Birds
Commercial Ross 308 broiler chicks of both sexes, purchased from one hatchery and one hatch, were used in the experiment. The trial was conducted in isolated pens of the Pavilion of Experimental Poultry Infections, at the Department of Avian Diseases, University of Warmia and Mazury in Olsztyn, which are maintained at a biosafety PCL 3 conditions. Water and feed were given to birds ad libitum. Rearing conditions were consistent with Aviagen (Aviagen, USA) recommendations.
Experiment Layout
The experiment was carried out with 24 commercial broiler chickens. The birds were reared until day 24 of life, after which they were divided into two groups (n = 12). One served as the control, and the other was infected with a proventriculus homogenate originating from a confirmed field TVP case. Before the infection (0 days post infection – dpi), 4 birds selected randomly from each group were euthanized and subjected to autopsy examination to check for macroscopic lesions typical of TVP. Afterwards, proventriculi were collected for histopathological examination, and samples of them and of other internal organs (intestines, spleen, liver, and bursa of Fabricius) were taken for molecular analysis. For histopathological examination, the proventriculus samples were fixed in a 10% formaldehyde solution. Samples for molecular analysis were frozen at − 20 °C until they were analyzed. The remaining birds from both groups were weighed and blood samples were collected for serological analysis. Blood was centrifuged at 1 500 × g for 15 minutes and the serum obtained was frozen at − 20 °C until it was analyzed. After infection, the birds were reared for the next 14 days. At 14 dpi, blood was sampled from all birds from both groups for serological analysis. Next, the birds were weighed and euthanized. Pathological lesions in the proventriculus were investigated and recorded during the anatomopathological examination, after which samples of this organ were collected for histopathological and molecular examinations. Production results were presented as the mean body weight (kg) +/− standard deviation (SD).
For euthanasia, chickens were placed in a chamber with Carbogen (95% O2 + 5% CO2). After 1 min, Carbogen was slowly replaced by 100% CO2. This method of euthanasia is responsible for stress reduction in the birds.
Proventriculi Homogenate And Infection
Proventriculi from the first Polish case of TVP [9] were used for the experimental infection. After homogenization, proventriculus samples were processed through three freeze–thaw cycles. After centrifugation (2 000 × g, 15 minutes), the supernatant was stored and used for the infection. Broiler chickens from the TVP group were infected with 5 mL of the supernatant per bird. The supernatant was given to birds per os, directly with a probe to the crop. At the same time, the birds from the control group were given PBS in the same way.
Serological Analysis
A commercial kit of ELISA Ab Tests (IDEXX Laboratories, USA) was used to determine the titer of anti-IBV, anti-REO, anti-FAdV, and anti-IBDV specific IgY in broiler serum. Particular stages of the tests were performed with an Eppendorf epMotion 5075 LH automatic pipetting station (Eppendorf, Germany), a BioTek ELx405 automatic multi-well plate washer (BioTek, USA), and a BioTek ELx800 plate reader. Sample to positive (S/P) ratio, plus/minus standard deviation (SD) were computed for each group in each sampling period.
Gross Lesion Evaluation
The birds were investigated for the presence of lesions typical of TVP in the proventriculus during an anatomopathological examination. Enlargement of the proventriculus coupled with thickening of its walls and their spotty discoloration in the cross-section were recorded. Results were collated as the number of birds with recorded pathological lesions relative to the total number of birds examined.
Histopathology
During necropsy, samples of the central part of the proventricular wall were embedded in 10% formalin (pH 7.4) and processed for histopathological examination. After passing the samples through intermediate liquids (increasing concentrations of alcohol and xylene) they were embedded in paraffin blocks. Sections of the examined samples 4 µm thick were stained with hematoxylin–eosin and microscope samples were scanned with a Pannoramic MIDI scanner (3DHISTECH, Budapest, Hungary). Samples were considered TVP positive if the following histopathological lesions were registered during examination: necrosis of glandular epithelium (necrosis), hypertrophy and hyperplasia of ductal epithelium, replacement of glandular epithelium by hyperplastic ductal epithelium (hyperplasia) and multifocal-to-severe infiltration of lymphoid cells (infiltration). For PCR analysis, five 4 um sections from each paraffin block (each bird) were put into xylene and prepared for PCR analysis.
