First Evidence of Fowl Adenovirus Induced Inclusion Body Hepatitis in Chicken in Bangladesh

Background The livestock sector contributes 1.90% to the GDP in Bangladesh during 2021–22. Poultry is one of the important subsectors struggling with diseases. Fowl adenoviruses (FAdVs) cause numerous diseases resulting in economic losses to the poultry industry worldwide. Several FAdV serotypes cause inclusion body hepatitis in chicken. Although FAdV infection was suspected, there was no confirmatory report from Bangladesh. The study was conducted to investigate the FAdV infection and antibodies in chicken. Methods A total of 50 samples, each composed of liver and spleen, were collected from different chickens of Gazipur, Dinajpur, and Panchagarh district. Each location belongs to A, B, and C poultry zones of Bangladesh, respectively. Viruses were detected by real-time PCR and conventional PCR. Blood samples (n = 303) were collected at the beginning and after the recovery from infection and tested by indirect ELISA. Sequencing of PCR products was done for serotyping and phylogenetic analysis. Results Clinical signs were observed including anorexia, drowsiness, ruffled feathers, reduced body weight, lack of uniformity, and high mortality (15–25%). Enlarged friable liver with yellow to tan color mottled with the focal soft area, fluid in pericardial sac, swollen and hemorrhagic kidneys, enlarged congested spleen and pancreas, etc. were found on postmortem examination. FAdVs were detected in 90% of the flocks except commercial layer flock from Dinajpur. Three serotypes, namely, 8b (70%), 11 (10%), and 5 (10%) were detected. Anti-FAdV antibody was detected in 80% flocks at the beginning of infection and in 90% of the flocks after recovery from infection. The antibody titer increases significantly (p < 0.05) after recovery from infection. Phylogenetic analysis revealed that the Bangladeshi FAdVs have close identity with viruses from Asia, Europe, and South and North America. Conclusions These findings suggested that several introductions of FAdVs were taken place in Bangladesh. To combat the disease, vaccination along with maintenance of biosecurity is essential.

