Recombinant Fowl aviadenovirus E (FAdV-E) penton base vaccination fails to confer protection against inclusion body hepatitis (IBH) in chickens.

Inclusion body hepatitis (IBH) is a metabolic disease affecting chickens, associated with different serotypes of fowl adenovirus (FAdV). Experimentally tested vaccines against IBH include several capsid-based subunit vaccines, but not the penton base protein. In the present study, specific pathogen-free chickens were vaccinated with recombinant penton base expressed from each of two different FAdV serotypes (FAdV-7 and FAdV-8b), followed by challenge with a virulent IBH-causing strain. No protection was observed with either vaccine, possibly due to the low immunogenicity of each protein and their inability to induce neutralizing antibodies in the host.

Local cellular immune response plays a key role in protecting chickens against hepatitis-hydropericardium syndrome (HHS) by vaccination with a recombinant fowl adenovirus (FAdV) chimeric fiber protein.

Fowl adenovirus (FAdV)-induced diseases hepatitis-hydropericardium syndrome (HHS) and inclusion body hepatitis (IBH) have been affecting the poultry industry with increasing severity in the last two decades. Recently, a subunit vaccine based on a chimeric fiber protein with epitopes from different fowl adenovirus serotypes (named crecFib-4/11) has been shown to confer simultaneous protection against both HHS and IBH. However, the underlying immune mechanisms in chickens are still enigmatic, especially because of frequently absent neutralizing response despite high levels of protection. In this study, we investigated the kinetics of the humoral and cellular immune responses in specific pathogen-free chickens after vaccination with crecFib-4/11 and/or challenge with a HHS-causing strain, on a systemic level, as well as locally in target and lymphoid organs. The humoral response was assessed via enzyme-linked immunosorbent assay (ELISA) and virus neutralization test in serum, while the cellular immune response was determined by phenotyping using flow cytometry. Although vaccination induced serum antibodies, as confirmed by ELISA, such antibodies exhibited no pre-challenge neutralizing activity against FAdV-4. Nevertheless, immunized birds experienced a significant B cell increase in the liver upon challenge, remaining high throughout the experiment. Furthermore, vaccination stimulated the proliferation of cytotoxic T lymphocytes, with earlier circulation in the blood compared to the challenge control and subsequent increase in liver and spleen. Overall, these findings imply that protection of chickens from HHS after crecFib-4/11 vaccination relies on a prominent local immune response in the target organs, instead of circulating neutralizing antibodies.

Special Issue: Avian Adenoviruses.

For years, research on avian adenoviruses, here fowl adenoviruses (FAdVs), received less attention, mainly due to limited clinical relevance in poultry production [...].

Species Fowl aviadenovirus B Consists of a Single Serotype despite Genetic Distance of FAdV-5 Isolates.

Fowl adenoviruses (FAdVs) are infectious agents, mainly of chickens, which cause economic losses to the poultry industry. Only a single serotype, namely FAdV-5, constitutes the species Fowl aviadenovirus B (FAdV-B); however, recently, phylogenetic analyses have identified divergent strains of the species, implicating a more complex scenario and possibly a novel serotype. Therefore, field isolates of the species were collected to investigate the contemporary diversification within FAdV-B, including traditional serotyping. Full genomes of fourteen FAdV-B strains were sequenced and four strains, possessing discriminatory mutations in the antigenic domains, were compared using virus cross-neutralization. Essentially, strains with identical antigenic signatures to that of the first described divergent strain were found in the complete new dataset. While chicken antiserum against FAdV-5 reference strain 340 could not neutralize any of the newly isolated viruses, low homologous/heterologous titer ratios were measured reciprocally. Although they argue against a new serotype, our results indicate the emergence of escape variants in FAdV-B. Charge-influencing amino acid substitutions accounted for only a few mutations between the strains; still, these enabled one-way cross-neutralization only. These findings underline the continued merit of the cross-neutralization test as the gold standard for serotyping, complementary to advancing sequence data, and provide a snapshot of the actual diversity and evolution of species FAdV-B.

High Phenotypic Variation between an In Vitro-Passaged Fowl Adenovirus Serotype 1 (FAdV-1) and Its Virulent Progenitor Strain despite Almost Complete Sequence Identity of the Whole Genomes.

