Obihiro University of Agriculture and Veterinary Medicine
OAK Obihiro University Archives of KnowledgeNot a member yet
5082 research outputs found
Sort by
Combined Effect of Feed and Housing System Affects Free Amino Acid Content of Egg Yolk and Albumen in Brown Layer Chickens
Get PDFapplication/pdfIn recent years, the market share for cage-free eggs has gradually increased. Because commercially available cage-free eggs are often produced not only by several housing systems but also with different feed crude protein (CP) levels, there are combined effects of feed and housing systems between cage-free and cage eggs. Therefore, using field data, this study aimed to determine the combined effects of feed and housing systems on egg traits and yolk and albumen amino acids in table eggs. Brown layers (n = 40) at the middle laying stage under two feed and housing systems (cage, CP 15.5% diet; barn, CP 17.0% diet) were used. One-way analysis of variance and Pearson’s correlation analysis were used to evaluate 10 egg traits, 19 yolk amino acid traits, and 20 albumen amino acid traits. We observed significant effects of feed and housing on two egg traits (yolk weight and eggshell color redness), 16 yolk amino acids (Asp, Glu, Asn, Ser, Gln, His, Arg, Thr, Ala, Tyr, Met, Cys, Ile, Leu, Phe, and Lys), and 14 albumen amino acids (Asp, Asn, Ser, Gln, Gly, His, Arg, Thr, Ala, Val, Met, Cys, Ile, and Leu). This study revealed that eggs from the barn system (CP 17.0%) contained higher levels of free amino acids in 15 yolk and nine albumen amino acid traits. Phenotypic correlations among the 49 egg traits indicated similar correlation patterns in both systems, which implies that the balance of free amino acid content in yolk and albumen is similar in each system. Although some potential confounding factors may be present for comparing egg content between cage (CP 15.5%) and barn (CP 17.0%) systems, this study suggests that commercially available cage-free eggs may be different from cage eggs not only in external egg traits but also yolk and albumen amino acid traits.journal articl
Impact of nutrient biofortification as a crop nutrient management strategy against insect pest infestation in potato (Solanum tuberosum L.)
Get PDF帯広畜産大学博士（農学）2022application/pdfLow or excessive soil fertility as well as pests and diseases are a major constraint to potato production. The influence of each individual nutrient element on potato plant growth, nutrient uptake and interactions, and interaction with herbivorous insect pests under field studies remain ambiguous due to the influence of environmental variations. Creating an in vitro model plant with deficient or excessive nutrient content will provide a more controlled study and allow for a better understanding of how the concentration of one element can affect the uptake of other elements. Also, how the nutrient status of each element in the plant interact with insect pests can well be elucidated under a controlled environment.
Here, we first designed a tissue culture-based nutrition control system to systematically analyze the effects of essential nutrients on potato plants. Insufficient or excessive nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg) contents were created by modifying the Murashige and Skoog (MS) medium. Deficient to toxic plant nutrient statuses were successfully defined by the evaluation of dry biomass and morphological symptoms. The N supplies of 20, 60-80, and 100 mM were defined as insufficient, optimum and toxic, respectively, whereas the P supplies of 0.4, 1.25, and 3.5 mM were defined as insufficient, optimum, and excessive, respectively, and K supplies of 13.5, 20, and 30 mM were defined as insufficient, optimum, and excessive, respectively. The Ca supplies of 1 and 10 mM were defined as insufficient and optimum, respectively, whereas Mg supplies of 1.5 and 9 mM were defined as optimum and excessive, respectively. The results showed that plant shoot growth, nutrient uptake and content, and nutrient interactions were all significantly impacted by the changes in the MS media nutrient concentrations. The increase in MS medium N supplies significantly increased shoot N uptake up to a toxic level where it drastically decreased. The changes in shoot N uptake had significant positive interactions with the changes in shoot P, K, Ca, and Mg uptakes. Increases in P supplies showed a steady increase in shoot P uptake up to a point where it became excessive. The changes in shoot P uptake induced a significant increase in shoot Mg content. The increase in MS medium K supplies showed slight increases shoot K uptake and this was associated with a significant decrease in shoot N uptake at both insufficient and excessive K supplies. The shoot Ca uptake remained stable between 1 and 3 mM of Ca supplies but drastically increased at 10 mM, and the changes in shoot Ca uptake was associated with significant steady increases in shoot N, K, and Mg uptakes. Increases in MS medium Mg supplies showed a steady increase in shoot Mg uptake with 9 mM having a huge uptake, and the changes in shoot Mg uptake had a significant positive interaction with shoot N uptake. Therefore, each nutrient would need to be carefully balanced with other elements because changes in their uptake can occur and have an unnoticed influence on the experimental results.
