Search Results (105)

Enteric viral infections represent a major concern for poultry production, causing growth retardation, impaired feed conversion, and increased mortality, particularly in young birds. To investigate the involvement of RNA and DNA enteric viruses in flocks exhibiting growth problems, seven poultry farms in southern Germany, including broiler, pullet, and breeder operations, were examined for the presence of chicken astrovirus (CAstV), avian reovirus (ARV), and fowl adenovirus-1 (FAdV-1) by means of RT-PCR. All farms exhibited growth retardation, diarrhea, and enteritis-associated lesions. Histopathology revealed features of runting–stunting syndrome in most of the broiler farms and depletion of lymphatic tissue in most of the pullet farms. CAstV was detected in all flocks, ARV in six, and FAdV-1 in four farms. To further characterize the viral agents, metagenomic sequencing of cecal tonsils from one severely affected broiler flock confirmed the presence of a CAstV strain identical (100%) to CAV/Belgium/4134_001/2019. In addition, the complete genome of avian Sicinivirus was assembled for the first time in Germany, showing 96.8% nucleotide identity with a Dutch strain (Chicken/NLD/2019/V_M_056_picorna_2). These findings demonstrate the widespread circulation and co-infection of enteric viruses on German poultry farms and underline the transboundary nature of these infections, emphasizing the need for enhanced surveillance and biosecurity measures to mitigate their impact on poultry health and productivity. Full article

Marek’s disease (MD), caused by pathogenic Marek’s disease virus serotype 1 (MDV-1), is one of the most important avian immunosuppressive and neoplastic diseases and has led to huge economic losses to the poultry industry worldwide. Rapid and accurate clinical diagnosis is of great significance for efficient control of the disease. Herein, we have established a multiplex PCR (mPCR) method to simply differentiate all of the three types of MDV, using five specific primers targeting to MDV-1 oncogene meq or MDV-2 and MDV-3/HVT gB genes. Simultaneously, it can detect any type of virulent or vaccine MDV strains in one PCR reaction, with amplicons of the short (S) and long (L)-meq of MDV-1 strains, and the gB of MDV-2 and HVT vaccine strains. Non-specific amplifications of avian leukosis virus (ALV), reticuloendotheliosis virus (REV), or fowl adenovirus virus 4 (FAdV-4) were not observed, indicating a good specificity of this method. A total of 522 clinical samples of tumor-bearing or suspected diseased birds collected from 30 poultry farms were detected. The results demonstrated that the newly developed mPCR method accurately detected and differentiated epidemic MDV-1 infections and vaccine strains, and provided nearly 100% consistency for detecting clinical wild-type infections compared with conventional PCR amplification of the meq gene. Collectively, our data has provided a highly efficient method for early differential diagnosis of MD clinical cases, virus identification and future evaluation of vaccination efficacy in healthy chicken flocks, which would be meaningful for efficient control of the disease. Full article

Fowl adenovirus serotype 4 (FAdV-4) has been identified as the primary pathogen responsible for hydropericardium-hepatitis syndrome (HHS), resulting in significant economic losses in major poultry-producing countries since 2015. Timely and accurate diagnosis of FAdV-4 infection is essential for the effective prevention and control of HHS. In this study, the two nonstructural genes of FAdV-4, 100K and 22K, were inserted into the expression vector pET-32a (+) respectively. The expressed recombinant proteins were used as coating antigens to develop two indirect ELISA methods, designated as 100K-ELISA and 22K-ELISA. Both ELISAs demonstrated high specificity, showing no cross-reactivity with serum samples positive for other avian diseases. Both ELISAs yielded positive results when applied to 50 serum samples from SPF chickens experimentally infected with FAdV-4 and negative results when applied to 50 serum samples from SPF chickens immunized with an inactivated FAdV-4 vaccine. Similarly, the field sample testing results demonstrated a significant ability to distinguish between vaccinated and infected samples. The 100K-ELISA and 22K-ELISA, which are based on nonstructural proteins, may be effective tools for differentiating between FAdV-4 infection and vaccination, offering a promising approach for differentiating infected from vaccinated animals (DIVA) strategies in poultry. Full article

