Virus
The FAdV-1 strain used in the experiment was isolated from a pool of gizzards collected from an outbreak of gizzard erosion in Germany [8]. The virus was identified as a European “pathogenic” FAdV-1, by comparing nucleic acid sequences of long and short fiber genes according to a method described by Marek et al. [5]. The virus was propagated on primary chicken embryo liver (CEL) cells and used to infect chickens at the fifth passage.
Animal experiment, clinical signs and cloacal swab samples
SPF broiler eggs were obtained from Animal Health Service, Deventer, The Netherlands. Commercial broiler eggs were obtained from a FAdV-1 seropositive broiler breeder flock. Following hatching, the birds were divided into two groups, respectively. Each group comprised 25 birds. The birds were housed separately in isolator units under negative pressure (Montair Andersen bv, HM 1500, Sevenum, The Netherlands) until termination of the study. The broilers were individually marked by Swiftack™ (Heartland Animal Health, Inc., Missouri, USA). Feed and water were provided ad libitum during the animal experiment. One group of SPF broilers (NSPFB) and one group of commercial broilers (NCB) were kept as negative controls and none of the birds were infected at any time. In one group of SPF broilers (SPFB) and one group of commercial broilers (CB), day-old birds were inoculated orally with 0.1 mL of the virulent FAdV-1 isolate, each bird receiving 107.8 mean tissue culture infective dose (TCID50). The birds were monitored daily for any clinical signs. The body weight of all chickens was measured on the first day of life and at 3, 7, 10, 14 and 17 days post infection (dpi). In addition, at each of these sampling points 5 birds per group were euthanized and necropsied. Cloacal swabs were taken at intervals of 3 to 4 days from the 5 birds per group that lived until 17 dpi. Blood samples were taken weekly from all birds.
The experiment was approved by the institutional ethics committee, the Advisory Committee for Animal Experiments (§12 of the Law for Animal Experiments, Tierversuchsgesetz – TVG) and the Federal Ministry for Science and Research under license no. 68.205/0179-II/10b/2009.
Post mortem examination
During post mortem examination gizzards in particular were investigated for pathological changes. A scoring scheme from Nakamura et al. [12] was applied to assess gross lesions of the koilin layer. Changes of the gizzard mucosa were investigated and scored in the same manner: no lesions = 0; mild lesions (less than one-third of the koilin layer/gizzard mucosa was affected) = 1; moderate lesions (one-third to one-half of the koilin layer/gizzard mucosa was affected) = 2 and severe lesions (more than one-half of the koilin layer/gizzard mucosa was affected) = 3.
For virological and histological investigations, tissue samples of the gizzard, liver and spleen were collected.
Histology
Samples of gizzard, liver and spleen were fixed in 3.5% neutral buffered formalin and then embedded in paraffin for histological investigations. Using a Microm HM 360 microtome (Microm Laborgeräte GmbH, Walldorf, Germany) tissue slices of 3 μm were prepared and routine staining using hematoxylin and eosin (H&E) was performed. The scoring scheme from Nakamura et al. [12] was used to assess intranuclear inclusions in the gizzard glandular epithelium: no lesions = 0; mild lesions (1 to 10 inclusions in one section) = 1; moderate lesions (10–20 inclusions in one section) = 2 and severe lesions (more than 20 inclusions in one section) = 3.
In order to demonstrate FAdV-1 DNA in paraffin-embedded tissue samples in-situ hybridization was performed according to a protocol described earlier [8], using a DNA probe based on the FAdV-1 long fiber gene.
Virus isolation and SYBR Green based real-time PCR
Cloacal swabs were placed in 1 mL of an antibiotics-phosphate buffered saline (PBS) solution (1 mg/mL streptomycin and 100 000 IU/mL penicillin). Tissue samples from gizzard, liver and spleen of the birds were homogenized in antibiotics-PBS solution. Samples were filter sterilized using syringe filters with a pore size of 0.2 μm (VWR, Vienna, Austria). Cloacal swab samples and tissue homogenates (20%) were used for virus isolation and for DNA extraction followed by real-time PCR.
For virus isolation CEL cell cultures were prepared according to a method described by Schat and Sellers [15] with some modifications. 100 μL of a cloacal swab sample or tissue homogenate were inoculated on nearly confluent CEL cells. Each sample was passaged up to three times or until a cytopathic effect was observed. A sample was considered negative when no cytopathic effect was noticed after three blind passages.
Viral DNA was extracted from 100 μL of the homogenized organ samples and from cloacal swabs with DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. The SYBR Green based real-time PCR was performed as described recently in order to quantify virus DNA in sample material [14]. Briefly, primers were designed to anneal within the highly conserved 52 K region. Using a double-stranded DNA-binding dye method with a Rotor-Gene SYBR Green PCR kit (Qiagen, Hilden, Germany) the real-time PCR was performed on a Rotor-Gene Q thermal cycler (Qiagen, Hilden, Germany). No template controls were included throughout sample preparation and PCR runs, in order to avoid cross contamination. To confirm real-time PCR product specificity melting curve analysis was done and amplification products were separated by electrophoresis in a 2% agarose gel. Threshold cycle (CT) values of investigated samples were compared with a well-defined standard curve and the number of copies of FAdV DNA per reaction mixture was calculated.
Serology
Prior to testing, all sera were inactivated for 30 min at 56°C in a thermomixer (Eppendorf, Vienna, Austria). The serum samples were tested for FAdV-1 specific antibodies by virus-neutralization test (VNT). The VNT was performed according to a constant virus diluted serum method using 100 TCID50/100 μL of the FAdV-1 strain used in the animal experiment. Respective titers were calculated according to Kärber [16]. An antibody titer below or equal to 3 log2 was regarded as negative.
Statistical analysis
The average body weight of orally infected broilers and uninfected controls at 0, 3, 7, 10, 14 and 17 dpi was recorded and analyzed by the student t-test. Statistical differences with P < 0.05 were considered to be significant. Data were analyzed with the statistical software package SPSS Version 17 (IBM SPSS Statistics; IBM Corporation, Somer, New York, USA).