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イヌ歯周病の原因細菌であるPorphyromonas gulaeの遺伝子型に関する研究 〜イヌの年齢、品種、疾患との関連について〜
https://az.repo.nii.ac.jp/records/2000484
https://az.repo.nii.ac.jp/records/200048450cbd92b-e05c-4ffb-9cf6-e0f3ad5051be
| 名前 / ファイル | ライセンス | アクション |
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diss_dv_kou190.pdf (2.2 MB)
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diss_dv_kou190_jab&rev.pdf (207 KB)
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| Item type | 学位論文 / Thesis or Dissertation(1) | |||||||
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| 公開日 | 2025-12-15 | |||||||
| タイトル | ||||||||
| タイトル | イヌ歯周病の原因細菌であるPorphyromonas gulaeの遺伝子型に関する研究 〜イヌの年齢、品種、疾患との関連について〜 | |||||||
| 言語 | ja | |||||||
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| 言語 | jpn | |||||||
| 資源タイプ | ||||||||
| 資源タイプ | doctoral thesis | |||||||
| アクセス権 | ||||||||
| アクセス権 | open access | |||||||
| アクセス権URI | http://purl.org/coar/access_right/c_abf2 | |||||||
| その他（別言語等）のタイトル | ||||||||
| その他のタイトル | Investigation of canine periodontal diseases caused by Porphylomonas gulae and its genotype distribution -Association of Porphylomonas gulae with age, breed, and diseases in dogs- | |||||||
| 言語 | en | |||||||
| 著者 |
安田, 隼也
× 安田, 隼也
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| 抄録 | ||||||||
| 内容記述タイプ | Abstract | |||||||
| 内容記述 | 歯周病は歯周病原因菌の感染により生じる口腔内の炎症性疾患で、成犬の約80%が発症すると言われている。歯周病の進行は歯周溝の形成、歯周病原性細菌の定着そして歯肉炎の発生が端緒となり、歯肉炎が慢性化することで炎症部位が拡大し、歯肉ならびに歯周溝内に不可逆的な歯周ポケットが形成される。その結果、歯牙の喪失や歯槽骨の融解につながり、慢性の歯周病は口腔内に留まらず直接的・間接的に他の全身疾患との関連する可能性が示唆されている。 ヒトでは、Porphyromonas gingivalis、Treponema denticola、Tannerella forsythensisと言われるRed Complexに分類される細菌が歯周病原因菌として高い病原性を有する事が知られているが、イヌでもPorphyromonas gingivalisの近縁種であるPorphylomonas gulae（以下、P. gulae）が歯周病進行に重要な役割を示すことが明らかになりつつある。P. gulaeの病原因子の一つに、41kDaのタンパク質が重合した糸状構造体であるFimAがあり、A、B、Cの3種類の遺伝子型（以下FimA線毛遺伝子型）に分類できる。中でもC型は病原性が高く、重度の歯周病罹患犬で有意な感染が確認できている。歯周病に伴う歯槽骨の融解や歯牙の喪失は、不可逆的な変化であることから、早期発見および早期対処が必要であり、歯周病の発症時期と機序の解明が急務である。 これまでに実施されたイヌにおける歯周病の実態調査は、海外で実施されたものが多く、主に中～大型犬を対象としている事から、小型犬が主体となる日本の実態に当てはめる事が難しい。その方法も人間の疫学調査と比較すると不十分なものが多く、Red Complexに該当する菌は、偏性嫌気性である事から単離培養の困難さも疫学調査が難しかった要因である。近年では、ポリメラーゼ連鎖反応（以下PCR法）を用いた、感度の高い検出法が確立されている。 そこで本研究は、日本の現状に合致した小型犬を対象とした大規模調査をより感度の高い方法で実施する事を目的とした。その際、イヌでの歯周病との関連が示唆されるP. gulaeに着目し、その定着時期、FimA線毛遺伝子型の保有状況および歯周重症度との関連、既存疾患と歯周重症度・P. gulaeの発現状況・FimA線毛遺伝子型の保有状況の関連について調査を実施した。 本研究では、国内にある77の動物病院に2015年から2022年の間に来院した小型犬665頭を対象とした。歯と歯肉の境界からマイクロブラシを用いて採材した歯垢サンプルからDNAを抽出し、P. gulaeの検出およびFimA線毛遺伝子型をPCR法によって決定した。FimA線毛遺伝子型は非検出、A型単独、B型単独、C型単独、A型およびB型、A型およびC型、B型およびC型、A型とB型およびC型の全てが検出された8つの群に分類した。歯周重症度はスコア1～4に分類し、スコアが低い順に歯周病無し、軽度、中等度、重度と定義した。 第1章では、体重10kg以下のイヌの各年齢におけるP. gulaeの検出、FimA線毛遺伝子型および歯周重症度との相関を調査した。その結果、P. gulaeは1歳齢以降で665頭中554頭（81.8%）に検出された。歯周重症度スコアは0歳齢および1歳齢では軽度を表すスコア2以下、1～5歳齢までは症状がやや進行し軽度と中等度の間である平均2.5以下であった。6歳齢以降では14歳齢および17歳齢を除いて平均が2.5以上3.0未満に収まったことから、6歳になると症状の進行が緩やかになることが示唆された。FimA線毛遺伝子型はA型が単独検出される割合以外は全ての組合わせが20%以下であり、年齢の上昇に伴い各線毛遺伝子型の検出率は単独・複数型に関係なく有意に上昇した。P. gulae非検出群に対してB型単独、C型単独群が歯周重症度スコアが有意に高かった。P. gulae非検出群とA型・B型・C型を含む群との比較、単独検出・複数検出・3種類検出群との歯周重症度スコアの比較においても有意差が見られた。P. gulae非検出群は年齢の上昇に伴い有意に減少する一方、FimA線毛遺伝子型が検出される割合と歯周重症度スコアの平均値はいずれも年齢が上昇するにつれて上昇し、高い相関性が認められた。このことから体重10kg以下のイヌにおいて1歳齢以上でP. gulaeの検出が認められ、また同菌の定着状況を確認するために6歳齢以降までの定期的な検査を行う必要がある事が示唆された。 第2章では、品種の違いによるP. gulaeの検出、FimA線毛遺伝子型および歯周重症度との相関について解析した。特に日本での飼育頭数が多い、トイプードル、ダックスフント、チワワに着目して解析した。