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 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.

Gizzard Erosion and Ulceration Syndrome in Moroccan Poultry Flocks and Molecular Characterization of Fowl Adenoviruses (FAdV).

Gizzard erosion and ulceration syndrome (GEUS) is caused by a fowl adenovirus serotype 1 (FAdV-1) and was first reported in laying hens in Japan in 1993. This syndrome has emerged as an epizootic in Morocco since 2014, causing significant economic losses for the poultry industry, but no involvement of a FAdV has been confirmed. Thus, the objective of this work was to assess GEUS cases that occurred in the country and to determine the role of FAdVs in their occurrence. Investigations were based on a retrospective reassessment of tissue sections and paraffin blocks of gizzards and livers from GEUS cases between 2014 and 2021 coupled with a prospective search of cases in 2022. Gizzards and livers were fixed in 10% neutral buffered formalin for histopathologic examinations according to standard methods and stored at -20 C for molecular analysis. After deparaffinizing, 10-µm-thick tissue sections along with fresh organs were subjected to DNA extraction using a commercial kit. A primer pair specific for the Hexon gene of FAdVs was used in conventional PCR; in contrast, for real-time PCR, a primer pair targeting the 52K gene was employed. In total, 24 flock cases with characteristic GEUS were assessed between 2014 and 2022. They were nine broiler cases aged between 11 and 39 days, 11 layer cases with an age between 17 and 29 wk, two cases in meat-type breeders aged 10 and 27 wk, and two flock cases of turkey poults aged 22 and 23 days. In most cases, microscopic lesions were consistent with an ulcerative and lymphoplasmocytic ventriculitis, and pathognomonic viral intranuclear inclusion bodies within degenerate epithelial cells were identified in four broiler flock cases, four layer cases, and one case in breeders and hence were highly suggestive of a FAdV infection. Among these nine cases that were positive at the histopathologic examination, six cases were found to be FAdV-PCR positive; another four cases were negative to histology but FAdV-PCR positive. Furthermore, a sequencing analysis was conducted, providing the initial evidence of the implication of FAdV-1 from species A as the cause of GEUS in Moroccan poultry. Additionally, a phylogenetic analysis was executed to facilitate a comparison between the strains investigated in this study and those identified in diverse geographic regions and across various time periods.

Síndrome de erosión y ulceración de la molleja en parvadas avícolas marroquíes y caracterización molecular de los adenovirus aviares (FAdV). El síndrome de erosión y ulceración de la molleja (GEUS) es causado por un adenovirus del pollo de serotipo 1 (FAdV-1) y se reportó por primera vez en gallinas de postura en Japón en 1993. Este síndrome se ha convertido en una epizootia en Marruecos desde 2014, causando importantes pérdidas económicas a la industria avícola, pero no se ha confirmado la participación de ningún adenovirus del pollo. Por lo tanto, el objetivo de este trabajo fue evaluar los casos de GEUS ocurridos en el país y determinar el papel de adenovirus del pollo en su presentación. Las investigaciones se basaron en una reevaluación retrospectiva de secciones de tejido y bloques de parafina de mollejas e hígados de casos de GEUS entre 2014 y 2021, junto con una investigación prospectiva de casos en el año 2022. Las mollejas y los hígados se fijaron en formalina al 10% amortiguada y neutra para exámenes histopatológicos de acuerdo con métodos estándar y se almacenaron a -20 C para análisis moleculares. Después de la desparafinación, las secciones de tejido de 10 µm de espesor junto con órganos frescos se sometieron a extracción de ADN utilizando un estuche comercial. Para realizar un método de PCR convencional, se utilizó un par de iniciadores específicos para el gene de hexon de los adenovirus del pollo, mientras que, para el método de PCR en tiempo real, se empleó un par de iniciadores dirigidos al gene 52K. En total, se evaluaron 24 casos de parvadas con la presentación característica del síndrome de erosión y ulceración de la molleja entre los años 2014 y 2022. Se trató de nueve casos de pollos de engorde con edades comprendidas entre los 11 y 39 días, 11 casos de ponedoras con una edad de entre 17 y 29 semanas, dos casos en reproductoras pesadas de 10 y 27 semanas, y dos lotes de pavitos de 22 y 23 días. En la mayoría de los casos, las lesiones microscópicas fueron consistentes con una ventriculitis ulcerativa y linfoplasmocítica, y se identificaron cuerpos de inclusión intranucleares virales patognomónicos dentro de células epiteliales degeneradas en cuatro casos de parvadas de pollos de engorde, cuatro casos de ponedoras y un caso en reproductoras y por lo tanto, fueron altamente sugestivos de la infección por adenovirus de pollo. Entre los nueve casos que fueron positivos en el examen histopatológico, se encontró que seis casos fueron positivos para adenovirus de pollo mediante PCR; otros cuatro casos fueron negativos a la histología pero positivos a la presencia de adenovirus del pollo mediante PCR. Además, se realizó un análisis de secuenciación que proporcionó la evidencia inicial del papel de adenovirus del pollo especie A como causante del síndrome de erosión y ulceración de la molleja en la avicultura de Marruecos. Además, se realizó un análisis filogenético para facilitar una comparación entre las cepas investigadas en este estudio y las identificadas en diversas regiones geográficas y en varios períodos de tiempo.