FAdVs cause a variety of diseases in chicken, and inclusion body hepatitis (IBH), hydropericardium hepatitis syndrome (HHS), and gizzard erosion and ulceration are most important of them. FAdV-1 of species A and FAdV-4 of species C are the causative agents of HHS and have been isolated from most cases of gizzard erosion and ulceration [8,9]. IBH is an acute disease caused by FAdV-D serotype 2, 3, 9, and 11 and FAdV-E serotype 6, 7, 8a, and 8b [10,11]. Te virus is transmitted vertically as well as horizontally [12], distributed throughout the world [13], and causes infection mainly in broiler chickens of 3-7 weeks of age, resulting sudden increase of mortality which occasionally might be as high as 30% [14][15][16]. IBH outbreaks have been confrmed in diferent countries and serotypes 2, 4, 8a, 8b, and 11 have been reported as the most frequent causal agents [17][18][19]. Te pathogenicity of recently isolated strains, mainly of FAdV-8b and 11 have been investigated with inconsistent results. Some studies had shown less to severe clinical signs and mortalities when infecting chickens of diferent ages, like from 1 day to 3 weeks of age, with FAdV serotypes 8b and 11 [20,21].
Phylogenetic analyses of partial hexon gene sequences are an adequate and quick method for diferentiation and genotyping of FAdVs [22]. Polymerase chain reaction (PCR) with primer sequences based on the hexon gene or the 52 K gene is useful for virus identifcation [23][24][25][26]. Besides, PCR together with DNA sequencing and/or restriction enzyme analysis has been used for FAdVs typing [27]. Real-time PCR and subsequent high-resolution melting (HRM) curve analysis of three regions of the hexon gene were also reported and assessed for their potential in diferentiating 12 FAdVs reference serotypes [28]. Also, a SYBR green-based real-time polymerase chain reaction is reported for detection and quantifcation of all FAdVs [26].
Bangladesh is largely an agricultural country and livestock is an integral part of the complex farming system. Livestock is not only a source of meat protein but also a major source of farm power services as well as employment. Te livestock subsector provides full time employment for 20% of the total population and part-time employment for another 50% [29]. Poultry is a rapidly growing sector, and its expansion is being driven by the rising incomes and shift in industry structure. In Bangladesh, there are roughly 30.41 million chickens, and poultry farms are expanding at a pace of 15% annually. Poultry meat accounts for a sizable 37% of Bangladesh's total meat production [30]. At constant prices, the contribution of livestock sector to the GDP in the fnancial year 2021-22 was 1.90% and the contribution of livestock to the overall agricultural sector was 16.52 percent [31]. Te greatest impact of poultry in sustainable development goal designed to help the poor is enhancement of livestock-production systems [32]. However, the poultry farmers still facing several challenges including disease outbreak causing huge loss in poultry production due to high morbidity and mortality [33]. Outbreak of several diseases was like Newcastle disease (ND), infectious bursal disease (IBD), infectious bronchitis (IB), egg drop syndrome (EDS), and fowl cholera (FC), etc. occurred every year, resulting enormous economic losses [34].
Based on the results of an agar gel difusion test FAdV infection of broiler parents was reported from Bangladesh in 2002 [35]. However, no one reported the prevalence of this disease based on molecular analysis. Farmers and veterinary professionals in Bangladesh have noticed clinical signs including unexpected mortality, slower growth, and greater feed conversion ratio over the past few years, and they assume the IBH virus may be the cause [33]. But none of the suspected cases were confrmed by laboratory tests including molecular technique [33]. Te disease is thought to have invaded Bangladesh and is currently spreading throughout the poultry industry, especially among broiler chickens. Investigating the disease-causing agent and developing preventative measures are thus necessary. In this study, we have evaluated and reported the FAdVs in suspected chickens from three geographically distinct regions in Bangladesh using clinical, virological, and serological methods. Tis is the frst report to date that shown virological including sequencing and phylogenetic analysis as well as serological evidences of FAdV infection in chicken in Bangladesh.  [36] riven the country into three zones: A (105-1212 chickens per square kilometer), B (344-1108 chickens per square kilometer), and C (252-868 chickens per square kilometer) based on the density of chickens. Tis study includes Dinajpur Sadar upazila of Dinajpur district from zone A, Tetulia and Atwary Upazilas of Panchagarh district from zone B and Gazipur Sadar Upazila of Gazipur district from zone C ( Figure 1).

Collection of Samples.
A total of ten focks of fve different farms located in geographically distant three locations viz. Gazipur Sadar Upazila, Dinajpur Sadar Upazila, Tetulia, and Atwar Upazilas of Panchagarh district, were included in this study. Te focks include one commercial layer fock (5000 birds) from Dinajpur district, four broiler breeder focks (10,000 birds/fock) from Panchagarh district and three broiler focks (2000 birds/fock) and two broiler breeder focks (10,000 birds/fock) from Gazipur district ( Table 1 and Figure 1). From each fock fve samples were collected. Each sample composed of liver (∼2 gm) and whole spleen of a suspected dead chicken. So, from ten focks a total 50 samples were obtained for virological investigation. Besides, 303 blood samples, 156 samples at the beginning of suspected infection and 147 samples after recovery from suspected infection were collected from these ten focks to check the anti-FAdV antibody titer (Table 2).

Nucleic Acid (DNA) Extraction.
Tissue samples, consisting of liver and spleen, were homogenized in phosphatebufered saline (pH 7.0-7.4) with 1 : 10 w/v ratio. Te homogenates were kept at −80°C for 2-3 hours and thawed at room temperature for ∼30 min. Freezing and thawing cycle was repeated for three times and fnally centrifuged at 8000 × g for 3 min. Supernatant was collected and used for DNA extraction. PureLink genomic DNA Mini Kit (Invitrogen, USA) was used to extract DNA as per manufacturer's instruction. Te purity of the extracted DNA was checked by NanoDrop ™ 1000 Spectrophotometer (Termo Fisher Scientifc, USA) and preserved at −20°C until further analysis.

