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.

An Outbreak of Fowl Aviadenovirus A-Associated Gizzard Erosion and Ulceration in Captive Bobwhite Quail (Colinus virginianus).

A flock of captive bobwhite quail (Colinus virginianus) experienced loose droppings, depression, and increased mortality starting at 3 wk of age. Necropsy of the affected birds revealed intestines dilated with frothy and tan fluid. Irregular dark brown fissures within the koilin layer of the gizzard were found in 20%-30% of the birds. Histologically, gizzards showed multifocal koilin degeneration or fragmentation, degeneration and necrosis of the subjacent epithelial cells, and infiltration of macrophages, lymphocytes, and heterophils. Necrotic epithelial cells occasionally contained large, smudgy, basophilic intranuclear inclusion bodies with marginated nuclear chromatin. Adenoviral paracrystalline arrays composed of icosahedral virions (60-70 nm diameter) were seen on transmission electron microscopy in the nuclei of epithelial cells in the gizzard mucosa. Adenovirus was isolated from gizzard, liver, intestine, and trachea by inoculation of specific-pathogen-free embryonated chicken eggs. Homogenates of the gizzard, liver, and intestine were positive for the adenovirus hexon gene by PCR. Sequencing of PCR amplicons confirmed the virus as fowl aviadenovirus A. The study isolates showed more than 99% and 97% nucleotide identity with quail bronchitis virus and with aviadenoviruses from gizzard erosion and ulceration (GEU) in broilers, respectively. The viral isolates showed six substitutions (G1T, C174A, A229G, C513A, T579A, and G621C) of which two were nonsynonymous (G1T and A229G), resulting in a change in the translated amino acid as A1S and S77G, respectively. These results indicate that adenoviruses of the same type or species can cause different clinical presentations in quails, e.g., bronchitis or GEU.

Artículo regular­Brote de erosiones y ulceraciones de la molleja asociadas con Aviadenovirus A del pollo en codornices de Virginia en cautiverio (Colinus virginianus). Una parvada de codornices de Virginia en cautiverio (Colinus virginianus) mostró heces acuosas, depresión y aumento de la mortalidad a partir de las tres semanas de edad. La necropsia de las aves afectadas reveló intestinos dilatados con líquido espumoso y marrón. Se encontraron fisuras irregulares de color marrón oscuro dentro de la capa de koilin de la molleja en el 20% al 30% de las aves. Histológicamente, las mollejas mostraron degeneración o fragmentación multifocal de la capa de koilin, degeneración y necrosis de las células epiteliales subyacentes e infiltración de macrófagos, linfocitos y heterófilos. Las células epiteliales necróticas contenían ocasionalmente cuerpos de inclusión intranucleares basófilos grandes, con cromatina nuclear marginada. Se observaron matrices paracristalinas adenovirales compuestas de viriones icosaédricos (60-70 nm de diámetro) en el microscopio electrónico de transmisión en los núcleos de las células epiteliales de la mucosa de la molleja. Se aisló adenovirus de molleja, hígado, intestino y tráquea mediante la inoculación de huevos embrionados de pollo libres de patógenos específicos. Los homogeneizados de la molleja, el hígado y el intestino fueron positivos para el gene del hexon del adenovirus por PCR. La secuenciación de amplicones de PCR confirmó la presencia de Aviadenovirus A del pollo. Los aislamientos del estudio mostraron una identidad mayor del 99% y 97% en la secuencia de nucleótidos con el virus de la bronquitis de codorniz y con aviadenovirus asociado con erosión y ulceración de mollejas (con las siglas en inglés GEU) en pollos de engorde, respectivamente. Los aislados virales mostraron seis sustituciones (G1T, C174A, A229G, C513A, T579A y G621C) de las cuales dos eran no-sinónimas (G1T y A229G), lo que resultó en un cambio en el aminoácido traducido como A1S y S77G, respectivamente. Estos resultados indican que los adenovirus del mismo tipo o especie pueden causar diferentes presentaciones clínicas en codornices, por ejemplo, bronquitis o erosión y ulceración de mollejas.