Quantity of virulent fowl adenovirus serotype 1 correlates with clinical signs, macroscopical and pathohistological lesions in gizzards following experimental induction of gizzard erosion in broilers.

In the present study day-old specific-pathogen-free (SPF) and commercial broilers with maternally derived fowl adenovirus serotype 1 (FAdV-1) antibodies were orally infected with a European "pathogenic" FAdV-1, isolated from broilers showing signs of gizzard erosion. During the experiment, broilers were observed and weighed daily up to 17 days post infection (dpi). Clinically, both infected groups showed significant decrease of weight compared to respective negative control groups. Birds were examined by necropsy at 3, 7, 10, 14 and 17 dpi. Pathological changes in the gizzards were noticed in both experimentally infected groups from 7 dpi onwards. Macroscopically, erosion of the koilin layer and inflammation or ulceration of the gizzard mucosa were observed. Histologically, presence of FAdV-1 in intranuclear inclusion bodies of degenerated glandular epithelial cells was demonstrated by in-situ hybridization and inflammatory cell infiltration of the lamina propria, submucosa and muscle layer was detected. Tissue samples were investigated by a recently developed real-time PCR and the viral DNA load was calculated from gizzard, liver, spleen and cloacal swabs with the highest amounts of FAdV-1 DNA found in the gizzard. For the first time, successful reproduction of clinical signs in broilers as well as pathological lesions in the gizzard were achieved with a European FAdV-1 isolate displaying some genetic differences to so far reported virulent FAdV-1 from Japan. Furthermore, highest viral load in gizzards could be linked with macroscopical and histological lesions. Therefore, the conducted analyses provide important insights into the pathogenesis of adenoviral gizzard erosion.

Vertical transmission and clinical signs in broiler breeders and broilers experiencing adenoviral gizzard erosion.

The present report documents an outbreak of adenoviral gizzard erosion in 22 broiler flocks in Germany. The clinical picture was characterized by uneven growth of affected broilers that resulted in considerably lower than average weight at slaughtering. Fowl adenovirus serotype 1 (FAdV-1) was isolated from gizzard lesions and histological examinations demonstrated FAdV-1-positive intranuclear inclusion bodies in gizzard epithelial cells of affected broilers by in-situ hybridization. Birds from all affected flocks originated from one broiler breeder farm. During production of affected birds, broiler breeders were between 27 and 32 weeks old. Enzyme-linked immunosorbent assay and specific virus neutralization assay of sera from parent birds demonstrated an acute FAdV-1 infection within the first 5 weeks of the production cycle. Clinically, broiler breeders exhibited a moderate fall in the hatchability of their chicks, while egg production remained normal. No further clinical signs could be observed. Genetically identical FAdV-1 strains were isolated from gizzards of embryos at the lowest point of hatchability and from affected broiler flocks raised on independent farms. For the first time, direct detection of viable FAdV-1 from gizzards of embryos and progenies of one FAdV-1-seropositive broiler breeder farm in the course of an outbreak of adenoviral gizzard erosion could be demonstrated, highlighting the importance of vertical transmission of this disease. Additionally, growth retardation and subsequent reduced average weight at the time of slaughter of broiler chickens underline the economic impact of adenoviral gizzard erosion for poultry production.

