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Establishment of Wild-Derived Strains of Japanese Quail (Coturnix japonica) in Field and Laboratory Experiments
Get PDFapplication/pdfDomestic quail are used as both farm and laboratory animals. As the wild ancestor of quails is “endangered,” field studies are needed to conserve them. If wild-derived strains of quail are established, they will be unique genetic resources for both farm and laboratory animals. The purpose of the present study was to conduct a field study and create wild-derived quail strains using the breeding stocks in Tokachi, Hokkaido, Japan. Field observations from 2019 to 2022 indicate that wild quails migrate and stay at reproductive sites from late April to late October. Our estimations of the approximate ages of the observed and captured quails imply that adult males have intermittent reproductive opportunities from May to August. Morphological traits collected from adult and juvenile quails in the 2019–2022 population were similar to those previously reported for Japan’s 1914 and 1970 populations. Using natural mating of captured wild males and domestic (Dom) females, we established the W50 and W75 strains, which possessed 50% and 75% genetic contributions from the wild stocks. These unique genetic resources can be applied for future conservation and experimental use to understand the domestication history and genetic basis of quantitative traits. © 2023 by the authors.journal articl
The First Survey of Bovine Babesia Species Infecting Yaks (Bos grunniens) in Mongolia
Get PDFapplication/pdfYak (Bos grunniens) farming is an important part of Mongolia's livestock industry. Yaks survive in harsh mountain environments; provide meat, milk, and wool; and serve as a mode of transportation. In Mongolia, yaks are frequently raised alongside other livestock animals such as cattle, Bactrian camels, sheep, goats, and horses. Recently, we demonstrated that Babesia bovis, Babesia bigemina, and Babesia naoakii-parasites with the potential to cause clinical bovine babesiosis-infect not only cattle but also Bactrian camels in Mongolia. However, yaks have never been surveyed for Babesia infections in this country. In the present study, we surveyed yaks in 8 Mongolian provinces: Bayankhongor, Bayan-Ulgii, Khovd, Khovsgol, Omnogovi, Ovorkhangai, Uvs, and Zavkhan. Blood samples were taken and deoxyribonucleic acid (DNA) was extracted from 375 yaks. Furthermore, Giemsa-stained thin smears were prepared from 315 of the 375 blood samples and then examined for the microscopic detection of Babesia parasites. Microscopy revealed that 34 (10.8%) of 315 blood smears were positive for Babesia parasites. All 375 DNA samples were then tested for B. bovis, B. bigemina, and B. naoakii infection using specific polymerase chain reaction assays. We observed that 238 (63.5%) yaks in all surveyed provinces and 8 (2.1%) yaks in 3 provinces (Bayankhongor, Bayan-Ulgii, and Omnogovi) were positive for B. bovis and B. bigemina, respectively. However, all yaks tested were negative for B. naoakii. This epidemiological survey, the first to report Babesia infection in Mongolian yaks, suggests that disease management strategies for yaks in this country should further address bovine babesiosis. © American Society of Parasitologists 2023.journal articl
Estimating the nonlinear interaction between somatic cell score and differential somatic cell count on milk production by parity using generalized additive models