Adenoviral gizzard erosion is an emerging disease with negative impact on health and production of chickens. In this study, we compared in vitro and in vivo characteristics of a fowl adenovirus serotype 1 (FAdV-1), attenuated by 53 consecutive passages in primary chicken embryo liver (CEL) cell cultures (11/7127-AT), with the virulent strain (11/7127-VT). Whole genome analysis revealed near-complete sequence identity between the strains. However, a length polymorphism in a non-coding adenine repeat sequence (11/7127-AT: 11 instead of 9) immediately downstream of the hexon open reading frame was revealed. One-step growth kinetics showed delayed multiplication of 11/7127-AT together with significantly lower titers in cell culture (up to 4 log10 difference), indicating reduced replication efficiency in vitro. In vivo pathogenicity and immunogenicity were determined in day-old specific pathogen-free layer chicks inoculated orally with the respective viruses. In contrast to birds infected with 11/7127-VT, birds infected with 11/7127-AT did not exhibit body weight loss or severe pathological lesions in the gizzard. Virus detection rates, viral load in organs and virus excretion were significantly lower in birds inoculated with 11/7127-AT. Throughout the experimental period, these birds did not develop measurable neutralizing antibodies, prevalent in birds in response to 11/7127-VT infection. Differences in pathogenicity between the virulent FAdV-1 and the attenuated strain could not be correlated to prominently discriminate genomic features. We conclude that differential in vitro growth profiles indicate that attenuation is linked to modulation of viral replication during interaction of the virus with the host cells. Thus, hosts would be unable to prevent the rapid replication of virulent FAdV leading to severe tissue damage, a phenomenon broadly applicable to further FAdV serotypes, considering the substantial intra-serotype virulence differences of FAdVs and the variation of diseases.

Vaccination with a fowl adenovirus chimeric fiber protein (crecFib-4/11) simultaneously protects chickens against hepatitis-hydropericardium syndrome (HHS) and inclusion body hepatitis (IBH).

In the past decades, fowl adenovirus (FAdV)-related diseases became an increasing concern for the poultry industry worldwide. Various immunization strategies against FAdVs have been experimentally investigated, with a particular focus on subunit vaccines against hepatitis-hydropericardium syndrome (HHS), caused by FAdV serotype 4, and inclusion body hepatitis (IBH), caused by serotypes 2, 8a, 8b and 11. In this study, we extended our innovative concept of recombinant chimeric fiber proteins to design a novel chimera combining epitopes from two distinct serotypes, FAdV-4 and -11, and we investigated its efficacy to simultaneously protect chickens against HHS and IBH. Specific pathogen-free chickens were vaccinated with the novel recombinant chimeric fiber and subsequently challenged with either a HHS- or IBH-causing strain. Vaccinated/challenged birds exhibited a reduction of clinical signs, limited hepatomegaly and lower levels of AST compared to the respective challenge controls. Furthermore, the vaccine prevented atrophy of HHS-affected lymphoid organs, such as thymus and bursa of Fabricius, and viral load in the target organs was significantly reduced. Clinical protection was associated with high levels of pre-challenge antibodies measured on ELISA plates coated with the vaccination antigen. Interestingly, the development of neutralizing antibodies was limited against FAdV-11 and absent against FAdV-4, indicating that protection granted by such an antigen may be linked to different immunization pathways. In conclusion, we proved that the concept of chimeric fiber vaccines can be extended across viral species boundaries and represents the first single-component FAdV subunit vaccine providing comprehensive protection against different FAdV-associated diseases.

Recombinantly Expressed Chimeric Fibers Demonstrate Discrete Type-Specific Neutralizing Epitopes in the Fowl Aviadenovirus E (FAdV-E) Fiber, Promoting the Optimization of FAdV Fiber Subunit Vaccines towards Cross-Protection in vivo.

