Molecular confirmation of fowl adenovirus associated with inclusion body hepatitis in upgraded variety native chicken in Tamil Nadu

Introduction

Poultry farming contributes significantly to food security and rural livelihoods, particularly through the rearing of native (ungraded) chicken varieties, which are well adapted to local agro-climatic conditions and low-input management systems. Despite their perceived hardiness, native chickens are increasingly affected by infectious diseases that compromise productivity and survivability. Among these, inclusion body hepatitis (IBH) has emerged as an important viral disease affecting chickens across diverse production systems. Inclusion body hepatitis is an acute disease characterized by sudden mortality, depression, and hepatic lesions, primarily affecting young chickens. The disease is caused by fowl adenoviruses (FAdVs), which belong to the genus Aviadenovirus under the family Adenoviridae (Hess, 2000) [4] . FAdVs are double-stranded DNA and non-enveloped viruses, and are widely prevalent in worldwide in poultry populations. Several studies have demonstrated the association of FAdVs with IBH, hydropericardium-hepatitis syndrome, gizzard erosion, and other disease conditions in chickens (Sharma et al., 2023; Matos et al., 2024) [15,5] . Although IBH was traditionally considered a disease of commercial broiler and breeder flocks, recent investigations have reported its occurrence in backyard and native chicken populations, indicating wider circulation of FAdVs than previously recognized (Dutta et al., 2022; Ramakrishnan et al., 2024) [3,12] . Native chickens are often reared under semi-intensive or backyard systems with limited biosecurity and minimal vaccination coverage, which may facilitate both horizontal and vertical transmission of FAdVs (Smyth and McNulty, 2008) [16] . Subclinical infections in such flocks may serve as reservoirs, posing a risk to organized poultry production systems. Diagnosis of IBH is conventionally based on clinical signs, gross pathological changes, and histopathological demonstration of characteristic intranuclear inclusion bodies in hepatocytes. However, these methods are not definitive, as similar hepatic lesions may be caused by other viral or toxic etiologies. To overcome these limitations, molecular diagnostic techniques, particularly polymerase chain reaction (PCR), have been widely adopted for the sensitive and specific detection of FAdV DNA from clinical samples (Meulemans et al., 2004; Sharma et al., 2023) [6,15] . PCR targeting the hexon gene has proven especially useful for confirmation and molecular epidemiological studies of FAdVassociated IBH. Despite increasing reports of IBH in commercial poultry, molecular confirmation of fowl adenovirus infection in ungraded native chickens remains poorly documented, particularly in India. Therefore, the present study was undertaken to molecularly confirm fowl adenovirus associated with inclusion body hepatitis in ungraded variety native chickens, thereby contributing to the understanding of FAdV circulation and emphasizing the need for molecular surveillance in native poultry populations.

Materials and Methods

Study area and birds

The present investigation was carried out in an upgraded native chicken flock from Namakkal district, Tamil Nadu. Affected birds showed clinical signs such as reduced feed intake, ruffled feathers, and diarrhoea. The recorded mortality in the affected flock was 3.0%.

Sample collection and necropsy

A total of five recently dead birds were subjected to detailed necropsy examination. Gross lesions were recorded with special reference to liver changes suggestive of inclusion body hepatitis. Liver tissue samples were aseptically collected from all five birds for molecular and histopathological examination.

Histopathological examination

Representative portions of liver tissue were fixed in 10% neutral buffered formalin, processed routinely, embedded in paraffin, sectioned at 4-5 µm thickness, and stained with hematoxylin and eosin (H&E). The stained sections were examined under a light microscope for characteristic histopathological lesions, including basophilic intranuclear inclusion bodies and hepatic cellular degeneration.

Polymerase chain reaction (PCR)

Genomic DNA was extracted from liver tissue samples using a commercial Qiagen DNA extraction kit following the manufacturer's instructions. The extracted DNA was quantified and stored at -20 °C until further use. PCR was performed for the detection of fowl adenovirus (FAdV) targeting the L1 loop region of the hexon gene. Amplification was carried out using hexon A and hexon B primers, which amplify an expected product size of 897 bp. The PCR cycle conditions were standardized as per Meulemans et al., (2001) [8] . PCR products were resolved by electrophoresis on 1.5% agarose gel containing ethidium bromide. The amplified products were visualized under a UV transilluminator and documented using a gel documentation system.

Virus isolation in chicken embryo liver cells

Virus isolation was attempted from PCR-positive liver samples using primary chicken embryo liver (CEL) cell cultures (Barua and Rai, 2003) [1] . The inoculated cultures were observed daily for cytopathic effects (CPE) such as cell rounding, detachment, and honeycomb appearance of liver cells.

