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Successful Production of Offspring Derived from Phospholipase C Zeta-Deficient Sperm by Additional Artificial Activation
Get PDFDuring mammalian fertilization, repetitive rises of intracellular calcium called calcium oscillations are required for full activation of oocytes. Therefore, oocytes such as round spermatid injected or somatic cell nuclear transferred require additional artificial activation which mimics the calcium oscillations. It is well recognized that sperm specific phospholipase C (PLCζ) is a strong candidate as the sperm factor which can induce calcium oscillations and, at least in mammals, the genetic mutation of PLCζ in human causes male infertility due to the lack of calcium oscillations in the oocytes. Recent studies showed that the sperm lacking PLCζ (Plcz1−/−) still could induce rise(s) of intracellular calcium in the oocytes after IVF but not intracytoplasmic sperm injection (ICSI). In the ICSI oocytes, no pronuclear formation or development to the two-cell stage was observed. However, it is still unclear whether additional activation treatment can rescue the low developmental ability of Plcz1−/−-sperm-derived oocytes after ICSI. In this study, we examined whether oocytes injected with a Plcz1−/− sperm can develop to term by additional artificial activation. In oocytes injected a Plcz1−/− sperm and Plcz1−/− and eCS (another candidate of the sperm factor) double knockout sperm (Plcz1−/−eCS−/−), the rates of pronuclear formation were very low (2.0 ± 2.3% and 6.1 ± 3.7%, respectively) compared to control (92.1 ± 2.6%). However, these rates were dramatically improved by additional procedures of PLCζ-mRNA injection or SrCl2 treatment (Plcz1−/− sperm + PLCζ mRNA, Plcz1−/− sperm + SrCl2 and Plcz1−/−eCS−/− sperm + PLCζ mRNA; 64.2 ± 10.8%, 89.2 ± 2.4% and 72.6 ± 5.4%, respectively). Most of the oocytes were developed to the two-cell stage. After embryo transfer, healthy pups were obtained in all these groups (Plcz1−/− sperm + PLCζ mRNA:10.0 ± 2.8%, Plcz1−/− sperm + SrCl2:4.0 ± 4.3% and Plcz1−/−eCS−/− sperm + PLCζ mRNA: 10.0 ± 5.7%). The rate in Plcz1−/− sperm + SrCl2 group was significantly lower than that in control (26.0 ± 2.4%). Taken together, our present results show that additional activation treatment such as SrCl2 and PLCζ mRNA can fully support to develop to term even in oocyte injected Plcz1−/− sperm. In addition, PLCζ-induced oocyte activation is more suitable for successful development to term compared to that such as phenomenon induced by SrCl2. These findings will contribute to improvement for male-dependent human infertility and reproductive technologies in other mammalian species.journal articl
Dasatinib attenuates airway inflammation of asthma exacerbation in mice induced by house dust mites and dsRNA
Get PDFAsthma exacerbation is a significant clinical problem that causes resistance to corticosteroid therapy and elevated hospitalization risk. Src family kinases (SFKs) contribute to various steps of the immune response, such as airway inflammation in viral or bacterial infections and allergic asthma. Therefore, we determined the effects of dasatinib (DAS), a typical Src inhibitor, on a murine asthma exacerbation model induced by house dust mites (HDM) and synthetic analog of double-stranded RNA, poly(I:C). A/J mice were sensitized to intrapreneurial HDM twice every seven days and challenged with intranasal HDM once every second day for a total of six exposures, and/or exposed to poly(I:C) twice daily for three consecutive days. Drug treatments were performed twice daily for three days, starting one day after the last HDM challenge or 2 h before each poly(I:C) exposure. DAS improved poly(I:C)-induced acute inflammation dose-dependently. Both DAS and fluticasone propionate (FP) attenuated HDM-induced allergic airway inflammation. However, in HDM and poly(I:C) induced-asthma exacerbated mice, DAS significantly improved inflammatory cells in bronchoalveolar lavage fluid and histological changes in the lungs, whereas FP did not. Therefore, SFKs are important targets for controlling severe asthma refractory to conventional therapies.journal articl
Microhabitat use and artificial-light related occurrence of the brown widow spider Latrodectus geometricus in a newly invaded area of the inland Kanto region, Japan