Vaccines against inclusion body hepatitis in chickens are complicated by the involvement of antigenically diverse fowl adenovirus types. Though immunization with fiber protein confers robust protection, type specificity of fiber antibodies is an obstacle for the desired broad coverage. In this study, we utilized information on multiple linear epitopes predicted in the Fowl Aviadenovirus E (FAdV-E) fiber head (knob) to develop chimeric fibers with an exchange between two serotypes' sequences, each containing proposed epitopes. Two consecutive segments pertaining to amino acid positions 1 to 441 and 442 to 525/523 in the fibers of FAdV-8a and -8b, types of Fowl Aviadenovirus E that cause inclusion body hepatitis, were swapped reciprocally to result in novel chimeras, crecFib-8a/8b and crecFib-8b/8a. crecFib was indistinguishable from monospecific recombinant fibers in its eactivity with different FAdV antisera in Western blotting. However, contrary to the results for monospecific fibers, crecFib induced cross-neutralizing antibodies against both serotypes in chickens. This demonstrates three nonidentical epitopes in the FAdV-E fiber, the conserved epitope detected in Western blotting and at least two epitopes participating in neutralization, being type specific and located opposite residue position 441-442. Furthermore, we supply conformational evidence for a site in the fiber knob with accessibility critical for neutralization. With such an extended neutralization spectrum compared to those of individual fibers, crecFib was anticipated to fulfill and even extend the mechanistic basis of fiber-mediated protection toward bivalent coverage. Accordingly, crecFib, administered as a single-antigen component, protected chickens simultaneously against challenge with FAdV-8a or -8b, demonstrated by up-to-complete resistance to clinical disease, prevention of target organ-related changes, and significant reduction of viral load. IMPORTANCE The control of inclusion body hepatitis, a disease of economic importance for chicken production worldwide, is complicated by an etiology involving multiple divergent fowl adenovirus types. The fiber protein is principally efficacious in inducing neutralizing and protective antibodies in vaccinated chickens; however, it faces limitations due to its intrinsic type specificity for neutralization. In this study, based on an in silico-guided prediction of multiple epitopes in the fowl adenovirus fiber head's loops, we designed chimeric proteins, swapping N- and C-distal fiber portions, each containing putative epitopes, between divergent types FAdV-8a and -8b. In in vitro and in vivo studies, the chimeric fiber displayed extended properties compared to those of individual monotype-specific fibers, allowing the number, distribution, functionality, and conformational bearings of epitopes of the fowl adenovirus fiber to be characterized in more detail. Importantly, the chimeric fiber induced cross-neutralizing antibodies and protective responses in chickens against infections by both serotypes, promoting the advancement of broadly protective subunit vaccination strategies against FAdV.

Longitudinal Serological Monitoring of Commercial Broiler Breeders for Fowl Adenoviruses (FAdVs)-Presence of Antibodies Is Linked with Virus Excretion.

Currently, the poultry industry worldwide is facing an emerging trend of fowl adenovirus (FAdV)-associated diseases with a significant economic impact, especially in meat-type chickens. Vertical transmission is an important feature of all FAdVs; hence, preventive measures mostly revolve around breeding stocks. However, knowledge about temporal development of FAdV infections in modern commercial settings is rare or even nonexistent. In the present study, longitudinal monitoring for FAdV was conducted in broiler breeder flocks located in a confined geographical region with intensive poultry production in Iran. For this, the antibody status of birds from 4 to 32 wk of age was monitored with a commercial FAdV-ELISA and virus neutralization test (VNT). In parallel, fecal shedding of FAdV was determined at the peak of egg production with real-time PCR and virus isolation. Overall, the commercial ELISA showed seroconversion of flocks before onset of production. VNT resolved in detail infection patterns of individual serotypes with a primordial FAdV-D (FAdV-2/-11) infection, frequently followed by FAdV-E (FAdV-8a, -8b) superinfection. FAdV-A (FAdV-1) was traced in half of the investigated flocks, while no evidence of infection with FAdV-C (FAdV-4, -10) was noted. Common serological profiles between different houses of the same farm indicate an overarching biosecurity. Serological profiles coupled with virological findings at the peak of egg production indicated that higher antibody levels, determined by ELISA, correlated with lower amounts of viral DNA in fecal excretion. Simultaneously, the number of isolated FAdVs belonging to distinct serotypes declined in accordance with a rise of neutralizing antibodies in birds, underlining the significance of serotype-specific antibodies in the epidemiology of FAdV in breeders. Investigations in breeders were complemented with screening of FAdV-associated diseases in local broilers over a 3-yr period; 26 cases of inclusion body hepatitis with dominant involvement of FAdV-11/FAdV-8b, one outbreak of adenoviral gizzard erosion related to FAdV-1, and no evidence of hepatitis-hydropericardium syndrome suggest that identical serotypes are maintained in the local poultry industry.