Detection of FAdVs Molecular
Test. Samples were principally tested by realtime PCR (Rt-PCR). However, some samples were also tested by conventional PCR. Rt-PCR was also done for serotyping of FAdV serotype-4 and 8b. For Rt-PCR, Taqman chemistry based FAdV Pockit kit (Gen-eReach Biotechnology Corp, Taiwan) was used for detection of FAdV-A to FAdV-E and FAdV-4 and FAdV-8b serotype specifc kits were used for serotyping. Rt-PCR was carried out on a 7500 FAST Real-Time PCR System (Applied Biosystems) with ABI 7500 software (Version 2.3). Te reaction was set in 25 μL scale and consisted of 2 μL template DNA and 23 μL of premix reagents. Te thermal profle consisted of the 40 cycles of 3 min at 95°C, 15 sec at 93°C and 1 min at 60°C.
In case of conventional PCR, fragment of loop 1 (L1) region of the hexon gene of FADV was amplifed by using the primers-F: 5′-ATGGGAGCSACCTAYTTCGACAT-3′ and R: 5′-AAATTGTCCCKRAANCCGATGTA-3′ described earlier [37]. Te PCR reaction mixture was in volume of 25 μL containing 12.5 μL of master mix (Termo Scientifc, Waltham, MA, USA), 2 μL (10 μM) of each forward and reverse primer, 6.5 μL of nuclease free water and 2 μL of template DNA. Negative control, all reagents except test sample, was run along with test sample and total volume was adjusted with nuclease free water. Amplifcation was carried out in a 2720 thermal cycler (Applied Biosystems, UK). Te thermal profle was 10 min at 95°C for initial denaturation followed by 40 cycles of denaturation at 95°C for 15 sec, annealing at 56°C for 30 sec, and extension at 72°C for 45 sec, and a fnal extension at 72°C for 5 min. Amplicons were analysed in ultrapure 1.5% agarose gel (Invitrogen), containing SYBR Safe DNA gel nucleic acid stain (Termo Fisher Scientifc) using 1X TBE electrophoresis running bufer.

Sequencing and Phylogenetic Analysis.
Sequencing was done with Applied Biosystems 3130 Genetic Analyzer using 20 μL reaction volume containing approximately 10 ng purifed PCR product as template, 5X ready reaction premix 4.0 μL, Big Dye terminator bufer 2.0 μL, primer 0.32 μL and ultrapure water to make 20 μL. Te primers used for amplifcation was also used for sequencing. Te sequence was read from both directions. Te electropherogram analysis and multiple sequence alignment (MSA) were done using MEGA-11 software [38]. Te evolutionary history was   inferred using the Neighbor-Joining method and Tamura-Nei model [39]. Initial tree for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-Nei model, and then selecting the topology with superior log likelihood value. Evolutionary analyses were conducted in MEGA11 [38].

GenBank Accession Number.
Nucleotide sequences generated in this study were submitted to GenBank and obtained accession number (Table 1).

Detection of Anti-FAdV Antibody by Indirect ELISA.
Serum samples were investigated for the detection of antibody to FAdV by indirect ELISA using a commercial FAdV Group-1 antibody test kit (Biochek, Reeuwijk, Holland) following the manufacturer's instructions. Initially, 5 µL serum was mixed with 245 µL of sample diluent which gives serum a dilution of 1 : 50. Ten, 50 µL diluted serum was added in to the antigen coated palate having 50 µL sample diluent (1 : 100) except two negative and two positive control wells. About 100 µL/well positive and negative control was added. Te plate was covered with lid and incubated at room temperature (22-27°c) for 30 minutes. Ten, the mixture of each well of the plate was aspirated and washed four times with wash bufer (350 µL per well). Te plate was the inverted and tap frmly on absorbent paper to remove residual wash bufer. Ten, 100 µL of conjugate was added and incubated at room temperature (22-27°c) for 30 minutes after covering with lid. Up on incubation, the contents of wells were aspirated and washed four times with wash bufer (350 µL per well). Residual bufer was removed as mentioned earlier.
Ten, 100 µL of substrate was added and incubated at room temperature (22-27°c) for 15 minutes after covering with lid followed by addition of 100 µL of stop solution into each wells. Te absorbance value was taken at 405 nm. Te sample/positive (S/P) ratio was employed using following formula to proceed for interpretation of results.
Moreover, the following equation was used to relate the S/P of a sample at 1 : 100 dilutions to an end point titer.
Samples with an S/P ratio of 0.5 or greater were considered as positive for anti-FAdV antibody. In other words, samples having titer 1071 or greater are considered as positive.