Get PDFThe invasion of the brown widow spider into Japan was first recorded in 1995. Since then, its establishment has been recognized mainly in major port areas, but further invasions are recently found from inland areas in the Kanto region of Japan. We thus attempted to clarify the actual status of its invasion by conducting a pilot extermination in a suspected area between October 13, 2022 and January 20, 2023. A total of two males, 39 females, and 83 egg sacs were recovered from 46 nests during our extermination. The nesting sites were typically found on or inside inconspicuous part of artificial outdoor objects, especially behind plastic benches and in recesses on the outer walls of buildings. These microhabitat uses were concentrated in environments where the surroundings were lit up by street and building lights at night. Artificial lights have a function to attract phototaxis insects so that the distribution of nesting sites is likely to be affected by the availability of these potential food sources. As this species has the potential to expand its distribution far inland more, additional surveys are required to predict future invasions and ecological impact.journal articl
Suppression of the excitability of rat nociceptive secondary sensory neurons following local administration of the Phytochemical, (-)-Epigallocatechin-3-gallate
Get PDFThe phytochemical, polyphenolic compound, (-)-epigallocatechin-3-gallate (EGCG), is the main catechin found in green tea. Although a modulatory effect of EGCG on voltage-gated sodium and potassium channels has been reported in excitable tissues, the in vivo effect of EGCG on the excitability of nociceptive sensory neurons remains to be determined. Our aim was to investigate whether local administration of EGCG to rats attenuates the excitability of nociceptive spinal trigeminal nucleus caudalis (SpVc) neurons in response to mechanical stimulation in vivo. Extracellular single unit recordings were made from SpVc neurons in response to orofacial mechanical stimulation of anesthetized rats. The mean firing frequency of SpVc wide-dynamic range neurons following both non-noxious and noxious mechanical stimuli was significantly inhibited by EGCG in a dose-dependent and reversible manner. The mean magnitude of inhibition by EGCG on SpVc neuronal discharge frequency was similar to that of the local anesthetic, 1% lidocaine. Local injection of half-dose of lidocaine replaced the half-dose of EGCG. These results suggest that local injection of EGCG suppresses the excitability of nociceptive SpVc neurons, possibly via the inhibition of voltage-gated sodium channels and opening of voltage-gated potassium channels in the trigeminal ganglion. Therefore, administration of EGCG as a local anesthetic may provide relief from trigeminal nociceptive pain without side effects.journal articl
Studies on expression of obesity-related gene in canine health status and adipocytes
Get PDF麻布大学博士(獣医学)Canine obesity has been increasing in recent years and is an inducer of various diseases such as arthritis and diabetes. Moderate exercise and dietary restriction can eliminate obesity, but its management is difficult. In humans, activation of brown fat cells has attracted attention as a new method of obesity reduction, and deconjugated protein (Ucp) 1 has been reported to hold the key, but not many studies have been conducted in dogs.
The expression of UCP1 in adipose tissues of various animal species showed high expression in dogs. Furthermore, the expression of UCP1 in RNA from various canine tissues revealed that UCP1 is expressed in trace amounts in tissues other than adipose tissue, such as brain and heart, and that there is an exon2 variant of UCP1.
Adipose tissue was collected during surgery at a veterinary clinic, and the expression of obesity-related genes in fat cells of a total of 129 dogs was examined. We found that the body condition score (BCS), a measure of obesity, was not a reliable multiple regression equation when used as the objective variable; positive correlations were found between the expression levels of most genes except Ucp1 and 3. BCS increased with age and was negatively correlated with the expression levels of Pparγ and Fasn and with the expression levels of Leptin and Opn3 showed a positive correlation. Differences in expression were also observed between sexes and between healthy and diseased groups.