Artículo regular­Monitoreo serológico longitudinal en reproductores pesados comerciales para detectar adenovirus del pollo (FAdV): la presencia de anticuerpos está relacionada con la excreción de virus. Actualmente, la industria avícola en todo el mundo enfrenta a una tendencia emergente de enfermedades asociadas con adenovirus del pollo (FAdV) con un impacto económico significativo, especialmente en pollos de engorde. La transmisión vertical es una característica importante de los adenovirus del pollo, por lo que las medidas preventivas giran principalmente en torno a las poblaciones de reproductores. Sin embargo, el conocimiento sobre el desarrollo temporal de las infecciones por adenovirus del pollo en los entornos comerciales modernos es escaso o incluso inexistente. En el presente estudio, se llevó a cabo un seguimiento longitudinal de adenovirus del pollo en parvadas de reproductoras pesadas ubicadas en una región geográfica confinada con producción avícola intensiva en Irán. Para ello, se evaluó el estado de anticuerpos de las aves de 4 a 32 semanas de edad con una prueba comercial de ELISA para adenovirus del pollo y por la prueba de virus neutralización (VNT). En paralelo, se determinó la eliminación fecal de adenovirus del pollo en el pico de producción de huevos mediante un método de PCR en tiempo real y por aislamiento del virus. En general, la prueba de ELISA comercial mostró seroconversión de parvadas antes del inicio de la producción. La prueba de virus neutralización reveló en detalle los patrones de infección de los serotipos individuales con una infección primordialmente por FAdV-D (FAdV-2/-11), seguida frecuentemente por una superinfección por FAdV-E (FAdV-8a, - 8b). Se rastreó FAdV-A (FAdV-1) en la mitad de las parvadas investigadas, mientras que no se observó evidencia de infección por FAdV-C (FAdV-4, -10). Los perfiles serológicos comunes entre las diferentes casetas de la misma granja indican una bioseguridad generalizada. Los perfiles serológicos junto con los hallazgos virológicos en el pico de producción de huevo indicaron que los niveles más altos de anticuerpos, determinados por ELISA, se correlacionaron con cantidades más bajas de ADN viral en la excreción fecal. Simultáneamente, el número de adenovirus de pollo aislados pertenecientes a distintos serotipos disminuyó de acuerdo con un aumento de anticuerpos neutralizantes en aves, lo que subraya la importancia de los anticuerpos específicos de serotipo en la epidemiología del adenovirus del pollo en reproductores. Las investigaciones en reproductoras se complementaron con la detección de enfermedades asociadas a adenovirus en pollos de engorde locales durante un período de 3 años; 26 casos de hepatitis por cuerpos de inclusión con participación dominante de FAdV-11/FAdV-8b, un brote de erosión de molleja adenoviral relacionado con FAdV-1 y ninguna evidencia de síndrome de hepatitis-hidropericardio sugieren que se mantienen serotipos idénticos en la industria avícola local.

Reduced Performance Due to Adenoviral Gizzard Erosion in 16-Day-Old Commercial Broiler Chickens in Iran, Confirmed Experimentally.

Adenoviral gizzard erosion (AGE) in broilers is an emerging infectious disease with negative impact on flock productivity. Despite of known primary etiological role of fowl adenovirus serotype 1 (FAdV-1) in AGE, there are a limited number of field reports worldwide, possibly because the disease is less noticeable and clinically difficult to assess. The present study documents an outbreak of AGE in 16-day-old broiler chickens on a farm in the north of Iran and the reproduction of the disease in an experimental setting. In the field, a sudden onset of mortality was noticed in affected broilers resulting in 6% total mortality and decreased weight gain leading to approximately 1-week delay to reach the target slaughter weight. Necropsy findings in dead broilers revealed black colored content in crop, proventriculus and gizzard together with severe gizzard erosions characterized by multiple black-brown areas of variable size in the koilin layer and mucosal inflammation. Microscopic examination revealed necrotizing ventriculitis marked with severe dissociation of koilin layer and degeneration of glandular epithelium with infiltration of mononuclear inflammatory cells. FAdV-1 was isolated from affected gizzards. Phylogenetic analysis of the hexon loop-1 (L1) sequence of the isolated virus showed 100% identity with pathogenic FAdV-1 strains previously reported from broiler chickens with AGE. Subsequently, an in vivo study infecting day-old commercial layer chickens with the field isolate demonstrated characteristic lesions and histopathological changes of AGE together with decreased weight gain in the infected birds. For the first time, the progress of a natural outbreak of AGE in Iran is described and experimental reproduction of the disease is demonstrated. The findings highlight the economic impact of the disease for regional poultry production due to mortality and impaired weight gain of the affected broilers.

Spotlight on avian pathology: fowl adenovirus (FAdV) in chickens and beyond - an unresolved host-pathogen interplay.

Fowl adenovirus (FAdV) infections in chickens have undergone substantial changes in recent decades, driven by host and pathogen factors. Based on the pathogenesis of inclusion body hepatitis (IBH) and hepatitis-hydropericardium syndrome (HHS), modern broilers are much more inclined to have difficulties keeping the metabolic homeostasis, whereas adenoviral gizzard erosion (AGE) is noticed equally in broilers and egg-layers. Defining the importance of certain serotypes for specific FAdV diseases is a major achievement of recent years but the isolation of viruses from clinically healthy birds remains unexplained, as virulence factors are hardly known and continue to be a "black box". Together with further studies on pathogenesis of FAdV-induced diseases, such knowledge on virulence factors would help to improve protection strategies, which presently mainly concentrate on autogenous vaccines of breeders to prevent vertical transmission.