Second, we evaluated the influence of these variations of each individual nutrient element on aphid Aphis gossypii host settling preference, reproduction and feeding behaviors. To assess the aphid settling preference, dual host settling choice tests were conducted using intact potato tissue culture plants. The reproduction tests were conducted by infesting aphids to freely move on the whole plant and reproduce, and the number of nymphs were counted. The EPG analyses were conducted using a single aphid on each plant where the time, intensity and frequency of different feeding behaviors such as non-probing, intracellular stylet puncture, salivation into the sieve elements, and phloem sap ingestion. Our findings showed that host settling preference, reproduction, and feeding behaviors of aphid were all (except for Ca and Mg on reproduction) significantly influenced by the changes in the potato nutrition status. Increases in N nutrition potato plants significantly increased aphid host settling preference, but the number of nymphs were significantly reduced. The aphids spent significantly shorter total non-probing time on potato plants with insufficient N nutrition compared to plants with optimum N nutrition. The insufficient N nutrition significantly increased the phloem sap feeding duration. Increase in P nutrition significantly increased aphid host settling preference with aphids showing more preference on excessive P potato plants compared to those with insufficient P nutrition. The number of nymphs significantly increased with the increase in P nutrition. The time to first intracellular stylet puncture, number of intracellular punctures, and total duration of intracellular puncture were all significantly shortened in potato plants with insufficient P nutrition when compared to potato plants with optimum and excessive P nutrition. Insufficient P nutrition significantly shortened the total duration of salivation in the sieve elements. The increase in potato plants’ K nutrition significantly reduced aphid host settling preference when insufficient and excessive K plants were compared. The number of nymphs significantly increased with the increase in K nutrition up to optimum where it drastically decreased at excessive K nutrition. The total duration of aphid’s phloem sap feeding was significantly shortened in potato plants supplied with excessive K nutrition compared to those with insufficient K nutrition. The aphids significantly preferred settling on potato plants with insufficient Ca than on those with optimum Ca nutrition and did not show any influence on number of nymphs. The aphid non-probing time was significantly shorter in potato plants with insufficient Ca compared to those with optimum Ca nutrition, whereas the time spent in phloem sap feeding was significantly longer in potato plants with insufficient Ca nutrition compared to those with optimum Ca nutrition. The aphids showed significantly higher preference for potato plants with excessive Mg nutrition than those with optimum Mg nutrition, but no significant change was observed on the number of nymphs. The time for aphid’s first intracellular stylet puncture was significantly longer in potato plants with optimum Mg nutrition compared to those with excessive Mg nutrition. The duration for aphid salivation in the sieve elements was significantly longer in potato plants with optimum Mg nutrition compared to those with excessive Mg nutrition. These results showed that the influence of plant nutrition in potato plants against aphid feeding behavior is at leaf surface, epidermis or mesophyll, and phloem sieve elements cell layers. The influence on each layer varies with each nutrient. Together, these tissue culture systems can be successfully used for further investigations of how each nutrient affects the efficiency of aphids to inoculate viruses, and the morphological and molecular mechanisms associated with each nutrient in vitro.土壌栄養の不足や過剰、および病害虫による被害は、ジャガイモ生産の重要な制限因子となっている。ジャガイモの植物体の生長や、栄養の吸収およびその相互作用、植食性昆虫との相互作用に対する各種栄養元素の影響に対する理解は、環境変動の影響を受ける圃場試験では曖昧な状態である。そこで、栄養状態が不足または過剰なモデル植物を作出すれば、より制御された環境下での実験を可能にし、特定の元素濃度が他の元素の吸収にどのように影響を与えるか、また、植物体中の元素含量が害虫とどのように相互作用するかをより深く理解することができることが期待される。