Get PDFapplication/pdfThis observational study aimed to use somatic cell score (SCS) and differential somatic cell count (DSCC), the combined proportion of polymorphonuclear leukocytes and lymphocytes in somatic cells, to investigate how mastitis affected milk production. Using generalized additive models, we analyzed 50,618 test-day records from 8,081 lactations from 7,912 cows in 197 herds between January 2021 and March 2022 to estimate the nonlinear interaction between SCS and DSCC, and the effects of lactation stages and seasons on milk yield, milk component percentages, and milk component yields by parity of cows. The results show that the interaction between SCS and DSCC on these traits was significant, nonlinear, and complex. When DSCC was high, the negative effects of SCS were minimal, even when SCS reached 8 (i.e., 3,200,000 somatic cells/mL). Cows with high DSCC could have milk yields similar to healthy cows, implying that these cows may have been in the early stages of mastitis and that the milk yield had yet to be affected. Contrastingly, when DSCC was low, milk loss due to high SCS was drastic, especially for cows in third or later lactations, whose milk yield could reduce from more than 35 kg/d to less than 15 kg/d (−59.9%). This tremendous milk loss in high-parity cows was likely due to their higher milk yield and higher risks of chronic mastitis. High SCS and low DSCC also led to a pronounced change in milk composition. The decrease in the percentage of lactose can be directly related to the damage of inflammation to the mammary gland, while the increase in fat and protein percentages was more attributable to the concentration effect resulting from the reduced milk yield. Compared with analyses based on categorized SCS and DSCC values, modeling these 2 indices directly helps us more precisely assess mastitis effects on milk yield and milk composition. For efficient milk production, our results indicate that we should prevent high-parity cows from entering a state of high SCS and low DSCC. © 2023 American Dairy Science Associationjournal articl
10ヶ月間の「ちくだいKIP体操プログラム」がコロナ禍の児童の身体形態と体力に及ぼす影響 : 第2期
Get PDFapplication/pdf本研究は一般社団法人ちくだいKIPによる10ヶ月間の体操教室が上士幌町在住の小学1年生から3年生の身体形態と体力に及ぼす影響について調査することを目的とした。6歳から9歳までの26名中24名がプロジェクトの参加を達成した。介入前後で身体形態データとして身長、体重、大腿長、大腿周囲径、大腿前部筋厚の測定、体力データとして新体力テストを実施した。プログラムは前年を踏襲した体操教室にスプリントトレーニング等を組み合わせた独自の内容となった。また、1ヶ月に約1回程度、上士幌町スポーツ少年団により様々なスポーツの体験会が実施された。介入前後で形態データのうち身長、体重、大腿長、大腿周囲径が有意に増加した(p<0.01)ものの、大腿前部筋厚には統計的な差は認められなかった。体力データはすべての測定値が有意に向上し(p<0.01)、全国平均値を上回った。このことは本プロジェクトの実施が体力低下傾向にある上士幌町の子どもの体力向上に継続的に寄与でき得ることを示している。一方、6歳から9歳時には大腿前部筋厚は変化しにくく、長軸が成長して筋量が増加している可能性が考えられる。今回、前年度よりも想定以上に体力が向上した理由は、様々なスポーツ体験がなされたことと、２人体制で、かつトレーニングの専門的な知識を有する人材が指導に入ったことであると考えられる。The purpose of this study was to investigate the effectiveness of a 10-months special exercise program in modifying the morphological and physical fitness data of children who lives in Kamishihoro Town. The subjects included 24 children aged 6~9years participated in this project. Height, weight, thigh length, thigh circumference, and anterior thigh muscle thickness as morphological data, and “the New physical fitness test” as physical fitness data were measured before and after the intervention. The program was original that combined sprint training with gymnastics that followed the previous year. In addition, about once a month, “the Kamishihoro Sports Shonen-dan” held various sports experience sessions. The morphological data, height, weight, thigh length, and thigh circumference increased significantly before and after the intervention (p<0.01), but there were no statistical changes in anterior thigh muscle thickness. All physical fitness data improved significantly(p<0.01) and exceeded the national average. This indicates that the implementation of this project can continuously contribute to the improvement of the physical strength of children in Kamishihoro Town, whose physical strength is declining. On the other hand, between 6~9years, the thickness of the anterior thigh muscle does not change easily, and it is possible that the long axis grows and the muscle mass increases. In this case, the reason why the physical strength improved more than expected compared to the previous year was that various sports experiences were done, and a two-person system and a person with specialized knowledge of training.departmental bulletin pape
