Adenoviral gizzard erosions in Italian chicken flocks.

TYPE 1 avian adenoviruses belong to the genus Aviadenovirus within the adenovirus family. Five species of fowl adenovirus (fAdV) (designated by the letters A to E) are recognised within the Aviadenovirus genus based largely on molecular criteria, in particular restriction enzyme fragmentation patterns and sequencing data (McFerran and McConnel Adair 2003). fAdV are common infectious agents in poultry. Most of these viruses replicate in healthy birds with little or no apparent signs of infection. Nevertheless, they are also associated with naturally occurring outbreaks of different diseases including hydropericardium syndrome, necrotising pancreatitis, inclusion body hepatitis and gizzard erosion (McFerran and McConnel Adair 2003). Sporadic outbreaks of adenoviral gizzard erosion were described in layer chickens and broilers in North America in the early 1990s (Goodwin 1993). More recently, adeno viral gizzard erosion in broiler chickens has been observed repeatedly in Japan (Ono and others 2001, 2003a) where fAdV are considered the most important cause of gizzard erosion and are responsible for considerable economic losses. Most of the cases have been associated with fAdV A serotype 1 (fAdV-1) (Ono and others 2003b). The condition has been experimentally reproduced in commercial broilers and specific pathogen free layer chickens (Okuda and others 2001a, b, Ono and others 2003a). There is a lack of information about the occurrence of this pathological condition in Europe. This short communication describes the pathological and virological findings from 12 outbreaks of adenoviral gizzard erosion in chicken flocks in Northern Italy, from 1995 to 2006. From each outbreak, five to 35 gizzards with erosions were collected during routine diagnostic postmortem examination (Table 1, cases 1 and 2) or submitted for examination after being condemned at the slaughterhouse (Table 1, cases 3 to 12). Except for one outbreak in layers and one outbreak in backyard chickens, affected gizzards were from slaughtered broiler chickens ranging in age from 42 to 63 days. Tissue samples were fixed in 10 per cent formalin and processed for routine histology. The paraffin-embedded tissues were sectioned at 4 μm and stained with haematoxylin and eosin. For immunohistochemistry, additional sections at 4 μm were cut from selected samples from each outbreak. For antigen retrieval, deparaffinised and hydrated sections were incubated with pepsin working solution (Dako) for 13 minutes at 37°C in a humidified chamber. The rabbit serum antiserotype 1 fAdV-A (Istituto Zooprofilattico Sperimentale, Pavia) was used as the primary antibody at a dilution of 1:2000 overnight at 4°C, following the avidin/biotin complex immunoperoxidase procedure. 3,3 ́-diaminobenzidine (Dako) with 0·5 per cent hydrogen peroxide in buffer (pH 7·2, 0·1M) was used as the chromogen and the sections were counterstained with Mayer’s haematoxylin. Portions of frozen gizzards from six outbreaks (Table 1, cases 5, 8 to 12) were processed for DNA extraction and PCR examination using primers (H1-H2) specific for the conserved region of the fAdV hexon gene (Raue and Hess 1998). Frozen gizzards were cut into 25 mg pieces and DNA extracted using the QIAamp DNA Mini Kit (Qiagen), according to the manufacturer’s instructions. Purified DNA stocks were quantified and stored at –80°C until use. The viral sequence from a 1219 bp region of the hexon gene was amplified by PCR, using primers designed based on the published DNA sequence of the fAdV-1 genome (GenBank accession number U46933). PCR amplification was carried out using 0·5 μg DNA incubated through 35 cycles at 95°C for 60 seconds, 60°C for 60 seconds and 72°C for 1·5 minutes. After amplification, PCR products were separated by electrophoresis in a 2 per cent agarose gel containing 1 μg/ml ethidium bromide. The PCR product of the expected length was sequenced, using both primers, by CRIBI Services on an ABI-PRISM 377 DNA sequencer using the ABI-PRISM BigDye Terminator Cycle Sequencing Kit (Applied Biosytems) with FIG1: Broiler chicken gizzards with severe erosions caused by fowl adenovirus