These findings suggest that canine UCP1 may differ from other animal species, and that gene expression in canine adipose tissue in relation to BCS, age, and tumor may provide a basis for elucidating the etiology of canine obesity. We believe that the gene expression in canine adipose tissue in relation to BCS, age, and tumor will provide basic knowledge for the elucidation of the etiology of canine obesity.獣医療において肥満は深刻な問題である。アメリカの報告では 2018 年時に飼育されていた犬の 56%、猫の 60%が肥満であるとされており、最も一般的な栄養障害とされている。肥満はヒト同様に糖尿病や循環器疾患、関節疾患、尿路感染症を引き起こすこと、さらに肥満は QOL の低下につながることが報告されている。また近年ではヒト医療において大腸癌や乳癌は肥満関連癌と呼ばれ、腫瘍と肥満の関連も注目されている。イヌにおいても腫瘍の発生率は増加しており、腫瘍と肥満の関連も報告されているため、これらの予防の観点からも肥満の予防・解消は重要であると考えられている。
肥満とは、食物摂取量が慢性的にエネルギー消費量を超えると発生するもので、白色脂肪細胞の過形成と肥大を特徴とする。脂肪細胞には、白色、褐色、ベージュ脂肪細胞の 3 種類がある。白色脂肪細胞は余剰なエネルギーを脂肪として蓄積する。一方、褐色・ベージュ脂肪細胞はエネルギーを熱として産生することができる。褐色・ベージュ脂肪細胞の熱産生には脱共役タンパク質(Ucp)1が関わっている。ヒトやマウスではこの Ucp1 と肥満に関する研究が多く行われており、肥満解消のためのサプリメントとして Ucp1 を増加させる作用を持つものも発売されている。しかしイヌにおける肥満の解消法は食事のコントロールと運動療法に頼るのが一般的である。食事のコントロールと運動療法は効果を得るまでに時間がかかるため、成功率が低いのが現状である。イヌにおいてもUcp1 を持つベージュ脂肪細胞を増やすことで肥満解消・予防、さらには腫瘍などの疾患の予防ができるのではないかと考えている。そこで今回、イヌの脂肪組織に注目し、肥満やその他の健康状態における脂肪細胞での遺伝子発現、つまり Ucp1 をはじめとする脂肪細胞の分化や機能に関する遺伝子発現を調べた。
第１章 イヌ Ucp1 遺伝子の発現
褐色・ベージュ脂肪細胞が持つ Ucp1 はミトコンドリア内膜に存在する脱共役タンパク質である。Ucp1 を高発現させたマウスでは肥満を予防できることが報告されており、Ucp1 は肥満の解消において重要な因子と考えられている。本章ではまず、ウシ、ラット、マウス、イヌ、ネコの 5 種類の動物種の脂肪組織における Ucp1 の発現を RT-PCR により比較した。その結果、イヌは脂肪組織における Ucp1 の発現量が他の動物種よりも多いことがわかった。さらに、イヌの各組織（脳、心臓、肺、肝臓、胃、脾臓、腎臓、骨格筋、皮膚、精巣）由来の RNAを用いて Ucp1 遺伝子の発現を調べたところ、2nd PCR による微量検出ではあるものの、脂肪組織だけではなく心臓や脳など多くの臓器で Ucp1 が発現していることがわかった。また、目的とするサイズの Ucp1 mRNA（バリアント 1）のほかに、やや小さいサイズの Ucp1 バリアント 2 が存在していることもわかった。このバリアント 2 の塩基配列を調べたところ、本来の大きさをもつ Ucp1mRNA（バリアント 1）の Exon2 領域を欠失したバリアントであった。バリアント 2 の推定アミノ酸配列はナンセンス変異が生じるなど、Ucp1 として機能していないタンパク質を生じていると考えられた。このようなバリアントはヒトやマウス、ラットなど他の動物種では報告がなく、イヌに特徴的であるかもしれない。存在意義については今後の検討が必要である。イヌの Ucp1 遺伝子が他の動物種より高発現していることや脂肪組織以外の臓器にも発現していることは、イヌにおいて Ucp1 遺伝子の発現制御は、肥満予防やその他の健康状態に影響を与えることが推察される。
第２章 イヌ白色脂肪細胞における肥満関連遺伝子発現
