Gizzard erosions in broiler chickens in Sweden caused by fowl adenovirus serotype 1 (FAdV-1): investigation of outbreaks, including whole-genome sequencing of an isolate.

The present paper describes the investigation of the first outbreaks of adenoviral gizzard erosions (AGE) in Sweden, in five broiler flocks. The investigation included whole viral genome sequencing and investigation of genomic organization and sequence relationships with other adenoviruses. All five flocks had a history of decreased growth and uneven size of birds since 9-10 days of age. Macroscopically, lesions consistent with AGE (detached koilin layers, discolouration, bleeding, erosions) were identified in gizzards in all five flocks. In four flocks histology was performed, and degeneration and inflammation of the koilin layer and gizzard mucosa were identified in all four. In one flock, intranuclear inclusion bodies typical for fowl adenovirus (FAdV) were detected in trapped epithelial cells in the koilin layer. In four flocks in situ hybridization was performed, and cells positive for FAdV serotype 1 (FAdV-1) were demonstrated in the koilin layer and gizzard mucosa. FAdV species A (FAdV-A) was detected in gizzard, liver, caecal tonsils and bursa of Fabricius by polymerase chain reaction (PCR) and sequencing. Ten out of ten examined parent flocks of the affected chickens were seropositive for FAdV, indicating former or on-going infection. However, FAdV was not detected in embryos from seropositive parent flocks and thus vertical transmission was not demonstrated. The entire nucleotide sequence of one sample was determined and found to be 43,856 base pairs (bp) in length. The genome sequence and organization were found to be similar to that of the reference apathogenic avian adenovirus "chicken embryo lethal orphan" (CELO). RESEARCH HIGHLIGHTSAGE in Swedish broilers: necropsy, histopathology, ISH, PCR, whole-genome sequencing.Whole FAdV-genome analysed: 43,856 bp, found to be most similar to CELO (U46933.1).Multiple point mutations, site insertions and deletions identified compared to CELO.Paper adds knowledge about European disease situation and pathogenic FAdV-strains.

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.

Pathogenicity of field strain of fowl aviadenovirus serotype 11 isolated from chickens with inclusion body hepatitis in Morocco.

Outbreaks of inclusion body hepatitis have emerged in Morocco since 2013 and has resulted in significant economic losses to poultry farms. Three isolates of the causative virus, Fowl adenonovirus (FAdV)were characterized from chickens with IBH, but their pathogenicity has never been investigated. In this work, the pathogenicity of an isolate FAdV 11 (MOR300315 strain) was evaluated by inoculating a group of 40 SPF chickens at 3 days of age by oral route. A group of 40 chicks injected with phosphate-buffered saline solution was used as a control group. The infected chickens showed decreased weight gain from 3dpi. Necropsy displayed pallor and enlargement in liver, swelling and slight hemorrhage in kidney and spleen at 6 dpi. Histopathological changes were mainly characterized by severe and extensive hepatic necrosis associated with the presence of basophilic intra-nuclear inclusion bodies within hepatocytes. The FAdV was reisolated in chicken embryo fibroblast cell culture from liver tissue homogenate of infected chicken from 3 to 6 dpi. Viral DNA was detected by PCR in liver, kidney, spleen and cloacal swabs from 3 to 13 dpi. Antibody response against inoculated FAdV was appeared from 9 dpi. These results confirmed that the FAdV 11 strain is pathogenic in chicken. This study is the first experimental infection of FAdV 11 in chicken in Morocco, which increase our understanding of its pathogenicity in chickens and indicate that preventive measures against FAdV infection in poultry farms should be implemented in Morocco.

Genetic Characterization and Pathogenicity Analysis of Recently Isolated Fowl Adenovirus 8b in Korea.