トイプードル226頭、ダックスフント192頭、チワワ76頭であった。解析の結果、歯周病重症度1の個体は全体の5%であった。歯周重症度スコアはダックスフントが最も高く、トイプードルとの間に有意差が見られた。チワワではFimA線毛遺伝子型B型が単独で検出された群とP. gulae非検出群との間に有意差が見られた。トイプードルではP. gulaeのFimA線毛遺伝子型の検出種類が増えるほど歯周重症度スコアが上昇し、FimA線毛遺伝子型A、B、C全ての型が検出される場合はP. gulae非検出群に比べて歯周重症度スコアが有意に増加した。ダックスフントではP. gulaeの検出およびFimA線毛遺伝子型と歯周重症度スコアには相関性が見られなかった。これらの結果により、品種によって歯周病の悪化状態とP. gulaeの検出状況に差が見られることが分かった。今回は体重10kg以下の小型犬かつ日本国内で著名な品種という分類で解析を行った。チワワは上顎の歯並びが他の品種とは異なる短頭種に分類されることもある。一方でダックスフントのように上顎が長く歯並びに影響が少ない品種もある。今後は小型犬における品種だけでなく、解剖学的な特徴に着目して歯周病を研究する必要があるかもしれない。 第3章では、僧帽弁閉鎖不全症の既往歴を持つイヌおよび歯牙を喪失したイヌについて、P. gulaeの検出、FimA線毛遺伝子型および歯周重症度との相関について解析した。 ヒトでは歯周病原性細菌が弁膜に到達し感染性心内膜炎を起こすことで僧帽弁閉鎖不全症に進行することが報告されている。本研究では前章までの665頭を含む1710頭まで母集団を増やし、そのうちトイプードル385頭、ダックスフンド273頭、チワワ135頭の8歳～15歳齢の範囲で僧帽弁閉鎖不全症とP. gulaeの検出および歯周重症度スコアの間の相関性を解析した。僧帽弁閉鎖不全症の有無については既往歴の申告をもとに分類した。その結果、解析を行った3品種における僧帽弁閉鎖不全症の有病率とP. gulaeの保有率との間には有意差は見られなかった。歯周重症度スコアとの関連についてトイプードルでは歯周重症度スコア3以上の群は2以下の群と比べて僧帽弁閉鎖不全症の有病率が有意に高かった。この結果は、イヌの僧帽弁閉鎖不全症の発症にはP. gulaeの関与は低いものの、品種によっては歯周状態が影響を与えることを示唆した。ヒトではP. gulaeの近縁種であるP. gingivalisをはじめとした歯周病原性細菌が引き起こす口腔環境の問題が、僧帽弁閉鎖不全症といった循環器系の疾患だけでなく糖尿病などの様々な全身疾患にも関与していることが報告されている。今後はイヌにおいても全身疾患との関連を検討する。 歯牙の喪失については、用いた665頭のうち残存歯数の記録がある188頭について喪失本数0本、1～10本、11～20本、21本以上の群に分類した。各群の個体数はそれぞれ74頭、69頭、24頭、21頭であった。これらの個体の歯周重症度スコアと歯牙喪失との関連について解析した結果、歯周重症度スコア3の個体では有意差はつかなかったものの、重症度スコア1、2、4のいずれにおいても歯牙喪失が無い群もしくは1～10本の喪失群と比べて11～20本及び21本以上の喪失群では有意に多かった。このことから歯周重症度スコアは歯牙の喪失と深い関係があることが示唆された。 FimA線毛遺伝子型と歯牙喪失との関連について、C型単独検出群では11～20本の歯牙欠損群が有意に増加し、A・B・C型の全てが検出された群では歯牙喪失20本以下の3群と比べて21本以上の歯牙喪失群が有意に多かった。このことから、歯牙喪失にはC型の影響が強く、また3つの線毛遺伝子型が揃うことでさらにその影響が強くなることが考えられた。 本研究では歯が残存している部位からP. gulaeの検出を行ったため歯牙喪失との相関を見出せなかった可能性がある。今後は歯牙を喪失した部位からの採材やP. gulaeが検出された歯牙が実際に喪失するのかといった研究などを通じてP. gulaeが歯牙喪失に与える影響の調査が必要だろう。 本研究では歯周病原性細菌の代表的細菌であるP. gulaeに注目して、小型犬におけるその定着時期について詳細に研究を行い、6歳齢頃までに定着し歯周状態にも影響を及ぼすことが確認できた。また、品種によってP. gulaeの存在と歯周病重症度に差が見られることが判明した。P. gulaeの存在と歯牙喪失および僧帽弁閉鎖不全症については相関性が認められなかった。今後は他の疾患とP. gulaeの存在との関連を調べる。 |
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| 言語 | ja | |||||||
| Abstract | ||||||||
| 内容記述タイプ | Abstract | |||||||
| 内容記述 | Periodontal disease is an oral inflammatory disease caused by infection with periodontopathogens and affects approximately 80% of adult dogs. The progression of periodontal disease begins with the formation of periodontal furrows, followed by the establishment of periodontopathogens, and the development of gingivitis. As gingivitis becomes chronic, the inflammatory area expands, and irreversible periodontal pockets are formed in the gingiva and periodontal furrow. As a result, tooth loss and alveolar bone degradation occur, suggesting that chronic periodontal disease is not confined to the oral cavity but may be directly or indirectly associated with other systemic diseases. In humans, bacteria classified in the Red Complex, namely Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythensis, are known to be highly pathogenic as periodontal disease-causing bacteria. It is becoming clear that Porphyromonas gingivalis and its close relative Porphylomonas gulae (hereafter referred to as P. gulae) play an important role in the progression of periodontal disease in dogs. One of the virulence factors of P. gulae is FimA, a polymerized filamentous structure of 41 kDa protein, which can be classified into three genotypes: A, B, and C (hereafter referred to as FimA lineage genotypes). Genotype