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.

Novel avian bornavirus in a nonpsittacine species (Canary; Serinus canaria) with enteric ganglioneuritis and encephalitis.

A canary bird (Serinus canaria) died with nonsuppurative ganglioneuritis of the proventriculus and gizzard and encephalitis, lesions comparable to proventricular dilatation disease (PDD) of psittacine birds. Recently, several genotypes of a novel avian bornavirus have been linked to PDD. In the canary, bornaviral antigen was detected by immunohistochemistry in both neural and extraneural tissues. The widespread viral dissemination was confirmed by reverse transcription-PCR. Sequence analysis revealed a unique genotype of avian bornavirus. This observation suggests that bornaviruses are natural pathogens of several avian species and that the family Bornaviridae comprises more viral genotypes (or viral species) than previously assumed.

Successful reproduction of adenoviral gizzard erosion in 20-week-old SPF layer-type chickens and efficacious prophylaxis due to live vaccination with an apathogenic fowl adenovirus serotype 1 strain (CELO).

Recently, outbreaks of adenoviral gizzard erosion (AGE) have been documented in pullets and layers housed free range and in enriched cage systems characterized by increased mortality and a negative impact on egg production. In the present study the pathogenicity of a fowl adenovirus serotype 1 (FAdV-1) field strain as well as the aetiological role of a FAdV-8a strain, both isolated from AGE affected pullets, were investigated in vivo in 20-week-old specific-pathogen-free (SPF) layer-type chickens. Furthermore, the efficacy of a single (week 17) and double (week 14 and 17) application of a live vaccine consisting of an apathogenic FAdV-1 (CELO strain) against challenge with virulent FAdV-1 was investigated. For the first time, AGE was successfully reproduced in adult birds after oral infection of 20-week-old SPF birds with a virulent FAdV-1 field isolate, characterized by pathological changes of the gizzard from 7 days post challenge onwards. In addition, a negative impact of the FAdV-1 infection on the development of the reproductive tract was observed. Thus, confirming the pathogenicity and aetiological role of FAdV-1 in the development of AGE and economic losses due to AGE in layers. In contrast, no pathological changes were observed in birds infected with FAdV-8a. Independent of a single or double application of the live FAdV-1 vaccine strain CELO, no gross pathological changes were observed in gizzards post challenge with the virulent FAdV-1, indicating that complete protection of layers against horizontal induction of AGE was achieved. Nonetheless, virulent FAdV-1 was detected in cloacal swabs and gizzards in both vaccinated groups post challenge determined by the application of an amplification refractory mutation system quantitative PCR used to differentiate between vaccine and challenge strains.

Detection and localization of a goose adenovirus in experimentally infected goslings, using indirect immunofluorescence with paraffin-embedded tissue sections.

To establish an ideal method for the detailed definition of the tropism of the goose adenovirus new-type gosling viral enteritis virus (NGVEV) in conventional paraformaldehyde-fixed paraffin-embedded gosling tissue sections, an indirect immunofluorescence assay was established and optimized in this study for the first time. Three-day-old goslings orally inoculated with the CN strain of NGVEV were killed from 0.5 h to 30 days post-infection (p.i.) at intervals, and organs were collected and prepared for immunofluorescence assay and ultrastructural observation. NGVEV antigens could be detected in the gastrointestinal tract and lymphoid organs as early as 24 h p.i. The lymphoid organs (bursa of Fabricius, thymus, Harderian gland and spleen), the digestive organs (proventriculus, gizzard, duodenum, jejunum, ileum, caecum, rectum and liver), kidney, myocardium and brain of NGVEV-infected goslings showed extensive evidence of presence of viral antigens. Lymphocytes, macrophages, mesenchymes, endothelial cells, epithelia, superficial and crypt mucosal cells, glandular cells, and fibrocytes served as the principal site for NGVEV localization. The number of positive cells and intensity of fluorescence increased until 15 days p.i. Ultrastructural observation showed that NGVEV particles could be seen from 3 days p.i. onwards.

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.

Adenoviral Gizzard Erosions in Two Belgian Broiler Farms.

