Elsevier Acta Medica Okayama 0032-5791 104 3 2025 An ultra-simplified protocol for PCR template preparation from both unsporulated and sporulated Eimeria oocysts 104810 EN Aruto Takano Departments of Veterinary Immunology, Graduate School of Veterinary Medical Sciences, Osaka Metropolitan University Dennis V. Umali Department of Veterinary Clinical Sciences, College of Veterinary Medicine, University of the Philippines Los Baños, College April H. Wardhana Research Center for Veterinary Science, National Research and Innovation Agency Dyah H. Sawitri Research Center for Veterinary Science, National Research and Innovation Agency Isao Teramoto Departments of Virology and Parasitology, Graduate School of Medicine, Osaka Metropolitan University Toshimitsu Hatabu Laboratory of Animal Physiology, Graduate School of Environmental, Life, Natural Science and Technology, Okayama University Yasutoshi Kido Departments of Virology and Parasitology, Graduate School of Medicine, Osaka Metropolitan University Akira Kaneko Departments of Virology and Parasitology, Graduate School of Medicine, Osaka Metropolitan University Kazumi Sasai Departments of Veterinary Immunology, Graduate School of Veterinary Medical Sciences, Osaka Metropolitan University Hiromitsu Katoh Departments of Veterinary Immunology, Graduate School of Veterinary Medical Sciences, Osaka Metropolitan University Makoto Matsubayashi Departments of Veterinary Immunology, Graduate School of Veterinary Medical Sciences, Osaka Metropolitan University Molecular biological techniques have enabled the accurate identification of the avian Eimeria parasite, however, the preparation of PCR template remains a bottleneck due to contaminants from feces and the robust oocyst's wall resistant to chemical and mechanical force. Generally, the preparation of PCR template involves three main steps: (1) pretreatment of oocysts; (2) disruption of oocysts; and (3) purification of genomic DNA. We prepared PCR templates from both unsporulated and sporulated E. tenella oocysts using various protocols, followed by species-specific PCR to define the limit of detection. Our data revealed that whereas neither pretreatment of oocysts with sodium hypochlorite nor purification of genomic DNA with commercial kits improved the limit of detection of PCR, disruption of oocysts was a critical step in the preparation of PCR templates. The most sensitive PCR assay was achieved with the template prepared by disrupting oocysts suspended in distilled water, followed by bead-beating and heating at 99°C for 5 min, which detected 0.16 oocysts per PCR. This ultra-simplified protocol for preparation of PCR template, which does not require expensive reagents or equipment, will significantly enhance the sensitive and efficient molecular identification of Eimeria. It will improve our understanding of the prevalence of this parasite at the species level and contribute to the development of techniques for the control in the field. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 0034-5288 139 2021 Reduction of macrophages by carrageenan decreases oocyst output and modifies local immune reaction in chick cecum with Eimeria tenella EN Dung Thi Ho Laboratory of Animal Physiology, Graduate School of Environmental and Life Science, Okayama University Hung Hoang Son Pham Laboratory of Animal Physiology, Graduate School of Environmental and Life Science, Okayama University Wataru Aota Laboratory of Animal Physiology, Graduate School of Environmental and Life Science, Okayama University Makoto Matsubayashi Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University Naotoshi Tsuji Department of Parasitology and Tropical Medicine, Kitasato University School of Medicine Toshimitsu Hatabu Laboratory of Animal Physiology, Graduate School of Environmental and Life Science, Okayama University This study aimed to evaluate the disease severity and local immune responses in macrophage-depleted chicks with Eimeria tenella. Macrophages were reduced by intraperitoneal injection of a carrageenan solution at 12, 13, and 16 days old, whereas the control group received intraperitoneal phosphate-buffered saline. Both chick groups were orally inoculated with E. tenella sporulated oocysts at 14 days old. Feces were collected daily, which were then quantified for oocysts. The chicks were sacrificed on day 5, and the ceca were collected for histopathological observation. The gene expression levels were measured using real-time quantitative reverse transcription-polymerase chain reaction. Macrophage-depleted chicks have been observed to shed a significantly reduced number of fecal oocysts compared to the infected control group. The parasite burden score in cecum specimens of macrophage-depleted chicks was significantly lower than those of infected control on day 5 after infection. Furthermore, macrophage reduction yielded significantly lower cecum histopathological scores and CD4 expression than those of the infected control group. The expression of interleukin (IL)-18, IL-22, interferon-γ, and inducible nitric oxide synthase was also noted to be significantly upregulated in both infected control and macrophage-depleted chicks compared to uninfected chicks. IL-4, IL-13, IL-17, and perforin expressions were also higher with macrophage depletion than in both control groups. These results suggest that macrophages serve as an invasive gate or a transporting vehicle to the site of first merogony. Furthermore, mononuclear phagocytes may play an important role in local immune responses, thus contributing to parasite development during early E. tenella infection. