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.

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.

Prevalence of Fowl Adenovirus Serotype 4 and Co-Infection by Immunosuppressive Viruses in Fowl with Hydropericardium Hepatitis Syndrome in Shandong Province, China.

Fowl adenovirus serotype 4 (FAdV-4) is the pathogenic agent of hydropericardium hepatitis syndrome (HHS) in chickens and ducks, which has caused huge economic losses for the Chinese poultry industry since 2015. In order to objectively determine the prevalence and co-infection status of the virus in Shandong province in China, we analyzed a total of 679 clinical cases of chickens and ducks from 36 farms in the province. The results showed that the FAdV-4 infection rate was 65.2% (443/679), and the rate in breeder ducks was almost two-fold higher than that in breeder chickens (68.57% vs. 34.30%). Notably, co-infection by H9N2 avian influenza virus, infectious bursal disease virus, and/or chicken infectious anemia virus was very common in the 443 FAdV-4-positive cases. Furthermore, phylogenetic analysis of the hexon genes of four Shandong FAdV-4 isolates revealed that these strains clustered into Indian reference strains, indicating that the Shandong FAdV-4 strains might have originated in India. These findings provide the first data on the prevalence and co-infection status of FAdV-4 in Shandong province, which may serve as a foundation for the prevention of FAdV-4 in the field.

Molecular characterization of fowl adenovirus isolate of Malaysia attenuated in chicken embryo liver cells and its pathogenicity and immunogenicity in chickens.

Fowl adenovirus (FAdV) is the causative agent of inclusion body hepatitis (IBH) in chickens with significant economic losses due to high mortality and poor production. It was objectives of the study to attenuate and determine the molecular characteristic of FAdV isolate (UPM1137) of Malaysia passages in primary chicken embryo liver (CEL) cells. The cytopathic effect (CPE) was recorded and the present of the virus was detected by polymerase chain reaction (PCR). Nucleotide and amino acid changes were determined and a phylogenetic tree was constructed. The pathogenicity and immunogenicity of the virus at passage 35 (CEL35) with virus titre of 106.7TCID50/mL was determined in day old specific pathogen free (SPF) chicks via oral or subcutaneous route of inoculation. The study demonstrated that the FAdV isolate was successfully propagated and attenuated in CEL cells up to 35th consecutive passages (CEL35) with delayed of CPE formation within 48 to 72 post inoculation (pi) from CEL20 onwards. The virus caused typical CPE with basophilic intranuclear inclusion bodies, refractile and clumping of cells. The virus is belong to serotype 8b with substitution of amino acid at position 44, 133 and 185 in L1 loop of hexon gene and in knob of fiber gene at position 348 and 360 at CEL35. It is non-pathogenic, but immunogenic in SPF chickens. It was concluded that the FAdV isolate was successfully attenuated in CEL cells with molecular changes in major capsid proteins which affect its infectivity in cell culture and SPF chickens.

Hexon and fiber gene changes in an attenuated fowl adenovirus isolate from Malaysia in embryonated chicken eggs and its infectivity in chickens.

Fowl adenovirus (FAdV) is distributed worldwide and causes economic losses in the poultry industry. The objectives of this study were to determine the hexon and fiber gene changes in an attenuated FAdV isolate from Malaysia in specific pathogen-free chicken embryonated eggs (SPF CEE) and its infectivity in commercial broiler chickens. SPF CEE were inoculated with 0.1 mL FAdV inoculum via the chorioallantoic membrane (CAM) for 20 consecutive passages. The isolate at passage 20 (E20), with a virus titer of 108.7TCID50/mL (TCID50, 50% tissue culture infective dose), was inoculated (0.5 mL) into one-day-old commercial broiler chicks either via oral or intraperitoneal routes. The study demonstrated that 100% embryonic mortality was recorded from E2 to E20 with a delayed pattern at E17 onwards. The lesions were confined to the liver and CAM. Substitutions of amino acids in the L1 loop of hexon at positions 49 and 66, and in the knob of fiber at positions 318 and 322 were recorded in the E20 isolate. The isolate belongs to serotype 8b and is non-pathogenic to broiler chickens, but it is able to induce a FAdV antibody titer. It appears that molecular changes in the L1 loop of hexon and the knob of fiber are markers for FAdV infectivity.

Co-infection of fowl adenovirus with different immunosuppressive viruses in a chicken flock.

