c.2) Fowl Adenovirus-4 Specific Immunoperaxidase Test
The avidin biotin complex peroxidase (ABC-P) method was performed
according to manufacturer’s instructions (ab64264, Abcam, HRP/DAB, MA,
USA). Briefly, tissue sections were deparaffinized, hydrated and then
boiled in antigen retrieval solution (citrate buffer pH 6.0) for 20
minutes. The sections were incubated in 3% hydroperoxide-methanol
mixture at room temperature for 20 minutes before blocking was performed
using normal goat serum at 37°C for 7 minutes. The sections were
incubated with polyclonal rabbit anti chicken FAdV-4 hyperimmune serum
(kindly provided from Prof. Dr. Hafez) diluted at 1:40 at 37°C for 1
hour. After this, the sections were incubated with a biotinylated, HRP
labelled anti-rabbt secondary antibody (Abcam kit, USA) followed by
exposure to the AEC substrate chromogen solution (Dako). For
counterstaining, sections were kept in Gill’s haematoxylin (Sigma) for 2
minutes and cover slipped with aquous mounting medium. Washing
procedures were carried out with Tris-HCl twice for 5 minutes excluding
the blocking step. As a negative control, the sections were treated with
Phosphate Buffered Saline (PBS) instead of primary antibodies. As a
positive control, the same protocol was performed on chicken liver and
brain sections that are obtained hyperimmun serum from ‘’Hafez Mohamed
Hafez in Freie Universität Berlin-Germany”, against FAdV-4 virus
infection study in chickens.
DNA extraction and PCR
DNA was extracted from 200 μL of supernatant from two groups of samples
separately, using a High Pure Viral Nucleic Acid Kit (Roche) according
to the manufacturer’s protocol. The extracted DNAs were stored at -20 °C
until PCR was performed.
PCR for PiAdV-A and PiCV was carried out in a total volume of 25 μl
containing 5 μl of DNA, 0.4 μM of each primer, and PCR master mix
(Grisp, Portugal). The PCR for PiAdV-A was carried out in a thermocycler
(Techne, UK) with an initial denaturation at 95 °C for 5 min, followed
by 35 cycles of 95 °C for 20 seconds, 58 °C for 30 seconds, and 72 °C
for 1 min with a final elongation step at 72 °C for 5 min. The PCR for
PiCV was carried out in the same thermocycler with an initial
denaturation at 95 °C for 5 min, followed by 35 cycles of 95 °C for 20
seconds, 62 °C for 30 seconds, and 72 °C for 30 seconds with a final
elongation step at 72 °C for 5 min. The PCR amplicons were visualised by
agarose gel electrophoresis. The PCR amplicons were purified according
to the manufacturer’s protocol (ExoSAP-IT PCR Product Cleanup Reagent,
USA), before sequencing.
The PiAdV-A primers (F1-s and F2-as) and PiCV (PiCV2-s and PiCV2-as)
primers were used in the study (Table 1) bind to a conserved region of
the fiber 2 gene of PiAdV-A and capsid gene of PiCV. The samples were
also tested by other primers which are listed in Table. 1, to
investigate FAdV, PiAdV-B and PiHV to detect the presence of other
possible viruses.
TABLE 1. Primers used in this study