Statistical Analysis of Data. Te Statistical Package for
Social Science (SPSS) version 20 was used to analyze the collected data. Te descriptive results were presented as mean and standard error. Unpaired T test was used for two variables (BE: Beginning of Exposure and AE: After Exposure). Te diferences were considered statistically signifcant at p values < 0.01 or < 0.05.

Clinical Findings.
Ten focks of broilers (n = 3), broiler breeders (n = 6), and commercial layer (n = 1) from fve farms of three geographically distant locations, Gazipur, Dinajpur, and Panchagarh districts, of Bangladesh were investigated. Veterinary practitioners and farmers reported that they observed drowsiness, rufed feathers, high mortality, etc. in their farms and taken veterinary care with history of no response to treatment even with diferent antibiotics. Broiler focks B1, B2, and B3 were derived from one farm and broiler breeder focks BB1 and BB2 were derived from another farm in the Gazipur district. On the other hand broiler breeder focks BB3, BB4 and BB5, BB6 were originated from the Atwary and the Tetulia Upazila, respectively, in the Panchagarh district. Distances among the multiple focks of each of the farms were within 50-80 feet. Only single commercial layer fock (CL01) derived from Dinajpur Sadar Upazila. Clinical signs were like anorexia, depression, reluctant to move, drowsiness, reduced body weight gain, lack of uniformity, respiratory distress, etc. in chicks of up to fve weeks of age were also noticed during farm visit (Figure 2). Among the studied focks (n = 10) morbidity was found 10%-65% whereas mortality varies from 5% to 30%. However, morbidity and mortality in FAdV positive focks were within 23%-65% and 12%-30%, respectively. Higher morbidity (65%) and mortality (30%) was found in a broiler breeder fock of Tetulia Upazila, Panchagarh, and later serotype 11 was detected from that fock. Flocks afected with serotype 8b had 23%-45% morbidity and 15%-25% mortality among the focks. Lowest morbidity (10%) and mortality (5%) was found in the commercial layer chicken fock of Dinajpur Sadar. However, FAdV was not detected from this fock. Comparatively, higher morbidity (35%-65%) and mortality (12%-30%) was found in broiler breeder focks than broiler focks where morbidity 25%-30% and mortality was 15%-20%, respectively. Te mortality reduced gradually 2-3 weeks later.

Postmortem Lesions.
Hemorrhages at skeletal muscle, an enlarged, friable liver with a yellow to tan color that was mottled with a localized soft area, and even petechial and ecchymotic hemorrhages beneath the capsule and parenchyma were discovered during the postmortem examination. Accumulations of fuid in pericardial sac were also found. Swollen and hemorrhagic kidneys were frequent, and the enlarged congested spleen and pancreas were also noticed. Figure 2 shows the representative images of postmortem lesions of diferent ages of chickens died of infection of FAdVs.

Prevalence of FAdVs.
FAdVs were investigated in ffty samples originated from diferent broiler focks (n � 3), broiler breeder focks (n � 6) and commercial layer fock (n � 1) from Gazipur (n � 5), Dinajpur (n � 1), and Panchagarh (n � 4) districts of Bangladesh. At farm level the prevalence of FAdVs was 80% (4/5), whereas prevalence was 90% (9/10) at fock level. By using Rt-PCR, 37 out of 50 samples were confrmed to be positive for FAdVs, representing a 74% prevalence rate ( Table 1). Some of the samples were also tested by conventional PCR. By conventional PCR, ∼600 bp DNA fragment from loop-1 region of the hexon gene was amplifed and a representative image is shown in Figure 3. Tere was complete agreement between the two tests' results (data not shown). Viruses were detected in samples of all broiler and broiler breeder focks collected from Gazipur and Panchagarh districts of Bangladesh. Ages of virus positive chickens were within 7-15 days. FAdV could not be detected in samples of commercial layer fock collected from Dinajpur district.