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.

Feed-borne Bacillus cereus exacerbates respiratory distress in chickens infected with Chlamydia psittaci by inducing haemorrhagic pneumonia.

Chlamydia psittaci is an important zoonotic pathogen and its oral route of infection plays an important role in the transmission and persistence. Bacillus cereus (B. cereus) strain, a common contaminant of animal feed and feedstuffs, can cause severe diarrhoea and malnutrition in poultry. In our previous study, a B. cereus strain (Dawu C), isolated from the haemorrhagic lungs of infected chickens, was shown to harbour two virulence genes (hblC and cytk) and was able to induce haemorrhagic lesions in the lungs, as well as gizzard erosion and ulceration (GEU) syndrome in broilers. In the present study, we tested the hypothesis that B. cereus-induced GEU would aggravate C. psittaci infection. Our results showed that SPF chickens exposed to B. cereus developed a severe GEU syndrome. More interestingly, prior infection with B. cereus facilitated C. psittaci infection, and aggravated GEU and respiratory distress, which were accompanied by high chlamydial loads in the lungs and severe lesions in respiratory organs. Moreover, levels of local inflammatory cytokines were elevated and T cell responses were impaired in the infected birds. In conclusion, GEU caused by B. cereus may facilitate chlamydial transmission from the ventriculus to the lungs.RESEARCH HIGHLIGHTS Bacillus cereus contributes to the gizzard erosion and ulceration syndrome in chickens.Exposure to Bacillus cereus exacerbates pneumonia in birds following chlamydial infection.Bacillus cereus facilitates persistent chlamydial infection and exacerbates immune responses.

Nosema apis and Nosema ceranae Tissue Tropism in Worker Honey Bees (Apis mellifera).

The microsporidia Nosema apis and Nosema ceranae are major honey bee pathogens that possess different characteristics in terms of the signs they produce, as well as disease development and transmission. Although the ventricular epithelium is generally considered the target tissue, indirect observations led to speculation that N. ceranae may also target other structures, possibly explaining at least some of the differences between these 2 species. To investigate the tropism of Nosema for honey bee tissues, we performed controlled laboratory infections by orally administering doses of 50 000 or 100 000 fresh mature spores of either species. The fat body was isolated from the infected bees, as well as organs from the digestive (esophagus, ventriculus, ileum, rectum), excretory (Malpighian tubules), circulatory (aorta, heart), respiratory (thoracic tracheas), exocrine (hypopharyngeal, mandibular and labial, cephalic, thoracic salivary glands), and sensory/nervous (brain, eyes and associated nerve structures, thoracic nerve ganglia) systems. Tissues were examined by light and electron microscopy at 7, 10, and 15 days postinfection. Both Nosema species were found to infect epithelial cells and clusters of regenerative cells in the ventriculus, and while the ileum and rectum contained spores of the microsporidia in the lumen, these structures did not show overt lesions. No stages of the parasites or cellular lesions were detected in the other organs tested, confirming the high tropism of both species for the ventricular epithelium cells. Thus, these direct histopathological observations indicate that neither of these 2 Nosema species exhibit tropism for honey bee organs other than the ventriculus.

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.

Erosive Enteritis and Intestinal Obstructions Caused by Decomposed Granite in a Flock of Lesser Flamingos (Phoeniconaias minor).

