Effects of avian infectious bronchitis with Newcastle disease and Marek's disease vaccinations on the expression of toll-like receptors and avian β-defensins in the kidneys of broiler chicks

Abstract

The aim of this study was to determine the effect of vaccinations for avian infectious bronchitis with Newcastle disease (IB/ND) and Marek's disease (MD) on the expression of toll-like receptors (TLR) that recognize viral RNA and microbial DNA, and AvBD in chick kidneys. Day-old chicks were vaccinated with MD or IB/ND vaccines or received no treatment (control group). The gene expression of TLR and AvBD in the kidneys of 3-day-old chicks and 10-day-old chicks was examined using real-time PCR. The localization of AvBD2 and AvBD4 was examined by immunohistochemistry at day three only. At 3 days of age, the expression of TLR7 and TLR21 was significantly higher in the IB/ND group (but not in the MD group) than in the control group. Conversely, at 10 days of age there was no significant difference in the expression of the three TLR between groups. In the 3-day-old chicks the expression levels of AvBD4, 5, 6, and 7 were higher in the MD group than in the control group. Furthermore, at this age, the expression levels of other AvBD were not significantly different between the control and vaccination (MD and IB/ND) groups. At 10 days of age, no AvBD expression was affected by MD and IB/ND vaccinations. Immunohistochemistry results localized AvBD2 in the leukocytes in the interstitial tissue and AvBD4 in the surface of microvillus epithelial cells of renal tubules, and in the epithelial cells of the collecting ducts and ureter. The localization of AvBD2 and AvBD4 was identified in all chicks. We suggest that the expression of innate immune molecules (including TLR and AvBD) in kidneys could be modulated by MD and IB/ND vaccination when performed at the day-old stage. Although the effects of both vaccinations may subside within 10 days, the enhanced expression of those innate immune molecules may support the innate immunodefense function in the kidneys of young chicks.

Keywords: AvBD; broiler chick kidney; toll-like receptors; vaccination.

Figure 1

Effects of Marek's disease (MD) and avian infectious bronchitis with Newcastle disease (IB/ND) vaccinations on the expression of toll-like receptors (TLR) 3, 7, and 21 (A−C: 3-day-old; D−F: 10-day-old) in the chick kidneys. Day-old chicks were vaccinated with IB/ND or MD vaccines, and the TLR expression was examined at 3 and 10 d of age using real-time PCR. Chicks in the control group (Con) received no vaccines. Values are the fold changes in the expression of target genes calculated using the 2^−ΔΔCT method and expressed as a ratio to the ribosomal protein S17. The solid bar represents the median value within each group. ∗, ∗∗Significantly different at P < 0.05 and 0.01, respectively (one-way ANOVA and Dunnett's test). The numbers of chicks in the control, MD, and IB/ND groups were 6, 7, and 7 in the 3-day-old analysis and 7, 6, and 7 in the analysis performed on 10-day-old chicks, respectively.

Figure 2

Real-time PCR products of avian β-defensins (AvBD) in the kidneys of 3-day-old chicks. Equal amount of PCR product solution (5 μL) for each AvBD was loaded. The amplified products of AvBD2, 4–7, and 9–11 show dense bands.

Figure 3

Effects of MD and IB/ND vaccinations on the expression of avian β-defensins (AvBD) in the kidneys of 3-day-old chicks. (A–H) These panels show AvBD2, 4−7, and 9−11. Values are the fold changes in the expression of target genes (n = 6, 7, and 7 in the Con, MD, and IB/ND groups, respectively). ∗Significantly different at P < 0.05 (one-way ANOVA and Dunnett's test). See Figure 1 for further explanations. Abbreviations: Con, control; IB/ND, infectious bronchitis with Newcastle disease; MD, Marek's disease.

Figure 4

Effects of MD and IB/ND vaccination on the expression of avian β-defensins (AvBD) in the kidneys of 10-day-old chicks. (A–H) These panels show AvBD2, 4−7, and 9−11. Values are the fold changes in the expression of target genes (n = 7, 6, and 7 in the Con, MD, and IB/ND groups, respectively). ∗Significantly different at P < 0.05 (one-way ANOVA and Dunnett's test). See Figure 1 for further explanations. Abbreviations: Con, control; IB/ND, infectious bronchitis with Newcastle disease; MD, Marek's disease.

Figure 5

Immunolocalization of avian β-defensin 2 and 4 (AvBD2 and 4) in the kidneys of 3-day-old chicks with or without MD and IB/ND vaccinations. See Figure 1 for the explanation of vaccination. (A) Localization of AvBD2 and 4. [a–c] Immunoreactive AvBD2 is localized in the leukocytes (arrows) in the interstitial tissues of control and vaccinated (MD and IB/ND groups) chicks. [d–f] Immunoreactive AvBD4 is localized on the surface of microvillus epithelial cells of renal tubules (arrowheads) and in the cytoplasm of epithelial cells of collecting ducts (arrows) in all chick groups. [g–i] Strong immunoreaction signals for AvBD4 is localized in the epithelial cells in the ureter (arrows) in all chick groups. [j–l] A magnified view of AvBD4 staining confirms the presence of AvBD4 in the cytoplasm of the epithelial cells in the ureter (arrows) in all chick groups, and on the surface of microvillus epithelial cells of the renal tubule (arrowhead on Figure 5l). (B) Negative control staining for AvBD 2 and 4 in MD vaccine group. [m–o] Negative-control staining for AvBD2 and AvBD4 does not show positive staining, except for only a faint staining in the nucleus of some ureter epithelial cells on the AvBD4 negative-control sections (thin arrows in Figure 5o), suggesting that staining of AvBD2 in the leukocytes and AvBD4 in the surface of microvillus epithelial cells of renal tubules and in the cytoplasm of epithelial cells of the collecting ducts and ureter was specific. Abbreviations: IB/ND, infectious bronchitis with Newcastle disease; MD, Marek's disease.