Fowl adenovirus infections are widely prevalent in poultry. Many of the viruses can infect chickens without resulting in overt disease. Nevertheless, some fowl adenoviruses can cause important disease complexes in chickens such as inclusion body hepatitis, hydropericardium syndrome, necrotic pancreatitis, and gizzard erosion. Adenoviral gizzard erosions have been regularly reported from Japan, but detailed reports from Europe are scarce and available only from Italy, Poland, Hungary, and Germany. This case report describes two concurrent outbreaks of gizzard erosions caused by fowl adenovirus A in two Belgian broiler farms. Clinical signs observed were signs of depression, reduced feed intake, reduced weight gain, and lack of uniformity of the flocks. At necropsy, typically multiple erosions within the koilin layer of the gizzard were observed. Histopathological examination showed a multifocal, erosive ventriculitis with basophilic intranuclear inclusions in the epithelium. PCR analysis confirmed the diagnosis of fowl adenovirus. These findings suggest that outbreaks of adenoviral gizzard erosion can also lead to significant economic losses in Belgium.

Putative host cell receptor for fowl adenovirus detected in gizzard.

This work was done to identify a fowl adenovirus (FAV) binding protein in the gizzard, a known target organ for certain strains of FAV serotype 1. By using a virus overlay protein binding assay (VOPBA), a putative FAV binding protein of approximately 200 kDa expressed in the gizzard was detected.

Reproduction of adenoviral gizzard erosion by the horizontal transmission of fowl adenovirus serotype 1.

The horizontal transmission ability of fowl adenovirus (FAV) serotype 1 99ZH strain, isolated from chickens exhibiting gizzard erosion, was investigated. Twelve 13-day-old specific pathogen-free chickens were inoculated orally with 10(6) TCID(50)/0.05 ml of the strain. An in-pen contact group (chickens in the same pen with inoculated chickens), hedge contact group (chickens in a pen connected with pens housing inoculated chickens), non-contact group (chickens in a separate pen placed at a distance of 70 cm from the connected pens), human exposure group (chickens in the next room and attended last every day) and negative control group were examined. Each group consisted of 11 or 12 uninoculated chickens. Gizzard lesions were grossly or histologically observed from 10 days after exposure (DAE) in the in-pen contact group, and from 15 DAE in the hedge contact and non-contact groups. The FAV gene was detected by polymerase chain reaction performed on cloacal swabs taken on 5 and 13 DAE from chickens in both contact groups, and on 20 and 26 DAE from those in the non-contact group. Serum neutralizing antibodies against FAV serotype 1 were detected in chickens from 13 and 26 DAE in both contact groups and in the non-contact group, respectively. In the human exposure and negative control groups, no infection was observed. We conclude that FAV-99ZH strain spreads rapidly through direct contact with inoculated chickens, and slowly through non-contact transmission, and that adenoviral gizzard erosion is reproduced by this horizontal transmission.

Whole genome sequencing of Fowl aviadenovirus A - a causative agent of gizzard erosion and ulceration, in adult laying hens.

Gizzard erosion and ulceration (GEU) caused by fowl aviadenovirus serotype 1 (FAdV-1) of the species Fowl aviadenovirus A (FAdV-A) represents an economically important problem in poultry production. The disease affects mostly young chicken broilers or layers before production. In this study, an unusual GEU outbreak in a flock of laying hens at 38weeks of age is described. The affected flock showed elevated mortality rates, with the highest number of dead birds appearing between the 39th and 40th week of life, with a subsequent reduction in laying performance and decreased total egg weight. Post-mortem examination showed the presence of erosion in multiple areas of the gizzard, with wall perforation in the proximity of the interventriculus. FAdV antibodies were detected in all examined sera with an ELISA assay. The virus was isolated from pathologically altered gizzards. PCR, subsequent sequencing and phylogenetic analysis of the partial hexon gene confirmed the presence of FAdV-A DNA. To investigate the molecular background of FAdV-A which causes GEU in adult hens, whole genome sequencing was performed on two FAdV-A strains - strain W-15, obtained from the outbreak described in this study and strain 61/11z, isolated from a GEU outbreak in 3-week-old broiler chickens in 2011. The genome size of FAdV-A W-15 is 43,849bp. Genome sequence and genome organization resembles those of the reference, apathogenic CELO strain and the newly sequenced GEU strain, 61/11z. Most amino acid changes, between CELO and GEU strains, were observed in ORF0, ORF1, ORF14, IVa2, polymerase, pIIIa, penton base and fiber-2. Analysis conducted on the translated ORFs revealed that W-15 and 61/11z are nearly identical, with the highest rate of amino acid mutations in pTP, 100K, ORF9 and ORF10. In this study, the occurrence of GEU, caused by FAdV-1 infection, in adult layer chickens and the effects of such infection on egg production parameters are described in detail. Moreover, the whole genome sequences of two pathogenic, GEU inducing FAdV-A strains have been provided and characterized for the first time, which in the future will help to pinpoint the viral factors involved in pathogenicity.

