1. Immunosuppressive disorders in poultry pose a significant threat to health and productivity. Ochratoxin A (OTA), a mycotoxin and fowl adenovirus serotype 4 (FAdV-4), the causative agent of hydropericardium syndrome and inclusion body hepatitis, are each known for their immunopathological effects. However, little is known about their combined impact on the immune system of poultry.2. This study investigated the synergistic immunosuppressive and pathological effects of OTA and FAdV-4 co-exposure in broiler chickens. A total of 144, one-day-old broiler chicks were randomly divided into six groups and exposed to OTA (200 ppb or 400 ppb), FAdV-4, or their combinations for 35 d. The FAdV-4 inoculum was PCR-confirmed and administered subcutaneously.3. Birds were monitored for clinical signs, mortality, feed intake and weight gain. Immune function was assessed via SRBC antibody titres, PHA-P - induced lymphoproliferation and carbon clearance assay. Gross and histopathological changes in lymphoid organs were also evaluated.4. The OTA exposure led to dose-dependent immunosuppression, with significant reductions (p ≤ 0.05) in humoral and cell-mediated responses, lymphoid organ atrophy and phagocytic dysfunction. The FAdV-4 infection alone caused immunosuppressive effects, which were significantly exacerbated when combined with OTA. Co-exposed birds exhibited severe lymphocytic depletion, pronounced histological lesions and increased mortality.5. The results demonstrated a synergistic immunosuppressive interaction between OTA and FAdV-4 in broiler chickens. This highlighted the need for routine mycotoxin screening in feed and molecular surveillance of adenoviral infections to prevent compounded immunosuppressive effects and economic losses in commercial poultry production.

The diseases caused by genotype VII Newcastle disease virus (NDV), H9N2 avian influenza virus (AIV), and fowl adenovirus serotype 4 (FAdV-4) continue to threaten the global poultry industry. However, no broad-spectrum vaccines provide simultaneous protection against these three pathogens. This study employed bioinformatics and immunoinformatics approaches to design a multi-epitope vaccine, named NFAF, which consists of B-cell, cytotoxic T lymphocyte (CTL) epitopes, and helper T lymphocyte (HTL) epitopes derived from hemagglutinin-neuraminidase (HN) and fusion (F) proteins of genotype VII NDV, hemagglutinin (HA) protein of H9N2, and Fiber2 protein of FAdV-4. The vaccine candidate was predicted to have non-allergenic properties, non-toxicity, high antigenicity, and favorable solubility. Each of its constituent antigenic epitopes has a high degree of conservation. Molecular docking demonstrated stable binding between NFAF and chicken Toll-like receptor (TLRs) and major histocompatibility complex (MHC) molecules. NFAF was expressed in soluble form in Escherichia coli and purified. Polyclonal antibodies against all three target viruses showed specific binding to NFAF. In vitro experiments revealed that NFAF effectively stimulated chicken peripheral blood mononuclear cells (PBMCs) and induced Th1, Th2, and pro-inflammatory cytokine production, confirming its immunogenicity, and increased the mRNA expression of the key signaling molecules MyD88 and NF-κB. These results suggested that NFAF could therefore be an efficacious multi-epitope vaccine against genotype VII NDV, H9N2, and FAdV-4 infections.

Molecular and pathological characterization of fowl adenovirus serotype 2 linked to inclusion body hepatitis in broiler chickens.