A Korean field strain of fowl adenovirus (FAdV) 8b was isolated from chickens showing high mortality. Isolated FAdV-8b strains with the hexon and fiber genes were genetically analyzed. The Korean FAdV-8b (K194/19) strain isolated in 2019 showed higher sequence identity with the FAdV-8b strain isolated in China but lower sequence identity with the Korean FAdV-8b (K187/08) strain isolated in 2008. The K194/19 strain formed a distinct subcluster within the FAdV-8b cluster in a phylogenetic tree based on hexon and fiber genes. FAdV can infect day-old chicks through vertical transmission, and so blood samples were obtained from 54-, 60-, and 63-wk-old parent chickens. FAdV-specific antibody levels were investigated with ELISA and virus neutralization (VN) tests with the K194/19 and K187/08 strains as antigens. In VN tests, all sera neutralized the K187/08 strain. However, the K194/19 strain was neutralized by sera collected from 60- and 63-wk-old chickens but not sera obtained from 54-wk-old chickens, indicating natural infection. Finally, to determine the pathogenicity of the K194/19 strain, 1-day-old and 4-wk-old specific-pathogen-free birds were infected with the K194/19 and K187/08 strains. No significant difference in pathogenicity was observed between the two strains. Although the K194/19 strain showed similar pathogenicity with the K187/08 strain, differences in nucleotide and amino acid sequences of the hexon and fiber genes may determine the evasion ability of the K187/08 neutralizing antibody, indicating the need for development of a novel FAdV vaccine.

Nota de investigación­Caracterización genética y análisis de patogenicidad de un adenovirus del pollo 8b aislado recientemente en Corea. Se aisló una cepa de campo coreana de adenovirus del pollo (FAdV) 8b de aves que mostraban una alta mortalidad. Se analizaron genéticamente cepas de FAdV-8b aisladas mediante los genes de hexón y de la fibra. La cepa coreana FAdV-8b (K194/19) aislada en 2019 mostró una mayor identidad de secuencia con la cepa FAdV-8b aislada en China, pero una menor identidad de secuencia con la cepa coreana FAdV-8b (K187/08) aislada en 2008. La cepa K194/19 formó un subgrupo distinto dentro del grupo de adenovirus del pollo 8b en un árbol filogenético basado en los genes de las fibras y hexones. El FAdV puede infectar a pollitos de un día a través de la transmisión vertical, por lo que se obtuvieron muestras de sangre de pollos reproductores de 54, 60 y 63 semanas de edad. Los niveles de anticuerpos específicos de FAdV se investigaron con ELISA y pruebas de neutralización de virus (VN) con las cepas K194/19 y K187/08 como antígenos. En las pruebas de neutralización, todos los sueros neutralizaron a la cepa K187/08. Sin embargo, la cepa K194/19 fue neutralizada por sueros recolectados de pollos de 60 y 63 semanas de edad, pero no por los sueros obtenidos de pollos de 54 semanas de edad, lo que indica una infección natural. Finalmente, para determinar la patogenicidad de la cepa K194/19, se infectaron aves libres de patógenos específicos de un día y cuatro semanas de edad con las cepas K194/19 y K187/08. No se observaron diferencias significativas en la patogenicidad entre las dos cepas. Aunque la cepa K194/19 mostró una patogenicidad similar con la cepa K187/08, las diferencias en las secuencias de nucleótidos y aminoácidos de los genes del hexón y de la fibra pueden determinar la capacidad para evadir los anticuerpos neutralizantes K187/08, lo que indica la necesidad de desarrollar una nueva vacuna contra adenovirus del pollo.

Characterization of a fowl adenovirus 9 (FAdV-9) early promoter and its application in generating dual expression FAdV-9s.

The CMV immediate early promoter from the EGFP expression plasmid pEGFP-N1 was replaced with the very left end of the fowl adenovirus 9 (FAdV-9) genome (ntds 73-574) to demonstrate and delineate the promoter function of this sequence. Expression of an EGFP ORF which replaced ORF1 and ORF2 demonstrated that the native promoter can drive down stream foreign gene expression. Replacement of ORF1 and ORF2 with a bicistronic cassette, incorporating a 493 bp IRES from an Ontario strain of avian encephalomyelitis virus (AEV) separating an EGFP ORF and mCherry ORF allowed for expression of both ORFs from a recombinant FAdV. These results provide an additional platform for multivalent vaccines development based on a native FAdV-9 promoter and an avian virus IRES.

