The course of pigeon circovirus infection in young pigeons experimentally kept under conditions mimicking the One Loft Race rearing system

The experiment was performed on 15 pigeons aged 4–5 weeks, which were bought as 3 birds from each of five private breeding facilities located in the Warmińsko-Mazurskie Voivodeship in Poland. Those facilities had had a documented PiCV infection in the five years prior to the experiment. All the pigeons used for the experiment were clinically healthy and free of pathogenic fungi, bacteria and parasites; they were also positive for pigeon circovirus genetic material in the blood or cloacal swabs.

After selection, the experimental birds were reared in one unit of the Pavilion of Experimental Infections belonging to the Department of Poultry Diseases in the Faculty of Veterinary Medicine of the University of Warmia and Mazury in Olsztyn (UWM), Poland, for six weeks, which mimicked the conditions of the OLR system and promoted natural cross-infection with PiCV. In addition, four pigeons negative for PiCV genetic material and necessary to the experiment background blood sample collection for flow cytometry analyses were kept in a separate unit. During the experiment, 1mL blood samples were collected into Vacutainer ethylenediaminetetraacetic acid tubes (BD, Franklin Lakes, NJ, USA) from each bird every 7 d. The blood samples were divided into two parts: (A) 20 μL used for DNA extraction to quantify PiCV viral loads, and (B) 500 μL used for the isolation of mononuclear cells for flow cytometry analyses. The B part was used for evaluation of the percentage of apoptotic and early necrotic cells in the B IgM⁺ lymphocyte subpopulation. On day 42 of the experiment, all birds were euthanised to enable the collection of their spleen samples for mononuclear cell isolation and flow cytometry analyses.

Total DNA extraction was performed with an Extractme Genomic DNA kit (Qiagen, Hilden, Germany) according to the manufacturer’s guidelines. The concentration and purity of eluted DNA were measured with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). The samples were then stored at –20°C until further analysis.

A ddPCR was performed in accordance with the protocol developed in previous studies (15, 16). Prior to the analysis, the quantification cycle (Cq) value was determined for each sample with a qPCR according to the method developed by Duchatel et al. (2) targeting a 139 bp fragment of the replication-associated gene (rep). The reaction mixture was as follows: 10 μL of Power Sybr Green PCR Master Mix (Life Technologies, Carlsbad, CA, USA), respectively 0.1 and 0.25 μL of forward and reverse primers (concentration of 10 μM), 7.65 μL of RNase-free water and 2 μL of eluted DNA. The reaction was carried out in a LightCycler Real-Time PCR System (Roche, Basel, Switzerland) under the following conditions: an initial denaturation step at 95°C for 10 min followed by 40 cycles of three-stage amplification: at 95°C for 15 s, 64°C for 30 s and 72°C for 30 s. Next, all samples with a Cq lower than 22 were diluted with distilled water according the rule that one single tenfold dilution increases the Cq by 3. This step was necessary to avoid the oversaturation of PCR droplets, which could make proper quantification impossible. After this step, the ddPCR reaction mixture was prepared with the following composition: 10 μL of QX200 ddPCR EvaGreen Supermix (Bio-Rad, Hercules, CA, USA), 0.22 μL of 10 μM forward and reverse primers (the same as in qPCR), 2.2 μL of template DNA, and 9.36 μL of nuclease-free water. The reaction mixture was mixed with 70 μL of QX200 Droplet Generation Oil for EvaGreen (Bio-Rad, Hercules, CA, USA) in a QX 200 droplet generator (Bio-Rad). The obtained droplet emulsions (40 μL) were further transferred into a semi-skirted PCR-clean 96-well plate and then heat-sealed with pierceable foil, and finally the PCR amplification was carried out in a C1000 Touch thermal cycler (Bio-Rad, Hercules, CA, USA). The ddPCR conditions were as follows: an initial denaturation step at 95°C for 5 min followed by 40 cycles of 95°C for 30 s, and 64°C for 1 min with a ramp rate of 2°C/s; the next cycle was a final hold performed at 4°C for 5 min. After the thermal cycling, the plate containing the droplets was chilled to room temperature and placed in a QX 200 droplet reader (Bio-Rad) for analysis. All samples were investigated in duplicate. The results were expressed as mean PiCV genome copy number +/– standard deviation per specified volume of the sample on each day of sampling.

To isolate mononuclear cells from peripheral blood, 1 mL of blood sample suspended in 1 mL of phosphate buffered saline (PBS) was applied onto Histopaque-1077 gradient medium (Merck, Darmstadt, Germany) and then centrifuged at 450 × g and 20°C for 10 min. The resultant buffy coat of mononuclear cells was collected into sterile test tubes, washed twice and suspended in 1 mL of PBS (Merck, Darmstadt, Germany). The absolute lymphocyte count (ALC) and lymphocyte viability were determined for each sample with an automatic Vi-cell XR Cell Viability Analyzer (Beckman Coulter, Brea, CA, USA).