C is the most virulent of the three, and significant infection has been observed in dogs with severe periodontal disease. Since the alveolar bone melting and tooth loss associated with periodontal disease are irreversible changes, early detection and early treatment are necessary, and clarification of the timing and mechanism of periodontal disease onset is urgently needed. Most of the surveys of canine periodontal disease have been conducted overseas and have targeted mainly medium- to large-sized dogs, making it difficult to apply them to the actual situation in Japan, where many are small-sized dogs. The methods used were often inadequate compared to those used in human epidemiological surveys, and the difficulty of isolating and culturing the Red Complex bacteria, which are biochemically anaerobic, was also a factor in the difficulty of epidemiological surveys. Recently, a highly sensitive detection method using polymerase chain reaction (PCR) has been established. Therefore, the purpose of this study was to conduct a large-scale survey of dogs weighing 10 kg or less in a more sensitive manner that is consistent with the current situation in Japan. We focused on P. gulae, which has been suggested to be associated with periodontal disease in dogs and investigated the relationship between the timing of its establishment, possession of the FimA lineage genotype and periodontal severity, and the relationship between existing diseases and periodontal severity, P. gulae expression status, and possession of the FimA lineage genotype. In Chapter 1, we investigated the detection of P. gulae and its correlation with FimA lineage genotype and periodontal severity at each age in canines weighing 10 kg or less. Periodontal pathology and P. gulae infection status were investigated and classified at each age in 665 dogs that visited 77 veterinary hospitals in Japan between 2015 and 2022. The detectability of P. gulae and FimA lineage genotypes were identified by PCR method using a microbrush to collect dental plaque samples from the tooth-gingival border. FimA lineage genotypes were classified into eight groups, including non-detects: type A alone, type B alone, type C alone, type A and B, type A and C, type B and C, and all types A, B, and C were detected. Periodontal severity was classified into scores 1 through 4, defined as no periodontal disease, mild, moderate, and severe in descending order of score. The results showed that P. gulae was detected in 554 of 665 (81.8%) of the animals after the age of 1 year. Periodontal severity scores averaged less than 2 at ages 0 and 1 year, and slightly advanced symptoms at ages 2 to 5 years, averaging less than 2.5, which is between mild and moderate. At 6 years or older ages, the mean scores were between 2.5 and 3.0, except for the 14- and 17-year-olds, suggesting that the progression of symptoms slowed down after a certain age. All combinations of FimA lineage genotypes were detected in less than 20% of cases except for the proportion of single A and B genotypes detected in some age groups, and the detection rate increased significantly with increasing age. Periodontal severity scores in the P. gulae non-detected group were significantly lower than those in the detected group. Both the detection rate of the FimA lineage genotype and the mean periodontal severity score increased with advancing