わが国の屋内塵性ダニと人家周辺の蚊の生息・分布調査と物理的防除に関する実験的研究
Get PDF帯広畜産大学博士（獣医学）2022application/pdf衛生害虫という言葉の概念は幅広く、様々な定義が存在するが、法的には「医薬品、医療機器等の品質、有効性及び安全性の確保に関する法律」に基づく感染症を媒介する害虫で、ゴキブリ、ハエ（ 成虫・幼虫 ）、蚊（ 成虫・幼虫 ）、ノミ、シラミ、トコジラミ、イエダニ、屋内塵性ダニ、マダニがこれに該当する。わが国では、これまで、マラリア、日本脳炎、デング熱、フィラリアなど様々な害虫媒介性感染症に悩まされ、これらを制圧してきた歴史があるが、常に新たな害虫媒介性疾病の問題が生じている。例えば、住宅の気密化によって、屋内塵性ダニ、特にヒョウヒダニのアレルゲンとして重要性が認識された。これに対しては、居住空間へのくん煙製剤が有効であることが知られているが、重要な生息場所である布団については、布団内の生息分布が不明なため、その対策は明確になっていない。また、物流の発達や地球温暖化により、わが国でのデング熱の再興やチクングニア熱の侵入の懸念が増⼤している。
以上のような背景のもと、本研究では重要な衛生害虫でありながら生息分布や生態的習性の詳細が不明な、屋内塵性ダニと、新興・再興感染症の媒介者として重要性が高いと想定される人家周辺生息蚊を対象として、その生息分布と生態的習性を明らかにし、適切な防除、特に物理的な対策に焦点をあて、その実用可能性を検討した。
第１章では、屋内塵性ダニの主要な生息場所である敷布団のダニ相、密度および空間分布を明らかにした。全国から20 サンプル収集し、従来の電気掃除機による表面の塵採集法ではなく、布団の5 カ所から一部 ( 5 cm×5 cm ) を切り取り、上層、中層、下層の3 層に分けて洗い出し法によってダニの全数調査を行った。その結果、敷布団の主要なダニは、アレルゲンとして重要であるヤケヒョウヒダニとコナヒョウヒダニであり、2 種の構成比率は布団サンプルによって⼤きく異なった。脱皮殻を含めたダニ密度は72,088 個体/ m2 で、学校における管理基準の100 個体/ m2 に比べ、非常に高い密度であった。また、5 カ所の採集ポイント間ではダニ数に有意な差は認められなかったが、布団の上端と下端では中層に多く、布団中央部3 カ所では下層に多くのダニが認められた。中層と表層（ 上層と下層 ）のダニの分布比率は布団サンプルによって⼤きく異なり、表層に分布するダニの割合は15.6%から94.3%であった。以上から、布団内のダニ種構成や空間分布は各家庭の変異が非常に⼤きく、各家庭における微細気象、室内温湿度、布団干しや掃除機掛けなどの生活習慣によって⼤きく影響されていると考えられた。
敷布団の表層だけでなく薬剤が浸透しにくい中層にもダニが分布していることが明らかになったことから、簡便に密封空間を低酸素状態にする脱酸素剤の殺ダニ効果について検討した。鉄系とアスコルビン酸系の2 タイプの脱酸素剤を用いてバリア袋内でのヤケヒョウヒダニとコナヒョウヒダニの致死効果を調べた結果、鉄系のもので2日間以上密封すれば、両種のダニの成虫だけでなく卵も100％致死させることができたことから、布団内部のダニに対する脱酸素剤の有用性が示された。
第2 章では、デング熱媒介蚊であるヒトスジシマカ成虫の住宅地における移動範囲を明らかにした。緑地が点在する住宅地で個体マーキング法を用いて調査を行った結果、異なる地点で再捕獲された個体は、平均で75.3 m、最⼤で187 m を移動していることが明らかになった。このことは、デング熱やチクングニア熱がわが国で発生した場合、発生地域での重点的な生息緑地を把握し、そこからおおよそ半径100 m の範囲の緑地に殺虫剤を優先的に処理することが重要であることを示唆している。最近、我が国で約70 年ぶりにデング熱が国内発生したが、本知見を基にして行政の蚊成虫対策範囲が決定された。
加えて、夜間に屋内に侵入し、ヒトから吸血を行うチカイエカに対する物理的な防除方法を検討した。蚊の休息場所として暗がりを選好する習性を利用したBox Trap について、捕獲数を高めるための構造や色、設置場所、設置個数について検討を行い、それぞれの要因について捕獲率を増加させる形状や色（ 開口部：9 cm L× 2〜9 cmW、遮光板角度：45°、天板色：赤か⿊ ）があることを明らかにした。また、トラップの設置位置は床面から80 cm くらいまでの高さが適切であることが分かった。更に、期待される捕獲（防除）効果を得るために必要なトラップ設置個数を、実験的に算出できる可能性が示されたことから、防除への実用可能性が示唆された。
以上、本研究では、屋内塵性ダニの布団内での分布が明らかにするとともに、これにより、薬剤を浸透させることが難しい布団内部のダニに対する対策の端緒として、脱酸素剤による物理的防除の有効性を示した。また、ヒトスジシマカの市街地における移動範囲を明らかにした研究結果は、デング熱国内発生時の薬剤散布範囲の策定に直接的に寄与した。加えて、チカイエカの屋内侵入を想定し、捕獲率を最⼤にするBox Trap の構造や色、配置場所を明らかにし、物理的防除の可能性を提示することができた。Although the concept of "sanitary pest" is broad and various definitions exist, legally, they are pests that transmit infectious diseases based on the "Act on Quality, Efficacy and Safety Assurance of Drugs and Medical Devices," and include cockroaches, flies (adults and larvae), mosquitoes (adults and larvae), fleas, lice, bed bugs, house tropical red mites, house dust mites, and ticks fall into this category. Japan has suffered from a variety of pest-borne infectious diseases such as malaria, Japanese encephalitis, dengue fever, and filariasis, and has a history of controlling these diseases, but new pest-borne disease problems are constantly emerging. For example, the airtightness of housing has led to the recognition of the importance of house dust mites, especially the Dermatophagoides mite, as an allergen. Although it is known that a smoke formulation in the living space is effective against this problem, measures for Japanese mattress, an important habitat, are not clear because the distribution of the habitat within the Japanese mattress is unknown. In addition, the development of logistics and global warming have increased concerns about the resurgence of dengue fever and the invasion of chikungunya fever in our country.