Comparison of the polymerase chain reaction-restriction fragment length polymorphism pattern of the fiber gene and pathogenicity of serotype-1 fowl adenovirus isolates from gizzard erosions and from feces of clinically healthy chickens in Japan.

The fiber gene sequence and pathogenicity of the serotype-1 fowl adenovirus (FAdV-1) isolated from gizzard erosions and from clinically normal chickens were compared among isolates. The FAdV-99ZH strain, which induced gizzard erosions, had a nucleotide sequence of the long fiber gene that was different from that of the Ote strain, which did not induce gizzard erosions. The differences could be distinguished by use of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The long fiber gene of 16 FAdV-1 isolates from gizzard erosions and 10 FAdV-1 isolates from the feces of clinically normal chickens was examined by use of PCR-RFLP analysis. All 16 FAdV-1 isolates from gizzard erosions had the same restriction patterns as those of strain 99ZH; however, 10 FAdV-1 isolates from normal chickens were classified into 3 groups. Specific-pathogen-free (SPF) chickens were inoculated orally with 2 FAdV-1 isolates from gizzard erosions or 3 FAdV-1 isolates from clinically normal chickens to determine the pathogenicity of each strain. Two of 2 FAdV-1 isolates from gizzard erosions induced gizzard erosions. Two of 3 FAdV-1 isolates from normal chickens had the same PCR-RFLP patterns as those of the Ote strain, but did not induce any gizzard erosions. However, 1 FAdV-1 isolate from clinically normal chickens had the same PCR-RFLP pattern as that of strain 99ZH and induced gizzard erosions. These results indicate that there are FAdV-1 strains that have different pathogenicity; one strain induces gizzard erosions, and the other does not. Use of PCR-RFLP analysis of long fiber genes may be able to distinguish between these two strains.

Characterization of monoclonal antibodies against fowl adenovirus serotype 1 (FAV1) isolated from gizzard erosion.

Six clones of monoclonal antibodies (Mabs) to fowl adenovirus (FAV) serotype 1 were produced. All Mabs reacted positively by enzyme-linked immunosorbent assay. Three Mabs recognized the putative 100-kD hexon protein and reacted to serotype 1 specifically by western blot analysis but did not react to other FAV serotypes (2, 3, 4, 5, 6, 7, and 8a). These Mabs will be useful for immunodiagnosis of FAV serotype 1 infection in chickens with gizzard erosion and in further research studies involving the genomes and proteins of FAV serotype 1.

Pathogenicity of fowl adenovirus isolated from gizzard erosions to immuno-suppressed chickens.

Pathogenicity of a fowl adenovirus (FAV), JM1/1 strain of serotype 1 derived from gizzard erosions of a broiler chicken, was examined to specific pathogen-free (SPF) chickens pre-treated with infectious bursal disease viruses (IBDVs) or cyclophosphamide (CY). Virulent IBDVs, classical type, were inoculated orally at 3 days of age of SPF chickens. CY was treated subcutaneously for 3 days after hatch. FAV was given orally at 30 days of age. At 40 days of age, all chickens were bled and autopsied for serology and gross observation. Gizzard lesions were ranked by the scores depending on their severities. IBDV- or CY-treated chickens showed significantly higher gizzard lesion scores than non treated birds. There were no gross lesions in any other organs except for bursal atrophy. Serologically, antibody production against FAV was highly suppressed by IBDV infection or CY treatment.

Gastrointestinal pathogenicity of adenoviruses and reoviruses isolated from broiler chickens in Alabama.