In poultry, fowl adenovirus (FAdV) and immunosuppressive virus co-infection is likely to cause decreased egg production, inclusion body hepatitis, and pericardial effusion syndrome. In this study, fowl adenovirus infection was found in parental and descendent generations of chickens. We used quantitative polymerase chain reaction (PCR) and dot blot hybridization to detect the infection of reticuloendotheliosis (REV), avian leukosis virus (ALV), and chicken infectious anemia virus (CIAV) in 480 plasma samples. The test samples were 34.58% FADV-positive, 22.29% REV-positive, 7.5% CAV-positive, and 0.63% ALV-positive. Sequence analysis showed that FADV belonged to serotype 7, which can cause inclusion body hepatitis. The ALV strain was ALV-A, in which the homology of gp85 gene and SDAU09C1 was 97.3%. The positive rate was lower because of the purification of avian leukemia, whereas the phylogenetic tree analysis of REV showed that the highest homology was with IBD-C1605, which was derived from a vaccine isolate. Through pathogen detection in poultry we present, to our knowledge, the first discovery of fowl adenovirus type 7 infection in parental chickens and found that there was co-infection of FAdV and several immunosuppressive viruses, such as the purified ALV and CIAV. This indicates that multiple infection of different viruses is ever-present, and more attention should be given in the diagnosis process.

Flock prevalence of exposure to avian adeno-associated virus, chicken anemia virus, fowl adenovirus, and infectious bursal disease virus among Ontario broiler chicken flocks.

Samples from 231 randomly selected commercial broiler chicken flocks in Ontario were tested at slaughter for exposure to chicken anemia virus (CAV), fowl adenovirus (FAdV), and infectious bursal disease virus (IBDV). Fifteen blood samples per flock were collected and analyzed for the presence of antibodies against CAV, FAdV, and IBDV by ELISA or agar gel immunodiffusion test. Fifteen cecal tonsils and cloacal swabs per flock were analyzed for the presence of CAV, FAdV, and IBDV by PCR. The prevalence of exposure to avian adeno-associated virus (AAAV) was estimated by a PCR test on a subset of FAdV-PCR-positive samples from 178 flocks. Genotypes of FAdV and IBDV were identified on a subset of isolates (n = 353 and 45, respectively). The flock-level period prevalence of exposure to AAAV, CAV, FAdV, and IBDV during grow-out were 88.76% (95% CI: 84.08-93.45%), 77.06% (95% CI: 71.59-82.52%), 96.54% (95% CI: 94.16-98.91%), and 48.92% (95% CI: 42.42-55.41%), respectively. Results of a multivariable logistic regression model showed a significant association of exposure to FAdV with exposure to AAAV (OR = 18.57, 95% CI: 3.67-93.86, P = 0.004) but not with exposure to CAV (P = 0.7752) or exposure to IBDV (P = 0.2274). Pathogenic FAdV genotypes (FAdV-02, FAdV-08, and FAdV-11) constituted 39.38% of the isolates. The most-common IBDV genotypes identified were IBDV NC171 (60%) and IBDV 05SA8 (28.89%). This is the first large-scale study to estimate the baseline flock prevalence of exposure to AAAV, CAV, FAdV, and IBDV in commercial broiler flocks in Canada. Potentially pathogenic genotypes of FAdV and IBDV that can guide vaccine development and disease control efforts in Ontario were identified.

Molecular differentiation and pathogenicity of Aviadenoviruses isolated during an outbreak of inclusion body hepatitis in South Africa.

Fowl adenovirus (FAdV) is a member of the genus Aviadenovirus and causes a number of economically important poultry diseases. One of these diseases, inclusion body hepatitis (IBH), has a worldwide distribution and is characterised by acute mortality (5% - 20%) in production chickens. The disease was first described in the United States of America in 1963 and has also been reported in Canada, the United Kingdom, Australia, France and Ireland, but until now, not in South Africa. Adenoviruses isolated from the first outbreak of IBH in South Africa were able to reproduce the disease in chicken embryo livers. The aim of the present study was to characterise the viruses and determine the pathogenicity of the FAdV strains responsible for the first reported case of IBH in South Africa. Polymerase chain reaction (PCR) amplification of the L1 loop region of the fowl adenovirus hexon gene using degenerate primer pair hexon A/B was used to identify the viruses that were isolated. Restriction fragment length polymorphism (RFLP) of the amplification products was used for the differentiation of 14 isolates of fowl adenovirus. Sequencing of the PCR products followed by amino acid comparison and phylogenetic analysis using the L1 loop region of the hexon protein was done to determine the identity of the isolates. Amino acid sequences of the hexon genes of all the South African isolates were compared with those of reference strains representing FAdV species. Amino acid comparison of 12 South Africa field isolates to FAdV reference strains revealed a high sequence identity (> 93.33%) with reference strains T8-A and 764. Two of the isolates had high sequence identity (93.40%) with reference strains P7-A, C2B and SR48. Phylogenetic analysis of the L1 loop region of the hexon protein of all 14 South African isolates was consistent with their RFLP clusters. The mortality rates of embryos challenged with 106 egg infective doses (EID50) FAdV 2 were 80% - 87% and mortality rates for embryos challenged with 105.95 (EID50) FAdV 8b were 65% - 80%.