Against this background, this study focused on house dust mites, which are important sanitary pests but the details of their habitat distribution and ecological habits are unknown, and mosquitoes living around human dwellings, which are assumed to be highly important as vectors of emerging and reemerging infectious diseases. The practical feasibility of appropriate control measures, especially physical measures, was discussed in this study.
In Chapter 1, the fauna, density, and spatial distribution in Japanese mattress, the primary habitat of house dust mites, were clarified. Twenty samples were collected from all over Japan, and instead of using the conventional surface dust collection method with an electric vacuum cleaner, a portion ( 5 cm x 5 cm ) was cut from five locations on the Japanese mattress and divided into three layers (upper, middle, and lower) to investigate the total number of mites by the washing out method. The results showed that the major mites in the Japanese mattress were the mites Dermatophagoides pteronyssinus and the D. farinae , which are important allergens, and the composition ratio of the two species varied greatly among the mattress samples. The density of mites, including exuviae, was 72,088 individuals/m2, which was very high compared to the control standard of 100 individuals/m2 at schools in Japan. Although there was no significant difference in the number of mites among the five collection points, more mites were found in the middle layer at the upper and lower ends of the mattress, and in the lower layer at the three collection points in the center of the mattress. The distribution ratio of mites in the middle and surface layers (upper and lower layers) varied greatly among the mattress samples, with the percentage of mites in the surface layer ranging from 15.6% to 94.3%. These results suggested that the mite species composition and spatial distribution in the mattress vary greatly from household to household, and are greatly influenced by the microclimate, indoor temperature and humidity, and lifestyle habits of each household, such as drying and vacuuming the mattress.
Since it was found that mites were distributed not only in the surface layer of the mattress but also in the middle layer, which is difficult for the agent to penetrate, the mite-killing effect of oxygen absorbers that can easily create a low-oxygen state in the sealed space was examined. The results showed that the iron-based oxygen absorber was effective in killing 100% of both adult and egg mites in the barrier bag, indicating that the oxygen absorber is effective in killing mites inside the mattress. This shows the usefulness of oxygen absorbers against mites inside mattresses.
In Chapter 2, the migratory range of adult Aedes albopictus, a dengue vector mosquito, in residential areas was clarified. The results of a survey using the individual marking method in a residential area dotted with green spaces revealed that individuals recaptured at different locations moved an average of 75.3 meters and a maximum of 187 meters. This suggested that if dengue or chikungunya fever occurs in our country, it is important to identify priority habitat green areas in the outbreak area and prioritize insecticide treatments in green areas within an approximate radius of 100 m from these areas. Recently, Japan experienced its first domestic outbreak of dengue fever in about 70 years, and the scope of mosquito adult control measures by the government was determined based on this knowledge.
In addition, physical control methods for Culex pipiens form molestus, which invade indoors at night and suck blood from humans, were studied. The structure, color, location, and number of Box Traps, which take advantage of the mosquitoes' preference for dark places as resting places, were examined to determine which shapes and colors (opening: 9 cm L x 2 to 9 cm W, shading plate angle: 45°, top plate color: red or black) would increase the capture rate for each of the factors. It was found that it is appropriate to place the trap at a height of up to about 80 cm from the floor. Furthermore, the possibility of experimentally calculating the number of traps required to achieve the expected capture (control) effect was demonstrated, suggesting the practical feasibility of pest control.
In conclusion, this study clarified the distribution of indoor dust mites in futons, and demonstrated the effectiveness of physical control with oxygen scavengers as a beginning of countermeasures against mites inside futons, which are difficult to penetrate with chemicals. In addition, the results of this study, which clarified the movement range of Aedes albopictus in urban areas, directly contributed to the formulation of the scope of chemical spraying in the event of a domestic outbreak of dengue fever. In addition, the structure, color, and location of box traps that maximize the capture rate were clarified, assuming indoor infestation by Cx pipiens form molestus and the possibility of physical control was presented.博士学位論文
大学院畜産学研究科 獣医学専攻
Doctoral Program of Veterinary Sciencedoctoral thesi
Morphological studies on the genital organ in the raccoon (Procyon lotor)
Get PDF帯広畜産大学博士（獣医学）2022application/pdfThe raccoon (Procyon loter) is a member of Procyonidae belonging to Carnivora, which is a middle-sized mammal and skillfully use the forelimbs. The raccoon native to North America was introduced into Japan in 1970s and has been naturalized in almost every prefecture of Japan. In Hokkaido, Japan, their population has increased, and many problems are induced by them.