筆者が勤務する動物病院を中心に複数の動物病院で、開腹手術を実施した129 頭のイヌの皮下または内臓の脂肪組織を採取し、cDNA を作製した。脂肪細胞分化と機能に関わる調節遺伝子、アディポカイン、褐色脂肪細胞形成や脱共役タンパク質（Ucp）、BMP シグナルに関わる遺伝子の 4 つのカテゴリーに分類した合計 23 遺伝子の発現量を定量的 RT-PCR を用いて測定した。発現量について、各遺伝子間および BCS や年齢、健康状態との関連を解析した。その結果、まず各個体でのそれぞれの遺伝子発現についてみてみると、調べた 23 遺伝子のうち Ucp1 と Ucp3 の 2 つの遺伝子において、顕著に高い値を示す個体がみられた。褐色・ベージュ脂肪細胞の熱発生に関与する Ucp1 遺伝子の発現が平均より20 倍高い検体であったものの、その個体の BCS は 3 であり特別痩せてはいなかった。ウシにおいて、Ucp1 の発現量が通常より 1500 倍以上高かった個体では飼料効率が低く、体重が増加しにくいことが報告されており、今回のイヌの 20倍程度では、BCS に影響を与える変化ではないのかもしれない。次に、肥満の指標であるボディコンディションスコア(BCS)を目的変数として解析したところ、信頼性の高い重回帰式は得られなかった。Ucp1 と Ucp3 を除く 21 の遺伝子間の発現量に正の相関が認められた。BCS は年齢とともに増加し、Pparγ および Fasn の遺伝子発現量と負の相関を示し、Leptin および Opn3 の発現量と正の相関を示した。加齢に伴い、脂肪細胞分化と機能に関わる調節遺伝子のカテゴリーに属する Pparγ、Fabp4、Fasn、Hsl と Insr 遺伝子、およびアディポカインの一つである Adipoq 遺伝子の発現量は減少した。これらに加えて加齢は、褐色脂肪細胞形成に関与する Prdm16 遺伝子や BMP シグナルに関わる遺伝子に分類される Bmp4、Alk3、Actr2a、Actr2b 遺伝子の発現量と負の相関を示した。一方で、加齢により Leptin と Ucp2 の遺伝子発現量は増加した。雌雄について見てみると、Pparγ、Adipoq、Bmp4 遺伝子発現はメスで高く、Ucp2 と Ucp3 遺伝子発現はオスで高かった。腫瘍疾患のイヌについて見てみると、健康なイヌに比べ Pparγ と Fasn の遺伝子発現は低く、Leptin、Ucp2、Ucp3 および Inhβb の発現は高かった。また、体格で見ると、Tnfαと Opn3 の遺伝子発現は小型犬に比べ大型犬で高かった。
肥満は、脂肪細胞数の増加と脂肪細胞の肥大化を特徴とするが、成人での肥満は脂肪細胞の肥大化が主な原因と考えられている。そしてその肥大化した脂肪細胞はより多くの Leptin 遺伝子を発現していることが知られている。今回のイヌの加齢による BCS の増加では、脂肪細胞の肥大化が起こっており、そのため Leptin 遺伝子の増加がみられたと考える。一方、Pparγ と Fasn の遺伝子発現の減少は脂肪細胞の肥大化に対する防御的反応と推測する。
最近、褐色脂肪細胞に光を照射すると光受容体である Opn3 遺伝子産物を介して熱発生が活性化されることが報告された。Opn3 遺伝子発現制御は不明であるが、今回、BCS に伴い Opn3 遺伝子の発現が増加をしたり、大型犬で高い発現量がみられたりしており、肥満との関連で今後注目をする必要がある。
BMP シグナルは褐色・ベージュ脂肪細胞分化の誘導に関与しており、またPrdm16 遺伝子産物は褐色・ベージュ脂肪細胞分化に必要な因子であることから、今回のイヌでの加齢によるこれら遺伝子の発現の減少は、加齢に伴う褐色・ベージュ脂肪細胞活性の減少を引き起こし、それが BCS の増加へつながっているのかもしれない。
肥満と腫瘍の関連については、アディポカインである Leptin の増加や Adipoq の低下などが炎症反応に影響を与え腫瘍を発生していることが知られている。今回のイヌの腫瘍群においても、Leptin は有意に増加しており、Adipoq は減少傾向であった。
本研究の結果からは BCS と調べた 23 の遺伝子の脂肪細胞における発現との明確な関連を示すことはできなかった。しかし、多くの遺伝子間では正に相関をしていた。Leptin や Opn3、Pparγ、Fasn は BCS と関連していることから、肥満の予防・解消には Ucp1 以外にも多くの遺伝子が関与している可能性も考えられた。ヒトではゲノムワイド関連解析から肥満に関連する遺伝子が報告されており、それらの遺伝子発現が、高脂肪食を与えたマウスの脂肪組織で変化することも知られている。今後イヌにおいても大規模なゲノムワイド関連解析を実施してそこから得られた関連遺伝子の脂肪細胞における発現解析を行うことが必要になるかもしれない。本研究は BCS、年齢、腫瘍などイヌのプロファイルと脂肪組織における遺伝子発現の関連を調べた。これらの結果はイヌの肥満の予防や治療に向けた候補遺伝子同定のための基礎的情報となる。doctoral thesi
Epidemiological investigation of canine periodontal pathology caused by Porphyromonas gulae infection and investigation of the effect of periodontal disease preventive drug.