本研究では、まず、ジャガイモに対する必須元素の影響を解析するために、組織培養ジャガイモの栄養含量制御系を確立した。ムラシゲ・スクーグ（MS）培地を改良し、窒素（N）、リン（P）、カリウム（K）、カルシウム（Ca）、マグネシウム（Mg）が欠乏または過剰の場位置を作成した。また、植物の栄養状態を、欠乏・不足・最適・過剰・有毒の５段階に分け、これらは植物体の乾燥重量および病徴の観察によって判断した。培地中のN濃度は、20mMで不足、60-80mMで最適、100mMで過剰となった。P濃度は、0.4mMで不足、1.25mMで最適、3.5mMで過剰となった。K濃度は、13.5mMで不足、20mMで最適、30mMで過剰となった。Ca濃度は、1mMで不足、10mMで最適となった。Mg濃度は、1.5mMで最適、9mMで過剰となった。これらの結果から、植物の生長および栄養吸収・含量、元素間の相互作用の全てが、MS培地の元素濃度の変化によって有意な影響を受けることが明らかとなった。MS培地中のN濃度の増加により、植物体中のN吸収量は有毒レベルにまで増加した。Nの吸収量の変化は、植物体中のP、K、Ca、Mgの吸収量を増加させた。MS培地中のP濃度の増加により、植物体中のP吸収量が過剰になるまで増加した。Pの吸収量の変化は、植物体中のMg吸収量を増加させた。MS培地中のK濃度の増加により、K吸収量はわずかに増加した。また、K濃度が不足および過剰の植物において、N吸収量は減少した。Ca濃度の吸収量は、MS培地中のCa濃度が1〜3mMの濃度では一定であったが、10mMで急激に増加した。Caの吸収量の変化は、N、K、Mgの吸収量を増加させた。MS培地中のMg濃度の増加により、9mMで急激に吸収量が増加した。また、Mg吸収量の変化は、植物体中のN吸収量を増加させた。以上の結果から、特定の元素の吸収量の変化により、他の元素吸収量に影響を与えることが明らかとなった。したがって、意図しない元素吸収量の変化が起こる可能性を考慮し、他の元素とのバランスを取る必要がある。博士学位論文大学院畜産学研究科 畜産科学専攻
Doctoral Program of Animal Science and Agriculturedoctoral thesi
Molecular epidemiological studies on livestock tick-borne parasitic diseases in the Philippines
Get PDF帯広畜産大学博士（獣医学）2022application/pdfBabesiosis, theileriosis, and anaplasmosis, alongside other TBDs, are significant parasitic diseases that inflict adverse health effects on livestock animals. This impact directly translates to million worth of financial losses for livestock raisers globally. The use of molecular tools to uncover the epidemiology of TBDs has been instrumental in their prevention and control. Considering the economic impact, elucidating the presence of tick-borne infections in livestock using appropriate diagnostic tools is vital not only in improving herd health but also in formulating and implementing countermeasures, which are expected to help farmers to recuperate the losses from TBDs. In this dissertation, I was able to elucidate the major TBPs of horses (Chapter 1), goats (Chapter 2), and cattle (Chapter 3) in the Philippines through the application of molecular diagnostic tools.
In Chapter 1, the presence of equine tick-borne infections in a racehorse park from Cavite, Philippines, was evaluated. A total of 124 thoroughbred horses specifically raised for racing were sampled. Babesia caballi (12.10%; 15/124), Theileria equi (0.81%; 1/124), Anaplasma phagocytophilum (10.48%;13/124), Borrelia burgdorferi sensu lato (Bbsl) (38.71%; 48/124), A. marginale (0.81%; 1/124), and Coxiella burnetii (0.81%; 1/124) were detected in racehorses. Of the 60 positive samples, 42 were single infections and 18 were multiple infections, the most frequent of which are coinfection with Bbsl and B. caballi. Sex (p = 0.026) was found to be a significant risk factor for B. caballi infection, with female horses 5.77 times more likely to be infected with B. caballi. Sequencing analysis revealed that seven partial 18S rRNA B. caballi isolates shared 98.63–100% identity and were classified as genotype A, whereas the single T. equi sequence had 99.77% identity with GenBank isolates and was confirmed as genotype E. Eight Anaplasma 16S rRNA partial sequences were highly identical to A. phagocytophilum and A. ovis, while partial sequences of Borrelia 5–23S rRNA were most closely related to Bo. japonica and Bo. garinii-like isolates. The first molecular detection of Borrelia and Anaplasma is reported in this study, as well as the first genotyping of B. caballi and T. equi in racehorses in the Philippines.