Raccoons are considered seasonal breeders, and in Hokkaido, the mating season peaks in February. Mated female raccoons stays pregnant for about 63 days and give birth to an average of 3-4 cubs。However, information on morphological changes in the raccoon genital organs with postnatal development, with seasonal change and during pregnancy is limited. In this study, therefore, morphological and functional changes were examined in the testes and prostate glands of male raccoons, during developmental and seasonal changes, and in female, morphological and functional dynamics of the uterus, especially cervix during pregnancy were investigated.
In the examination of the raccoon testes, the regression of spermatogenesis was detected from June to August, although not all individuals. Moreover, the decline in the functions of Leydig cells was suggested. In the regression of spermatogenesis, there were no changes in the proliferative activity of the germ cells. Therefore, the regression was thought to be caused by shedding of germ cells. In the postnatal testicular development, testes began to the canalize at 6 months of age, and all individuals reached sexal maturation over 12 months old.
In the studies on the prostate glands, the seasonal dynamics of glandular structure, p63 and AR expression and proliferation in raccoon prostate glands were examined. Raccoon prostates exhibit seasonal variation of regression and regeneration. Moreover, in the postnatal development, it was revealed that histological aspects in the postnatal development resemble those of the reorganization in adult regressed prostate glands.
In the examination of the prostatic utricle, in the prostate glands of the raccoont, the prostatic utricle with various shapes was recognized. The prostatic utricle of raccoons had saclike and tubular glands and goblet cells. The saclike and tubular glands first appeared in juvenile stage, but not infantile. The lumen had secretions with PAS- and AB-positive in infantile stage, on the other hand, in adults, the secretions with PAS- and AB-positive, and PAS-positive and AB-negative secretion were secreted. Therefore, the prostatic utricle showed morphological and functional changes along with maturation.
In the studies on the cervix, histological changes was revealed during pregnancy. As pregnancy progressed, the intraepithelial cysts with mucous were expanded, and secretion increased to close the cervical orifice. It is suggested that the rupture of intraepithelial cysts may form the epithelial folds by the ripped surface epithelium.アライグマ（Procyon loter）は, 食肉目アライグマ科に属する中型の哺乳類で, 水辺を好み器用に前肢を使用することが知られている。アライグマは, 1970年代に北アメリカから移入され, 現在野生化して日本全国にその分布を広めている。北海道においても, アライグマはその分布を広めており, 様々な問題を引き起こしている。
アライグマは季節繁殖動物と考えられており, 北海道では交尾期のピークが2月にあると報告されている。アライグマの妊娠期間は63日で, 3から4頭の子を出産する。しかし, 生後発達, 季節変化, そして妊娠といった期間におけるアライグマの生殖器の形態学的な変化の情報はほとんどない。本研究では, 雄のアライグマにおいて, 生後発達および季節変化における精巣や前立腺の形態学的および機能学的変化を調べ, さらに雌では, 妊娠にともなう子宮頸の形態学的および機能学的動態を検索した。
アライグマの精巣の検索では, 全ての個体ではないが6月から8月の間に精子形成の退行が認められ, さらにライディッヒ細胞の機能の低下を示唆することができた。精子形成の退行において, 生殖細胞の増殖能には変化がなかったことから, 生殖細胞の脱落が精子形成の退行を引き起こしていると考えられた。また, 生後発達においてアライグマの精巣では, 生後6か月で精細管の管腔形成が生じ, また12か月以上で全ての個体が性成熟に達することが明らかになった。
前立腺では, 管腔構造, ｐ63やARの発現, そして細胞増殖能の季節動態が調べられ, アライグマは, 前立腺の退縮と再発達にともなうこれらの変化を示すことを明らかになった。さらに, 前立腺の生後発達の解析では, 生後発達の組織学的様相が, 成体の退縮した前立腺が再発達する際の特徴と類似することを明らかにした。
前立腺小室の検索では, アライグマの前立腺に様々な形の前立腺小室が認められた。アライグマの前立腺小室には, 管状腺, 嚢状腺そして杯細胞といった腺構造が認められ, 管状腺と嚢状腺は幼若個体には認められず, 若年個体になって初めて出現する。幼若個体では, 前立腺小室の管腔内にPASとABに陽性を示す分泌物が認められ, 成体ではPAS陽性とAB陰性の分泌物がさらに分泌されるようになる。このように, 前立腺小室は成長にともなって形態学的, 機能学的な変化を示した。
雌の生殖器である子宮頸の組織学的検索では, 妊娠期に組織学的変化が認められた。妊娠の進行にともなって, 粘液を保有した上皮内嚢胞は拡張し, 子宮頸の分泌は増加して子宮口は塞がれた。また, 上皮内嚢胞の破裂が上皮ヒダの形成に関与している可能性が示唆された。博士学位論文
大学院畜産学研究科獣医学専攻
Doctoral Program of Veterinary Sciencedoctoral thesi