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.

Adenoviral gizzard erosion in broiler chickens in Germany.

Avian adenovirus infections cause important disease complexes in chickens, but many of the viruses also infect chickens without resulting in overt disease. Previously several outbreaks of gizzard erosions caused by a fowl adenovirus A serotype-1 (FAdV-1) were reported from Japan. Here we report an outbreak of gizzard erosions in 12 broiler flocks in Germany in 2011. Chickens had a reduced daily weight gain and a higher total mortality rate of up to 8%. The birds showed a severe detachment of the koilin layer and ulcerative to necrotizing lesions of the underlying mucosa. Histopathologically, necrotizing ventriculitis with basophilic, intranuclear inclusion bodies in epithelial cells was diagnosed. Immunohistochemistry, egg culture, and electron microscopic examination revealed adenovirus-like particles in the samples. No concurrent infectious agent could be identified. The virus was genotyped as FAdV-1 by PCR and subsequent sequencing. Phylogenetic analysis of the hexon loop L1 gene yielded 100% sequence identity to the chicken embryo lethal orphan strain. These findings suggest that outbreaks of adenoviral gizzard erosion can lead to significant economic losses in Germany and may be caused by an unusual virulent FAdV-1 strain.

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.

Fowl adenovirus (FAdV) in India: evidence for emerging role as primary respiratory pathogen in chickens.

Adenoviruses have been isolated from both clinically healthy and diseased birds worldwide. The pathogenic role of most of the FAdVs is still questionable. They can quickly take on the role of opportunistic pathogens when additional factors, particularly concurrent infections, adversely affect the health of the avian host. Immnosuppressing agents especially chicken infectious anemia and infectious bursal disease viruses are known to enhance the pathogenicity of FAdVs upon coinfection. The aim of the present study was to screen for the involvement of FAdV in poultry flocks affected with respiratory disease complex by RT-PCR. The samples were also screened by RT-PCR/PCR for other respiratory pathogens. Thirty two commercial poultry flocks with the history of respiratory disease complex from various parts of India. FAdV nucleic acid could be detected in tissue samples of 13 out of 34 farms investigated. Out of 13 FAdV positive farms, FAdV and CIAV were alone detected in 4/13 (31%) whereas, in other farms more than two respiratory pathogens were detected together. CIAV was detected in all the farms (34/34) investigated. Eosinophilic intranuclear inclusion bodies were noticed in FAdV infected laryngeal and tracheal epithelium under light microscopy. The findings of the study assert that FAdV can play the role of primary respiratory pathogen in immunocompromised birds and also in the presence of other respiratory pathogens.

Molecular characterization of fowl adenoviruses isolated from chickens with gizzard erosions.

Broiler chickens with clinical signs of uneven growth, depression, and dull feathers were submitted to our laboratory and, at necropsy, lesions in proventriculus, gizzard, and intestines were detected. Fowl adenovirus serotype 1 (FAdV-1) was isolated from digestive tissues. The virus, assigned as FAdV-PL/G068/08, showed 99.5% nucleotide homology and 99.2% amino acid homology in hexon gene with chicken embryo lethal orphan (CELO) strain classified as the European reference of FAdV-1. One-day-old and 21-d-old SPF chickens were inoculated with FAdV-PL/068/08 by both nasal and ocular routes and then observed daily and examined by necropsy at 6, 10, and 14 d postinoculation. Experimental infection with isolated virus was fatal for younger chickens and major lesions occurred in the gizzards. No clinical or pathological changes were observed in chickens infected at 21 d of age, but the presence of intranuclear inclusion bodies in gizzard epithelial cells was detected. Molecular characterization was based on the long and short fibers genes sequencing and comparison of obtained sequences with other FAdV-1 strains. The homology between FAdV-PL/G068/08 and other sequences available in GenBank was between 98.9 and 99.8% (short fiber region) and 99.0 and 99.7% (long fiber region) at nucleotide level and between 98.4 and 100% (short fiber region) and 99.3 and 99.9% (long fiber region) at amino acid level. No correlation between identified amino acid changes in short and long fiber proteins and pathogenicity of studied FAdV-1 strains was observed. Although short and long proteins were indicated as factors influencing virus pathogenicity, the role of identified sequence differences in infectivity determination remain unclear.