This study investigated the molecular characteristics and genetic diversity of Fowl adenovirus (FAdV) strains circulating in commercial broiler and layer flocks in the Southern Marmara and Aegean regions of Türkiye between January and December 2025. Liver samples (n = 120) collected from twelve flocks with increased mortality and clinical signs compatible with adenoviral infection were analyzed. Detection was performed using circular amplification technology and PCR targeting the hexon L1 region, and positive samples were sequenced for molecular characterization. BLAST analysis showed that all isolates belonged to Aviadenovirus hepatitidis and were identified as serotype 8b. Pairwise comparisons showed high nucleotide identity among isolates (97.4–100%) and 98.1–100% similarity with the Turkish reference strain MK937075. Only three isolates displayed nucleotide substitutions, while most sequences were identical within the analyzed region. Amino acid similarity ranged from 95.2% to 100%. Phylogenetic analysis revealed that all isolates clustered within a single monophyletic group together with previously reported Turkish FAdV-8b strains. Necropsy findings included hepatomegaly, multifocal hepatic pallor, petechial hemorrhages, gizzard erosion, and serous pericardial involvement. The detection of genetically closely related isolates across multiple provinces suggests regional circulation of a common viral lineage. These findings demonstrate that FAdV-8b is currently the predominant serotype associated with inclusion body hepatitis outbreaks in this major poultry production area and highlight the importance of molecular surveillance and targeted control strategies, including breeder monitoring and region-specific vaccine development. Full article

Avian reovirus (ARV) is a ubiquitous pathogen in commercial poultry, traditionally associated with viral arthritis, malabsorption syndrome, and growth retardation. In recent years, the rapid genetic diversification of ARV has raised increasing concerns regarding vaccine mismatch, immune dysregulation, and complex disease outcomes in vaccinated flocks. In this study, an integrated investigation combining large-scale field surveillance, molecular characterization, and controlled animal experiments was conducted to elucidate the epidemiological features of ARV and its impact on heterologous vaccine-induced protection. Epidemiological surveillance revealed widespread ARV circulation in commercial poultry flocks, with marked genetic divergence between contemporary field isolates and classical vaccine strains. Phylogenetic analysis based on the σC gene demonstrated that the majority of circulating strains clustered within emerging genotypes that were genetically distinct from vaccine-related lineages. Using a controlled infection–vaccination–challenge model, prior ARV infection was shown to significantly impair humoral immune responses induced by an inactivated Aviadenovirus hydropericardii (fowl adenovirus serotype 4, FAdV-4) vaccine, as evidenced by reduced FAdV-4-specific antibody levels. Importantly, ARV pre-infection compromised vaccine-mediated protection and was associated with enhanced FAdV-4 pathogenicity following challenge, resulting in increased mortality, aggravated clinical manifestations, and more pronounced pathological lesions. These findings indicate that prior ARV infection is associated with reduced FAdV-4 vaccine-induced humoral responses and partial loss of protective efficacy under controlled experimental conditions. Importantly, this study provides quantitative experimental evidence using a defined infection–vaccination–challenge interference model rather than proposing a previously unrecognized virus-virus interaction. These results underscore the necessity of enhanced ARV surveillance and optimized immunization strategies in modern poultry production systems. Full article

In recent years, outbreaks of inclusion body hepatitis (IBH) have been reported with increasing frequency worldwide. In Austria, the presence of fowl aviadenoviruses (FAdVs) and/or IBH has been documented sporadically. The present report describes a surge of natural IBH cases in fifteen Ross 308 broiler flocks. The clinical picture was characterized by the sudden onset of increased mortality in birds 3 to 15 days of age, persisting for approximately one week, resulting in cumulative flock mortality ranging from 2% to 27%. The main pathological findings were hepatitis with diffuse necrotic foci and petechial hemorrhages with intranuclear inclusion bodies by histology. FAdV was detected in liver samples from all affected flocks. Based upon partial hexon sequence analysis, FAdV serotype-11, species-D (FAdV-11, FAdV-D) was identified, showing complete sequence identity among broiler-derived strains and high identity with previous isolates from Austria and globally IBH-associated isolates. Furthermore, FAdV-11 DNA was detected in dead-in-shell embryos collected during the ongoing outbreak, originating from one suspect broiler breeder flock, confirming vertical transmission. Serological investigations of the suspect breeders confirmed the FAdV-11 infections within the studied timeframe. In conclusion, investigations confirmed vertical transmission of FAdV-11 and subsequent manifestation of IBH in broilers under field conditions, highlighting the epidemiological importance of broiler breeders in IBH outbreaks. Full article