Studies on modification of miracidium hatching technique (MHT) for preparation of single-genome DNA for use in population structure analysis of Schistosoma japonicum and development of ELISA for diagnosis of S. mekongi infection in humans
Get PDF帯広畜産大学博士（獣医学）2022application/pdfSchistosomiasis is a disease caused by blood flukes belonging to the genus Schistosoma. These parasites are endemic in 78 countries throughout the world wherein more than 240 million people suffer from the infection among over 700 million people at risk. Asian zoonotic schistosomiasis is caused by S. japonicum and S. mekongi infections. Their zoonotic nature leads to enhanced disease transmission, making schistosomiasis management challenging. Understanding the epidemiology and dynamics of the parasite’s transmission between humans and reservoir animal hosts in the field will be aided by knowledge of the genetic diversity of S. japonicum. In addition, there is no vaccine available for schistosomiasis, and thus, disease control is mainly based on MDA with praziquantel. Diagnostic tools with high sensitivity and specificity are needed to accurately assess the status of the endemicity within a community and to monitor the efficacy of the MDA program. The general aim of this study is to develop a means to obtain this information to promote MDA towards elimination of Asian zoonotic schistosomiasis.
In Chapter 1, the previous MHT protocol was modified to apply the technique for
preparation of S. japonicum single-genome DNA. The information regarding the genetic diversity of S. japonicum is indispensable to obtaining an accurate understanding of the epidemiology of schistosomiasis and the transmission dynamics of schistosomes among their definitive hosts. DNA samples originating from a single genome should be prepared to accurately examine the parasite’s genetic diversity. The miracidium, a larval stage of the parasite, can be useful material for single-genome DNA preparation. In this study, the previous protocol for the MHT was modified with 96-well plastic ELISA plates to individually collect a miracidium for single-genome DNA preparation. In addition, the effects of light conditions on hatching rates were evaluated. The results showed that the highest hatching rate was observed under the sunlight condition (92.4%), followed by fluorescent light (88.0%). The lowest hatching rate was recorded under the dark condition (4.7%). These results indicated for the first time, to our knowledge, that sunlight was the most efficient light source for the MHT. Furthermore, the study confirmed that a 0.85% NaCl solution and the dark condition prevented miracidium hatching and could be used to store the eggs until the MHT is conducted. In addition, successful amplification of 18S rRNA gene and microsatellite markers from DNA isolated from a single miracidium also confirmed the quality of the single-genome DNA for subsequent application.
In Chapter 2, recombinant antigen-based serology from S. japonicum was evaluated for the diagnosis of S. mekongi human infections. To date, no recombinant antigen has been assessed using ELISA for the detection of S. mekongi infection due to the lack of genome information of this parasite species. In this study, several recombinant S. japonicum antigens that had been developed previously were evaluated for the detection of S. mekongi infection. ELISA results showed that S. japonicum SEA at low concentration showed better diagnostic performance than the recombinant antigens tested using the archived serum samples from Cambodia. Because recombinant antigen has several advantages over crude antigen in the application with ELISA, further optimization of the recombinant antigens should be done in future studies to improve their diagnostic performance for detecting S.mekongi infection in humans.
