Samples
The intestine and muscle samples from 22 wild boars were collected between September 4 and March 2, 2020, in Namie town in Fukushima prefecture. Furthermore, control intestine samples were collected from three wild boars in Hyogo prefecture. Each location is depicted in Fig. 1. In each case, after the licensed hunters slaughtered the wild boar to be exterminated, only the tissue was transferred to the study.
Measurement of radioactivity
Radioactivity in the muscle samples was determined by gamma-ray spectrometry using high-purity germanium (HPGe) detectors (Ortec Co., Oak Ridge, TN, USA), as described in our previous report3. Gamma rays from 137Cs were observed.
Exposure dose estimation
In order to estimate internal and external dose rates of the wild boars according to the ICRP publication 10826, we supposed the shapes of wild boars as prolate spheroids whose long axis was to be their body lengths. The short axis was given from their weight assuming their specific gravities were the same
as water. The dose rates were calculated from the contribution of 137Cs, not including
natural radionuclides. The energy deposition to the spheroids by beta and gamma rays from radionuclides were calculated by the numerical simulation with the use of the Particle and Heavy Ion Transport code System (PHITS)27. For the sake of simplicity, we supposed the spheroids consisted of only muscle, which would give overestimated values because muscle contains more radio cesium than other organs. The external exposure dose was calculated from the air dose rates which were observed from the monitoring post near the boars captured place. The average values of the air dose rates were obtained from fitting observed data of two years with decay curve. The background due to the natural radionuclides was estimated to be 0.05 µGy/h which was observed before the Fukushima Daiichi accident, and was removed before the fittings. The half-lives of the air dose rates were 2000–3000 days depending on the environment. Assuming the external exposure dose was ascribed to the 137Cs included in the surface of the ground. The amount of the 137Cs was calculated so as to reproduce the observed air does rates. Since the maximum range of the beta ray from 137Cs is a few millimeters, almost all of the beta ray from inside the body should be absorbed in the boar’s body, but the beta ray from outside the body would stop in its fur. The beta rays contribute 100% to internal exposure dose but 0% to external one. Since the linear attenuation coefficient for gamma rays from 137Cs is 0.084 cm−1 = (12 cm)−1, some of the gamma rays cannot stop in the body depending on the size of the body. The numerical simulation suggested that 65–90 percent of the gamma rays from 137Cs inside the body would go out, and 40–65 percent of the gamma rays from 137Cs outside would go through the body.
Pathological analysis
A piece of the small intestine was fixed in 10% neutral formalin at 4 °C for 24–48 h. Then, paraffin blocks were prepared for pathomorphological examination using hematoxylin and eosin (HE) staining.
Gene expression analysis
Total RNA was extracted from the whole tissue of the intestine using TRIzol Reagent (Life Technologies, Inc., Frederic, MD, USA) according to the manufacturer’s instructions. RNA concentration was measured using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and cDNA was synthesized using random primers and SuperScript II (Life Technologies, Inc.). Real-time PCR for IFN-γ, TLR3, and CyclinG1 was performed using Brilliant SYBR Green QPCR Master Mix III (Stratagene, La Jolla, CA, USA) with an AriaMx system (Agilent Technologies, Santa Clara, CA, USA). Primer sequences were designed using Primer-BLAST with sequences obtained from GenBank as described in the previous report4. Amplification conditions were 95 °C for 3 min, 40 cycles at 95 °C for 5 s, and 60 °C for 20 s. Fluorescence signals measured during the amplification were analyzed. Ribosomal RNA primers were used as an internal control, and all data were normalized to constitutive rRNA values. Quantitative differences between the groups were analyzed using the AriaMx software (Agilent Technologies).
Statistical analysis
All data are presented as mean ± standard error (SE) for each treatment group. Differences in mRNA expression among the groups were determined using the unpaired t-test with Welch’s correction. (Prism: GraphPad Software Inc., La Jolla, CA, USA). Differences were considered to be statistically significant at a P value of < 0.05.
Ethics approval
No animals were killed for this research. Use of all animals was secondary to control wildlife pests. We conducted the investigation with the permission of the local government.
Source: Ecology - nature.com