age, and the correlation between the two increased with advancing age. This suggests that dogs weighing less than 10 kg should be tested for P. gulae at least 1 year of age, and that P. gulae should be tested periodically until 6 years of age or older to check for the presence of the organism. In Chapter 1, various breeds weighing 10 kg or less were evaluated, however, it is conceivable that there may be cases in which marked differences appear among the breeds, and conversely, cases in which they do not. In Chapter 2, we focused on toy poodles, dachshunds, and chihuahuas, which are particularly numerous in Japan, and investigated the correlation between P. gulae detection, FimA lineage genotype, and periodontal severity, and analyzed differences by breed. There were 226 toy poodles, 192 dachshunds, and 76 chihuahuas used in the analysis. The analysis showed that 5% of the animals had no periodontal disease, that is score 1. The periodontal severity score was highest in dachshunds, with a significant difference between dachshunds and toy poodles, and the correlation between FimA lineage genotype and periodontal severity score was significant in chihuahuas, where FimA lineage genotype B was detected alone, compared to the group without P. gulae detection. In toy poodles, periodontal severity scores increased as the number of FimA lineage genotypes of P. gulae increased, and when all FimA lineage genotypes A, B, and C were detected, there was a significant difference in periodontal severity scores compared to the P. gulae non-detection group. No correlation was found between P. gulae detection and FimA lineage genotype and periodontal severity score in dachshunds. These results indicate that there are differences in the status of periodontal deterioration and detection of P. gulae among breeds and suggest the need for different methods of coping with periodontal disease. In Chapter 3, the detection of P. gulae in individuals with missing teeth and its correlation with FimA lineage genotype and periodontal severity were investigated based on the classification methods used in Chapters 1 and 2. Tooth loss was classified with 0, 1-10, 11-20, and 21 or more teeth lost. The number of individuals in each group was 74, 69, 24, and 21, respectively. The results of the analysis showed that almost all individuals with periodontal severity score 1 concentrated on 0 tooth loss. The number of individuals with a periodontal severity score of 2 decreased as the number of teeth lost increased. Among the periodontal severity score 2 individuals, the proportion of dogs with 0 tooth loss was significantly higher than those with 11 to 20 teeth loss and those with 21 or more teeth loss. The number of dogs with periodontal severity score 4 increased with the number of missing teeth. There were no significant differences between groups except for periodontal severity score 3, where P. gulae was more frequently detected in the group with less tooth loss. The number of FimA genotypes was significantly higher in the missing tooth group with 11 to 20 missing teeth for the group in which only the C genotype was detected, and in the group with 21 or more missing teeth for the group in which all the A, B, and C genotypes were detected. The reason for the lack of significant differences in the detection of FimA genotypes in this study may be that the number of dogs in