Domain in Fiber-2 interacted with KPNA3/4 significantly affects the replication and pathogenicity of the highly pathogenic FAdV-4.

The outbreaks of hepatitis-hydropericardium syndrome (HPS) caused by the highly pathogenic serotype 4 fowl adenovirus (FAdV-4) have caused a huge economic loss to the poultry industry globally since 2013. Although the Fiber-2 has been identified as a key virulent related factor for FAdV-4, little is known about its molecular basis. In this study, we identified the efficient interaction of the Fiber-2 with the karyopherin alpha 3/4 (KPNA3/4) protein via its N-terminus of 1-40aa. The analysis of the overexpression and knockout of KPNA3/4 showed that KPNA3/4 could efficiently assist the replication of FAdV-4. Moreover, a fiber-2-edited virus FAV-4_Del with a deletion of 7-40aa in Fiber-2 was rescued through the CRISPR-Cas9 technique. In comparison with the wild type FAdV-4, FAV-4_Del was highly attenuated in vitro and in vivo. Notably, the inoculation of FAV-4_Del in chickens could provide full protection against the lethal challenge with the wild type FAdV-4. All these findings not only give novel insights into the molecular basis for the pathogenesis of Fiber-2 but also provide efficient targets for developing antiviral strategies and live-attenuated vaccine candidates against the highly pathogenic FAdV-4.

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.

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.

Fatal necrotic tracheitis by Aviadenovirus in captive Alagoas curassows (Pauxi mitu) extinct from the wild.

Extinct from nature, captive young Alagoas curassows (Pauxi mitu) were found agonizing or dead with respiratory disease. Intranuclear inclusion bodies were found in the epithelia of the trachea, associated with marked necrotic tracheitis. An Aviadenovirus was isolated in chicken eggs and characterized genetically with 99% identity to the fowl Aviadenovirus A, as based on the hexon protein gene. This is the first report of respiratory disease caused by Aviadenovirus in any cracid species in Brazil, recommending for stricter biosecurity in the conservation premises. RESEARCH HIGHLIGHTS Fatal tracheitis in curassows extinct from nature was associated with Aviadenovirus A. Seven-month-old Alagoas curassows (Aves: Cracidae) died with haemorrhagic tracheitis. Aviadenovirus A with 99% identity to fowl adenovirus 1 was detected in dead curassows. Fatal tracheitis by Aviadenovirus was described in Pauxi mitu (Aves: Cracidae).

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.

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.

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.

Development of a TaqMan-based real-time PCR assay for the detection of Novel GPV.

The newly emerged disease, duck beak atrophy and dwarfism syndrome (BADS), is caused by novel goose parovirus (N-GPV). Although N-GPV infection has severe consequences, few methods for detecting this virus have been developed. Therefore, the availability of rapid and reliable molecular diagnostic methods would aid future studies of this novel virus. Clinical specimens from 138 suspected cases of N-GPV infection and 120 cloacal swabs from breeding ducks were used in this study. The targeted sequence of N-GPV cloned into the pMD18-T vector was used to generate the N-GPV DNA standard curve. The specificity of the assay was validated using duck plague virus, GPV, duck hepatitis virus, avian influenza virus, duck reovirus, tembusu virus, and fowl adenovirus. The lowest limit of detection was 8.8×101copies/µL with a good linear standard curve (Y=-3.3682X+37.220, R2=0.9953) over a wide range of input DNA, of which the concentration was between 8.8×101 to 8.8×108copies/µL. The results show that the real-time PCR assay is a highly sensitive, specific, reproducible, and versatile method for quantitatively detecting N-GPV DNA, and thus can be used to detect this virus, thereby facilitating epidemiological investigations of animals with BADS.

Phylogenetic analysis of fowl adenoviruses isolated from chickens with gizzard erosion in Japan.