Fowl adenoviruses (FAdVs) are widespread viruses in poultry populations, responsible for several severe diseases, including Inclusion Body Hepatitis (IBH), Adenoviral Gizzard Erosion (AGE), and Hepatitis-Hydropericardium Syndrome (HHP). These diseases have been associated with significant economic and health impacts on poultry industries. Accurate detection and genotyping play a key role in the diagnosis of these infections, as different FAdV genotypes are associated with distinct disease syndromes and epidemiological patterns. In this study, we aimed to evaluate the clinical, analytical, and genotyping performance of the Hex L1 PCR combined with High-Resolution Melting (HRM) Curve analysis for investigating recent IBH and AGE outbreaks in Morocco. The study involved 26 clinical samples collected from broiler and layer poultry farms suspected with IBH or AGE. These samples were amplified using conventional PCR, real-time PCR/52 K test, and the Hex L1 PCR/HRM test. Field samples were also sequenced and compared with HRM curve analysis results to validate the genotyping accuracy of the Hex L1 PCR/HRM method. Phylogenetic analysis of the sequenced samples revealed several FAdV genotypes, including FAdV-11 and FAdV-8b in IBH cases, and FAdV-1 and FAdV-8a in AGE cases, highlighting the genetic diversity of circulating strains. The Hex L1 PCR/HRM method successfully amplified all 12 FAdV serotypes, demonstrating excellent reproducibility and repeatability, with coefficients of variation ranging from 0.19% to 1.82%. Moreover, this method showed a strong correlation with the real-time PCR/52 K test, achieving a high correlation coefficient of 0.9077. The HRM curve analysis accurately genotyped all the field samples, with results consistent with sequencing outcomes. In conclusion, this method provides a fast, sensitive, and reliable alternative for FAdV detection and genotyping. It enables universal detection, quantification, and genotyping in a single step, overcoming the limitations of traditional techniques, making it an ideal tool for sample screening, while sequencing validation is necessary for confirmation.

A novel species-specific multiplex PCR assay for the differentiation of five Fowl adenovirus species (A to E): Application to field surveillance.

Fowl adenovirus serotype 4 ORF1B protein suppresses type I interferon production by inhibiting IRF7 nuclear translocation.

ABSTRACT This paper describes the investigation of the epidemiology of fowl adenoviruses (FAdVs) among fast-growing meat-type breeder parent chickens in Sweden, based on serology, PCR and partial genome sequencing of samples collected during rearing and egg production. Blood samples (n = 1910) from breeder flocks at 16 (79 flocks), 24 and 36 (52 flocks) weeks of age were analysed for FAdV antibodies. Ninety-four percent of the flocks had seroconverted at 16 weeks of age, and at 36 weeks of age all 52 flocks were seropositive. From 35 of these flocks, dead-in-shell chicks (DIS, n = 949) from eggs laid at 26–27 and 36–37 weeks of age were sampled, and pooled liver and caecal tonsils were analysed by PCR. Nucleic acid from FAdV was not detected. Negative PCR results were confirmed by testing tissues from 62 broiler breeders and 80 DIS from four additional seropositive flocks, collected at age 24–26 weeks. From six other breeder flocks, liver and caecal tonsils were sampled from one bird (when available) per day during rearing from 1–112 days of age. From 6 weeks of age, in 4/6 flocks, FAdV species A and/or D were detected by PCR and partial hexon gene sequencing. In conclusion, FAdVs were detected during rearing in breeder birds but not in DIS. This suggests that vertical transmission of FAdVs was prevented by immunity acquired early in life. However, it should be noted that high antibody titres in breeder birds does not necessarily confer protection against vertical transmission. RESEARCH HIGHLIGHTS The majority of 16-week-old parent flocks were seropositive to FAdV. FAdV was not detected by PCR in dead-in-shell chicks from seroconverted flocks. FAdV-A and D were detected in broiler breeders during rearing from 6 weeks of age. Results suggest that vertical spread was prevented by immunity in breeder birds.