Get PDF麻布大学博士(獣医学)Periodontal disease is the infection with the highest incidence in small companion animals such as dogs and cats, and the plaque buildup, gingivitis, and periodontitis found in the process were concluded to be the most important health problems in a study of 31,484 dogs in the United States. In addition, 70% of cats and 80% of dogs have periodontal disease by the age of two, with small dogs and toy breeds being particularly susceptible. Canine periodontal disease is a chronic dental condition in which periodontogenic bacteria form biofilms in periodontal pockets, causing gingival injury and alveolar bone fusion. Periodontal disease is formed by the association of many periodontogenic bacteria. In humans, Porphyromonas gingivalis (P. gingivalis), Treponema denticola, and Tannerella forsythia are considered to be the three species of Red complex and are considered to be particularly virulent. In dogs and cats, Porphyromonas gulae (P. gulae), a close relative of P. gingivalis, has been isolated from the gingival sulcus and is known to have an important role in the development and exacerbation of periodontal disease. Fimbrilin, the pathogenic protein that forms the surface linear hairs of this bacterium, is called FimA. the gene encoding FimA, or the fimA gene, is classified into three main subtypes, type A, type B, and type C, and these genotypes are closely related to the severity of periodontal disease. Among these fimA genes, the C-type P. gulae is considered the most pathogenic because it predominates in the oral cavity of dogs with severe periodontitis. However, the association between fimA genotype and clinical features of periodontal disease in dogs remains to be elucidated. The relationship between fimA genotype trends with age, fimA genotype and severity of periodontal disease, and clinical features of periodontal disease is unknown. In addition, periodontal disease is difficult to recover from once periodontitis and alveolar bone fusion have been induced. Prophylactic dental care at home by owners is highly important. In this study, an epidemiological survey was conducted in Chapter 1 on the actual age-specific possession of each fimA genotype of P. gulae in Japanese small dog breeds and the association between periodontal pathology (periodontal disease score) and clinical signs (number of permanent teeth). In Chapters 2 and 3, the efficacy of a canine periodontal disease prevention product CPC and Pt-containing formulation was investigated in vitro and in vivo based on epidemiological studies in dogs.
Chapter 1: Percentage of P. gulae fimA genotypes in dogs and correlation of periodontal disease grade by fimA genotype.
The subjects were 225 periodontally diseased dogs aged 6-211 months who visited Primo Animal Hospital Sagamihara Chuo, Atsugi Primo Animal Hospital, Sagamiono Primo Animal Hospital, and Yasuda Veterinary Clinic. The dogs were divided into three groups according to their age in months: less than 50 months old (n = 50), between 50 and 100 months old (n = 60), and more than 100 months old (n = 115). The prevalence of each fimA genotype in P. gulae, the severity of periodontal disease, and the number of permanent teeth as clinical signs supporting periodontal pathology were compared among the fimA genotypes. The results showed that the P. gulae-positive rate in all 225 dogs was 76.9%, and the percentage increased with age. In particular, the percentage of dogs with P. gulae of the C-type fimA gene increased significantly with age (20% in the group less than 50 months of age, and more than 40% in the groups between 50 and 100 months of age and more than 100 months of age) compared to the other genotypes. This suggests that P. gulae may be involved in the increase and worsening of periodontal disease with aging. A comparison of the severity of periodontal disease between dogs with and without P. gulae showed that the severity of periodontal disease was higher in P. gulae positive dogs compared to P. gulae negative dogs in all age groups. When comparing the severity of periodontal disease among genotypes, significantly worse severity of periodontal disease was observed in P. gulae fimA carrying dogs of types A and C compared to P. gulae negative dogs in the <50 month old group. In the group between 50 and 100 months of age, a significant worsening of the severity of periodontal disease was observed with P. gulae fimA-bearing dogs of genotype C compared to P. gulae-negative dogs.
In the over 100-month-old group, significant worsening of periodontal disease severity was observed with all genotypes of P. gulae fimA carriers compared to P. gulae-negative dogs. And, possession of P. gulae fimA type C significantly worsened periodontal disease severity at all ages. Next, a comparison of the number of permanent teeth among genotypes showed that the number of dogs with complete dentition (42 teeth) decreased with age in the P. gulae-positive group in the groups between 50 and 100 months of age and in the group over 100 months of age. In addition, the number of dogs with complete dentition was significantly reduced in the group older than 100 months of age due to the possession of P. gulae fimA of types B and C, compared to the P. gulae-negative dogs. These results indicated a significant association between the presence of P. gulae and the number of permanent teeth. The fact that injured periodontal ligaments and alveolar bone are irreversible also suggests that protection against infection by P. gulae, especially those carrying the C-type fimA gene, is important to prevent periodontal disease.