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 0165-2427 240 2021 Relationship between Eimeria tenella associated-early clinical signs and molecular changes in the intestinal barrier function 110321 EN Hung Hoang Son Pham Laboratory of Animal Physiology, Graduate School of Environmental and Life Science, Okayama University Makoto Matsubayashi Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University Naotoshi Tsuji Department of Molecular and Cellular Parasitology, Kitasato University Graduate School of Medical Science Toshimitsu Hatabu Laboratory of Animal Physiology, Graduate School of Environmental and Life Science, Okayama University The major clinical signs of coccidiosis in chickens due to Eimeria parasite are diarrhea and bloody feces. Previous studies showed that the impairment of the intestinal epithelial barrier and the elevation of the intestinal permeability are causes of clinical signs associated with coccidia challenges. Nevertheless, the information about molecular changes of the epithelial barrier at the early stage of the infection with a specific Eimeria species has not been mentioned. Hence, this study aims to elucidate the temporal relationships between epithelial barrier conditions and clinical signs in chickens infected with Eimeria tenella over the time from the earliest stages of infection.<br> White Leghorn chickens were inoculated with 1 × 104 oocysts of E. tenella. Thereafter the chickens were monitored for their daily clinical signs through observation, and between 5 dpi to 10 dpi, feces were collected for oocysts counting. Chickens were then administrated with fluorescein isothiocyanate-dextran (FITC-d) for gastrointestinal permeability test and tissues were collected each day for histopathological observation and total RNA extraction. Finally, the mRNA expression levels of the tight and adherens junction genes and cytokine genes were evaluated using the quantitative real-time polymerase chain reaction (qRT-PCR).<br> In this study, clinical signs such as diarrhea and bloody feces were observed concurrently from 3 to 8 dpi. Histopathology changes such as severe inflammation, hemorrhage, and epithelial desquamation were identified in the cecum specimens. The FITC-d level in the E. tenella-infected group was significantly higher than in the control group. In the infected group, the expression of claudin-2 gene was also upregulated, whereas the expressions of claudin-3 and E-cadherin genes were decreased as compared to the control group. These results implied that clinical signs of avian coccidiosis were associated with the intestinal barrier disruption via changes in expression levels of claudins and E-cadherin at the intestine. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 2051-817X 8 22 2020 Alteration of chemokine production in bovine endometrial epithelial and stromal cells under heat stress conditions e14640 EN Shunsuke Sakai Laboratory of Reproductive Physiology, Graduate School of Environmental and Life Science, Okayama University Toshimitsu Hatabu Laboratory of Animal Physiology, Graduate School of Environmental and Life Science, Okayama University Yuki Yamamoto Laboratory of Reproductive Physiology, Graduate School of Environmental and Life Science, Okayama University Koji Kimura Laboratory of Reproductive Physiology, Graduate School of Environmental and Life Science, Okayama University After parturition, cows frequently develop uterine bacterial infections, resulting in the onset of endometritis. To eliminate the bacteria, bovine endometrial cells secrete chemokines, such as IL-6 and MCP1, which attract macrophages (M Phi s) to the subepithelial stroma. These attracted M Phi s are not only involved in bacterial elimination but also the orchestration of inflammation and tissue repair. These immune responses aid in the recovery from endometritis; however, the recovery from endometritis takes longer in summer than in any other season. Based on these findings, we hypothesized that heat stress (HS) affects the chemokine production in endometrial cells. To confirm this hypothesis, we compared IL-6 and MCP1 production induced by lipopolysaccharide (LPS) in bovine endometrial epithelial and stromal cells under normal (38.5 degrees C) and HS conditions (40.5 degrees C). In the endometrial epithelial cells, IL-6 production stimulated by LPS was significantly (p < .05) suppressed under HS conditions. MCP1 production in endometrial epithelial cells was not detected under both the control and HS conditions regardless of the presence of LPS. Moreover, LPS significantly (p < .05) stimulated IL-6 and MCP1 production in endometrial stromal cells. Moreover, HS significantly (p < .05) enhanced their production compared to that under the control conditions. In addition, HS did not affect the migration ability of M Phi s; however, the supernatant of the endometrial stromal cells cultured under the HS condition significantly (p < .05) attracted the M Phi s when compared to the control condition. These results suggest that HS disrupts chemokine production in two types of endometrial cells and alters the distribution of M Phi s in the endometrium during the summer. No potential conflict of interest relevant to this article was reported.