Fowl adenovirus (FAdV) fiber-based vaccine against inclusion body hepatitis (IBH) provides type-specific protection guided by humoral immunity and regulation of B and T cell response.

A recombinant fowl adenovirus (FAdV) fiber protein, derived from a FAdV-8a strain, was tested for its efficacy to protect chickens against inclusion body hepatitis (IBH). FAdV-E field isolates belonging to both a homotypic (FAdV-8a) and heterotypic (-8b) serotype were used as challenge. Mechanisms underlying fiber-induced protective immunity were investigated by fiber-based ELISA, virus neutralization assays and flow cytometry of peripheral blood mononuclear cells, monitoring the temporal developments of humoral and cellular responses after vaccination and challenge exposure. Birds were clinically protected from the homologous challenge and showed a significant reduction of viral load in investigated target organs, whereas fiber-based immunity failed to counteract the heterologous serotype infection. These findings were supported in vitro by the strictly type-specific neutralizing activity of fiber immune sera. In protected birds, fiber vaccination prevented a post-challenge drop of peripheral B cells in blood. Furthermore, fiber immunization stimulated CD4+ T lymphocyte proliferation while moderating the CD8α+ T cell response and prevented challenge-induced changes in systemic monocytes/macrophages and γδ+ T cell subpopulations. Both vaccinated and adjuvant-only injected birds experienced a priming of systemic B cells and TCRγδ+ T lymphocytes, which masked possible pre-challenge effects due to the antigen. In conclusion, within FAdV-E, recombinant fiber represents a vaccine candidate to control the adverse effects of homotypic infection by eliciting an effective humoral immunity and regulating B and T cell response, whereas the failure of heterotypic protection suggests a primordial role of humoral immunity for this vaccine.

Gizzard Erosion Associated with Fowl Adenovirus Infection in Slaughtered Broiler Chickens in Iran.

Gizzard erosions have been noticed in slaughtered broiler chickens during inspection at a processing plant in Iran. The condition was detected in piled gizzards derived from seven commercial broiler farms brought to slaughter on the same day. In total, 48 gizzards with lesions underwent thorough pathologic and virologic investigation. Perforation, roughening, and discoloration of the koilin layer as well as inflammation of the mucosa were observed macroscopically. Histologic examination showed dissociation of and cellular debris in the koilin layer accompanied by a loss and degeneration of glandular epithelium with mild to marked infiltration of inflammatory cells in the mucosa, submucosa, and muscular layer. Fowl adenovirus serotypes 1 (FAdV-1), 11 (FAdV-11), and 8a (FAdV-8a) were found in 13, 12, and 1 gizzard(s), respectively. Therein included were two gizzards that showed mixed infections with FAdV-1 and FAdV-11. Detailed analysis of the hexon gene revealed that the Iranian FAdV-1 isolates could be divided into two subclusters, more closely related to either the European (CELO) or the Asian (Ote) FAdV-1 reference strains. The present study, for the first time, describes not only the appearance of gizzard erosion but also the isolation of FAdV-1 and FAdV-8a from broilers in Iran and offers insights on the epidemiology of FAdV infection in Iranian flocks.

Erosión de molleja asociada con infección por adenovirus del pollo en pollos de engorde procesados en Irán. Se han observado erosiones de molleja en pollos de engorde sacrificados durante la inspección en una planta de procesamiento en Irán. La condición se detectó en mollejas apiladas derivadas de siete granjas comerciales de pollos de engorde que fueron sacrificados el mismo día. En total, 48 mollejas con lesiones se sometieron a una exhaustiva investigación patológica y virológica. Se observó macroscópicamente perforación, rugosidad y la decoloración de la capa de queratina, así como inflamación de la mucosa. El examen histológico mostró disociación y restos celulares en la capa de queratina acompañada por una pérdida y degeneración del epitelio glandular con infiltración leve a marcada de células inflamatorias en la mucosa, la submucosa y la capa muscular. Se encontraron aviadenovirus del pollo de los serotipos 1 (FAdV-1), 11 (FAdV-11) y 8a (FAdV-8a) en trece, doce y una molleja (s), respectivamente. Se incluyeron dos mollejas que mostraban infecciones mixtas con FAdV-1 y FAdV-11. El análisis detallado del gene de la proteína del hexon reveló que los aislamientos iraníes del serotipo FAdV-1 se dividieron en dos subgrupos, más estrechamente relacionados con las cepas de referencia del serotipo 1 de Europa (CELO), o de Asia (Ote). El presente estudio describe por primera vez, no solo la aparición de la erosión de la molleja, sino también el aislamiento de FAdV-1 y FAdV-8a de los pollos de engorde en Irán y ofrece información sobre la epidemiología de la infección por FAdV en parvadas iraníes.

