Molecular Epidemiology of Fowl Adenovirus (FAdV) from Inclusion Body Hepatitis (IBH) Incidences from Indian Broilers Revealed the Prevalence of Serotypes of FAdV-D and E

Fowl adenoviruses (FAdVs) are implicated in wide range of diseases in poultry including inclusion body hepatitis (IBH), hydropericardium syndrome (HPS), gizzard erosions (GE), respiratory and enteric conditions causing severe economic loss worldwide (Adair and Fitzgerald, 2008). FAdVs are double stranded DNA viruses and belongs to the genus Aviadenovirus of Adenoviridae family. As per the current International Committee on Taxonomy of Viruses (ICTV) classification, there are five species of Aviadenoviruses (A-E) and are further subdivided into 12 serotypes (FAdV-1 to 8a, 8b, 9-11) based on their restriction enzyme digestion pattern and cross-neutralization tests (Meulemans et al., 2004).

FAdV serotypes vary in their pathogenicity and are associated with different disease conditions. Hydropericardium syndrome (HPS), popularly known as Angara or litchi disease is primarily associated with FAdV-4 serotype (Choi et al., 2012), whereas gizzard erosions (GE) and inclusion body hepatitis (IBH) are mainly associated with FAdV-2, 8a, 8b and 11 serotypes (Mittal et al., 2014). The temporal and geographical distribution of different serotypes also varies (Kiss et al., 2021). Molecular serotyping based on the hexon coding sequence is widely used in recent times replacing the conventional cross-neutralization assays and restriction fragment length polymorphism assays owing to the latter’s tediousness and complexity.

All the serotypes are reported in Indian poultry associated with IBH-HPS with a predominance of FAdV-4 and 8 serotypes. Inactivated FAdV-4 vaccines are used widely in broiler parents in recent years to pass on the passive immunity to commercial broiler chicks. However, outbreaks are often reported from commercial broiler flocks (Shinde et al., 2020). Continuous molecular surveillance for identifying the circulating serotypes and serotype specific vaccination is needed for effective control of the disease and its spread. Hence, in the present study we investigated that the serotypes associated with IBH currently circulating in broilers.

Sample collection
 
Tissue samples were collected from clinically affected and IBH suspected commercial broiler flocks from different states of India between April 2019 and June 2021. A total of 98 flocks covering all four zones (North, South, East and West) of India were included in the study. All the birds were reared under deep litter system in open houses. Broilers varying in the age group of 2 and 5 weeks from flocks experiencing 5-30% mortality were sampled. The samples were analysed at ICAR-Directorate of Poultry Research, Hyderabad during 2020-21.
 
Histopathological examination
 
The liver tissues collected in 10% neutral formal saline were processed for tissue sections and slides were prepared following standard protocol. The tissue sections were stained by Hematoxylin and Eosin (HandE) stain and visualized under light microscope.
 
Genomic DNA extraction
 
DNA was isolated from all tissue samples from 98 flocks by following standard procedure. Briefly, the samples (50 mg) were lysed in lysis buffer containing proteinase K at 37°C for 30 min. DNA was extracted with phenol-chloroform-isoamyl alcohol (25:24:1) twice, followed by precipitation of DNA with absolute isopropanol, washed with 70% ethanol, dried at room temperature and resuspended in nuclease-free water. The extracted DNA was stored at -20°C until further use in PCR.
 
PCR screening of samples for FAdV by PCR
 
The PCR was performed with the hexon gene specific primers as described by Meulemans et al., (2001) with slight modifications. Briefly, the reaction was carried out in 25 µl reaction using 2 µl of template DNA with 2.5 µl of 10 × Taq buffer with MgCl₂, 0.5 µl of each primer (20 pmol), 0.5 µl of dNTPs (10 mMol) and 0.25 µl of Taq DNA polymerase (5 U/ml) and nuclease free water. The PCR conditions were as follows: 95°C for 10 min, 35 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 1 min and final elongation at 72°C for 10 min.  The presence of 897 bp product after amplification in the samples were considered FAdV positive and further processed for sequencing.
 
Sequencing and phylogenetic analysis
 
The amplified PCR products (hexon L1, 897 bp) from 37 positive flocks were sequenced in both directions using ABI automated sequencer. The obtained sequences were edited and aligned in EditSeq and MegAlign programme of DNA Star software. The nucleotide sequences of field FAdV strains were submitted in NCBI database (Table 1).



A total of 23 reference hexon gene sequences representing all 12 FAdV serotypes were retrieved for analysis (Table 2). The reference sequences along with 37 sequences of the present study were aligned by using Clustal W method and annotated with MEGA X version 10.0.5 (www.megasoftwae.net) (Kumar et al., 2018). The best-fit model for analysis was selected by comparing the Bayesian information criterion (BIC) scores. Phylogenetic tree was constructed by using the hexon gene sequences of both reference and current strains by following the Neighbour-joining method with 1000 bootstrap replicates based on Tamura-Nei model (Tamura et al., 2011). All positions containing gaps and missing data were eliminated. The FAdV strains were serotyped based on the clustering of the sequences with reference strains in the phylogenetic tree.

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Screening for other immunosuppressive viruses by PCR (MD, ALV, REV and CAV)
 
The DNA samples were used for screening other immunosuppressive viruses by PCR. MD, ALV and REV in the samples were screened by multiplex PCR using primer sets to target genes developed by our lab as described earlier (Kannaki et al., 2021). The samples were screened for CAV in a separate PCR using the primers targeting the VP3 region as described earlier (Yilmaz et al., 2001).

