Detection of Inclusion Body Hepatitis Virus in broilers at Sharkia Province, Egypt

| During the last 10 years, many inclusion body hepatitis (IBH) outbreaks across Egypt were reported causing high economic losses to poultry industry. The current study aimed the detection and molecular characterization of IBH in broilers at Sharkia governorate. For identification and genotyping of fowl adenoviruses related to IBH in broiler flocks, liver samples representing 40 broiler flocks were collected and tested for FAdVs based on hexon gene detection using polymerase chain reaction (PCR). We recorded most loses among broilers at 3-5 weeks of age. Only depression, ruffled feather were the observed signs with varied mortality (8-14%). On necropsy, liver was yellowish, friable, enlarged and hemorrhagic. For identification and genotyping of Fowl adenoviruses related to inclusion body hepatitis (IBH) in broiler flocks at Sharkia governorate, liver samples representing 40 broiler flocks were collected and tested for FAdVs based on hexon gene detection using polymerase chain reaction (PCR Out of the forty examined broiler flocks, three flocks (7.5%) were recorded PCR positive for Aviadenoviruses. Based on hexon loop-1 gene analysis, the nucleotide sequence identity of the three positive samples (EG101/2018, EG102/2018 and EG103/2018) was >97.7% and genetically was closer to reference strains serotype 2 (FAdV-2), the three examined strains shared a high degree of nucleotide identity (97.6-99.6%). Using neighbor joining for the phylogenetic tree construction depending on hexon gene nucleotides and amino acids sequences, our strains were clustered in the same clade with FAdV type D. This study help to extend the current knowledge about the currently circulating IBH viruses and to develop vaccination and control strategy.


nuclEIc acId ExtractIon
Extraction of DNA was carried out using DNeasy® Blood and Tissue Mini Kits (QIAGEN, USA) according to instructions of manufacture. Purified DNAs were preserved at -20°C for PCR and Sequencing.

molEcular dEtEctIon oF IBh VIrus usIng pcr
The extracted DNAs were tested to detect FAdVs using HotStartTaq® Plus Master Mix Kit (QIAGEN, USA). 2µl of extracted DNAs was amplified in 20 µl of the final volume of PCR Master Mix containing 1.5 mM MgCl 2 , 200 µM of each dNTP, 1 unit HotStartTaq Plus DNA polymerase, and 10 µM of each primers set (Table 1). Thermo-cycling profile was at 95°C for 5 min for enzyme activation and initial denaturation followed by 35 cycles at 94°C for 30 sec, 60°C for 30 sec and 72°C for 45 sec and a final extension step at 72°C for 10 min. The amplified PCR products were electrophoresed in 1.2% agarose gel, stained with ethidium bromide and photographed under UV light. Molecular identification of viral IBH via Gene sequencing: The entire ORF of Hexon gene was amplified using three pairs of primers that were chosen according to sequence database and analyzed by Oligo Analyze 3.1 IDT Technologies, Germantown, MD, USA. These primers were synthesized by Macrogen, Korea (Table 1).
For entire Hexon gene ORF amplification, the extracted DNA was amplified using HotStartTaq® Plus Master Mix Kit (QIAGEN, USA). 2µl of extracted DNAs was am-plified in 20 µl of the final volume of PCR Master Mix containing 1.5 mM MgCl 2 , 200 µM of each dNTP, 1 unit HotStartTaq Plus DNA polymerase, and 10 µM of each primers set (Table 1). Thermo-cycling profile was at 95°C for 5 min for enzyme activation and initial denaturation followed by 35 cycles at 94°C for 30 s, the annealing temperatures were 61°C, 52°C, 58°C for 30 s (HexA, B, C respectively) and 72°C for 45 sec and a final extension step at 72°C for 10 min. The amplified PCR products were electrophoresed in 1.2% agarose gel stained with ethidium bromide and documented using ultraviolet gel documentation system (BIORAD®, California, USA).
PCR products specific for Hexon gene were purified from the agarose gel using Montàge DNA gel extraction kit (Millipore, USA) and sequenced in an automated ABI 3730 DNA sequencer (Macrogen®, Korea). The sequences were aligned by the Clustal W method and compared with corresponding sequences available in Genbank by BLAST web tool of the Genbank. A phylogenetic tree was made using MEGA version 6.0 software.