Fowl Adenovirus (FAdV) Recombination with Intertypic Crossovers in Genomes of FAdV-D and FAdV-E, Displaying Hybrid Serological Phenotypes.

After analyzing 27 new genomes from fowl adenovirus (FAdV) field isolates and so-far unsequenced prototypes, we report the first evidence for recombination in FAdVs. Recombination was confined to species FAdV-D and FAdV-E, accommodating the largest number of, and the intraspecies-wise most differentiated, types. The majority of detected events occurred in FAdV-E, involving segments with parental origin of all constitutive types. Together with the diversity of breakpoints, this suggests widespread recombination in this species. With possible constraints through species-specific genes and diversification patterns, the recombinogenic potential of FAdVs attains particular interest for inclusion body hepatitis (IBH), an important disease in chickens, caused by types from the recombination-prone species. Autonomously evolving, recombinant segments were associated with major sites under positive selection, among them the capsid protein hexon and fiber genes, the right-terminal ORFs 19, 25, and the ORF20/20A family. The observed mosaicism in genes indicated as targets of adaptive pressures points toward an immune evasion strategy. Intertypic hexon/fiber-recombinants demonstrated hybrid neutralization profiles, retrospectively explaining reported controversies on reference strains B3-A, T8-A, and X11-A. Furthermore, cross-neutralization supported sequence-based evidence for interdomain recombination in fiber and contributed to a tentatively new type. Overall, our findings challenge the purported uniformity of types responsible for IBH, urging more complete identification strategies for FAdVs. Finally, important consequences arise for in vivo studies investigating cross-protection against IBH.

Fiber-based fluorescent microsphere immunoassay (FMIA) as a novel multiplex serodiagnostic tool for simultaneous detection and differentiation of all clinically relevant fowl adenovirus (FAdV) serotypes.

The recent emergence of fowl aviadenovirus (FAdV) induced disease outbreaks in chicken flocks worldwide, with distinct aetiologies confined to particular FAdV species and serotypes, is increasingly urging the need for specific and mass-applicable antibody screening systems. Despite this exigency, there are to date no available serological procedures which satisfactorily combine the criteria for sensitive detection of antibodies against FAdVs, diagnostic reliability in face of cross-reactions and requirements for a rapid and large-scale application. In order to address this gap, a multiplexed fluorescent microsphere immunoassay (FMIA) based on recombinant FAdV fiber proteins from six different serotypes FAdV-1, -2, -4, -8a, -8b and -11 was developed, which enabled simultaneous detection of antibodies against all clinically relevant serotypes in a single reaction within a high throughput setting. Based on a panel of >300 monospecific antisera raised against each of the 12 FAdV serotypes, 100% serotype-specificity was demonstrated for FAdV-1 (FAdV-A) and FAdV-4 (FAdV-C) fiber-based analytes. Analytes based on serotypes affiliated to FAdV-D and FAdV-E exhibited moderately lower specificities of 91.2-95.7%. This was attributed almost exclusively to mutual recognition between FAdV-2 and -11 field strains and to a much lesser extent to reference strains, supporting earlier proposals to merge them into a single serotype. Similarly, extensive cross-reactions between FAdV-8a and -8b were noted. Altogether intraspecies cross-reactions can be attributed to viruses with a close etiological intersection. Antisera against other important avian viruses remained negative by the FMIA, further validating its specificity. Compared to the virus-neutralization (VN) test, FMIA and individual fiber-based enzyme-linked immunosorbent assays (ELISAs) were equally sensitive in the detection of sera against FAdV-2 and -11, as well as FAdV-8a and -8b field strains, while they were even superior to VN test in detection of FAdV-1 and FAdV-4 responses, likely attributed to a relative abundance of fiber antibodies early upon infection. Moreover, application of the FMIA on field samples comprising a diversified response against all 12 FAdV serotypes further consolidated its specificity and agreement with VN test.

Development of sensitive indirect enzyme-linked immunosorbent assays for specific detection of antibodies against fowl adenovirus serotypes 1 and 4 in chickens.