FAdVs have slowly emerged as primary pathogen associated with IBH, HPS and GE conditions in young broilers causing major economic threat worldwide in recent decade (Kiss et al., 2021). In India also, increasing mortality and poor growth rate were observed along with hepatitis outbreaks in young broilers in recent times (Shinde et al., 2020). Mortality rate ranged from 5 to 30% in the affected farms. Necropsy findings revealed enlarged pale, friable, yellow coloured liver and haemorrhages on liver in most of the farms. Hydropericardium was not observed in affected farms. Histopathological observation revealed the presence of basophilic intranuclear inclusion bodies in hepatocytes, vacuolation, degeneration and necrosis of hepatocytes, engorgement of sinusoids and haemorrhages in liver. Only two flocks showed gizzard erosions and unthriftiness.

Hexon is the major surface protein of adenoviruses containing the neutralizing epitopes. Hexon gene comprises of conserved pedestal regions P1 and P2 and variable loop regions L1, L2 and L4 (Toogood et al., 1989). Hence, gene sequencing of loop 1 of hexon is routinely used for FAdVs serotyping in recent times. 42 out of 98 flocks screened by PCR were positive by hexon gene specific PCR. Remaining 56 samples were negative by PCR.

The phylogenetic analysis based on the hexon loop-1 gene nucleotide sequences of FAdV strains revealed the presence of three serotypes (11, 8b and 8a) in the flocks with IBH (Fig 1). FAdV-11 was the most prevalent 67.56% (25/37) serotype followed by FAdV-8b 29.72% (11/37). The current FAdV-11 strains closely clustered with earlier reported Indian FAdV-11 serotypes (EU847633 and AY581276) from North India. FAdV-8a was observed in only one flock 2.70% (1/37). It clustered with TR59 and 8565 strains of 8a serotype from Belgium and USA respectively. These serotypes belong to D (FAdV-11) and E (FAdV-8a and FAdV-8b) FAdV species respectively.



FAdV-4, 8a, 8b and 11 are considered as potentially virulent serotypes causing severe clinical disease conditions. FAdV 2 and 11 were most widely associated with IBH in broiler with recent increasing incidences associated with 8a and 8b also (Kajan et al., 2013). Recently, serotypes 8a, 8b and 11 were also implicated in enteric disease and malabsorption in broilers (Torre et al., 2018). Other serotypes cause disease or mortality in the presence of other immunosuppressive agents or co-factors.

At least three species of FAdVs were known to cause clinical disease in many countries including India. Among them, serotypes belonging to D and E species are the most prevalent in many countries. In the present study, FADV-D species is the most prevalent associated with IBH indicating the geographical variation. While the pathogenicity of FAdV-4 in causing HPS is well established with experimental infection studies (Zhao et al., 2015), pathogenicity of the other serotypes is scanty (Steer et al., 2015) and yet to be established.

The samples tested negative for other immunosuppressive viral pathogens viz., MD, ALV, REV and CAV upon screening by PCR. In the present study, we did not observe any HPS condition in affected flocks either alone or with hepatitis. Further sequencing and phylogenetic analysis also shown the absence of FAdV-4 serotype in the outbreaks. Extensive vaccination followed in Indian broiler breeder flocks with inactivated FAdV-4 serotype with targeted passive immunity in broiler chicks may be implicated in significant control of HPS in field conditions and supported by the evidence that FAdV-8b and 11 serotypes become prevalent followed by drastic decline in FAdV-4 cases in South Korea after the introduction of FAdV-4 vaccines (Lai et al., 2021). In addition, FAdV-8b has emerged as the dominant serotype recently with initial high prevalence of FAdV-11 for two years followed by the introduction of 4 serotype specific vaccine in China and South Korea also (Wang et al., 2018; Lai et al., 2021). Emerging 8b strains from different parts of world causes IBH and showed broad tissue tropism with less mortality (Huang et al., 2019). Similar trend is observed in the current study too.

Co-infection of FAdVs and other pathogens are often quite common and reported regularly (Brown et al., 2019). However, FAdVs have emerged as primary pathogens in causing clinical diseases in recent times. Absence of any co-infection with other immunosuppressive viruses such as MD, ALV, REV and CAV in the present study reiterates the fact that co-infection or presence of other viruses may not be critical or necessary for these serotypes (FAdV-11, 8b and 8a) in causing the clinical disease conditions as reported earlier by other workers (Steer et al., 2011). Challenge studies with these serotypes in SPF chicken have been shown to induce disease earlier.

Almost all the serotypes were reported from India associated with IBH-HPS from different parts of the country. Nevertheless, FAdV-4 and -8 were reported to be prevalent in commercial broilers. Identification of other serotypes in the current study, along with a recent report of FAdV-11 serotype in broilers (Shinde et al., 2020) indicates the dynamics of disease landscape. Little or no cross-protection was observed among the serotypes and protection unlikely to occur unless the serotypes belong to same FAdV species (Hess, 2020). Although a previous report showed cross-protection induced by inactivated FAdV-4 vaccine against 8b and 11 in an experimental study (Kim et al., 2014), under field conditions similar protection is not observed (Lai et al., 2021). Moreover, poor cross-protectivity between 8b and 11 FAdV serotypes was also reported. This reiterates the current observation and emphasis the use of multiple serotypes for vaccination for complete protection.

In summary, at least three serotypes of FAdV species D and E were circulating in Indian broilers with high prevalence of FAdV-11 serotype during 2020-2021. Continuous molecular surveillance for the FAdV serotypes and pathogenicity studies are further warranted. The current information would be useful while devising the biosecurity measures and vaccination strategy for effective IBH control.