Phylogenetic Analysis. Evolutionary analyses of FAdVs
were conducted in MEGA11 using the Neighbor-Joining method. Te viruses were separated into three distinct clusters representing three diferent serotypes of FAdVs (Figure 4). In the tree Bangladeshi FAdV serotype 8b, serotype 11 and serotype 5 were clustered separately with other viruses. About 99.91% nucleotide identity was found among Bangladeshi FAdV serotype 8b. On the other hand, 99.99% homology was found between two FAdV-5 serotypes. FAdV serotypes 5 of this study were found to have 100% homology at amino acid level. Similarly, 100% homology was also found in case of serotypes 8b. However, the distance between the serotype 5 and 8b was found 0.42%, between the serotypes 5 and 11 was 0.39% and between the serotypes 8b and 11 was 0.29%. Phylogenetic analysis revealed that the Bangladeshi FAdVs obtained from chicken has close identity with viruses from Asian (India, China 3.6. Anti-FAdV Antibody Prevalence. Anti-FAdV antibody was investigated by indirect ELISA in serum samples collected at the beginning of suspected infection (n � 156) and after recover from suspected infection (n � 147) and results are presented in Table 2. Irrespective of sampling time, the anti-FAdV antibody was found in 80% (8/10) and 90% (9/ 10) focks at beginning of infection and after recovery of infection, respectively. Samples originated from commercial layer fock of Dinajpur Sadar upazila was found negative for anti-FAdV antibody. At beginning of infection only 0-40% samples were found seropositive and their titer was very low (399 ± 81-885 ± 172). On the other hand after recovery of infection 53.33-100% samples were found seropositive and their titer was found very high and ranges from 1246 ± 172-9699 ± 832. Te proportional seroprevalence at sample level was higher (82.31%; 131/147) in case of samples obtained after recovery from suspected infection than that of samples taken at beginning of infection (16.67%; 26/156). Te anti-FAdV antibody titers difers signifcantly at 99% confdence level (p < 0.01) in one fock and at 95% confdence level (p < 0.05) in eight focks at beginning of infection and after recovery from infection ( Table 2).

