequency (37/82, 45.12%), and isolates from food samples showed the highest rate of 9/18 (50.00%), followed by human 17/35 (48.57%) and then poultry 11/29 (37.93%). The second drug with the highest resistance rate is ampicillin with 5/29 (17.24%) for poultry, 4/35 (11.43%) for human, and 0/18 (0.00%) for food isolates respectively. Similarly, a total of 19 antimicrobial resistance (AMR) genes corresponding to the nine drugs used in the disc diffusion assay were evaluated from the whole genome sequence data. The aminoglycoside resistance gene aac(6')-ly was detected in 79 of the 82 isolates (96.34%). While the phylogenetic analysis revealed distinct lineages isolated, the three sources indicating possible cross-contamination. In conclusion, the results showed that the genomic profile of Salmonella Enteritidis isolated from humans, poultry, and food samples share genetic traits, hence the need to institute measures at controlling the continuous spread of these resistant pathogens

Development of live attenuated fowl adenovirus isolate of Malaysia for vaccine production

Fowl adenovirus (FAdV) infection is one of the major threats among viral diseases in poultry industry worldwide. The disease caused severe economic losses due to high mortality and poor production. Malaysian`s FAdV isolate are highly pathogenic in chickens and has been identified as primary pathogen of inclusion body hepatitis (IBH) and gizzard erosion. It is indeed an urgent need to develop FAdV vaccines to prevent and control FAdV infection. It was objective of the study to develop live attenuated FAdV isolate of Malaysia for future production of vaccine against the disease. FAdV isolate (UPM1137) was obtained from liver sample of chickens during outbreak of IBH and gizzard erosion. The isolate was successfully isolated and identified in specific pathogen free (SPF) embryonated chicken eggs and Vero cells. Thickening and cloudy of chorioallantoic membrane (CAM) with pale, petechial haemorrhages and multifocal area of necrosis of the liver and hydropericardium of embryo were recorded at day 9 post-inoculation (pi). The cytopathic effect (CPE) in Vero cells revealed cytoplasmic extensions and formation of plaque at day 6pi. It was confirmed that the isolate belong to FAdV by PCR with expected PCR size product and through sequence analysis. Partial hexon gene region with 1166 base pair (bp) of nucleotide (nt) sequence from SPF embryos and Vero cells samples were assigned with Genbank accession number, KF866370 (UPM1137E2) and KF866371 (UPM1137V1), respectively. For fiber gene, 1094bp of nucleotide sequence were deposited into GenBank with accession number KY305950 (UPM1137E2) and KY364903 (UPM1137V1). The isolate is highly related to group E species under serotype 8b. The isolate is highly pathogenic in SPF embryonated chicken eggs with 100% mortality and typical gross and histological lesions associated with FAdV. The embryonic mortality pattern was delayed from 17th consecutive passages (E17) to E20. This is consistent with the molecular changes in both L1 loop and knob regions of the nucleotide bases and amino acid in hexon and fiber gene and may indicate virus attenuation. Molecular changes were also prominent at 5th consecutive passages of the virus in Vero cells (V5). However, the FAdV isolate was highly susceptible to be adapted and attenuated in primary chicken embryo liver (CEL) cells. Early CPE was observed at 2nd consecutive passages (CEL2) to CEL19 within 24 to 48 hours post inoculation (pi) in the form of cells rounding, refractile and clumping. The CPE formation was delayed from CEL20 to CEL35 within 48 to 72 hours pi. Nucleotide sequences of hexon and fiber gene from all propagated isolates were deposited to GenBank and assigned with the accession numbers KY305943 to KY305949 (hexon) and KY305951 to KY305957 (fiber). Molecular changes in L1 loop of hexon gene are minimal in nucleotide bases of CEL5 to CEL20 without caused any amino acid changes. All changes mainly occur in other part of hexon gene. However, changes noticeable from CEL25 to CEL35 propagated FAdV isolates. Sequence analysis in fiber gene revealed both nucleotide and amino acid changes in knob region are prominent at high passage level from CEL20 to CEL35. It was demonstrated that the marker gene for adaptation in CEL cells and SPF embryonated chicken eggs were identified in nucleotide of L1 loop hexon gene at position 90(T-C) and in knob of fiber gene at both nucleotide and amino acid at position 1078(G-C) and 360(A-P), respectively. Additionally, the marker gene for attenuation in CEL cells was detected in knob region of fiber gene at position 1062(A-C). Sequence analysis of hexon and fiber gene product of all the samples either from the SPF eggs, Vero cells or CEL cells passages revealed 1166bp and 1094bp, respectively belongs to FAdV group E serotype 8b. Phylogenetic tree constructions revealed that isolates were clustered and closed to each other. The pathogenicity, immunogenicity and efficacy of an attenuated FAdV isolate CEL35 (106.7TCID50/ml) was determined in 1-day-old SPF chickens via oral (0.1ml) and subcutaneous (SQ) (0.1ml) route of inoculation. On day 14 post-inoculation (pi), chickens in challenged groups were inoculated with 0.2ml pathogenic FAdV isolate, UPM11134 (108.3TCID50/ml) via intraperitoneal (IP) route. The study shown that the attenuated FAdV isolate is non-pathogenic and safe in SPF chickens without exhibit clinical signs associated with FAdV infection throughout the trial. The FAdV inoculated chickens in challenged group were normal without exhibit abnormal clinical signs, gross and histological lesions. However, chickens in non-inoculated group showed clinical signs of weakness, depression and recumbency at day 4 post-challenge (pc) prior recovery on day 8pc. The body weight was significantly lower (p0.05) when compared to the SQ and control groups. In challenged groups, the antibody titer was significantly increased (p0.05) between route of inoculation throughout the trial. Similarly, in chickens inoculated with E20 FAdV, the antibody titer in oral (4119 ± 792) and IP (4598 ± 871) group were equally distributed among the routes. There were no differences in antibody titer between attenuated isolates either through oral or IP routes (p>0.05). However, FAdV inoculated chickens with CEL35 (2348 ± 1800) and E20 (1833 ± 792) attenuated isolates via IP route have high antibody titer significantly (p<0.05) compared to control group (69 ± 34) at day 21pi. It was concluded that the attenuated FAdV isolate are non-pathogenic and immunogenic in commercial broiler chickens. The attenuated FAdV isolate in CEL cells and SPF embryonated chicken eggs induced similar antibody response either through oral or IP routes. It was concluded that FAdV is highly susceptible in SPF embryonated chicken eggs and primary CEL cells rather than in Vero cells with nucleotide and amino acid changes in hexon at L1 loop region and in fiber gene at knob region. The attenuated isolates are non-pathogenic in SPF and commercial broiler chickens with capability to induce FAdV antibody. It appears that parenteral route is an effective route of FAdV inoculation when compared to the oral route and the attenuated isolates are suitable to be used for future development of FAdV vaccine against the disease