key: cord-0967660-c8rm4f84
authors: Shimizu, Masahiro; Nii, Takahiro; Isobe, Naoki; Yoshimura, Yukinori
title: Effects of avian infectious bronchitis/Newcastle disease and Marek's disease vaccinations on the expression of Toll-like receptors and avian β-defensins in the kidneys of broiler chicks
date: 2020-09-12
journal: Poult Sci
DOI: 10.1016/j.psj.2020.08.071
sha: 076c12c361e53c163b4ea8b76499a243e4e88ea5
doc_id: 967660
cord_uid: c8rm4f84

The aim of this study was to determine the effect of vaccinations for avian infectious bronchitis/Newcastle disease (IB/ND) and Marek's disease (MD) on the expression of Toll-like receptors (TLRs) that recognize viral RNA and microbial DNA, and avian β-defensins (AvBDs) in chick kidneys. Day-old chicks were vaccinated with MD or IB/ND vaccines, or received no treatment (control group). The gene expression of TLRs and AvBDs 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) when compared to the control group. Conversely, at 10 days of age there was no significant difference in the expression of the three TLRs between groups. In the 3-day-old chicks the expression levels of AvBD4, 5, 6, and 7 were higher in the MD group when compared to the control group. Furthermore, at this age, the expression levels of other AvBDs 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 TLRs and AvBDs) 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 immuno-defense function in the kidneys of young chicks.

Many reports have demonstrated that the avian infectious bronchitis (IB) virus and 52 Newcastle disease (ND) virus infect the kidneys of immature and mature chickens, causing 53 pathological changes and disease (Chen and Itakura, 1996; Cong et al., 2013; Bande et al., 54 2016; El-Bahrawy et al., 2017) . Inoculation of chickens with Marek's disease (MD) virus 55 isolated from CVI988/Rispens-vaccinated chickens caused tumors, mainly in the spleen, liver, 56 and kidney (Cui et al., 2016) . Experimentally inoculated pathogenic Escherichia coli and 57 Salmonella typhimurium infected various organs, including the kidneys (Barrow et al., 1987; 58 Pourbakhsh et al., 1997) . Thus, the kidney is an organ susceptible to many pathogenic 59 microbes. Since the adaptive immune functions are not mature during the first few weeks of a 60 chicken's life, the innate immune system plays an essential role in defense against infection 61 by pathogenic microorganisms.

Toll-like receptors (TLRs) recognize microbe-associated molecular patterns to initiate 63 the innate immune response, such as the synthesis of proinflammatory cytokines and 64 antimicrobial peptides (Yoshimura, 2015) . TLR3 and TLR7 recognize the dsRNA and ssRNA were given mixed vaccines of IB and ND containing IB virus H120 strain and ND virus B1 102 strain (Poulvac COMBI; Kyoritsu Seiyaku Co.) through eye drops. The chicks in the control 103 group received no treatment. All chicks were maintained in a brooding room with a lighting 104 schedule of 23 h light: 1 h dark. They were given a commercial starter diet (Nichiwa Sangyo 105 Co. Ltd., Kobe, Japan) and water ad libitum. Tissue collection was performed at two different 106 ages, 3-d-old and 10-d-old (2 days and 9 days after vaccination respectively). Chicks were 107 euthanized with carbon dioxide prior to tissue collection (only the caudal renal division was 108 collected to make the sample tissue uniform). The left kidney was used for gene expression 109 analysis, and the right kidney was collected for histology. The number of 3-d-old chicks 110 totaled 20, including 6 chicks in the control, 7 in the MD, and 7 in the IB/ND groups. The Scientific., Waltham, MA, USA). The RNA samples were reverse-transcribed using Rever-Tra 125 Ace (Toyobo Co., Ltd., Osaka, Japan) as per manufacturer's instructions. The reaction 126 mixture (10 μL) comprised 0.5 μg total RNA, 1 × reverse transcription buffer (Toyobo Co., inactivation at 99°C for 5 min using a programmable thermal controller. Finally, the cDNA 131 samples were stored at -30°C until use.