Fowl adenoviruses (FAdVs) represent a major threat to poultry health, with serotypes FAdV-1 and FAdV-4 causing adenoviral gizzard erosion (AGE) and hepatitis-hydropericardium syndrome (HHS), respectively. A wide variety of afflicted birds, including chicken, pigeon, and psittacine species, have been reported to carry aviadenoviruses. The disease is highly contagious and spreads rapidly between flocks and farms through vertical and horizontal transmission. In this study, we implemented a multi-stage computational drug-discovery pipeline to identify natural inhibitors of the viral fiber proteins for both FAdV-1 and FAdV-4. A curated library of 7523 natural compounds from the African Natural Products Database (ANPDB) and the South African Natural Compounds Database (SANCDB) was subjected to ADMET-based filtering, molecular docking, ADMET prediction, and 500 ns molecular dynamics simulations against four structural targets: Fiber-1 and Fiber-2 of FAdV-4, and the Short and Long Fibers of FAdV-1. Three ligands, ANPDB_6449 (−10.3 kcal/mol), ANPDB_2908 (−10.2 and −10.0 kcal/mol), and SANCDB_245 (−9.2 kcal/mol), consistently emerged as strong candidates across the entire computational workflow. While ANPDB_2908 demonstrated notable multi-target capability by binding to fiber proteins from both FAdV-1 and FAdV-4, ANPDB_6449 and SANCDB_245 exhibited strong serotype-specific potential, supported by stable interaction profiles and favorable drug-likeness characteristics. Together, these compounds highlight promising natural scaffolds for the development of targeted antiviral interventions against pathogenic FAdV serotypes. Full article

Fowl adenovirus serotype 4 (FAdV-4) is the major causative agent of hydropericardium-hepatitis syndrome (HHS), a disease responsible for considerable economic losses in poultry production. Although inactivated and live-attenuated vaccines reduce mortality, continued outbreaks highlight the need to optimize vaccination strategies. To address these limitations, we developed and evaluated a bivalent subunit vaccine composed of recombinant hexon-L1 and fiber-2 proteins, two major antigenic determinants associated with neutralization and pathogenicity. The proteins were expressed in Escherichia coli, purified under native conditions, confirmed for purity and antigenicity, and emulsified into a water-in-oil formulation. Chickens were immunized with either 10 μg or 20 μg doses, boosted after 14 days, and challenged with the homologous virulent FAdV-4 strain SDLC202009. The 20 μg dose conferred complete survival, eliminated histopathological lesions, prevented viral detection in tissues by PCR and immunohistochemistry, and fully blocked viral shedding. Similarly, the 10 μg dose induced a good protection with only minor pathological differences compared to the group treated with 20 μg. These results demonstrate that a bivalent hexon-L1 and fiber-2 subunit formulation elicits strong, dose-dependent humoral and tissue-level protection against homologous FAdV-4 challenge under the conditions tested. The experimental design did not include a monovalent fiber-2 comparator; therefore, conclusions regarding the relative contribution of each antigen are not drawn. Full article

Fowl adenoviruses are opportunistic emerging viruses that spread widely in fowls, infecting birds of all ages, including young broiler chicks. This study aims to genotype the current adenovirus strains associated with inclusion body hepatitis hydropericardium syndrome (IBH-HPS) among infected broilers in Upper Egypt and to evaluate their pathogenic features. In 2024, 100 tissue samples were collected across Assiut and Sohag governorates in Upper Egypt for genetic characterization and pathogenicity evaluation. FAdVs were detected in 22% (11/50) of flocks. Typical FAdV lesions of dead embryos were observed after seven days post egg inoculation. Regarding the PCR assay of the hexon gene, only 8 of 30 samples were confirmed positive at 897 bp, yielding a 26.6% positivity rate. The remaining samples were considered negative using established RT-qPCR protocols for other viral pathogens. Partial sequencing of the hexon gene revealed that FAdV isolates (n = 4) clustered within FAdV species-D serotype 2/11, as determined by phylogenetic analysis. The four isolates shared (98–99%) and (94–100%) nucleotide and amino-acid similarities to FAdV-D of Israeli strains (2019–2020) and contemporary Egyptian isolates (2022), respectively, and low genetic divergence (54–81%) in comparison with other documented species. The amino acid sequence alignment and 3D structure indicate that the four immunogenic HVRs are located in the L1 region of the hexon protein, and that the highly conserved ⁹¹GQMTT⁹⁵, a specific region for FAdV-D serotype 2/11, is present. Regarding pathogenicity, the gross and histopathological findings observed clearly demonstrate the systemic pathogenicity of FAdV-2/11 in the infected group, with a final mortality rate of 30% at seven days post-infection (dpi). The FAdV DNA in hepatic tissues and cloacal swabs was confirmed by the PCR method at 3 dpi and 5 dpi. These results emphasize the circulating of FAdV-2/11 species D in Upper Egypt and highlight the significant need for a single inactivated vaccine that effectively targets the relevant FAdV serotypes to achieve broader and more efficient protection. Full article