Coding potential and transcript analysis of fowl adenovirus 4: insight into upstream ORFs as common sequence features in adenoviral transcripts.

Recombinant fowl adenoviruses (FAdVs) have been successfully used as veterinary vaccine vectors. However, insufficient definitions of the protein-coding and non-coding regions and an incomplete understanding of virus-host interactions limit the progress of next-generation vectors. FAdVs are known to cause several diseases of poultry. Certain isolates of species FAdV-C are the aetiological agent of inclusion body hepatitis/hydropericardium syndrome (IBH/HPS). In this study, we report the complete 45667 bp genome sequence of FAdV-4 of species FAdV-C. Assessment of the protein-coding potential of FAdV-4 was carried out with the Bio-Dictionary-based Gene Finder together with an evaluation of sequence conservation among species FAdV-A and FAdV-D. On this basis, 46 potentially protein-coding ORFs were identified. Of these, 33 and 13 ORFs were assigned high and low protein-coding potential, respectively. Homologues of the ancestral adenoviral genes were, with few exceptions, assigned high protein-coding potential. ORFs that were unique to the FAdVs were differentiated into high and low protein-coding potential groups. Notable putative genes with high protein-coding capacity included the previously unreported fiber 1, hypothetical 10.3K and hypothetical 10.5K genes. Transcript analysis revealed that several of the small ORFs less than 300 nt in length that were assigned low coding potential contributed to upstream ORFs (uORFs) in important mRNAs, including the ORF22 mRNA. Subsequent analysis of the previously reported transcripts of FAdV-1, FAdV-9, human adenovirus 2 and bovine adenovirus 3 identified widespread uORFs in AdV mRNAs that have the potential to act as important translational regulatory elements.

Comparison of the fibers of Fowl adenovirus A serotype 1 isolates from chickens with gizzard erosions in Europe and apathogenic reference strains.

A total of 18 samples from 4 outbreaks of gizzard erosions in broiler chickens in Europe were used in the current study. Fowl adenoviruses were found in samples from all 4 outbreaks, and isolates were identified as Fowl adenovirus A (FAdV-A) serotype 1. As described earlier, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the long fiber gene was conducted. However, all 18 samples showed the same pattern as apathogenic FAdV-1 strains: Ote and chicken embryo lethal orphan (CELO) viruses. Nucleotide and amino acid sequences of the long and short fiber of several isolates from broiler chickens with gizzard erosions were analyzed, and 100% identity between the field isolates on the protein level was revealed. Only 1 nonsynonymous mutation (T→A) was present in the long fiber of studied isolates compared to the CELO strain. The same mutation was also present in the Ote strain. Four nonsynonymous mutations were present in the long fiber of studied isolates compared to Ote strain. In the short fiber, 6 nonsynonymous mutations were found in the studied isolates compared to the CELO strain. However, the short fiber of pathogenic isolates was 100% identical to apathogenic Ote strain. In conclusion, the usefulness of PCR-RFLP analysis of the long fiber gene of FAdV-1 isolates in distinguishing between those that induce gizzard erosions and those that do not remains questionable for the isolates obtained from European poultry flocks. The role of certain FAdV-1 strains with their long and short fiber in pathogenicity regarding gizzard erosions is still not clear.

Classification of fowl adenoviruses by use of phylogenetic analysis and high-resolution melting-curve analysis of the hexon L1 gene region.