In Chapter 2, a total of 396 goat samples were collected from six Philippine provinces and were molecularly screened for the presence of Babesia/Theileria and Anaplasma. A detection rate of 77.02% (305/396) and 38.64% (153/396) were noted for the respective TBPs. Six samples were positive for B. ovis (1.52%). Sex and age group were associated with higher Babesia/Theileria and Anaplasma detection rates, respectively, while significantly higher rates for both pathogens were observed in exotic goat breeds. The representative Babesia/Theileria sequences shared 89.97–97.74% identity and were most closely related to T. orientalis, T. annulata, and Theileria sp. On the other hand, Anaplasma 16S rRNA sequences were related to A. odocoilei, A. platys, and A. phagocytophilum. This is the first molecular identification of B. ovis, Theileria spp., and Anaplasma spp. in goats from the Philippines.
In Chapter 3, a new molecular tool was evaluated for the characterization of piroplasma species diversity in bovines. Of 162, 58.64% tested positive for piroplasma using a conventional RLB-PCR assay that targets the V4 hypervariable region of the 18S rRNA gene. The positive cattle samples were from Bohol (83.33%; 20/24), Cavite (70.97%; 44/62), and Cebu (40.79%; 31/76). By leveraging the AMPtk pipeline, the merged reads generated a total of 2,179 ASVs. The BLAST non-redundant database assigned the taxonomy of 175 ASVs, which were then dereplicated into 97 ASVs. Further filtering yielded a final count of 79 distinct ASVs. The taxonomy hits of the 79 ASVs corresponding to 10 species were B. bovis (n = 37), B. bigemina (n = 18), T. orientalis (n = 13), Babesia sp. (n = 3), Hepatozoon canis (n = 2), Sarcocystis cruzi (n = 2), T. annulata (n = 1), T. equi (n = 1), T. mutans (n = 1), and Theileria sp. Thung Song (n = 1). Based on the phylogenetic analysis, ASVs assigned to B. bovis and Babesia sp. showed three major subclades and were 90.76–100% identical with B. bovis isolates deposited in GenBank. On the other hand, the evolutionary inference suggested that most of the B. bigemina and Babesia sp. ASVs were phylogenetically distinct from previously reported isolates, as evidenced by the strong statistical support, and shared 90.23–99.77% identity with isolates from the database. Thirteen T. orientalis ASVs (91.72–100% identical to other T. orientalis isolates) clustered in the T. orientalis complex subclade, 4 of which formed a subgroup with high nodal support. As anticipated, ASVs taxonomically recognized as T. annulata, T. equi, and T. mutans were separated into groups based on the species to which they belong. Furthermore, the Hepatozoon ASVs were distinctly divergent from previously detected Philippine canine H. canis isolates, while phylogenetic analysis revealed that Sarcocystis ASVs were closely related to bovine S. cruzi isolates from Malaysia and India.
The fundamental purpose of this research was to map out the different TBPs infecting economically important livestock in the Philippines. The presented findings from the three chapters of this dissertation attest to the attainment of this purpose. The circulating TBP populations and the extent of tick-borne infections in horses, goats, and cattle in the Philippines were revealed through the use of molecular detection tools, proving the applicability of these platforms in identifying tick-borne infections in different livestock. These results are vital in ascertaining the TBD status in the Philippines, where infections are widely present but often overlooked and neglected. The findings from these studies shall be beneficial in crafting and implementing livestock tick and TBD control and prevention programs in the country.バベシア症、タイレリア症、アナプラズマ症は、他のマダニ媒介感染症（TBD）と並んで、家畜の健康に悪影響を与える重要な寄生虫病である。こんら影響は、世界中の畜産農家が被る甚大な経済的損失に直結する。このような経済的損失を低減するために、適切な分子診断手法を用いて家畜のマダニ媒介性感染症の流行実態の一端を明らかにすることは、家畜の健康状態を改善するだけでなく、対策の立案・実施に不可欠である。そこで、本研究ではフィリピンにおけるウマ（第1章）、ヤギ（第2章）、およびウシ（第3章）の主要なマダニ媒介性病原体（TBP）の分子疫学調査を実施した。