BMFH Press Acta Medica Okayama 21866953 39 3 2020 Oral administration of the probiotic bacterium Lactobacillus acidophilus strain L-55 modulates the immunological parameters of the laying hen inoculated with a Newcastle disease virus-based live attenuated vaccine 117 122 EN Dung Thi Ho Graduate School of Environmental and Life Science, Okayama University Toshimitsu Hatabu Graduate School of Environmental and Life Science, Okayama University Yosuke Sunada Research & Development, Ohayo Dairy Products Co., Ltd. Yasuhiro Kondo Graduate School of Environmental and Life Science, Okayama University Probiotic supplements containing living bacteria have attracted interest as a potential source of health benefits for humans and livestock. The aim of this study was to determine whether administration of Lactobacillus acidophilus strain L-55 (LaL-55) enhances the immune response among chicks exposed to a Newcastle disease virus (NDV)-based live attenuated vaccine. Oral administration of LaL-55 augmented the elevation in the total numbers of leukocytes and lymphocytes following inoculation with the NDV-based live attenuated vaccine. Monocyte counts increased after LaL-55 administration independent of inoculation with the NDV vaccine. Among chicks that were administered LaL-55, there was a dose-dependent increase in the NK cell activity measured by a 51Cr release assay at 2 weeks after the secondary NDV vaccine inoculation. Two weeks after the secondary inoculation with the NDV vaccine, interferon (IFN)-γ-mRNA expression was significantly elevated in mononuclear splenocytes from chicks that were administered LaL-55. Meanwhile, LaL-55 administration did not change the mRNA levels of IFN-α, IFN-β, and interleukin-1β. These results may suggest that coadministration of LaL-55 with an NDV vaccine augments the immune response against the virus. Therefore, LaL-55 may help protect against viral diseases in poultry. No potential conflict of interest relevant to this article was reported.

The Pharmaceutical Society of Japan Acta Medica Okayama 0918-6158 43 3 2020 Daily Meal Supplemented with Astaxanthin-Enriched Yolk Has Mitigative Effects against Hypertension in Spontaneously Hypertensive Rats 404 408 EN Toshimitsu Hatabu Laboratory of Animal Physiology, Graduate School of Environmental and Life Science , Okayama University Takumi Harada Laboratory of Animal Physiology, Graduate School of Environmental and Life Science, Okayama University Yuri Takao Laboratory of Animal Physiology, Graduate School of Environmental and Life Science, Okayama University Ho Thi Dung Laboratory of Animal Physiology, Graduate School of Environmental and Life Science, Okayama University Akihiro Yamasato K.I Chemical Industry Co., Ltd. Tatsuya Horiuchi K.I Chemical Industry Co., Ltd. Atsuya Mochizuki K.I Chemical Industry Co., Ltd. Yasuhiro Kondo Laboratory of Animal Physiology, Graduate School of Environmental and Life Science, Okayama University The aim of this study was to investigate the effects of egg yolk powder enriched with astaxanthin (ASX-E) on blood pressure in spontaneously hypertensive rats (SHR) and to verify the benefits of ASX-E as a functional food. To investigate the antihypertensive effect, SHR were fed with an ASX-E mixed diet before hypertension development. Blood pressures were determined periodically during the study by the tail-cuff method. At the end of the study, animals were euthanized, and their thoracic aortas were collected to determine vascular conductance. The thoracic aorta tension was measured with a force displacement transducer. Concentration-dependent response relationships were determined by cumulative addition of 10−9–10−4 M Carbamoylcholine (Cch). Blood pressures of the SHR in the ASX-E mixed diet group were ASX-dose-dependently lower than that of those in the control group. In SHR fed with an ASX-E mixed diet, Cch induced vasorelaxation in the thoracic aorta with endothelium lining but not without endothelium. However, the antihypertensive effect of ASX-E was not observed on blood pressures in SHR that were fed with ASX-E only after the development of hypertension. Results suggest that ASX-E protects endothelial function and thereby prevents the development of hypertension. Hence, the results of our research indicate that daily consumption of ASX-E has a potential benefit on human health. No potential conflict of interest relevant to this article was reported.