each group was insufficient due to the segmentation of the study population, and that the samples were collected from the areas with remaining teeth. Therefore, it is necessary to increase the number of cases and reexamine the relationship between the detection of P. gulae and the FimA lineage genotype and tooth loss. In chapter 4, the correlation of preexisting disease with P. gulae detection, FimA lineage genotype, and periodontal severity was investigated. In the history of the 665 animals included in Chapter 1, mitral regurgitation (MR) was the most frequently described case (65 dogs) among the 490 animals with a stated history. The presence or absence of MR was classified based on the reported history. Periodontal severity scores, P. gulae detection, and FimA lineage genotype were as in previous chapters. The age of dogs affected by MR was significantly different from that of healthy dogs. Periodontal severity scores were higher than in healthy dogs, but there were no significant differences in either P. gulae detection or FimA lineage genotype detection status. In the present comparison, there was no correlation between periodontal status or periodontopathogens in the development of MR. This may be since there was a significant difference in the age between the onset and non-onset groups in the comparison of mitral regurgitation, the incidence of which increases with age, and that the number of n for each group was not necessarily large because each group was subdivided into smaller groups. In this study, the time of establishment of periodontopathogens was studied in greater detail than in the past, and it was confirmed that they are established by the age of 6 years and affect periodontal status. However, the effects of periodontopathogens on certain breeds of dogs were found to vary. No correlation was found between the presence of periodontopathogens and tooth loss or MR. Since it is becoming clear that the oral microflora in humans differs depending on the living environment and specific diseases, it is suggested that other bacteria other than P. gulae or factors on the host side may be influential in individuals who had both periodontal disease and other diseases. Therefore, it is necessary to conduct more detailed analysis for each breed, to conduct further large-scale surveys of cases with specific diseases as the main complaint, to reconsider the method of wood sampling, and to study host-side factors. |
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| 言語 | en | |||||||
| 学位名 | ||||||||
| 学位名 | 博士（獣医学） | |||||||
| 学位授与機関 | ||||||||
| 学位授与機関識別子Scheme | kakenhi | |||||||
| 学位授与機関識別子 | 32701 | |||||||
| 学位授与機関名 | 麻布大学 | |||||||
| 学位授与年月日 | ||||||||
| 学位授与年月日 | 2025-09-15 | |||||||
| 学位授与番号 | ||||||||
| 学位授与番号 | 甲第190号 | |||||||
| Rights | ||||||||
| 値 | 本論文の一部は、以下に公表した。 Yasuda J, Yasuda H, Nomura R, Matayoshi S, Inaba H, Gongora E, Iwashita N, Shirahata S, Kaji N, Akitomo T, Mitsuhata C, Uchiyama J, Fukuyama T, Matsumoto-Nakano M, Nakano K, Masaru Murakami M： Investigation of periodontal disease development and Porphyromonas gulae FimA genotype distribution in small dogs. Scientific Reports 14, Article number: 5360 (2024) https://doi.org/10.1038/s41598-024-55842-8 |
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| 出版タイプ | VoR | |||||||
| 出版タイプResource | http://purl.org/coar/version/c_970fb48d4fbd8a85 | |||||||