第1章では、フィリピンカビテ州の競走馬牧場におけるマダニ媒介性感染症について調べた。サラブレッド馬124頭から血液サンプルを採集し、各種寄生虫の核酸検出を行ったところ、Babesia caballi（12.10％；15/124）、Theileria equi（0.81％；1/124）、Anaplasma phagocytophilum（10.48％；13/124）、Borrelia burgdorferi sensu lato（Bbsl）（38.71％；48/124）、A. marginale（0.81％；1/124）、Coxiella burnetii（0.81％；1/124)が検出された。陽性60検体のうち、42検体が単独感染，18検体が混合感染であり、BbslとB. caballiとの重複感染が最も多かった。B. caballiとT. equiについて、遺伝子型を調べたところ、それぞれA型とE型に分類された。これらの結果のなかで、BorreliaとAnaplasmaの検出とB. caballiとT. equiの遺伝子型の特定は、いずれもフィリピンのウマにおいては初の報告である。
第2章では、フィリピン6州から計396頭のヤギ血液サンプルを採集し、マダニ媒介性病原体の核酸検査を行った。Babesia/Theileria属およびAnaplasma属の病原体の検出率はそれぞれ77.02%（305/396）、38.64%（153/396）であった。Babesia/Theileria属およびAnaplasma属の検出率は、それぞれ外来種と高年齢層で高かった。Babesia/Theileria の代表的な塩基配列は89.97-97.74%の同一性を持ち、T. orientalis、T. annulata、Theileria sp.に最も近縁であった。B. ovisは6検体（1.52％）で陽性であった。一方、Anaplasma 16S rRNA 塩基配列はA. odocoilei、A. platys、A. phagocytophilumに近縁であった。これらの結果のなかで、B. ovis、 Theileria spp.およびAnaplasma spp.は、フィリピンのヤギにおける初の知見である。
第3章では、ウシにおけるピロプラズマの種多様性を特徴付けるための新しい分子ツールを評価した。18S rRNA遺伝子のV4超可変領域を標的とした従来のRLB-PCR法で162頭中58.64%がピロプラズマ（Babesia属/Theileria属）陽性と判定された。陽性ウシは、ボホール（ 83.33%; 20/24）、カビテ（ 70.97%; 44/62）、セブ（ 40.79%; 31/76）由来であった。AMPtkパイプラインを用いると、マージされたリードから合計2,179個のASVが生成された。BLAST non-redundantデータベースにより175個のASVが分類され、さらに97個のASVに分割された。フィルタリングを行った結果、最終的に79個の異なるASVを数えることができた。79個のASVのうち10種に対応する分類のヒットは、B. bovis (n = 37) 、 B. bigemina (n = 18) 、 T. orientalis (n = 13) 、 Babesia sp. (n = 3）、Hepatozoon canis（n = 2）、Sarcocystis cruzi（n = 2）、T. annulata（n = 1）、T. equi（n = 1）、T. mutans（n = 1）およびTheileria sp. Thung Song（n = 1）であった。 系統解析の結果、B. bovisおよびBabesia sp.に割り当てられたASVは3つの主要なサブクレードを示し、GenBankに登録されているB. bovis分離株と90.76～100％の同一性を有していた。 一方、進化推論では、B. bigeminaとBabesia sp.のASVの多くは、既報告分離株とは系統的に異なることが示唆された。 13種のT. orientalis ASV（他のT. orientalis分離株と91.72〜100%同一）はT. orientalis complexサブクレードにクラスター化し、そのうち4種は高いノードサポートを持つサブグループを形成していた。予想通り、分類学的にT. annulata、T. equi、T. mutansと認識されているASVは、その属する種に基づきグループに分けられた。さらに、Hepatozoon ASVsは、これまでに検出されたフィリピン由来イヌのH. canis分離株とは明確に分岐しており、Sarcocystis ASVsはマレーシアおよびインド産由来のS. cruzi分離株に近縁であることが明らかとなった。
本研究の総括的な目的は、フィリピンにおける経済的に重要な家畜に感染している様々なマダニ媒介性病原体を特定することであった。特に第3章で得られた知見は、この目的の達成を証明するものである。フィリピンのウマ、ヤギ、およびウシにおけるマダニ媒介性病原体の流行と分布が分子検出ツールによって明らかになり、異なる家畜におけるマダニ媒介性感染症の特定にこれらのプラットフォームが適用可能であることが証明された。これらの結果はフィリピンの家畜でこれまでに見過ごされがちであったマダニ媒介感染症の状況を把握する上で極めて重要である。これらの研究結果は、今後フィリピンにおける家畜のマダニ媒介性感染症の管理・予防プログラムの策定・実施に有益なものとなるであろう。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
Disruption of a DNA fragment that encodes the microneme adhesive repeat domain-containing region of the BBOV_III011730 does not affect the blood stage growth of Babesia bovis in vitro
Get PDFapplication/pdfBabesia bovis, an intraerythrocytic hemoprotozoan parasite, causes the most pathogenic form of bovine babesiosis, negatively impacting the cattle industry. Comprehensive knowledge of B. bovis biology is necessary for developing control methods. In cattle, B. bovis invades the red blood cells (RBCs) and reproduces asexually. Micronemal proteins, which bind to sialic acid of host cells via their microneme adhesive repeat (MAR) domains, are believed to play a key role in host cell invasion by apicomplexan parasites. In this study, we successfully deleted the region encoding MAR domain of the BBOV_III011730 by integrating a fusion gene of enhanced green fluorescent protein-blasticidin-S-deaminase into the genome of B. bovis. The transgenic B. bovis, lacking the MAR domain of the BBOV_III011730, invaded bovine RBCs in vitro and grew at rates similar to the parental line. In conclusion, our study revealed that the MAR domain is non-essential for the intraerythrocytic development of B. bovis in vitro.journal articl