Conventional serological methods for detection and differentiation of antibodies against fowl aviadenoviruses (FAdVs) are laborious and time-consuming, therefore ELISAs based upon recombinant proteins were developed in the present study to overcome this limitation for clinically relevant serotypes FAdV-1 and FAdV-4. In order to develop serotype-specific ELISAs, the two distinct fibers, fiber-1 (fib-1) and fiber-2 (fib-2), characteristically present only in FAdV-1 and FAdV-4, were applied separately as coating antigens. Sera raised against each recombinant fib-1 and fib-2 of FAdV-1 and FAdV-4 did not react with any of the heterologous fiber ELISAs, as anticipated by the low degree of amino acid identity between those FAdV fibers (23.1-41.2%), indicating that heterologous fibers do not share common epitopes. Testing of 172 monospecific sera, raised against all FAdV serotypes (1-8a and 8b-11), retrieved specificities between 99.3% and 100.0% for the ELISAs, further substantiating the serotype-specificity of fibers. Investigating sera from chickens experimentally inoculated with different FAdV-1 or FAdV-4 strains revealed that ELISAs were equally or more sensitive than the virus-neutralization (VN) test. Furthermore, strong correlations were demonstrated between fiber antibody titres and neutralization activity. Particularly, sera directed against live virus showed a pronounced fiber antibody response, which might be explained by an excessive production of fibers during infection. Application of the newly developed fiber ELISAs on field sera with heterogeneous serological status demonstrated high sensitivity and serotype-specificity of this test system, providing for the first time a diagnostic tool for mass screening of chicken flocks against FAdV serotypes, namely FAdV-1 and FAdV-4.

Fowl aviadenovirus serotype 1 confirmed as the aetiological agent of gizzard erosions in replacement pullets and layer flocks in Great Britain by laboratory and in vivo studies.

An investigation into the aetiology and pathogenesis of adenoviral gizzard erosion has been conducted following three natural outbreaks affecting one flock of 6-week-old replacement pullets and two consecutive placements of free range layers at the age of 21 and 23 weeks. Affected flocks showed increased mortality (0.12-0.30% per week), and gizzard lesions were consistent with fowl aviadenovirus (FAdV) involvement. To substantiate the initial findings, a selection of archived formalin-fixed paraffin-embedded gizzard samples from another 12 pullet and layer flocks, for which macroscopic and histopathological diagnosis of the disease were recorded in Great Britain during the period 2009-2016, were also investigated. In situ hybridization (ISH), virology and/or PCR confirmed the presence of FAdV species-A, serotype-1 (FAdV-A, FAdV-1) DNA in gizzard samples of all 15 cases investigated. Co-infections with additional FAdV serotypes including FAdV-8a were detected by serology and/or virology in two of the pullet flocks. However, species-specific in situ hybridization revealed that pathological changes of affected gizzards were only associated with the detection of FAdV-A. A subsequent in vivo study infecting 21-day-old SPF pullets with FAdV-1 or FAdV-8a strains isolated from the 6-week-old replacement pullets revealed characteristic pathomorphological changes only in the gizzards from birds infected with FAdV-1. While infection with FAdV-8a was confirmed by virology and serology, infected SPF birds did not develop pathomorphological changes. Therefore, the aetiological involvement of the isolated FAdV-8a in the development of adenoviral gizzard erosion in commercial pullets has been ruled out.

Fowl adenovirus-induced diseases and strategies for their control - a review on the current global situation.

The stand-alone pathogenicity of fowl adenoviruses (FAdVs) had long been disputed, given the ubiquity of the viruses versus sporadic outbreaks, and variation between experimental studies. However, a globally emerging trend of FAdV-associated diseases has marked the past two decades, with hepatitis-hydropericardium syndrome mainly in Asia besides Arabian and Latin American countries, and geographically more disseminated outbreaks of inclusion body hepatitis. Finally, the appearance of FAdV-induced gizzard erosion (AGE) in Asia and Europe completed the range of diseases. Epidemiological studies confirmed serotype FAdV-4 as agent of hepatitis-hydropericardium syndrome, whereas inclusion body hepatitis is related to FAdV-2, -8a, -8b and -11. Members of the biologically more distant serotype FAdV-1 induce AGE. Urged by increasing problems in the field, numerous pathogenicity studies with FAdVs from outbreaks substantiated the primary aetiologic role of particular strains for distinct clinical conditions. Developments in the poultry industry towards highly specialized genetic breeds and rigorous biosecurity additionally contribute to the growing incidence of FAdV-related diseases. Confirming field observations, recent studies connected a higher susceptibility of broilers with their distinct physiology, implying the choice of bird type as a factor to be considered in infection studies. Furthermore, elevated biosecurity standards have generated immunologically naïve breeding stocks, putting broilers at risk in face of vertical FAdV transmission. Therefore, future prevention strategies should include adequate antibodies in breeders prior to production and - if necessary - vaccination, in order to protect progenies. This review aims to deliver a detailed overview on the current global situation about FAdV-induced diseases, their reproduction in vivo and vaccination strategies.

Genetic diversity of species Fowl aviadenovirus D and Fowl aviadenovirus E.