TAYLOR & FRANCIS Acta Medica Okayama 0916-8451 83 5 2019 Mulberry juice freeze-dried powder attenuates the disease severity by the maintaining of colon mucosa in mice with DSS-induced acute colitis 914 922 EN Yang Wang Graduate School of Environmental and Life Science , Okayama University Toshimitsu Hatabu Graduate School of Environmental and Life Science , Okayama University This study aimed to evaluate the microbial compositions and gene expression related to inflammation in dextran sodium sulfate (DSS)-induced acute colitis and the effect of mulberry supplementation. Male BALB/c mice received a diet supplemented with mulberry juice freeze-dried powder (MFP) or not for 3 weeks. After 3 weeks, the mice received water containing 5% (w/v) DSS or not for 1 week. The disease activity index score in mice fed MFP was significantly decreased. A significant decrease in Bifidobacterium spp. and the Clostridium perfringens subgroup was observed in mice not fed MFP. The number of goblet cell and NLRP6 expression were observed in mice fed a diet supplemented with MFP compared with mice not fed MFP. These results may indicate that mulberry mitigates DSS-induced acute colitis by a changing the gut microbial flora and by improving mucosal conditions. No potential conflict of interest relevant to this article was reported.

岡山実験動物研究会 Acta Medica Okayama 34 2018 鶏コクシジウム症：アイメリア・テネラ感染メカニズムの解明に向けて 17 20 EN Toshimitsu Hatabu Graduate school of Environmental and Life Sciences, Okayama University Avian coccidiosis is most important entero-parasitic disease in the world. Eimeria parasite is causative agent of this disease. In Japan, this parasite species were widely spread and the positive rates are about 50 in layer and 70 % in broiler. The symptoms of coccidiosis are diarrhea, bloody excretion, weight loss, and die. Eimeria tenella is the most pathogenic protozoa. The sporozoites, infection form of this parasite, entry to epithelial cells around the crypt of cecum in early infection. After infection, parasites proliferate in epithelial cells, and form to sexual stage finally. However, we have less information about the pathophysiology, especially invasive mechanisms and infection route, by E. tenella infection. We have focused to analyze the invasive mechanism and route of this parasite because this phenomenon is first event to cause the pathophysiological changes in the infection. I would like to inform about Eimeria parasite and introduce our research in this paper. No potential conflict of interest relevant to this article was reported.

岡山大学農学部 Acta Medica Okayama 2186-7755 102 2013 マラリア重症化関連新規宿主因子としてのScavenger receptor Aの同定 63 68 EN Toshimitsu Hatabu Severe falciparum malaria such as cerebral malaria and severe anemia is leading causes of morbidity and mortality. Plasmodium falciparum-infected red blood cells (pRBC) adhere to the endothelial cells via receptors expressed on the surface of the endothelial cells, and sequester in the microvasculature of several organs. Severe anemia, which may be due to a number of factors including rupture of the pRBC and phagocytosis of pRBC, is another cause of death. However, the molecular mechanism underlying both the cytoadherence and erythrophagocytosis related with severe malaria is not completely understood. Here, we report that the pRBC bind to the class A scavenger receptor, scavenger receptor A (SR-A), which is expressed on the surface of the activated phagocytes. 　First, we confirmed mRNA expression of scavenger receptors in the various tissues of P. berghei ANKA-infected mice. The expression of SR-A mRNA in all tissues was enhanced for 7 days postinfection. We also confirmed mRNA expression of SR-A in the human macrophage cell line, THP-1 cells, cultivated with pRBC. SR-A mRNA expression in THP-1 cells with pRBCs was observed after 24 hr cultivation, but not RBCs. Then, to identify cytoadherence of pRBCs to SR-A, human SR-A cDNA was transfected to CHO cells (CHO-SR-A cells). pRBC adhered to the CHO-SR-A cells, but not to the CHO-mock cells. Interestingly, the cytoadherence of both mature stage and ring form pRBCs to the CHO-SR-A cells was observed. Anti-SR-A antibody, but not Anexin V, efficiently blocked the cytoadherence of the pRBC to the CHO-SR-A cells. 　These results may suggest that SR-A acts as a host factor related with cytoadherence of the pRBC, which contributes to our present understanding of the pathology of severe falciparum malaria. No potential conflict of interest relevant to this article was reported.