Use of Transabdominal Ultrasound and Maternal Hormone Testing for the Prenatal Monitoring of Equine Fetal Enlarged Bladder
Get PDFapplication/pdfUltrasound examination during late gestation is one of the best methods for monitoring potential pregnancy risks. Enlarged bladder is a urological disorder rarely observed in equine fetuses. This clinical case report aimed to present a case illustrating the development of equine fetal enlarged bladder using transabdominal ultrasound examinations and maternal hormone evaluation during gestation. An 8-year-old Hokkaido native pony was impregnated by embryo transfer, and at 215 days of gestation, abnormalities of the fetal bladder were detected. The bladder volume increased with gestational age, and a second bladder was observed at 257 days of gestation. No abnormalities were observed in the fetal kidneys. Moreover, the maternal plasma progesterone concentration was measured throughout the gestation period. The progesterone concentration was elevated from 36 weeks of gestation until parturition. At 363 days of gestation, parturition induction was conducted, and a foal successfully delivered. This case report is the first to describe the development of equine fetal enlarged bladder and record the corresponding ultrasound and hormone profiles.journal articl
コモンマーモセットの進行性糸球体腎症に関する病理学的研究
Get PDF帯広畜産大学博士（獣医学）Doctor of Veterinary Medicine2023application/pdfコモンマーモセットの背景病変のなかでも，腎症は自然発生性に生じる病変としていくつか報告されているが，いまだに不明な点も多い．また，体系的な評価方法もないため，種々の実験において腎臓病変を評価する際の妨げとなる．そこで，本研究ではコモンマーモセットの進行性糸球体腎症について，光学顕微鏡学的検査で病理組織学的な病変スコアを評価し，そのスコアから腎症をグレーディングすることで，本腎症の体系的な評価を試みた．
第１章では，コモンマーモセットの進行性糸球体腎症を光学顕微鏡学的検査で糸球体病変，再生尿細管，硝子円柱，間質への炎症性細胞浸潤と線維化についてスコア化した．そしてこれらのスコアの合計値に基づき，腎症を5つのグレードに分類した．さらに，透過型電子顕微鏡学的検査により各腎症グレードにおける超微形態学的な特徴，糸球体病変と年齢の関係性および糸球体病変の初期変化とその病態の進行について調べた．その結果，腎症グレード1では，光学顕微鏡的に顕著な糸球体病変が認知できないにもかかわらず，電子顕微鏡学的検査では足細胞の足突起の消失が認められた．腎症グレード2では，光学顕微鏡的に糸球体門部から糸球体末梢側に向かって軸性のメサンギウム基質やメサンギウム細胞の増殖が明らかとなり，電子顕微鏡的にも腎症グレード1よりも糸球体足細胞の足突起の消失は進行していた．腎症グレード3では，糸球体メサンギウムの病変は門部から末梢側に向かってメサンギウム基質の増加やメサンギウム細胞の増殖が明らかとなり，糸球体基底膜とパラメサンギウム領域の血管内皮直下にマッソン・トリクローム染色で赤色に染色される沈着物がみられた．透過型電子顕微鏡観察では，同領域に高電子密度の沈着物が認められた．腎症グレード4 では，糸球体基底膜の走行や配列の不整が顕著となり，同部位には高電子密度の沈着物および細胞質成分が透過型電子顕微鏡検索で観察された．以上の結果より，本分類類方法を使用することで，コモンマーモセットの進行性糸球体腎症について病変の進行に合わせたグレーディングを可能とした．
第２章では，若齢のコモンマーモセット5例の腎臓について，光学顕微鏡学的，電子顕微鏡学的検査に合わせて免疫組織学的検査を実施し， 若齢で本腎症がまだ初期と考えられる段階での免疫グロブリンの関与について形態学的に調べた．その結果，若齢の動物で，光学顕微鏡的な病変が認められないにもかかわらず，電子顕微鏡的には足細胞の足突起の消失が認められ，さらにIgM の沈着がメサンギウム領域に認められた．腎症グレードが進行するとメサンギウム領域へのIgA の沈着が認められ，さらにメサンギウム領域や糸球体係蹄壁へのIgG の沈着も認められた．以上の結果により，若齢動物においても，すでにメサンギウム基質の増加やIgM，IgA の沈着が認められることが分かった．また，進行性糸球体腎症の病態進行において，病変初期ではIgM が沈着し， IgA やIgG の沈着が病変の進行に関与している可能性が示された．
コモンマーモセットの進行性糸球体腎症では， 病変初期の足細胞の足突起の消失が特徴的であることから， 第３章として足細胞の足突起のスリット膜を構成する重要な分子であるネフリンの発現状況について調べた．その結果，ネフリンの発現は腎症グレード0～2で連続的な線状パターンを示したが，腎症グレード3の例では，非連続性の線状パターンあるいは粗顆粒状パターンを示し，光学顕微鏡的な検査でもパターンの違いが明らかであった．さらに，形態学的解析により，コモンマーモセットの進行性糸球体腎症の足細胞におけるネフリン発現量は，腎症グレードが増加するに伴い低下することが明らかになった．また，硝子円柱の組織学的スコアが増加するに伴い，ネフリン発現量が低下する傾向も認められた．ネフリン発現パターンの変化はスリット膜構造の変化を反映していると考えられ，これらの結果は，病変の進行に伴ってスリット膜構造が変化し，この変化とネフリン発現の減少や硝子円柱の増加が密接に関連していることを示唆している．