Complete genomes of eight reference strains representing different serotypes within the species Fowl aviadenovirus D (FAdV-D) and Fowl aviadenovirus E (FAdV-E) were sequenced. The sequenced genomes of FAdV-D and FAdV-E members comprise 43 287 to 44 336 bp, and have a gene organization identical to that of an earlier sequenced FAdV-D member (strain A-2A). Highest diversity was noticed in the hexon and fiber genes and ORF19. All genomes sequenced in this study contain one fiber gene. Phylogenetic analyses and G+C content support the division of the genus Aviadenovirus into the currently recognized species. Our data also suggest that strain SR48 should be considered as FAdV-11 instead of FAdV-2 and similarly strain HG as FAdV-8b. The present results complete the list of genome sequences of reference strains representing all serotypes in species FAdV-D and FAdV-E.

Detailed molecular analyses of the hexon loop-1 and fibers of fowl aviadenoviruses reveal new insights into the antigenic relationship and confirm that specific genotypes are involved in field outbreaks of inclusion body hepatitis.

Forty-eight fowl aviadenoviruses (FAdVs) isolated from recent IBH outbreaks across Europe were investigated, by utilizing for the first time the two major adenoviral antigenic domains, hexon loop-1 and fiber, for compound molecular characterization of IBH-associated FAdVs. Successful target gene amplification, following virus isolation in cell culture or from FTA-card samples, demonstrated presence of FAdVs in all cases indicative for IBH. Based on hexon loop-1 analysis, 31 European field isolates exhibited highest nucleotide identity (>97.2%) to reference strains FAdV-2 or -11 representing FAdV-D, while 16 and one European isolates shared >96.0% nucleotide identity with FAdV-8a and -8b, or FAdV-7, the prototype strains representing FAdV-E. These results extend recognition of specific FAdV-D and FAdV-E affiliate genotypes as causative agents of IBH to the European continent. In all isolates, species specificity determined by fiber gene analysis correlated with hexon-based typing. A threshold of 72.0% intraspecies nucleotide identity between fibers from investigated prototype and field strains corresponded with demarcation criteria proposed for hexon, suggesting fiber-based analysis as a complementary tool for molecular FAdV typing. A limited number of strains exhibited inconsistencies between hexon and fiber subclustering, indicating potential constraints for single-gene based typing of those FAdVs. Within FAdV-D, field isolate fibers shared a high degree of nucleotide (>96.7%) and aa (>95.8%) identity, while FAdV-E field isolate fibers displayed greater nucleotide divergence of up to 22.6%, resulting in lower aa identities of >81.7%. Furthermore, comparison with FAdVs from IBH outbreaks outside Europe revealed close genetic relationship in the fiber, independent of the strains' geographic origin.

Recombinant FAdV-4 fiber-2 protein protects chickens against hepatitis-hydropericardium syndrome (HHS).

Virulent fowl adenovirus (FAdV) serotype 4 strains are the etiological agents of hepatitis-hydropericardium syndrome (HHS), a highly infectious disease in chickens with severe economic impact. In the present study, three different FAdV-4 derived capsid proteins, fiber-1, fiber-2, and hexon loop-1, were expressed in a baculovirus system and tested for their capacity to induce protection in chickens. Purified recombinant proteins were administered to day-old specific pathogen-free (SPF) chickens allocated in three separate groups and challenged with virulent FAdV-4 at 21 days of life. Two additional groups served as controls, a challenge control group with mock-vaccinated but infected birds and a negative control group with PBS injection substituting both vaccination and challenge. The fiber-2 vaccinated group displayed high resistance against the adverse effects of the challenge with only one dead bird out of 28, as compared to the challenge control group where the infection caused 78% mortality. A moderate protective effect resulting in 38% mortality was observed for fiber-1, whereas the hexon loop-1 vaccinated group was not effectively protected as manifested by 73% mortality. While a fiber-2 specific ELISA showed a gradual antibody increase after immunization of birds with the homologous protein, a commercial ELISA did not detect vaccination-induced antibodies in any of the groups but displayed a difference in challenge virus-directed response in protected and non-protected birds. Although immunoblotting confirmed the presence of specific antibodies in all vaccinated groups, the anti-protein sera did not exhibit neutralizing activity. Fecal excretion of challenge virus DNA was detected with a real-time PCR in the majority of tested birds until termination of the study independent of protection, indicating the prevention of clinical symptoms, but not infection, by vaccination. In conclusion, recombinant fiber-2 was identified as a protective immunogen and is proposed as an attractive candidate for a subunit vaccine to prevent hepatitis-hydropericardium syndrome in chickens.