A total of 44 fowl adenovirus (FAdV) samples from 6 European countries, Pakistan, India, Kuwait, Mexico, Peru and Ecuador were used in this study and the phylogenetic analyses based on the loop 1 (L1) region of hexon gene were performed. For comparison, available hexon sequences of representatives of different FAdV species were also used. At least 12 genotypes within the five FAdV species (A-E) were revealed and the existence of these genotypes was supported by high bootstrap values. Furthermore, three primer pairs binding to the conserved pedestal regions (HexL1s/HexL1as and HexA/HexB) and pedestal (P1) region and loop 2 (L2) region (HexF1/HexR1) of the FAdV hexon gene were used for high-resolution melting (HRM)-curve analysis and results were compared with those of phylogenetic analyses. HRM-curve analysis based on the HexL1s/HexL1as region grouped all tested field isolates and reference strains into 22 subgroups, consistently with phylogenetic analysis. This method is a rapid and cost-effective alternative to existing serotype identification methods and offers a possibility to classify FAdV isolates more precisely. However, it has limitations such as need for extensive interpretation of results and potential for indeterminate results. Gaining of hexon sequences of further field isolates offers the potential for novel and additional information in analysis of the molecular epidemiology of FAdV.

Characterization of fowl adenoviruses isolated in Ontario and Quebec, Canada.

Fowl adenoviruses (FAdV) are generally considered ubiquitous, but certain serotypes and strains are known to be associated with primary diseases, such as inclusion body hepatitis (IBH). Fifty-two FAdV isolates were collected from the provinces of Ontario and Quebec over a 4-year period. These 2 provinces have the largest poultry industries in Canada. Except for one virus, which originated from a guinea fowl, all other viruses were isolated from chicken samples. Most of these were from broilers, although some were from broiler breeders, and one was from layer pullets. Thirty-four isolates were from clinical IBH cases with the final laboratory diagnosis of IBH; however, for 18 isolates, the varied case diagnosis was seemingly unrelated to FAdV. All IBH-associated viruses had deoxyribonucleic acid (DNA) profiles compatible with FAdV species E (28 cases) or species D (6 cases), and the DNA fragment profiles of 26 species E viruses were indicative of serotype 8. Two viruses were serotype 6, as confirmed by virus neutralization. All species D viruses had a DNA profile similar to that of FAdV-2. The number of serotype 8 virus isolations has increased over the years, and by 2001 serotype 8 had become the dominant serotype in Ontario, and continues to be so. Moreover, this virus (FAdV-8) has shown a strong association with IBH.

Serologic monitoring of a broiler breeder flock previously affected by inclusion body hepatitis and testing of the progeny for vertical transmission of fowl adenoviruses.

The increasing number of clinical cases of inclusion body hepatitis (IBH) associated with fowl adenoviruses (FAdVs) is a growing concern in different parts of the world, including Canada. After an outbreak of IBH in a 10-d-old pullet broiler breeder flock, we serologically monitored the flock from 8 to 46 wk of age, using the agar gel precipitation test (AGPT) offered by diagnostic laboratories and an FAdV group-specific enzyme-linked immunosorbemt assay (ELISA) developed earlier. In addition, we tested 1-d-old progeny for possible vertical transmission of FAdV when the breeder flock approached the peak of egg production by performing virus isolation and polymerase chain reaction (PCR) procedures on target organs. As in previous studies comparing the 2 tests, ELISA was more sensitive than AGPT. With ELISA, a few birds had weakly positive results at 8 wk of age, and all the birds had strongly positive results from 12 wk of age until the end of the study. This group-specific ELISA is therefore a sensitive and practical way to monitor FAdV antibodies in commercial flocks. None of the 1-d-old chicks tested were positive by PCR, nor was FAdV isolated from the same tissues, indicating an absence of transmission of infectious virus to the progeny. The lack of virus production and transmission could be due to the presence of high antibody titers in the layers.

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.

Study of vertical transmission of fowl adenoviruses.

The vertical transmission of fowl adenoviruses (FAdVs) was studied by polymerase chain reaction (PCR) and virus isolation. Liver, spleen, kidney, and bursa of Fabricius were collected from 60 chicks 1 d old representing progenies hatched to 6 broiler breeder flocks in 6 geographically different premises in Ontario, Canada. The presence of FAdV DNA sequences was detected by PCR with the use of primers specific for the conserved pVI gene of FAdV-9 in 58 (24%) of the 240 samples tested. All samples from 1 flock were negative for FAdV sequences, and only a few samples were positive in 3 flocks, whereas 32% and 72% of the samples from the other 2 flocks were positive. Testing of 1 sample with primers designed to amplify the L1 region of the hexon protein gene and amino acid sequence analysis of the PCR product indicated that the sequences were similar to serotype-8a FAdV sequences. No fowl adenoviruses were isolated in chicken hepatoma cells from any of the 30 samples inoculated. These findings imply that vertical transmission and establishment of latent infection with FAdVs can occur in chickens.