ExtErnal ExamInatIon and post-mortEm lEsIons
Birds were depressed with mortalities lasts for 3-4 days.
Sometimes watery diarrhea. Friable, enlarged liver with echymotic haemorrhage were the most common lesion during necropsy (Figure 1).

molEcular dEtEctIon oF IBhV usIng pcr
Out of 40 tested clinical liver samples, only three were PCR positive using the screening primer sets targeting the hexon gene producing 890bp   The highest nucleotide similarity among the examined strains and reference FAdVs was 97.7% with FAdV-2 (FAdV-D). The divergence between field strains was very low, with sequence similarity ranged from 97.6-99.6% (Table 2). The highest nucleotide similarity between reference strains of the different types was 78.6% between different strains of FAdVs-D and strains belongs to FAdVs-6 (FAdV-E). Moreover, our tested aviadenoviruses showed high similarity and clustered with the Saudi FAdVs when aligned and assembled, Figure (2).
The recently isolated strains (EG/101, EG102 and EG/103) showed high divergence from the FAdVs strains MMR-T1 and MMR-B15 belong to FAdVs subtype E isolated during 2015 from Egypt. The similarity was low, it was varied from 59.9 and 61.5%. The higher percentage of identity was 93.1% between our three isolates and FAdV-D/kfr4 which isolated during 2018 from a geographically related region (Table 3).

dIScuSSIon
Recently, fowl aviadenoviruses are reported worldwide in many avian species including wild birds and domestic poultry (Hess, 2013;Mohamed et al., 2018). In Egypt, many authors suggested its incrimination in losses among broilers and more investigation become of necessary to highlight the role of the disease (Radwan et al., 2018;El bestawy et al., 2020). Clinically the diseases was first reported in Mainland China in broilers of three to five weeks age, and occasionally in eggs producing flocks layers) aged ten to twenty weeks of age and always described as HHS (Zhao et al., 2015). During the last 10 years, IBH outbreaks with multiple serotypes occurred all over the world (Schachner et al., 2016(Schachner et al., , 2017. In our research, we demonstrated IBH related FAdVs in clinically infected birds exhibited the typical signs and lesions of IBH infections in chickens (Shane, 2000;Chandra et al., 2000;Laanani et al., 2015). Three out of 40 tested samples were positive for Adenovirus serotypes using primers that target Hexon gene. Elbestawy et al. (2020) while working on 37 broiler flocks reported 17 as positive. Radwan et al. (2018) reported that two out of nine commercial broiler flocks (3-5 weeks old) were positive. Mittal et al. (2013) stated a positive relation between number of outbreaks and immune suppressive diseases.
In our study, three strains were detected in chickens with IBH infection, and the entire hexon gene; the major capsid protein and has groups and sub-group determinants (Harrach et al., 2012;Schachner et al., 2016) were sequenced and analyzed. In addition, neighbor joining based phylogenetic tree was constructed using assemble sequences from reference FAdV isolates representing different geographical localities, the analysis showed that the 3 isolates (EG101/2018, EG102/2018 and EG103/2018) were clustered into serotype-2, FAdV-D species.
The entire gene sequences of hexon gene were analyzed. The results revealed very low divergence among the 3 strains ranged from 0.4 to 2.6%. Marek et al. (2013) stated a closer genomic relationship among strains representing the same serotype. The highest nucleotide similarity among the tested and reference strains was 97.7% with FAdV-2 (FAdV-D). Elbestawy and collegue, (2020) characterized 17 strains isolated from different localities in Egypt as fowl adenoviruses D. The study also indicated that the virus is still circulating in the Egyptian poultry flocks and may causes huge losses.
The studied strains (EG/101, EG102 and EG/103) showed high divergence from the FAdVs strains MMR-T1 and MMR-B15 belong to FAdVs subtype E, isolated during 2015 from Egypt. The similarity was low, it was varied Journal of Animal Health and Production 2020 | Volume 9 | Special Issue 1 | Page 88 from 59.9 and 61.5%. The higher percentage of identity was 93.1% between our three isolates and FAdV-D/kfr4, which isolated during 2018 from a geographically related region. It indicating, the emergence of species D to Egypt in last two years.
Interestingly, the studied strains showed high similarity with very low distance with Saudi strains. The three isolates were clustered with FAdVs strains isolated from Europe, Australia, India and Saudi Arabia in the same clade; this increase the proposition that variances in hexon gene are serotypes related and not locality related (Schachner et al., 2016).
Other fowl adenoviruses type E and type D also reported in Egypt by Radwan et al. (2018) and Mohamed and Kamel, (2019) respectively. The FAdVs types D and E are slightly divergent (Marek et al., 2010), this may raise the need to bivalent or homogenous vaccine to prevent further loses among birds.

concluSIon
The FAdV-2 (species D) is the causative agents of IBH in broiler chickens with 3-5 weeks old age and continue causes periodical outbreaks. Continuous molecular and serological surveillance of FAdVs may provide a worthy database for vaccines and an efficient control strategy development. The variance and divergence among FAdVs types isolated from infected flocks in Egypt should be considered during vaccine selection and manufacturing.