Decomposed granite is a material commonly used in zoos as a base for flamingo exhibits. In 2012, the artificial turf over the decomposed granite in the Fort Worth Zoo flamingo exhibit was removed and pressure washed, the granite was replaced, and the turf was then restretched in place. Approximately 8 months later, 3 lesser flamingos (Phoeniconaias minor) showed signs of lethargy and hematochezia. Results of physical examination revealed poor body condition in all birds, with variable hematologic abnormalities, including anemia, hypoproteinemia, and electrolyte disturbances. Radiographs of birds showed large amounts of radiopaque material in the ventriculus and dilated intestinal loops. Two birds died either during treatment or surgery, and 1 bird was euthanatized. Postmortem examination of all 3 birds revealed large amounts of fine, granite-like material in the ventriculus and intestines, severe congestion of intestinal mucosa, and obstruction of intestinal lumen by caseous plugs surrounded by fecal material. Microscopic evaluation of the material removed from the ventriculus showed birefringent crystals that corresponded to microscopic silica grains, consistent with the decomposed granite from the exhibit. Histologic examination of tissues showed marked chronic erosive colitis, mild erosive proventriculitis, and lesions in the muscular layer of the ventriculus. Subsequently, 82 remaining flamingos in the flock were radiographed. Results showed the same material in the ventriculus of most birds, but no intestinal obstructions were identified. Different treatment techniques were attempted unsuccessfully to remove the material from the flamingos that had the largest amount of decomposed granite in their gastrointestinal tracts. Ultimately, all birds were treated with oral gastrointestinal protectants, adsorbents, laxatives, and lubricants, along with antibiotics administered in drinking water. No further mortalities occurred once medical treatments were implemented.

Contaminated feed-borne Bacillus cereus aggravates respiratory distress post avian influenza virus H9N2 infection by inducing pneumonia.

Avian influenza virus subtype H9N2 is identified in chickens with respiratory disease while Bacillus cereus (B. cereus) has been frequently isolated from chicken feed in China. However, the roles of co-infection with these two pathogens remain unclear. In the present study, SPF chicks were intragastrically administered with 108 CFU/mL of B. cereus for 7 days and then inoculated intranasally with 100 EID50 of H9N2 three days later. Alternatively, chickens were initially inoculated with H9N2 and then with B. cereus for one week. Post administration, typical respiratory distress persisted for 5 days in both co-infection groups. Gizzard erosions developed in the groups B. cereus/H9N2 and B. cereus group on 7th day while in group H9N2/B. cereus on 14th day. More importantly, both air-sac lesions and lung damage increased significantly in the co-infection group. Significant inflammatory changes were observed in the B. cereus group from day 7 to day 21. Moreover, higher loads of H9N2 virus were found in the co-infected groups than in the H9N2 group. Newcastle Disease Virus (NDV) specific antibodies were decreased significantly in the H9N2/B. cereus group compared to the B. cereus and the B. cereus/H9N2 groups. Nonspecific IgA titers were reduced significantly in the B. cereus group and the H9N2/B. cereus group compared to the control group. In addition to this, lower lymphocyte proliferation was found in the con-infection groups and the H9N2 group. Hence, feed-borne B. cereus contamination potentially exacerbates gizzard ulceration and aggravates H9N2-induced respiratory distress by inhibiting antibody-mediated immunity and pathogen clearance. Thus controlling the B. cereus contamination in poultry feed is immediately needed.

Anatomopathological changes, quantification and distribution of Libyostrongylus spp. in regions of the proventriculus and ventriculus of naturally- and experimentally-infected ostriches.

Nematodes of the genus Libyostrongylus parasitize ostriches, causing high mortality rates. These nematodes are found in the proventriculus and ventriculus of ostriches, but little is known about their distribution and the possible anatomopathological changes they cause in the various regions of these organs. This paper describes the distribution and quantification of Libyostrongylus and pathological changes found in regions of the proventriculus and ventriculus of ostriches with high and low levels of both natural and experimental infection. Ostriches were necropsied and tissue samples from the distinct regions of both organs were analysed based on nematode counts and histopathology after staining with haematoxylin and eosin, Masson's trichrome or Alcian blue/PAS. The cranial and glandular regions of the proventriculus were the most parasitized. The ventriculus contained more nematodes in the caudal region. No macro- or microscopic pathological changes were observed in either of these organs of experimentally-infected birds. However, naturally-infected birds with high levels of infection presented proventriculus with macroscopic lesions and heterophilic infiltrates surrounding nematodes. In the glandular region of this organ, nematodes were located in the adenomeres of the secretory ducts, causing altered architecture and erosions and ulcerative lesions with damaged epithelium. Nematode eggs were found in the koilin layer of the middle and caudal regions of the ventriculus only of these birds. The pH of the regions assessed by Alcian blue/PAS staining changed from acidic in the proventriculus to more alkaline in the caudal region of the ventriculus. These data add knowledge to the biology of Libyostrongylus. RESEARCH HIGHLIGHTS The most parasitized areas were the cranial and glandular regions of the proventriculus. Naturally-infected birds with high levels of infection presented macro lesions in the proventriculus and damaged epithelium. Nematode eggs were found in the ventriculus. The proventriculus had an acidic pH, which turned alkaline towards the ventriculus.