Thirty-four fowl adenoviruses (FAdVs) isolated from chickens with gizzard erosion (GE) from 1999 to 2010 were characterized phylogenetically together with foreign isolates. The phylogenetic analysis based on part of the hexon gene classified these 34 FAdV isolates into 3 groups: FAdV-1, -8a and 8b, thereby suggesting that FAdVs associated with GEs in chickens are diverse. All 30 FAdV-1 isolates were genetically identical, and they were also identical with FAdV-1 isolates from GEs in chickens in European countries (Germany, Poland, Austria, Hungary and Italy). Thus, the same type of FAdV-1 has been associated with outbreaks of GE in Japanese chickens for the past 10 years, which may have spread from a common ancestor, although the epidemiological relationship is unknown.

Infection with an apathogenic fowl adenovirus serotype-1 strain (CELO) prevents adenoviral gizzard erosion in broilers.

Gizzard erosion in broilers due to an infection with virulent fowl adenovirus serotype 1 (FAdV-1) is an emerging disease. Although experimental studies were performed, a possible prevention strategy was not reported so far. The present study was set up to determine (i) a possible influence of birds' age at time of inoculation on the pathogenicity of a European FAdV-1 field strain (PA7127), (ii) the virulence of a apathogenic FAdV-1 strain (CELO), and (iii) its capability to protect SPF broilers from adenoviral gizzard erosion caused by the field virus. Oral infection of birds with PA7127 at 1-, 10- and 21-days of life, resulted in reduced weight gain compared to non-infected birds, with significance for birds infected at day-old. Independent of the birds' age at time of inoculation, clinical signs appearing approximately one week after challenge coincided with gizzard lesions. Birds infected exclusively with CELO at the first day of life did not show any clinical signs or pathological changes in the gizzard, confirming the apathogenicity of this European FAdV-1. A similar result was obtained for birds orally infected at the first day of life with CELO and challenged three weeks later with the pathogenic PA7127 strain. Therefore, complete protection of adenoviral gizzard erosion in broilers by vaccination of day-old birds could be demonstrated for the first time, although virus excretion was detected post challenge. Establishment of an amplification refractory mutation system quantitative PCR (ARMS-qPCR) facilitated the identification of the FAdV-1 strain and presence of challenges virus was confirmed in one sample.

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 detection of avian pathogens in poultry red mite (Dermanyssus gallinae) collected in chicken farms.

Poultry red mite (PRM, Dermanyssus gallinae) is a blood-sucking ectoparasite as well as a possible vector of several avian pathogens. In this study, to define the role of PRM in the prevalence of avian infectious agents, we used polymerase chain reaction (PCR) to check for the presence of seven pathogens: Avipox virus (APV), Fowl Adenovirus (FAdV), Marek's disease virus (MDV), Erysipelothrix rhusiopathiae (ER), Salmonella enterica (SE), Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG). A total of 159 PRM samples collected between 2004 and 2012 from 142 chicken farms in 38 prefectures in Japan were examined. APV DNA was detected in 22 samples (13.8%), 19 of which were wild-type APV. 16S ribosomal RNA (16S rRNA) of MS was detected in 15 samples (9.4%), and the mgc2 gene of MG was detected in 2 samples (1.3%). Eight of 15 MS 16S rRNA sequences differed from the vaccine sequence, indicating they were wild-type strains, while both of the MG mgc2 gene sequences detected were identical to the vaccine sequences. Of these avian pathogen-positive mite samples, three were positive for both wild-types of APV and MS. On the other hand, the DNAs of ER, SE, FAdV and MDV were not detected in any samples. These findings indicated that PRM can harbor the wild-type pathogens and might play a role as a vector in spreading these diseases in farms.

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.

Species determination of fowl adenoviruses based on the 52K gene region.

In the present study, the classification of fowl adenoviruses (FAdVs) based on a part of the 52K gene region was described. A total of 44 FAdV field samples from different countries and sources were detected using a recently developed SYBR Green-based real-time PCR. Amplified products were sequenced, and phylogenetic analyses were conducted on the basis of the 116-bp region. For comparison, the already published sequences of the 52K gene region of aviadenoviruses were used in the analyses. The phylogenetic analysis allowed the grouping of the FAdVs into the established five different FAdV species: Fowl adenovirus A to Fowl adenovirus E. The existence of the species was supported by high bootstrap values (> 70%). This method provides the advantages of quantitation and high sensitivity for FAdV detection in combination with species assignment.