Chicken respiratory diseases represent multifactorial conditions resulting from viral, bacterial, mycoplasmal pathogens, and environmental factors, causing significant economic losses within the poultry industry. A specific respiratory disease characterized by breathing difficulties and bronchial occlusion due to caseous exudates is termed chicken bronchial obstruction. However, the absence of rapid, precise, and highly sensitive diagnostic methods for differentiation of primary respiratory disease pathogens or opportunistic pathogens, including avian influenza virus (AIV), infectious bronchitis virus (IBV), Pseudomonas aeruginosa (P. aeruginosa), and Escherichia coli (E. coli), constitutes a substantial challenge. This study developed a quadruplex droplet digital polymerase chain reaction (ddPCR) method that targeted the HA gene of H9 subtype AIV, the M gene of IBV, the Pal gene of P. aeruginosa, and the UidA gene of E. coli. Following the optimization of annealing temperature, sensitivity, and repeatability, the minimum detectable concentrations were determined as 3.02 copies/μL for the HA gene of H9 subtype AIV, 3.08 copies/μL for the M gene of IBV, 3.19 copies/μL for the Pal gene of P. aeruginosa, 3.39 copies/μL for the UidA gene of E. coli. No cross-reactivity was observed with Newcastle disease virus (NDV), H5 subtype AIV, H7 subtype AIV, fowl adenovirus serotype 4 (FAdV-4), infectious laryngotracheitis virus (ILTV), Avibacterium paragallinarum, Streptococcus, Salmonella, Pasteurella multocida, and Staphylococcus aureus. The method demonstrated excellent repeatability, with a coefficient of variation (CV) below 9%. The 185 clinical samples collected in Hebei Province China are tested by both quadruplex ddPCR and quadruplex qPCR method and the results compared. The sensitivity of the quadruplex ddPCR method (57.30%; 106/185) slightly exceeded that of the quadruplex qPCR method (49.73%; 92/185). Pathogens or opportunistic pathogens positive rates obtained via the quadruplex ddPCR were 40.00% for H9 subtype AIV, 33.51% for IBV, 24.32% for P. aeruginosa, and 27.57% for E. coli. In comparison, the positive rates of H9 subtypes AIV, IBV, P. aeruginosa, and E. coli from the quadruplex qPCR were 36.22%, 30.81%, 21.62%, and 24.32%, respectively. The coincidence rates between the two methods were 96.22% for H9 AIV, 97.30% for IBV, 97.30% for P. aeruginosa, and 96.76% for E. coli. These results demonstrated that the quadruplex ddPCR method represented a highly sensitive, specific, and rapid technique for identifying H9 subtype AIV, IBV, P. aeruginosa, and E. coli. Full article