In Chapter 3, recombinant antigen of S. mekongi TPx-1 (rSmTPx-1) was expressed, and the antigen was evaluated for its performance in detecting S. mekongi infection in humans by ELISA. Decades of use of numerous control measures, which include MDA using praziquantel, have resulted in a decrease in the prevalence of schistosomiasis mekongi. This, however, has led to a decrease in sensitivity of the Kato-Katz stool examination, which is considered the gold standard in the diagnosis of schistosomiasis mekongi. In this study, a gene coding for S. mekongi TPx-1 was cloned, and the recombinant antigen (rSmTPx-1) was expressed to develop a serological assay with high sensitivity and specificity that could replace the Kato-Katz technique. Diagnostic performance of rSmTPx-1 as an antigen in ELISA for detecting human schistosomiasis was compared with that of the recombinant antigen of S. japonicum TPx-1 (rSjTPx-1) using a panel of serum samples collected from endemic foci in Cambodia. The sensitivity and specificity of rSmTPx-1 in ELISA were 89.3% and 93.3%, respectively, whereas those of rSjTPx-1 were 71.4% and 66.7%, respectively. In addition, the higher Kappa value of 0.82, which was calculated between ELISA with rSmTPx-1 and Kato-Katz, confirmed better agreement between them than between ELISA with rSjTPx-1 and Kato-Katz (Kappa value 0.38). These results suggested that ELISA with rSmTPx-1 could be a potential diagnostic method for detecting an active human S. mekongi infection.
In conclusion, this study has proposed two useful tools for disease surveillance and assessment of the efficacy of an MDA program that can lead to the elimination of schistosomiasis. The modified MHT can be used in field studies for recovering miracidium to prepare single-genome DNA for population genetic studies in the future. Application of additional recombinant S. mekongi antigens with ELISA might have a potential role in developing a reliable and accurate diagnostic test for detecting S. mekongi infections in humans and reservoir animals in the future.住血吸虫症は，Schistosoma 属の吸虫によって引き起こされる寄生虫病で，世界78 ヶ国で流行が認められる。世界人口の7 億人以上がこの病気への感染の危険の下で日々の生活を営み，また，そのうちの2 億4,000 万人以上がこの寄生虫病の症状に苦しんでいる。アジアに認められる人獣共通性の住血吸虫症（アジア型住血吸虫症）は，日本住血吸虫（S. japonicum）及びメコン住血吸虫（S.mekongi）の感染によって引き起こされ，ヒトと保虫宿主動物の間でも感染が成立することから，病気の伝搬が加速され，対策がより難しくなっている。日本住血吸虫の遺伝的多様性に関する知見は，野外での寄生虫ライフサイクルにおけるヒトと保虫宿主の相互関係や，寄生虫病の疫学を理解する上で重要な情報になる。一方，住血吸虫症に対するワクチンは未だ開発されていないことから，第一選択薬Praziquantel （ PZQ ） を用いた流行地住民への集団投薬（ mass drug administration: MDA）が，住血吸虫症の流行が認められる国と地域での主要な寄生虫病対策になっている。MDA を成功裏に推進するためには，流行地住民における住血吸虫症の流行状態を正確に把握し，またMDA の効果を査定するための，高感度・高精度の診断法の導入が必要になる。そこでこの研究では，アジア型住血吸虫症の排除に向けた取り組みに必須となる情報を得るための手法として，寄生虫の集団構造解析に使用するシングルゲノムDNA の調整を目的としたミラシジウムふ化法の改良及び，メコン住血吸虫症の患者の診断を目的としたELISAの開発を行った。
第1 章では，日本住血吸虫の集団構造解析で使用するシングルゲノムDNAの調整を目的としたミラシジウムふ化法（MHT）の改良を行った。寄生虫の遺伝団的多様性に関する情報は，住血吸虫症の疫学及び，保虫宿主嗜好性など寄生虫のライフサイクルを理解するのに不可欠な情報になる。寄生虫の遺伝的多様性を正確に調べるには単一のゲノムから調整したDNA を準備する必要があり，虫卵内に形成される幼虫ミラシジウムが有用な材料になる。そこで，シングルゲノムDNA 調製用のミラシジウムを効率的に収集するため，大型の専用フラスコを用いる既存のラシジウムふ化法を，96 穴プラスチックELISA プレートでの簡易法に改良した。更に，ミラシジウムのふ化率に与える光照射の影響についても検討を加えた。その結果，日光（太陽光）下でミラシジウムのふ化を誘導した時に最も高い孵化率（92.4%）が，蛍光灯下で誘導した時に次に高いふ化率（88.0%）が，また，虫卵を暗所に置いた時に最も低いふ化率（4.7%）が得られた。これらから，通常は蛍光灯下行うMHT を日光下で行っても，効率的にミラシジウムのふ化が誘導できることが初めて明らかになった。また，虫卵を0.85% NaCl 溶液中で暗所に置くとミラシジウムのふ化が抑えられることも確認され，この条件が虫卵の保存に有用なことも解った。シングルゲノムDNA を鋳型にしたPCR では，18S rRNA遺伝子及びマイクロサテライトメーカー遺伝子が増幅され，MHT の改良法が寄生虫の集団構造解析の研究にも十分応用可能であることが確認できた。