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.

Heat stress as a predisposing factor for necrotic enteritis in broiler chicks.

Heat stress is a physical environmental stressor, which can affect performance, health and welfare of poultry. The present study investigates the effect of cyclic acute heat stress as a predisposing factor for necrotic enteritis in broiler chicks in an experimental challenge model. Two hundred and forty broiler chicks were randomly allocated to four treatment groups, as follows: group A served as negative control (25°C), group B birds were subjected to cyclic acute heat stress (35°C), group C birds were challenged and group D birds were both subjected to heat stress and challenged. From each bird, the intestine, gizzard and liver were collected and scored for gross lesions. The intestinal digesta was collected for pH and viscosity determination. One caecum from each bird was taken for microbiological analysis. The statistical analysis and evaluation of data revealed that the heat stress in challenged birds showed a relative trend to increase the severity and the incidence of necrotic enteritis lesions, although it was not considered as statistically significant (Ρ = 0.077). Additionally, the heat stress induced necrotic enteritis outbreak in unchallenged birds; the challenge of birds as well as its combination with the heat stress affected significantly (Ρ ≤ 0.05) the pH and viscosity of intestinal digesta and the caecal Clostridium perfringens counts. The study provides evidence that cyclic acute heat stress is an environmental stressor, which can significantly affect necrotic enteritis and gut health, and thus should be taken into account in warmer areas of the world where poultry farming becomes a major industry.

Comparison of gizzard and intestinal microbiota of wild neotropical birds.

Gut bacterial communities have been shown to be influenced by diet, host phylogeny and anatomy, but most of these studies have been done in captive animals. Here we compare the bacterial communities in the digestive tract of wild birds. We characterized the gizzard and intestinal microbiota among 8 wild Neotropical bird species, granivorous or frugivorous species of the orders Columbiformes and Passeriformes. We sequenced the V4 region of the 16S rRNA gene in 94 collected samples from 32 wild birds from 5 localities, and compared bacterial communities by foraging guild, organ, locality and bird taxonomy. 16S rRNA gene-based sequencing data were examined using QIIME with linear discriminant analysis effect size (LEfSe) and metabolic pathways were predicted using PICRUSt algorism. We identified 8 bacterial phyla, dominated by Firmicutes, Actinobacteria and Proteobacteria. Beta diversity analyses indicated significant separation of gut communities by bird orders (Columbiformes vs. Passerifomes) and between bird species (p<0.01). In lower intestine, PICRUSt shows a predominance of carbohydrate metabolism in granivorous birds and xenobiotics biodegradation pathways in frugivorous birds. Gizzard microbiota was significantly richer in granivorous, in relation to frugivorous birds (Chao 1; non-parametric t-test, p<0.05), suggesting a microbial gizzard function, beyond grinding food. The results suggest that the most important factor separating the bacterial community structure was bird taxonomy, followed by foraging guild. However, variation between localities is also likely to be important, but this could not been assessed with our study design.

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.

Evaluation of gastroscopy and biopsy of the proventriculus and ventriculus in pigeons (Columba livia).

