Farm identification, selection & definitions
In this study, three case farms were selected throughout March and April of 2017 from a list of broiler farms located in the Mornington Peninsula region, Victoria. The flocks were comprised of mixed-sex of Cobb and Ross broiler breeds between 36-50 days of age. Upon the onset of an outbreak of ILT in a poultry farm based on clinical diagnosis, the company veterinarian or service person notified the investigators. As soon as ILT case farms had been identified, an investigator visited the farm and collected air samples within 24h.
Air sampling procedure
All air samples were collected using the SKC Biosampler®; a liquid cyclonic impinging device (SKC Inc, USA) and BioLite pump (SKC Inc, USA) as shown in Figure 1. Briefly, the collection vessel contained 5 ml of alkaline polyethene glycol (PEG) solution ( 60% v/v PEG 200 (Sigma-Aldrich, USA), 20 mM KOH, pH 13.5) or phosphate buffer saline (PBS) (8.1 mM Na2HPO4, 137 mM NaCl, 1.4 mM KH2HPO4 and 2.6 mM KCl) in which material from the air was trapped. The air sampler was used for 20 min in three different locations within the shed at the height of either 45 cm or 120 cm off the floor at a rate of 12.5 L per min placed on card table. In between sampling, the glass collection vessel was disinfected with 80% ethanol. Each sample was transferred from the collection vessel to a clean sterile 15 ml conical centrifuge tubes for transport and storage in the laboratory at -20 0C.
DNA extraction of feather samples
Feather samples were gathered randomly off the floor inside the selected sampling shed. Feathers were collected and stored in 50 ml conical centrifuge tubes and stored at -20°C until processing. Approximately 5 mm of each collected feather shaft was cut off and were divided into groups of three, with four shafts per group. Total DNA was extracted from feathers tips using the Mouse Direct PCR Kit (Biotool, USA) following the manufacturer’s instructions.
DNA purification of air & feather samples
DNA purification from air and feather samples were performed as described by Li and Sheen with the following modifications . Briefly, 500 μl of DNA binding solution (6M NaI) is added to 300 ml of extracted DNA feather sample to which 10 μl of 100mg/ml silica dioxide (Sigma, USA) was added, vortexed then incubated at room temperature for 2 min. Samples were centrifuged for 10s, and the supernatant removed. Silica matrix was then washed with 500 μl of washing solution (50% v/v ethanol, 10 mM Tris-HCl pH 7.5, 100 mM NaCl and 1mM ethylenediaminetetraacetic acid (EDTA), centrifuged at 13,000 x g, discarded supernatant and repeated once. Silica matrix was resuspended in 30 μl of sterile water and incubated at 70°C for 2 min. The samples were centrifuged at 16,000 x g for 2 min and eluted DNA transferred to a fresh tube and stored at -20°C. A similar purification method was used for the air sample except the starting volume was 2.5 ml to which 3 ml of DNA binding solution and 100 ml of 100mg/ml silica dioxide (Sigma, USA) was added. DNA was eluted by the addition of 100 ml of sterile water.
PCR for detection of Gallid herpesvirus 1
Conventional PCR was used for the detection of gallid herpesvirus 1 by the presence of the thymidine kinase (TK) gene, as described by Mahmoudian et al. (2011), with the following modifications . Reactions were carried out in a final volume of 25 µL and amplified in a C-Master GT thermal cycler (Dynamica, Australia). The following amplification conditions consisted of an initial denaturation step of 94˚C for 3 min, 94˚C for 15 s, 60˚C for 45 s, 72˚C for 150 s, repeated for 35 times with a final extension at 72˚C for 3 minutes. Amplicon size was checked by agarose electrophoresis migration by loading directly on 1% (w/v) agarose (Lonza, USA) gel, prepared as per manufacturer’s instructions, with the addition of 0.5 µL of Sybr® Safe DNA Gel Stain (Life Technologies, USA). The gel was run at 110 V for 45 minutes and imaged using the GelDocTM XR+ (BioRad, USA) instrument and software.