Real-time PCR 134 Real-time PCR was performed using the Aria Mix Real-time PCR system (Agilent 135 Technologies Japan. Ltd., Tokyo, Japan). The reaction mixture (10 μL) consisted of 1 μL 136 cDNA, 1 × Brilliant III SYBR Green QPCR Mix (Agilent Technologies Japan, Ltd.), 0.25 μM 137 of each primer, and water. The primer sequences used in this study are shown in Table 1 . Two 138 different PCR protocols were used for the amplification. The first PCR protocol was 50 cycles 139 at 95°C for 5 s, and 60°C (RPS17, TLR3, 7 and 21), 62°C (AvBD2, 4, 6, 9 and 12) or 63°C 140 (AvBD5 and 10) for 10 s. The second protocol was 50 cycles at 95°C for 5 s, and 55°C 141 (AvBD1 and 7), 56°C (AvBD3), 60°C (AvBD11, 13 and 14), or 62°C (AvBD8) for 10 s, 142 followed by 72°C for 10 s each. The real-time PCR products using the samples from 3-d-old 143 chicks were examined by electrophoresis on 2% (w/v) agarose gel containing 0.025% (w/v) 144 ethidium bromide to confirm the products of amplification. For that electrophoresis, five μL 145 real-time PCR product solutions was loaded equally in each sample.

In the real-time PCR analysis, expression of TLR3, 7, and 21 were examined as they 147 are the receptors recognizing RNA and DNA virus molecular patterns. Expression of AvBD2, 148 4, 5, 6, 7, 9, 10 and 11 were examined because their real-time PCR products showed dense 149 bands on the agarose gel electrophoresis. Real-time PCR data were analyzed using the 2 -ΔΔCT 150 method to calculate the relative level of gene expression in each sample and were expressed 151 as ratios to the ribosomal protein S17 (RPS17) housekeeping gene (Livak and Schmittgen,

Samples from the right side caudal renal division from chicks in each group (control, 156 MD, and IB/ND) were fixed in 10% (v/v) formalin in PBS, and processed for paraffin 157 sections (4 μm in thickness). After deparaffinization, they were autoclaved at 110°C for 15 158 min in 10 mM Tris buffer (pH 10.0) for antigen retrieval. Then, the sections were incubated 159 with 1% (w/v) blocking reagent (Roche Co., Basel, Switzerland) for 1 h, followed by an Then, these sections were counterstained with hematoxylin and covered after dehydration.

The antibody to AvBD2 had been used to localize that protein in the chick intestine Sigma -Aldrich Japan Co., Tokyo, Japan. The AvBD4 antibody in the anti-serum was purified 177 using an affinity column (HiTrapTM NHS-activated HP, GE Healthcare Japan) conjugated 178 with AvBD4 synthetic peptide as described above. by replacing the primary antibodies with absorbed antibodies, which were prepared by 181 incubating AvBD2 or AvBD4 antibodies with corresponding peptides at a ratio of 1:5 by 182 weight.

Statistical analysis 185 The significance of differences in the real-time PCR data between control and vaccine 186 (MD and IB/ND) groups was examined using one-way ANOVA followed by Dunnett's test.

Differences were considered significant when the P-value was < 0.05. Figure 5 shows the localization of immunoreactive AvBD2 and 4. In the control group,

AvBD2 was identified in the leukocytes in the interstitial tissue among the renal tubules and 208 renal corpuscles (Fig. 5a) . The AvBD4 was localized on the surface of microvillus epithelial 209 cells of renal tubules (Fig. 5d ). Immunoreaction signals for AvBD4 was also localized in the We report here that TLRs and AvBDs are expressed in the chick kidney and their 221 expression is affected by MD and IB/ND vaccination performed at day-old of age. The major 222 findings were, at 3-d-old of age (2 d after vaccination), (1) the expression of TLR7 and 21 223 was higher in the chicks received IB/ND vaccination than in control, (2) expression of AvBD4, 224 5, 6 and 7 was higher in the chicks vaccinated by MD than in control, and (3) AvBD2 was 225 localized in the leukocytes and AvBD4 was in the epithelium of renal tubules and ducts.

The current results for the expression of TLR3, 7, and 21 support the report by Xu et The current study showed the expression of AvBD1 to 13 in the kidney of 3-day and 234 10-day-old chicks. There are differences in the expression profiles (which may be due to the 235 breed and age of chicks), but all three studies suggest that AvBDs are expressed in the chick 236 kidney.

Immunohistochemical results suggest that AvBD2 was synthesized by the 238 leukocytes in the interstitial tissues, and AvBD4 was synthesized by the epithelial cells of Day-old chicks were vaccinated with IB/ND or MD vaccines and the TLR expression was examined at 3 and 10 days 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-d-old analysis and 7, 6, and 7 in the analysis performed on 10-d-old chicks.

J o u r n a l P r e -p r o o f 

Expression of avian β-defensins in