To isolate mononuclear cells from the spleen, samples of organs collected during necropsy were ground mechanically in a manual glass homogeniser in the presence of a cell culture medium. Once a homogenous suspension had been achieved, the whole sample was passed through nylon filters with a mesh diameter of 70 μm (Corning, Corning, NY, USA). The resultant filtrate was centrifuged at 450 × g and 20°C for 10 min, and the cell pellet was resuspended in mononuclear cell isolation gradient. The layer of mononuclear cells obtained this way was rinsed twice and suspended in sterile PBS with 5% addition of activated bovine serum (Merck, Darmstadt, Germany). Afterwards, the ALC was determined for each sample with the Vi-cell XR Cell Viability Analyzer (Beckman Coulter, Brea, CA, USA).

Mononuclear cells isolated from blood and spleen samples and counted were stained with specific polyclonal goat anti-chicken antibodies (fluorescein isothiocyanate (FITC)- conjugated anti-B IgM; Bio-Rad, Hercules, CA, USA). Approximately 500,000 mononuclear cells isolated from each sample were stained and incubated in darkness on ice for 30 min. Then, the cells were rinsed with PBS twice (Merck, Darmstadt, Germany) and centrifuged, and the obtained cell pellets were resuspended in PBS and used for staining for apoptosis evaluation. The staining was performed according to the protocol described by Stenzel et al. (16). Cells stained for extracellular markers were washed on ice with Annexin V binding buffer (BD Biosciences, Franklin Lakes, NJ, USA). The supernatant was removed by centrifugation, and the cells were suspended in the same buffer. Next, the FITC-conjugated Annexin V (BD Biosciences, Franklin Lakes, NJ, USA) and 7-aminoactinomycin D (BD Biosciences, Franklin Lakes, NJ, USA) were added to the cells. After incubation, the cells were diluted with Annexin V binding buffer and analysed with flow cytometry within 1 h using a fluorescence-activated cell sorting FACS Canto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Data were acquired in FACSDiva Software 6.1.3. (BD Biosciences, Franklin Lakes, NJ, USA). Cells were analysed and immunophenotyped in FlowJo 7.5.5 software (BD Biosciences, Franklin Lakes, NJ, USA). Data were expressed as mean percentages of specific lymphocyte subpopulations +/– standard deviation.

The significance of differences in the percentage of the subpopulations of IgM⁺ B lymphocytes in blood and spleen between the experimental and control group was analysed using Student’s t-test. The significance of differences between the sampling dates in PiCV copy number in the blood was analysed using repeated measures analysis of variance followed by Tukey’s test. Differences were considered significant at the confidence level of 95 (P-value < 0.05). All analyses were performed with Statistica 13.3 software (Statsoft, Cracow, Poland).

At the beginning of the experiment, the average PiCV genome copy number (gcn) in the blood of the examined pigeons was 342 ± 859. The viraemia peak of 6,054 ±/14,556 PiCV gcn was noted on day 14 of the experiment. Thereafter, viraemia declined, falling to 5,500 and 4,600 ± 11,000 PiCV gcn on days 21 and 28 of the experiment, respectively. A remarkable decrease of viraemia was noted on day 35 of the experiment, and its lowest level of 202 ± 679 PiCV gcn was noted on the last sampling date (Table 1). The differences between sampling dates were found statistically insignificant (P-values of 0.57–1.00).

The results of the flow cytometry analyses are presented in Tables 2 and 3. As shown in Table 2, the percentage of B IgM⁺ lymphocytes in the population of blood mononuclear cells was very similar throughout the experiment in both experimental and control pigeons and reached 7.46 +/– 0.87 and 6.98 +/– 0.7 on average, respectively. The percentage of apoptotic B IgM⁺ lymphocytes of the analysed subpopulation was also similar and reached 2.12 +/– 0.87 % and 2.55 +/– 1.52 % on average in the experimental and control pigeons, respectively. The differences in results were significant only on day 0 of the experiment (P-value = 0.04). In turn, the average percentage of necrotic lymphocytes of the analysed subpopulation was 0.2 +/– 0.25 and 0.24 +/– 0.32 in the experimental and control pigeons, respectively. The differences were significant on day 14 (P-value = 0.03) and 42 (P-value = 0.02) of the experiment.

As shown in Table 2, there was no statistical difference between the percentages of the individual IgM⁺ B cell subpopulations in the population of splenocytes. The percentage of splenic B IgM⁺ apoptotic cells was ca. 40% higher in the experimental group than in the control one. However, this difference was statistically insignificant (P-value = 0.31). The representative histograms of the analysed blood and spleen samples are shown in Fig. 1.

Fig. 1.

Representative histograms of splenic B IgM⁺ flow cytometry analyses. A. Density plot of the extracellular staining for B IgM, Annexin V and 7-AAD. Three states of the cells were identified: viable cells, QIII (Annexin V/7-AAD = –/–); early apoptotic cells, QIV (Annexin/7-AAD = +/–); necrotic and late apoptotic cells, QI and QII (Annexin V/7-AAD = –/+ and +/+). B. Representative histogram of the sample with high apoptosis. C. Representative histogram of the sample with low apoptosis. The red color on the part B and C indicates on control cells (stained with anti-B IgM antibodies and 7-AAD), whereas the blue color indicates on apoptotic cells stained additionally with Annexin V