Results and Discussion

In this study, the affected upgraded native chicken flock showed clinical signs including reduced feed intake, ruffled feathers, and diarrhoea. The overall mortality recorded was 3.0%. These findings are consistent with earlier reports indicating that fowl adenovirus (FAdV) associated inclusion body hepatitis generally causes low to moderate mortality but significant economic losses due to poor performance and sudden deaths, particularly in young chickens (McFerran and Adair, 2003; Schachner et al., 2018) [7,13] . Necropsy of five dead birds revealed enlarged, pale, friable livers with focal to diffuse hemorrhages. Such hepatic lesions are considered characteristic of inclusion body hepatitis and have been widely documented in FAdV infections (Nakamura et al., 2011; Matos et al., 2016) [10,5] . The absence of significant lesions in other organs supports the primary hepatic tropism of the virus. Histopathological examination of liver sections revealed hepatocellular degeneration, necrosis, and the presence of prominent basophilic intranuclear inclusion bodies within hepatocytes (Fig. 1). These intranuclear inclusions are pathognomonic for adenoviral infection and represent sites of viral replication (Ojkic et al., 2008) [11] . Similar histological findings have been consistently reported in IBH outbreaks in commercial and native chicken flocks in India and other countries (Dahiya et al., 2002; Mittal et al., 2014) [2,9] . PCR amplification targeting the L1 loop region of the hexon gene yielded an expected amplicon size of 897 bp in all five liver samples, confirming the presence of fowl adenovirus infection (Fig. 2). The hexon gene is highly conserved and widely used for molecular detection and characterization of FAdV strains (Meulemans et al., 2001; Schachner et al., 2016) [8,14] . The detection of FAdV by PCR corroborated the gross and histopathological findings observed in the present study. [17,13] . The findings emphasize the need for regular surveillance, molecular diagnosis, and implementation of appropriate biosecurity and vaccination strategies to prevent economic losses in native and upgraded poultry systems.

Acknowledgement

AcknowledgementThe authors express sincere thanks to Tamil Nadu Veterinary and Animal Sciences University (TANUVAS), Chennai -51, for all the necessary support.

References

1. Barua S, Rai A. Cultivation of fowl adenovirus 4 in chick embryo liver cell culture and purification of the virus by ultracentrifugation. Indian J Comp Microbiol Immunol Infect Dis. 2003;24(2):195-196.
2. Dahiya S, Srivastava AK, Verma SC, Gupta NC. Inclusion body hepatitis in broiler chickens in India. Indian J Vet Pathol. 2002;26(2):95-98.
3. Dutta S, Chakraborty A, Das S, Bandyopadhyay S. Molecular detection and characterization of fowl adenovirus associated with inclusion body hepatitis in backyard chickens. Indian J Vet Pathol. 2022;46(2):8994.
4. Hess M. Detection and differentiation of avian adenoviruses: A review. Avian Pathol. 2000;29(3):195206.
5. Matos M, Grafl B, Liebhart D, Hess M. Fowl adenoviruses-A review of molecular diversity, pathogenicity and diagnosis. Viruses. 2024;16(2):215.
6. Meulemans G, Boschmans M, Van den Berg TP, Decaesstecker M. Polymerase chain reaction combined with restriction enzyme analysis for detection and differentiation of fowl adenoviruses. Avian Pathol. 2004;33(2):164-170.
7. McFerran JB, Adair BM. Avian adenoviruses-A review. Rev Sci Tech. 2003;22(2):589-602.
8. Meulemans G, Boschmans M, Van den Berg TP, Decaesstecker M. Polymerase chain reaction combined with restriction enzyme analysis for detection and differentiation of fowl adenoviruses. Avian Pathol. 2001;30(6):655-660.
9. Mittal D, Jindal SK, Tiwari SK, Narang RK. Molecular characterization of fowl adenovirus associated with inclusion body hepatitis in broiler chickens. Vet World. 2014;7(4):205-209.
10. Nakamura K, Mase Y, Yamaguchi M, Shibahara K, Yuasa M. Pathological study of inclusion body hepatitis caused by fowl adenovirus in broiler chickens. Avian Dis. 2011;55(2):273-280.
11. Ojkic D, Guo Y, Swinton E, Vallieres B, Brash SK. Characterization of fowl adenoviruses isolated from chickens with inclusion body hepatitis in Ontario, Canada. Avian Dis. 2008;52(1):26-34.
12. Ramakrishnan S, Balasubramaniam GA, Kumanan K, Porteen K. Molecular evidence of fowl adenovirus infection associated with inclusion body hepatitis in native chickens of South India. J Vet Anim Sci. 2024;55(1):12-18.
13. Schachner A, Hess M, Marek S. Avian adenovirus infections in chickens-A review. Viruses. 2018;10(12):701.
14. Schachner A, Marek S, Jaskulska C, Bilic M, Hess M. Detailed molecular analyses of the hexon loop-1 region allow precise classification of fowl adenoviruses and reveal potential pathogenicity markers. PLoS One. 2016;11(5):e0155374.
15. Sharma P, Singh SD, Kataria JM, Dhama K. Hexon genebased molecular detection of fowl adenovirus associated with inclusion body hepatitis in chickens. Indian J Anim Res. 2023;57(4):456-461.
16. Smyth JA, McNulty MS. Adenoviridae. In: Saif YM, Fadly AM, Glisson JR, McDougald LR, Nolan LK, Swayne DE, editors. Diseases of Poultry. 12th ed. Blackwell Publishing; 2008. p. 251-266.
17. Toro H, Gonzalez C, Cerda L, Hess S, Reyes E. Vertical induction of the inclusion body hepatitishydropericardium syndrome with fowl adenovirus and chicken anemia virus. Avian Dis. 2000;44(1):215-222.