第2 章では，日本住血吸虫由来の抗原を応用して，メコン住血吸虫症を診断するELISA の開発を行った。メコン住血吸虫症では，メコン住血吸虫のゲノム情報の不足から，未だ組換体抗原を応用したELISA が開発出来ていない。そこで，これまでに開発された日本住血吸虫由来の組換体抗原及び虫卵由来の可溶性粗抗原（SEA）の，メコン住血吸虫症を診断するELISA における有用性を評価した。カンボジア人患者及び虫卵検査（Kato-Katz 法）陰性の流行地住民から採取した血清のパネルを用いて検討した結果，低濃度SEA を抗原とするELISA に，組換体抗原を用いるELISA を上回る優れた診断性能が認められた。一方，組換体抗原を用いるELISA は，抗原の大量供給と品質管理が容易で，大規模調査への応用も可能になる。ELISA 用組換体抗原の改良と開発が待たれるところである。
第3 章では，メコン住血吸虫由来の組換体抗原を作製して，メコン住血吸虫症の診断を目的としたELISA に応用した。数十年に及ぶMDA の結果，メコン住血吸虫症流行地では，この寄生虫病の有病率が減少すると同時に感染の強度も低下したことで，Kato-Katz 法の感度が低下している。そこで，Kato-Katz 法に替わる高感度及び高特異性のELISA を開発するため，メコン住血吸虫のチオレドキシンペルオキシダーゼ-1（SmTPx-1）遺伝子を単離して，組換体タンパク質（rSmTPx-1）を大腸菌で作製した。カンボジア人患者及びKato-Katz 法陰性の流行地住民から採取した血清のパネルを用いて検討した結果，rSmTPx-1 抗原を用いたELISA の感度と特異性は，それぞれ，89.3%と93.3%であった。一方，日本住血吸虫由来の組換体タンパク質（rSjTPx-1）を抗原として用いたELISA の感度と特異性は，それぞれ，71.4%と66.7%であった。rSmTPx-1 抗原を用いたELISA とKato-Katz 法間のカッパ係数（κ係数）は0.82 で，両者には高い相関が認められた。一方，rSjTPx-1 抗原を用いたELISA とKato-Katz 法間のκ係数は0.38 であった。このことから，rSmTPx-1 抗原を用いたELISA では，虫卵を排泄するActiveな患者の検出が可能なことが示唆された。本研究では，住血吸虫症の排除に向けた，疾病流行状況の監視とMDA 効果の査定に有用な二種類の手法を提案することが出来た。改良型のMHT を野外材料からのシングルゲノムDNA の調整に応用することで，寄生虫のライフサイクル解明に有用な，遺伝団的多様性に関する情報の入手が可能になる。また，rSmTPx-1以外の組換体抗原をELISA に応用することで，メコン住血吸虫症の患者と保虫宿主動物での寄生虫感染を検出可能な，高感度及び高特異性の血清診断法の開発が可能になる。博士学位論文
大学院畜産学研究科 獣医学専攻
Doctoral Program of Veterinary Sciencedoctoral thesi
Vessel development in compound buds of interspecific hybrid grape induced by artificial deacclimation treatments
Get PDFapplication/pdfGrape compound buds adapt to subfreezing temperatures in winter by supercooling, but the supercooling ability is thought to be lost upon formation of xylem connections between canes and buds. It was reported that compound buds of the Vitis vinifera variety ‘Chardonnay’ lack xylem cells in mid-winter, and that vessels differentiate during deacclimation. However, the pattern of vessel formation in compound buds may differ in cold-hardy Vitis species and interspecific hybrid varieties grown in colder regions. We investigated vessel formation in compound buds of the interspecific hybrid variety ‘Yamasachi’, which were harvested in mid-winter, during artificial deacclimation treatments. Before these treatments, ‘Yamasachi’ buds had a high supercooling ability (approx. −30°C) and contained cells with characteristics of vessel elements, that is, secondary wall thickening and lignification, at the basal parts. However, the cells still contained organelles and did not have a hydraulic conductivity function. Xylem continuity between the canes and buds was established from day 7 of deacclimation at 20°C. The different pattern of seasonal vessel formation in compound buds of ‘Yamasachi’ from that of V. vinifera may reflect the rapid development traits of Vitis species growing in cold regions with short growing seasons.journal articl