IntroductionDucks rank among the most important sources of animal protein globally, yet hepatic and splenic hemorrhage and necrosis in Muscovy ducks present a critical challenge to the poultry industry. The causes behind such diseases are often multifaceted, involving both established and newly emerging pathogens. MethodsIn this study, we leveraged metatranscriptomic sequencing to profile the intestinal viral communities of healthy and diseased Muscovy ducks from a Guangdong Province farm that experienced a hepatic and splenic hemorrhage in June 2024. ResultsOur findings revealed marked differences in viral community profiles between the two groups, with the diseased cohort exhibiting higher α-diversity. Taxonomic analyses across multiple levels uncovered significant variations in viral composition, including shifts in phylums like Uroviricota and families such as Demerecviridae. At the genus and species levels, several bacteriophages and eukaryotic viruses displayed differential abundance. Notably, Avian orthoreovirus was detected exclusively in diseased ducks, with a specific novel duck reovirus (NDRV) validated via RT-qPCR as a potential contributor to hepatic and splenic pathogenesis. In contrast, known pathogens such as Duck hepatitis A virus (DHAV) and Fowl adenovirus serotype 4 (FAdV-4) were not detected. DiscussionThis study constitutes the first comprehensive analysis of the Muscovy duck gut virome, highlighting NDRV as a potential causative agent and emphasizing the utility of metatranscriptomics in pathogen discovery.

High-resolution structural studies have mainly focused on two out of the six adenovirus genera: mastadenoviruses and atadenoviruses. Here we report the high-resolution structure of an aviadenovirus, the poultry pathogen fowl adenovirus serotype 4 (FAdV-C4). FAdV-C4 virions are highly thermostable, despite lacking minor coat and core proteins shown to stabilize the mast- and atadenovirus particles, having no genus-specific cementing proteins, and packaging a 25% longer genome. Unique structural features of the FAdV-C4 hexon include a large insertion at the trimer equatorial region, and a long N-terminal tail. Protein IIIa conformation is closer to atadenoviruses than to mastadenoviruses, while protein VIII diverges from all previously reported structures. We interpret these differences in light of adenovirus evolution. Finally, we discuss the possible role of core composition in determining capsid stability properties. These results enlarge our view on the structural diversity of adenoviruses, and provide useful information to counteract fowl pathogens or use non-human adenoviruses as vectors.

A novel recombinant serotype 4 fowl adenovirus efficiently protects both serotype 4 and 11 fowl adenovirus.

Gut microbiota dysbiosis by antibiotics increases FAdV-4 susceptibility and alters antiviral immunity in chickens.

Molecular characterization, pathology and innate cytokine responses associated with fowl adenovirus 8b in commercial chicken flocks in Bangladesh

Inclusion body hepatitis is one of the disease that cause economic loses in poultry industry. Infection with the fowl adenovirus leads to the illness known as inclusion body hepatitis. Affected chickens showed dullness, depression, ruffled feathers and mild greenish diarrhea with low morbidity and high mortality. Seventy-five samples were collected from various areas around Iraq, including Diyala, Karbala and Tikrit, that were thought to be infected with the fowl adenovirus. The infected chickens ranged in age from 25 to 45 days. After that viral nucleic acid (DNA) was isolated from collected livers. This was followed by testing using conventional PCR by amplification of the Loop 1 gene, which yielded a positive result for the presence of fowl adenovirus. Avian species are at risk of inclusion body hepatitis (IBH) infection, with subclinical infection being the most common type. The study reveals that the liver is affected by a disease that can be diagnosed through necropsy findings such as the presence of multiple genotypes such as FAdV-4, FAdV-8b and E. Molecular tools like PCR offer superior accuracy and sensitivity compared to seroprevalence techniques, which are not specific to the species or type of adenoviruses or their co-infecting illnesses. The current research aimed to clinical diagnosis of adenovirus in chickens and confirmed diagnosis by conventional PCR.