本研究では，コモンマーモセットの進行性糸球体腎症の病理組織学的なグレーディングを可能にした．そして，超微形態学的特徴や免疫グロブリンの沈着，ネフリンの発現について，各腎症グレードの詳細を明らかにした．本研究の成果は，毒性試験を含む多くの研究で腎病変を判断する一助となるものと考える．Among the background lesions of common marmosets, some nephropathies have been reported as spontaneous lesions, but there are still many unclear points. In addition, the absence of a systematic evaluation method hinders evaluation of the renal lesions in various experiments. Therefore, in this study, I attempted to systematically evaluate spontaneous progressive glomerulonephropathy in common marmosets by evaluating the histopathological score by light microscopy and grading the nephropathy based on the score.
In Chapter 1, spontaneous progressive glomerulonephropathy in common marmosets was scored for glomerular lesions, regenerating tubules, hyaline casts, interstitial inflammatory cell infiltration, and fibrosis by light-microscopy. Based on the sum of these scores, nephropathy was classified into 5 grades. In addition, transmission electron microscopy (TEM) was used to examine the ultrastructural characteristics of each grade of nephropathy, the relationship between glomerular lesions and age, and the initial changes and progression of glomerular lesions. As a result, in nephropathy grade 1, effacement of podocyte foot processes was observed electron-microscopically, although there was no significant glomerular lesion light-microscopically. In grade 2 nephropathy, mesangial cell proliferation and mesangial matrix increasing from the glomerular hilum to the peripheral side of the tuft is revealed by light-microscopy, and glomerular basement membrane lesions are more advanced than in grade 1 nephropathy by electron-microscopy. In grade 3 nephropathy, glomerular mesangial lesions revealed mesangial cell proliferation and mesangial matrix increasing from the hilus to the peripheral side of the tuft. In Masson's trichrome-stained sections, red deposits were observed under the endothelium of the glomerular basement membrane and the paramesangial region. TEM examination revealed electron-dense deposits in the same regions. In nephropathy grade 4, irregular arrangement of the glomerular basement membrane was remarkable. At the same sites, electron-dense deposits or cytoplasmic components were observed by TEM. Based on these results, the use of this classification enabled grading according to the progression of lesions for progressive glomerulonephropathy in common marmosets.
In Chapter 2, immunohistochemical examination was performed to investigate about the involvement of immunoglobulin along with light-microscopy and electron-microscopy on the kidneys of 5 young common marmosets whose nephropathy is still considered to be in the early stages. As a result, in young animals, although no lesions were observed on light-microscopy, electron-microscopy showed effacement of podocyte foot processes. In addition, IgM deposition was observed in the mesangial region by immunohistochemical examination. As the grade of nephropathy progressed, IgA deposition was observed in the mesangial region, and IgG deposition was also observed in the mesangial region and the glomerular loop. From these results, it was found that an increase in the mesangial matrix and the deposition of IgM and IgA were already observed in young animals. In addition, in the progression of progressive glomerulonephropathy, the deposition of IgM occurred in the early stage of the lesion, and the deposition of IgA and IgG may be involved in the progression of the lesion.