CPNV Identification
Paraffin fixed sections of the proventriculi were used for CPNV identification. One mL of xylene was added to each sample comprising five paraffin-fixed proventriculus sections (each 5 µm thick) and these were incubated for 5 minutes at 50 °C in order to remove paraffin residues. Further RNA isolation steps were performed with an Isolate II FFPE RNA/DNA Kit (Bioline, London, UK) according to the manufacturer’s recommendations. Concentration and the purity of isolated RNA were established with the use of a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). A High-Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA, USA) was used to transcribe the RNA. The reaction was performed with 2 µL of 10X RT Buffer, 0.8 µL of 25X dNTP Mix (100 mM), 0.5 µL of 100 µM 5′- GGGCGGTAACCATTCAGATA- 3′ reverse primer, 1 µL of MultiScribe Reverse Transcriptase, 1 µL of RNase Inhibitor, 4.7 µL of nuclease-free water and 10 µL of RNA (previously incubated for 5 min at 99 °C). The PCR was performed as described previously [13]. The amplification of the target 171 bp CPNV gene was performed with the use of a HotStarTaq Plus Master Mix Kit (Qiagen, Hilden, Germany) and carried out with 10 µL of HotStarTaq Plus DNA Polymerase, 0.1 µL of each 100 µM primer, 2 µL of CoralLoad PCR Buffer, 5,8 µL of RNase-free water and 2 µl of cDNA. After pre-denaturation at 95 °C for 5 min, the denaturation step was performed at 94 °C for 1 min, followed by primer annealing at 55 °C for 1 min, product elongation at 72 °C for 1 min, and final elongation at 72 °C for 10 min. Thirty-five replication cycles were performed.
IBDV Identification
Total RNA was extracted directly from the homogenised internal organs of the examined birds. Briefly, 700 µL of sterile PBS was added to each sample (0.2 g) and then samples were homogenized with the use of a TissueLyser II (Qiagen). Samples were centrifuged at 1 500 × g for 30 s. After centrifugation, 200 µL of the supernatant was used for further steps of RNA isolation, which were performed with an RNeasy Mini Kit (Qiagen) according to the manufacturer’s recommendations. Reverse transcription was performed with the use of High-Capacity cDNA Reverse Transcription Kit (Life Technologies). IBDV was detected in the samples adopting the method described previously by Lin et al. [17] again with the use of a HotStarTaq Plus Master Mix Kit (Qiagen) and the following set of primers: IBDV F (sense primer): 5’ CCCAGAGTCTACACCATA 3’ and IBDV R (antisense primer): 5’ TCCTGTTGCCACTCTTTC 3’. The reaction was performed with 10 µl of HotStarTaq Plus DNA Polymerase, 0,1 µl of each 100 µM primer, 2 µL of CoralLoad PCR Buffer, 5,8 µl of RNase free water and 2 µl of cDNA. After pre-denaturation at 95 °C for 5 min, the denaturation step was performed at 94 °C for 1 min, followed by primer annealing at 55 °C for 1 min, product elongation at 72 °C for 1 min, and final elongation at 72 °C for 10 min. Thirty-five replication cycles were performed.
FAdV Identification
Total DNA was extracted directly from the internal organs of the examined birds. Extraction was performed using a DNA Mini Kit (Qiagen) according to the manufacturer’s procedure. DNA templates were stored at − 20 °C until further analysis. DNA obtained from the reference strain type/species 2/D served as a positive control.
The specific oligonucleotide primers were used for the amplification of the loop L1 region of the hexon gene of all FAdV types. The primers were synthesised at the Genomed company (Warsaw, Poland). The sequences of nucleotide primers were as follows: FAdV F (forward primer) – 5′ATGGGAGCSACCTAYTTCGACAT 3′ and FAdV R (reverse primer) – 5′AAATTGTCCCKR AANCCGATGTA 3′. The expected product size was 830 bp.
The reaction was conducted in a final volume of 25 µL, which contained 2.5 µL of 10X PCR buffer, 1 µL of dNTP (10 mM), 1.5 µL of each primer (10 µM), 2 µL of DNA template, and 11.5 µL of sterile water. After the pre-denaturation at 95 °C for 5 min, the denaturation step was performed at 94 °C for 45 s, followed by primer annealing at 55 °C for 1 min, product elongation at 72 °C for 2 min, and final elongation at 72 °C for 10 min. Thirty-five replication cycles were performed. Amplification was conducted in a basic gradient thermocycler (Biometra, Göttingen, Germany).
After amplification, electrophoresis was carried out in 2% agarose gel with 1 µg/mL of ethidium bromide. Electrophoresis was conducted in Tris borate EDTA buffer, pH 8.2, (150 V and 80 mA) for 50 min in a Mini-Sub Cell (Biorad, Hercules, CA, USA). After gel electrophoresis, the size of the amplicons was compared with the MassRuler Low Range DNA ladder to 1 031 bp (Fermentas/Thermo Fisher Scientific Baltics, Vilnius, Lithuania). The results were visualised using a UV transilluminator, then photographed and analysed.
The results were considered positive when the DNA product obtained had the predicted size of 830 bp.
Statistics Statistical analysis was performed with GraphPad Prism 6.05 with the use of Mann Whitney U test. Differences were considered statistically significant at p < 0.05.