Viral diseases are a significant challenge facing the poultry industry, negatively impacting productivity and the agricultural economy. Among these viruses is fowl adenovirus (FAdV). The study aims to identify adenovirus in broilers in the Basrah province, Iraq. The study concentrated on clinical symptoms, postmortem lesions, histological abnormalities, and polymerase chain reaction (PCR) to validate a laboratory diagnosis. From October 2023 to April 2024, the present investigation collected 100 sick 10-day-old chickens from an unusual field in the Abu Sakhir district of Basrah. The poultry laboratory at the College of Veterinary Medicine, University of Basrah, handled the samples. Euthanized were chicks showing clinical symptoms. Every case was reviewed for gross lesions related to viscera. The livers of infected birds showed microhemorrhages or bruises with localized or diffuse areas of necrosis. The hearts showed a straw-coloured, diffuse fluid accumulation in the pericardial sac. The surface of the kidneys was enlarged and hemorrhagic. Microscopically, the liver exhibited basophilic intranuclear bodies, a necrotic area, and mononuclear cell infiltration. The heart showed vascular congestion in the pericardium, accompanied by inflammatory cell infiltration between the muscle fibres. Renal showed hemorrhage and degeneration of the epithelium, mononuclear cell infiltration and basophilic intranuclear bodies. PCR confirmed the presence of the adenovirus genome from infected birds.The study emphasizes the importance of early identification and accurate diagnosis to minimize the impact of FAdV on poultry health and productivity.

Fowl adenovirus (FAdV) can cause different poultry diseases with economic losses in the broilers and layers commercial farms. FAdV is currently classified into five species and 12 serotypes, disseminated in poultry flocks worldwide. The present study aimed to identify FAdV species and serotypes in Brazilian poultry farms. A total of 678 chicken flocks from the main Brazilian poultry-producing regions were evaluated for FAdV infection between 2020 and 2023. FAdV was detected by a real-time PCR targeting 52K gene and further genotyped by partial sequencing of the hexon gene followed by phylogenetic analyses. The results demonstrated that FAdV was detected in 72 flocks (10.6%). In 46 of these samples, FAdV species and serotypes could be identified, including three main species: Aviadenovirus ventriculi (FAdV-A = 15), Aviadenovirus gallinae (FAdV-D = 15) and Aviadenovirus hepatitidis (FAdV-E = 16). Phylogenetic analysis based on 173 partial hexon sequences (including sequences from this study, 44 previously sequenced in Brazil, and 86 data from other countries) revealed five separate clades for FAdV species. All Brazilian FAdVs were classified into the same three species reported above (FAdV-A = 19, FAdV-D = 34, FAdV-E = 37), and also in well-supported subclades for each serotype: FAdV-A1 (n = 19), FAdV-D9 (n = 1), FAdV-D11 (n = 33), FAdV-E6 (n = 1), FAdV-E8a (n = 33), FAdV-E8b (n = 3). Amino acid substitutions in the hyper variable regions (1, 2 and 3) and conserved motifs of the Hexon protein were further analyzed, enabling discrimination between closely related serotypes. This study demonstrates the circulation of different FAdVs in Brazil, highlighting FAdV-A1, FAdV-D9, FAdV-D11, FAdV-E6, FAdV-E8a and FAdV-E8b. The findings reported here also indicate genetic and amino acid diversity in the Hexon protein of the FAdVs in Brazilian poultry farms, which are of importance for molecular surveillance and poultry diseases control strategies.

Duck viral enteritis (DVE) is an acute and highly contagious disease that affects waterfowl such as ducks, geese and swans. Duck enteritis virus (DEV) is the pathogen, causing huge economic losses to waterfowl farming in recent years. Establishing a rapid, simple, and visual detection should facilitate the early identification of DEV. After the amplification primers and reaction conditions were optimized, three multienzyme isothermal rapid amplification (MIRA) methods—basic MIRA, MIRA–quantitative PCR (MIRA–qPCR) and MIRA–lateral flow dipstick (MIRA–LFD)—were established to detect DEV. Specificity analyses showed that the three MIRA methods specifically detected DEV, with no cross-reaction with fowl adenovirus serotype 4, novel goose astrovirus, Muscovy duck reovirus, avian influenza virus subtype H9, or duck circovirus. The basic MIRA reaction was completed in 30 min at 35 °C, requiring only a pair of primers. Detection with MIRA–qPCR or MIRA–LFD was completed within 20 min, and the limits of detection were 1 × 101 copies/μL for both. MIRA–LFD required no specialized instruments, and the results could be viewed directly with the naked eye. Compared with the traditional PCR, MIRA assays are simple, rapid, and effective and therefore more suitable for the field detection of DEV.