Chlorobaculum tepidum ferredoxin: ${\mathrm{NADP}}^{+}$ oxidoreductase (CtFNR) is a dimeric thioredoxin reductase (TrxR)-type FNR, whose mechanism and redox properties are poorly characterized. In this work, we focused on the reoxidation mechanisms of its flavin adenine dinucleotide (FAD) cofactor using quinones (Q), nitroaromatics (${\mathrm{ArNO}}_2$), and other nonphysiological oxidants with different single-electron reduction midpoint potentials ($E_7^1$) and electrostatic charge. Like in other FNRs, the rate-limiting step of the reaction is the reoxidation of FAD semiquinone (${\mathrm{FADH}}^{•}$). However, only one FAD per dimer functions in CtFNR due to some nonequivalence of the NADP(H) binding domains in separate subunits. The reactivity of Q increases with increasing $E_7^1$, while ${\mathrm{ArNO}}_2$ form another analogous series of lower reactivity. The compounds are reduced in a dominant single-electron way. These data are consistent with an “outer sphere” electron transfer mechanism. On the basis of reactions with 3-acetylpyridine adenine dinucleotide phosphate, the two-electron reduction midpoint potential of FAD at pH 7.0 is −0.282 V. In CtFNR, 11% ${\mathrm{FADH}}^{•}$ was stabilized at equilibrium. Calculated electron transfer distances in reactions with Q and ${\mathrm{ArNO}}_2$ were in the range of 2.6–3.4 Å. Taken together with previous studies of Rhodopseudomonas palustris and Bacillus subtilis FNRs, this work allows us to generalize the information on the catalytic ant thermodynamic properties of TrxR-type FNRs. In addition, our data may be valuable from an applied perspective, e.g., the use of redox mediators in photobioelectrochemical systems or microbial cells based on anoxygenic phototrophic bacteria. Full article

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. Full article

Vaccination remains the primary strategy for controlling infectious diseases in farm animals. However, current conventional vaccines demonstrate clinical limitations including suboptimal immunogenicity and frequent booster requirements, which compromise disease management efficacy. This study presents an innovative vaccine platform combining yolk immunoglobulin (IgY) with inactivated antigens as co-immunization components. We developed two formulations targeting economically significant pathogens: avian Fowl Adenovirus Serotype 4 (FAdV-4) and Porcine Epidemic Diarrhea Virus (PEDV). For FAdV-4 vaccine evaluation in specific pathogen-free (SPF) chickens, the IgY-antigen complex demonstrated superior immunogenic properties compared to conventional inactivated vaccines. When administered as a single dose at 14 days of age, the experimental formulation elicited significantly stronger humoral responses as measured by both serum neutralization (SN₅₀) and ELISA. Notably, this vaccination strategy provided 100% protection against lethal FAdV-4 challenge from 0 h to 20 weeks post-vaccination, with complete absence of clinical disease manifestations. In PEDV assessment using mouse models, the IgY-antigen formulation induced significantly higher antibody titers than inactivated antigen alone at all post-immunization timepoints (p < 0.01). Comparative analysis revealed our dual-component platform enhanced both the intensity and rapidity of protective immune responses compared to traditional inactivated vaccines. These findings establish that the IgY-antigen co-immunization strategy represents a promising approach for developing new veterinary vaccines with improved protective efficacy. The platform’s ability to generate robust, rapid-onset immunity while maintaining single-dose effectiveness addresses critical limitations of current vaccine technologies. Full article

This study reports on the direct electron transfer (DET) ability of the enzyme spermidine dehydrogenase (SpDH) and its use in a DET-type enzymatic sensor for detecting spermine. SpDH was found to exhibit internal electron transfer from its cofactor, flavin adenine dinucleotide (FAD), to heme b. This was confirmed by observing the heme b-derived reduction peak at 560 nm in the presence of spermine, the substrate. SpDH was immobilized on a gold electrode via a dithiobis (succinimidyl hexanoate) self-assembled monolayer. The cyclic voltammetry analysis of the SpDH-immobilized gold electrode revealed an increased oxidation current in the presence of 0.1 mM spermine with an onset potential of −0.14 V vs. Ag/AgCl in the absence of an additional external electron acceptor. This result confirmed that SpDH is capable of DET. Chronoamperometric analyses were conducted using an SpDH-immobilized gold electrode with spermine as the substrate under a 0 V oxidation potential vs. Ag/AgCl using an artificial saliva matrix containing 10 µM ascorbic acid and 100 µM uric acid. The sensor exhibited good linear correlation between the current increase and spermine concentration from 0.2 to 2.0 µM, with a limit of detection of 0.084 µM, which encompasses the physiologically relevant spermine concentration found in the saliva. Primary structure alignments and 3D structure predictions revealed that all SpDH homologs possess two conserved histidine residues in the same location on the surface as the heme b ligand of SpDH. This indicates their potential for DET-ability with an electrode. Full article

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. Full article