Discussion
We have investigated three types of chicken-namely (i) Broiler, (ii) Broiler breeder and (iii) Commercial layer chicken. It is to be noted that usually broilers are reared in open-sided house with high density of birds. Generally crumble type feed is provided at starter stage and pellet at grower and fnisher stage. Biosecurity measures in broiler farms are limited and farmed very close to locality, highway, other commercial poultry operations, etc. On the other hand, broiler breeders are reared in controlled sheds and crumble feed is given from starter stage to whole production period. Te farm area is isolated from highway and other poultry operations. All in and all out system is usually followed with comprehensive biosecurity measures. In case of commercial layer, birds are reared in cage with crumble  feed as starter and mash feed in production period. Te farm area is usually isolated from highway and other poultry operations. Moderate biosecurity measures are ensured.
Te present study showed that FAdV infections have afected various ages of chickens with diferent clinical signs. However, clinical signs gradually declined after 2-3 weeks of frst appearance. Te virus might be transmitted by horizontally and/or vertically. We could not determine whether the route of virus transmission was vertical or horizontal. Mortality during IBH outbreaks may peaks within 3-4 days of clinical infection and can reach 10% and occasionally be as high as 30% [11,18,19,21]. Our fndings of 15-25% mortality, clinical signs and postmortem lesions ( Table 2) are in compliance with the fndings of above studies. Moreover, IBH induced 100% mortality in commercial broiler chickens was reported from Malaysia in 2005 [40]. We have found serotype 8b of genotype E as the most prevalent FAdV in studied chicken population in Bangladesh. Circulating other two serotypes included FAdV 5 and 11. It is reported that during last ten years, the most reported IBH-induced serotypes belong to species D and E [41]. Several other studies also reported that IBH outbreaks have been confrmed in diferent countries and serotypes 2, 4, 8a, 8b, and 11 was the most frequent serotype as a causal agent [15][16][17]. Serotype 11 of species E was found to cause higher morbidity and mortality in the present study. Tis fnding is also in line with the reports of Matos et al. [42] who reported that birds infected with FAdV serotype 8b and 11 showed more severe clinical signs and moralities than those inoculated with FAdV serotypes 2, 7, and 8a. Tough clinical signs like anorexia, reluctant to move, postmortem lesions viz. enlarged liver and mortality (5%) were found in commercial layer fock, we could not detect FAdV from this group of chicken. Even they were serologically negative at beginning of clinical sign or infection and after recovery from infection. Tese fndings suggest that other pathogen may be responsible for inducing those pathological conditions. Coinfection with FAdV serotype 8b and 5 was found in fock BB2. FAdV serotype 5 was detected from focks BB1 and BB2 of the same farm and the distance of the sheds of these two focks was only 50 feet. We could not detect serotype 5 from samples of other focks. So, there is no chance of contamination with serotype 5 from other focks. Additionally, there was little chance of laboratory contamination with serotype 8b. Because, the samples of fock BB2 were collected and tested in December 2021. Tere were no other samples collected and tested in the laboratory at nearby dates of December 2021. Hence, this coinfection was not due to contamination and laboratory originated. In a recent study, Liu et al. [43] showed coinfection with FAdV serotypes 4 and 8b. Tey reported similar clinical symptoms, mortality rates and degree of tissue lesions in coinfected birds as in single infection with serotype 4. However, they found that co-infection with FAdV-4 and FAdV-8a suppresses the replication and proliferation of FAdV-4 but enhances the replication and proliferation of FAdV-8a in the chicken liver. Tese means co-infection may worsen the clinical conditions of chicken.
Anti-FAdV antibody was detected in 80% of the focks at the beginning of infection and the ages of birds were within 7-16 days (Table 2). It indicates that this antibody is not due to exposure of the sampled birds to FAdVs. Tis antibody might be derived from mother. Exposure of parent birds to circulating feld FAdVs or use of FAdV vaccine either in combination with other vaccine or as single vaccine in parents might induced antibody in them and later passed to the chicks through egg yolk. Because Bangladesh does not formally use the FAdV vaccination. Antibody titer level was less at the beginning of infection and the titer increases signifcantly (p < 0.05) after recovery from infection (Table 2) is very usual fndings. Because exposed birds might develop high level of antibody after exposure. We were unable to determine whether the antibody is against serotype 8b, 11, or 5 due to the fact that we employed an indirect ELISA kit that only detects antibodies to Group-1 FAdVs and not specifc serotypes. We were unable to detect antibodies in the serum of samples taken from layer focks in the Dinajpur district, even after they had supposedly recovered from an infection. Tis result agrees with the results of the virus detection. Tus, additional causes may be responsible for the clinical signs, morbidity, and mortality seen in this layer fock.
Phylogenetically, Bangladeshi FAdVs were distinctly branched in to three clusters of serotypes 8b, 5, and 11 with viruses from Asian, European, and North American and South American countries of the world (Figure 4). Hence, it is assumed that multiple introduction of FAdV in Bangladesh may be happened. High homology among the serotypes may also suggest spread of viruses within the country. One FAdV-8 serotype of ours has been linked to an Indian virus ( Figure 4) and the virus was detected in the sample of broiler breeder fock BB2. Bangladesh's poultry industry was formed by the importation of chickens, eggs, feed materials, utensils, etc. from several countries where FAdVs were recorded. Te introduction of FAdVs in Bangladesh may be facilitated by the import of such materials. Te possibility of vertical transmission may is also existed because it is well known that FAdVs can be spread vertically through the embryonated egg and horizontally [12]. Strategies should be developed to tackle the disease as diferent FAdV serotypes are circulating in Bangladesh and infecting chicken. One of the control strategies may be vaccination of chicken with FAdV vaccine. In addition to vaccination, maintenance of farm biosecurity is essential to reduce the incidence of infection. We could not proceed for virus isolation, cytopathic efect (CPE) observation, histopathological studies of affected organs, etc. are the limitations of this study.

Conclusion
Tis study provided molecular proof that three FAdV serotypes were spreading in Bangladeshi poultry. Additionally, serological evidence was presented. Our results imply that FAdVs might be newly emerging pathogens. Preventive measures like immunization and the maintenance of strong biosecurity should therefore be taken into account in the fght against the disease.