The widespread prevalence of different serotypes of fowl adenoviruses (FAdVs) has led to diverse vaccine demands, especially for subunit vaccines targeting FAdV-4 and FAdV-11, which cause hydropericardium-hepatitis syndrome (HHS) and inclusion body hepatitis (IBH), respectively. Although the Fiber protein is known to elicit robust immune protection, further exploration is needed to enhance the production of cross-neutralizing antibodies. This study utilized structural prediction and homology modeling techniques, employing domain-swapping strategy to integrate neutralizing epitope-containing amino acid sequences (274-451aa and 364-543aa) into the shaft domain of the Fiber protein. Two novel chimeric proteins were recombinantly expressed and developed into subunit vaccines: Fiber-C4/D11 and Fiber-D11/C4. Immunogenicity assessments revealed that the Fiber-C4/D11 vaccine group rapidly induced an antibody response against FAdV-11 within 7 days post-vaccination. By 28 days post-vaccination (dpv), the Fiber-C4/D11 vaccine group exhibited significantly higher levels of cross-neutralizing antibodies compared to the Fiber-D11/C4 group (p < 0.05). Challenge experiments demonstrated that both vaccines effectively alleviated clinical symptoms and prevented mortality in SPF chickens. Compared to Fiber-D11/C4, Fiber-C4/D11 significantly reduced body weight loss, liver lesions, viral titers in tissues, and viral shedding. Notably, no cross-neutralizing antibodies were detected following FAdV-4 or FAdV-11 infection, indicating a lack of natural cross-protection between the two serotypes. The chimeric vaccine addressed this gap, offering a promising multivalent approach to mitigate FAdV infections and advancing fowl adenoviral subunit vaccine strategies.

From 2015 to 2024, inclusion body hepatitis (IBH) was diagnosed in a total of 346 chicken necropsy cases submitted to the California Animal Health and Food Safety (CAHFS) laboratory system. The majority of cases (68%; 237/346) originated from a single commercial broiler company with a high density of poultry premises within the California Central Valley region. Reported mortality varied between 0.5% and 4.8% per day, and morbidity ranged between 5% and 10%. Clinical signs observed in affected flocks included depression, reluctance to move, ruffled feathers, greenish diarrhea, and, occasionally, icterus of skin and adipose tissue. Typical histopathological lesions included extensive hepatic necrosis, multifocal pancreatic necrosis, gizzard erosions, and glomerulonephropathy. Intranuclear inclusions were identified, mainly within degenerating hepatocytes, followed by pancreatic acinar cells, glandular epithelium of proventriculus and gizzard, and enterocytes of the small intestines and renal endothelium. Diagnosis was based on microscopic changes characteristic of IBH and a positive conventional PCR targeting the hexon gene of fowl adenovirus (FAdV) on liver tissue pools. Sequence analysis was performed on 41 selected FAdV-positive samples from diagnostic cases. The majority of FAdV PCR-positive samples aligned with FAdV-8b (16/41). However, FAdV-7 (14/41), FAdV-4 (7/41), and FAdV-11 (4/41) were also detected from IBH cases. Disease management was successfully achieved by the administration of an autogenous vaccine to breeders, at 11 and 18 wk of age, to confer maternal antibody protection to the broiler progeny.

Acclimation of suspension LMH cell line for high-efficient inactivated FAdV-4 vaccine production.

FAdV-4 infection induces selective mitochondrial autophagy.