Adenoviruses and reoviruses isolated from commercial broiler chickens were evaluated for gastrointestinal pathogenicity in specific-pathogen-free Leghorn chickens. The viruses were originally isolated from either the proventriculus or a gastrointestinal pool of tissues of broiler chickens with proventriculitis or enteritis. Isolates were cloned by terminal dilution. Day-old chickens were inoculated by oral and ocular routes with undiluted tissue culture fluids (titers of 10[2]-10[4] TCID50/ml) and then examined at necropsy on days 5, 10, and 15 postinoculation. Chickens in all virus groups (but not the control group) developed wet, unformed fecal droppings that persisted for the duration of the study. Mild lesions occurred in reovirus-inoculated chickens and included hyperplasia of lymphocyte aggregates in various organs and mild gizzard erosions. Chickens inoculated with adenovirus isolates developed marked gizzard erosions and necrotizing pancreatitis as well as mild proventriculitis. Intranuclear viral inclusion bodies occurred in gizzard epithelium and pancreatic acinar cells at the sites of lesions. Lymphocytic atrophy occurred in the bursa of Fabricius. Respective viruses were reisolated from proventriculus and duodenum collected from chickens of each group; no viruses were isolated from controls. Under the conditions of this study, adenovirus isolates were more pathogenic than the reovirus isolates in the digestive system.

Experimental infection of specific-pathogen-free chickens with serotype-1 fowl adenovirus isolated from a broiler chicken with gizzard erosions.

Gizzard lesions were formed in specific-pathogen-free (SPF) white leghorn chickens inoculated with fowl adenovirus (FAV). The virus, serotype 1 FAV 99ZH strain (FAV-99ZH), was originally isolated from the gizzard mucosa of commercial broiler chickens exhibiting gizzard erosion with intranuclear inclusion bodies. Five-day-old and 53-day-old SPF white leghorn chickens were inoculated with FAV-99ZH by both oral and ocular routes and then examined at necropsy on days 3, 5, 7, 10, 14, and 21 postinoculation (PI). There were no clinical signs in any of the chickens after the inoculation. Focal gizzard lesions occurred macroscopically, however, in inoculated chickens at several experimental periods. FAV was recovered from tissue samples of the proventriculus, gizzard, pancreas, and rectum by day 10 or 7 PI but was not recovered from liver samples of any of the chickens. These results indicate that FAV isolated from gizzard erosion is able to reproduce gizzard lesions as necrosis and erosion in SPF white leghorn chickens and that it may have a greater degree of tissue tropism in gizzards and other digestive organs than in the liver.

Experimental gizzard erosions in specific-pathogen-free chicks by serotype 1 group I avian adenoviruses from broilers.

The pathogenicity of serotype 1 group I avian adenovirus (GIAAV) strains isolated from gizzard erosions for 1-wk-old specific-pathogen-free (SPF) chickens was investigated. In Experiment 1, SPF chickens inoculated orally with a serotype 1 strain (Tokushima2000/GE) of GIAAV isolated from gizzard erosion in broilers were euthanatized and necropsied from 3 to 12 days after inoculation. Clinically, there were no significant signs in the chickens inoculated with Tokushima strain. The gizzards were found macroscopically to be dilated with liquefied contents. The koilin layer of the gizzard exhibited multifocal white lesions with occasional erosions. Gross lesions of the koilin layer (white foci and erosion) appeared at 3 days after inoculation and persisted until 12 days after inoculation. The gross lesions were most severe at 9 days after inoculation. Histologically, multifocal degeneration, necrosis, and loss of the glandular epithelial cells with intranuclear inclusions, liquefactive change of koilin substance, and erosive lesions of koilin layers and surface epithelium of the gizzards were observed. Macrophages increased in the focal areas of the lamina propriabelow the liquefactive koilin substances. Immunohistochemically, intranudear inclusions were positive against GIAAV antigen. Histologic lesions of gizzards were most severe at 7 and 9 days after inoculation, but the frequency of intranuclear inclusions was greatest at 5 days after inoculation. In Experiment 2, the pathogenicities of four strains of serotype 1 GIAAV for 1-wk-old SPF chickens were compared. Three strains isolated from gizzard erosion had similar pathogenicities but one strain from a normal chicken was not pathogenic for chickens. These results suggest that gizzard erosions by serotype 1 GIAAV infection may occur in broiler chickens in Japan.