OBJECTIVE To evaluate the efficacy and safety of gastroscopy and biopsy of the proventriculus and ventriculus in pigeons (Columba livia). ANIMALS 15 adult pigeons. PROCEDURES Each pigeon was anesthetized, and the upper gastrointestinal tract (from the cervical portion of the esophagus to the ventriculus) was endoscopically evaluated by use of a rigid endoscope inserted orally. Saline (0.9% NaCl) solution was orally infused to achieve lumen dilation and visibility. Two mucosal biopsy specimens were collected from each of the proventriculus and ventriculus, histologically evaluated, and graded for crush artifacts and depth. Pigeons were monitored for adverse effects for 3 to 6 days after the procedure, after which they were euthanized for necropsy. RESULTS Gastroscopy via the oral approach provided excellent visibility of the lumen and mucosal surfaces of the proventriculus and cranial portion of the ventriculus and was safe provided that appropriate precautions were taken. Two intraoperative deaths occurred at the beginning of the study; following procedure refinement, no additional deaths occurred. No major adverse effects of the procedure were detected in the remaining 13 pigeons during the postoperative monitoring period or at necropsy. Diagnostic quality of proventriculus specimens was adequate for 10 of 13 pigeons. Eight of 13 ventriculus specimens were of inadequate quality, and only 3 were of adequate quality. CONCLUSIONS AND CLINICAL RELEVANCE Gastroscopy was useful for evaluating the lumen and mucosal surface of the proventriculus and ventriculus in pigeons, and biopsy of those organs was safely performed with the appropriate technique. Further evaluation of these techniques is needed in birds with clinical disease and birds of other species.

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.

High mortality in laying hen pullets caused by crop and gizzard impactions associated with ingestion of bale net wrap.

High mortality was observed in young replacement layers. Balls of bale net wrap strings were found in the crop and/or gizzard of birds causing impaction and traumatic injury. Some birds experienced loss of portions or the entire tongue secondary to ischemic necrosis. Mortality stopped with the removal of strings from the environment.

Mortalités élevées chez des poulettes pondeuses causées par des impactions du jabot et du gésier associées à l'ingestion de cordes d'attache de ballots. Une mortalité élevée a été observée chez les jeunes pondeuses de remplacement. Des balles de ficelle de cordes de ballots ont été trouvées dans le jabot et/ou le gésier des oiseaux causant ainsi une impaction et des traumatismes. Quelques oiseaux ont perdu des portions ou la totalité de la langue en raison d'une nécrose ischémique. Les mortalités ont cessé après l'enlèvement des ficelles de l'environnement.(Traduit par Isabelle Vallières).

Clinical signs and progression of lesions in the gizzard are not influenced by inclusion of ground oats or whole wheat in the diet following experimental infection with pathogenic fowl adenovirus serotype 1.

In the present study the effects of dietary gizzard stimulation on the development and severity of adenoviral gizzard erosion were investigated. For this purpose, specific pathogen-free broilers were divided into six groups, investigating the influence of an oat-containing diet with higher fibre content, a whole wheat-containing diet and a control diet of nearly identical composition, but containing ground wheat. For each feed administered, one group of birds was experimentally infected on the 10th day of age by the oral route with virulent fowl adenovirus serotype 1 (FAdV-1), recently proven to induce gizzard erosions, while the respective negative control groups remained uninfected. Experimental feed was administered from 2 days post-infection onwards. No significant differences on gizzard health or in weight gain could be detected between uninfected control groups or between FAdV-1 infected groups that received different experimental feed. However, independent of the supplied diet, a significantly reduced weight gain was noted from 7 days post-infection onwards in FAdV-1 infected broilers compared to uninfected birds that received the same diet. Macroscopically, discolouration and erosion of the koilin layer and inflammation of the gizzard mucosa were observed in all FAdV-1 infected groups. Histologically, necrosis, degeneration of gizzard epithelial cells and multiple basophilic intranuclear inclusion bodies were observed. In summary, after experimental infection with FAdV-1 development of gizzard erosion in chickens was not influenced by the feeding regimes investigated. Therefore, it is unlikely that dietary gizzard stimulation influences the outcome of adenoviral gizzard erosion in vertically infected broilers.

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.