In the progressive glomerulonephritis of the common marmoset, the effacement of the foot process of the podocyte in the early stage of the lesion is characteristic, and the expression of nephrin which is an important molecule constituting the slit membrane of the foot process of the podocyte was examined in Chapter 3. As a result, nephrin expression showed a continuous linear pattern in nephropathy grades 0-2, but showed a discontinuous linear pattern or a coarse granular pattern in patients with nephropathy grade 3. In this way, the difference in staining pattern of nephrin was clearly demonsterated by light microscopic examination. Furthermore, morphometrical analysis revealed that the expression of nephrin in the podocytes of common marmosets with progressive glomerulonephropathy decreased as the nephropathy progressed. In addition, nephrin expression tended to decrease as the histological score of hyaline casts increased. Because changes in nephrin expession pattern reflect changes in the slit membrane structure, these results suggest that the slit-membrane structure changes with the progression of the lesion and that this change is closely related to a decrease in nephrin expression and an increase in hyaline casts.
This study enabled histological grading of progressive glomerulonephropathy in common marmosets. And details of each nephropathy grade were clarified in terms of ultrastructural morphology, immunoglobulin deposition, and nephrin expression. I believe that the results will help determination of renal lesions in many studies including toxicologic studies.博士学位論文大学院畜産学研究科doctoral thesi
Molecular Identification of Piroplasmids in Ticks from Infested Small Ruminants in Konya Province, Turkey
Get PDFapplication/pdfTicks play a pivotal role in propagating a diverse spectrum of infectious agents that detrimentally affect the health of both humans and animals. In the present study, a molecular survey was executed of piroplasmids in ticks collected from small ruminants in four districts within Konya province, Turkey. Microscopic examination identified 1281 adult ticks, which were categorized into 357 pools based on their species, sexes, host animals, and collection site before DNA extraction. The infection rates were calculated by using a maximum likelihood estimate (MLE) with 95% confidence intervals (CI). Hyalomma detritum, H. excavatum, Rhipicephalus bursa, R. sanguineus, and R. turanicus were identified in this study. Among the five tick species identified here, R. turanicus exhibited the highest infestation rate in both goats and sheep. The presence of Babesia ovis and Theileria ovis based on 18S rRNA was confirmed using molecular assay. The overall MLE of infection rates for B. ovis and T. ovis was 2.49% (CI 1.72–3.46) and 1.46% (CI 0.87–2.23), respectively. The MLE of B. ovis and T. ovis infection rates in R. bursa was 10.80% (CI 7.43–14.90) and 0.33% (CI 0.02–1.42), respectively, while that in R. turanicus was 0.12% (CI 0.01–0.51) and 2.08% (CI 1.25–3.22). This study further confirms that R. turanicus and R. sanguineus can act as vectors for B. ovis, thus advancing our comprehension of tick-borne piroplasmids epidemiology and providing valuable insights for the development of effective control strategies for ticks and tick-borne diseases in Turkey. © 2023 by the authors.journal articl
A case of a dog with mandibular extraskeletal osteosarcoma after long-term puncture extirpation of the salivary gland cyst in the mandible
Get PDFapplication/pdfBackground: Extraskeletal osteosarcoma, unlike skeletal osteosarcoma, is a rare malignant mesenchymal tumor with a soft tissue primary that has been reported to occur in a variety of soft tissues. Case Description: The case is a 14-year-old, unneutered male Miniature Pinscher, weighing 6.7 kg, who had been treated medically for more than 5 years with a management strategy of puncture extirpation of a salivary gland cyst in the mandible; 1 month earlier, the fluid retention could not be removed, and after a computerized tomography scan showed no lesion in the mandible adjacent to the mass lesion, surgical resection was performed. Conclusion: Previous reports of extraskeletal osteosarcoma from the salivary glands in dogs have been rare. However, treatment of a salivary gland cyst in the mandible by long-term puncture extirpation may be a potential predisposing factor for the development of extraskeletal osteosarcoma around the mandible. © 2023, Faculty of Veterinary Medicine, University of Tripoli. All rights reserved.journal articl