Goslings and Muscovy ducklings
The infectious material for further study was taken from goslings and ducklings obtained by Department of Poultry Viral Diseases at NVRI during the post-mortem examination. All conducted autopsy examinations were done according to international guidelines and recommendations. All examinations conducted in the National Veterinary Research Institute were approved by National Bioethics Committee (Lublin, Poland). The liver and heart tissues were weighed and manually homogenised. The weight of each sample was 25 mg and was homogenised in PBS (phosphate saline buffer, Medlab, Lublin, Poland).
Correlation between age of birds and clinical symptoms
Correlation between the age of the examined birds and the clinical symptoms and lesions severity was calculated using Spearman's rank (Rs) correlation one-tail test with 5% with significance level. The choice of one-tailed test was due to the predicted nature of GPV and MDPV replication and incidence of clinical sympthoms or lesions. The Rs value was calculated according to the following formula:
where: d2 - square of differences between ranks and n - number of cases. Ranks for clinical symptoms and lesions were calculated as following: 0 - absence of clinical symptoms/lesions to 1-5 - one to five different clinical symptoms and lesions. The obtained RS value was compared with the critical p value for this level (0.5035). The Rs value was higher than the critical level which resulted in the rejection of the null hypothesis (H0) about no significant correlation between the age and symptoms in the examined birds. This promoted the alternative hypothesis (H1) about the observed correlation. Calculations were made in Microsoft Excel ver. 2007 (Microsoft, RedMond, Washington, USA) and plotted as graphs.
Total DNA was extracted from homogenates of liver and heart from goslings and Muscovy ducklings collected from 16 farms according to the procedure for tissue homogenates (Qiagen, Hilden, Germany). The numbers of the used field isolates of GPV stand for: 14/01, 24/03, 33/03, 232/06, 8/07, 14/07, 16/07, 18/07, 27/08, 47/08, 27/09, 30/09, 81/10. The isolates of MDPV stand for: 52/09, 52/10 and 21/11.
Standard strains of viruses and DNA templates
GPV 88v was obtained from strain collection of the Department of Poultry Viral Diseases at the National Veterinary Research Institute (NVRI) in Puławy, Poland. The MDPV FM standard strain was provided by CEVA-Phylaxia Corporate, Budapest, Hungary. Goose circovirus (GCV), avian adenovirus 1 (FAdV-1), goose hemorrhagic polyomavirus (GHPV) were used as negative controls and were chosen from the strain collection of the Department of Poultry Viral Diseases (NVRI) in Puławy, Poland. The DNA templates were extracted from 200 μL of viral stock solutions using Qiagen Mini Kit (Qiagen, Hilden, Germany).
PCR primer and probes
Oligonucleotide primers and Taqman probe specific for ITR inverted terminal repeats region of GPV and MDPV were designed using Primer Express version 2.0.1 (Applied Biosystems, Foster City, California, USA) on the basis of complete sequence of the VG31/1 GPV strain (GenBank Accession No. EU583392) and the sequence of MDPV FM ITR region (GenBank Accession No. X75093). Due to single nucleotide differences in ITR region between GPV and MDPV the degenerated Taqman probe was designed with single Y nucleotide complementary to cytosine or thymine (C or T). The localisation of primers and probe with complementary regions was presented at Figure . The sequences of the primers and probe used were listed in Table . There were two product sizes: 123 bp for GPV and 126 bp for MDPV. The fluorogenic probe was labeled with the reporter: 6-carboxyfluoroscein (FAM) at the 5' end and with tetramethylcarboxyrhodamine (TAMRA) as a quencher at 3' end of the sequence. The probes and primers were synthesized by Genomed Co. (Warsaw, Poland) and purified by high-performance liquid chromatography [HPLC] system.
Figure 5 Localisation of designed primers and probe for ITR region of GPV and MDPV. The fragment of ITR was shown (from 1st to 540th nucleotide). Complementary sequences for primer were highlighted in yellow while for probe in green. The degenerated nucleotide (more ...)
Optimization of quantitative real-time PCR
The reaction was performed in 7500 Applied Biosystems thermocycler in 96-well tray (Applied Biosystems, Foster City, California, USA). The test was optimised using 4 primer concentrations (0.1, 0.2, 0.4 and 0.5 μmol/μL) and 4 probe concentrations (0.1, 0.2, 0.3, 0.5 μmol/μL). The reaction was conducted in 25 μL reaction volume in 96-well optical plates (Applied Biosystems, Foster City, California, USA). The data were collected by Applied Biosystems software ver. 2.0.1 (Applied Biosystems, Foster City, California, USA).
The recombinant plasmid pITR was constructed by the PCR product insertion (123 bp) into pGEM-T Easy vector (Promega, Fitchburg, Wisconsin, USA). The primers used for the ITR fragment amplification were identical with the ones used for real-time PCR (Table ). The construct was cloned into DH5α cells (Invitrogen, San Diego, USA) with blue/white screening according to procedures recommended by the producer. A single white colony harboring the plasmid with the ITR fragment was used for the inoculation of 5 ml of liquid LB medium with ampicillin (100 μg/mL), then the culture was incubated for 18 h at 37°C with shaking 225 rpm. The construct purity was determined by picking a single bacteria colony to PCR reaction mixture in the conditions described above. The plasmid DNA was extracted from 5 ml of inoculated media using Plasmid Mini Kit (Qiagen, Hilden, Germany).
Construction of standard curve and regression analysis
Four ten-fold successive dilutions of the purified pITR plasmid with copy number ranging from 1.0 × 106 to 1.0 × 103 DNA/1 μL were used. The dilutions were prepared in PCR-grade purity water (Qiagen, Hilden, Germany). Each dilution was tested in triplicate and used for the construction of a standard curve by plotting a logarithm of copy number against cycle threshold values (CT). The standard deviation (SD) between replicates was also calculated and given on the standard curve plot. The values of regression coefficient R2, slope, intercept and reaction efficiency (E) were calculated by ABI 7500 system software ver. 2.0.1 (Applied Biosystems, Foster City, California, USA). The regression analysis was done using data exported to Microsoft Excel program ver. 2007 (Microsoft, RedMond, Washington, USA). For calculation of Y value used for the determination of viral copy number in the tested DNA samples the following equation was applied: Y = (slope)X + (intercept) where X - logarithm starting quantity and Y - threshold cycle. The viral load in the examined DNA samples extracted from the liver and heart of goslings and ducklings was calculated as a log10 from the mean of two replicates of each DNA sample per 1 gram of tissue.
Real-time PCR sensitivity and specificity
The sensitivity of real-time PCR was tested in standard reaction conditions on the basis of six ten-fold dilutions from 1.0 × 106 to 1.0 × 101 copies of pITR plasmid. The detection limit of the assay was determined as the highest dilution which resulted in the presence of the fluorescent curve detected as CT value. The specificity was determined using DNA of 88v strain, MDPV FM, GoCV, FAdV-1 and GHPV from the strains collection of the Department of Poultry Viral Diseases at NVRI.
Correlation between the age of birds and the copy number of GPV and MDPV
Correlation between the age of the examined birds and viral load in the examined DNA samples as a log10 per 1 gram of tissue was calculated using one-tailed Spearman's rank (Rs) correlation test according to the manner described above for the clinical symptoms. The data were presented as separate graphs in Microsoft Excel ver. 2007 (Microsoft, RedMond, Washington, USA).