Chapter 2: Investigation of the effect of cetylpyridinium chloride and platinum nanocolloids on P. gulae
We focused on cetylpyridinium chloride (CPC) and platinum nanocolloids (Pt), which are used as periodontal prophylactics for humans, to elucidate the medicinal effects of these drugs on canine periodontal disease.
In the field of human dentistry, CPC has been reported to reduce motile rods and spirochetes by cleaning periodontal pockets with a 0.05% CPC solution, and to inhibit plaque accumulation and improve gingivitis when used as an oral rinse (mouthwash).
The mechanism of action of Pt also removes reactive oxygen species at the site of inflammation and alleviates/eliminates local oxidative stress. This is thought to alleviate and suppress periodontal tissue damage and inflammatory reactions caused by reactive oxygen species, thereby promoting healing. Since biofilm dissolving action has also been observed, it has been expected in human dentistry to inhibit excessive foreign body invasion, leukocyte infiltration, and bacterial colonization under the biofilm during the tissue repair process.
To evaluate the effects of CPC and Pt-containing preparations on periodontal disease in dogs, the effects of CPC and Pt-containing preparations on growth inhibition of P. gulae, inhibition of halitosis-causing substances (hydrogen sulfide and methyl mercaptan) produced by P. gulae, and on inflammatory reactions in macrophage cells were examined in vitro. Q-ness Mouth Cleaner was used as the CPC and Pt-containing formulation, and its efficacy was evaluated at the recommended 1% concentration for actual use. Verification of the growth inhibitory effect of the actual concentration (1%) of CPC and Pt-containing preparations on P. gulae showed significant inhibition of P. gulae growth compared to the negative control group at 4 hours after incubation. Exposure of CPC and Pt-containing preparations to P. gulae at the actual concentration used (1%) significantly inhibited hydrogen sulfide production from the bacteria compared to the negative control group. Furthermore, cytokine (IL-1β, IL-6, TNFα) production was measured when macrophage cells (J774.1 cells) exposed to P. gulae were treated with CPC and Pt-containing preparations at the actual concentration used (1%), and a significant decrease in TNFα production was observed after exposure to CPC and Pt-containing preparations, compared to the negative control group. These results indicate that CPC and Pt-containing preparations at actual concentrations have growth inhibition, hydrogen sulfide production inhibition, and anti-inflammatory activity against P. gulae.
Chapter 3: Investigation of the effects of cetylpyridinium chloride and platinum nanocolloids on periodontal disease in domestic dogs.
Fifteen dogs with mild to moderate degrees of periodontal disease and halitosis were given CPC and Pt-containing formulations in drinking water for one month. The sensory evaluation of halitosis, degree of plaque adhesion, oral gas concentration, degree of gingivitis, oral thiol concentration, and periodontal bacterial activity were measured before and after the application of the formulation. Periodontal bacterial activity was evaluated using the BANA-degrading enzyme test (BANA test), which is an enzyme produced by anaerobic bacteria including periodontogenic bacteria (P. gulae, P. gingivalis, Treponema denticola, and Tannerella forsythensis). The activity of periodontogenic bacteria was evaluated by measuring this enzyme. As a result, no significant changes in periodontal pathology or halitosis were observed before or after administration of CPC and Pt-containing formulations, suggesting that the therapeutic effect of CPC and Pt-containing formulations on dogs with advanced periodontal disease was low.
Next, we investigated the auxiliary effect of CPC and Pt-containing preparations on dental procedures. Fifty dogs that had undergone dental procedures such as scaling under anesthesia at Primo Animal Hospital Sagamihara Chuo were divided into four groups: a group that received tooth brushing and CPC and Pt-containing formulation in their drinking water (n=15), a group that received only tooth brushing (n=16), a group that received only CPC and Pt-containing formulation in their drinking water (n=8), and an untreated group (n=11). The gingivitis index, tartar index, halitosis, oral thiol concentration, and periodontal bacterial activity were compared before dental treatment, 7 days, 1 and 3 months after treatment.
Periodontal bacterial activity was determined by measuring the enzymatic activity of N-benzoyl-DL-arginyl peptidase using ADplit®. The ADplit test for periodontal bacterial activity is simpler to perform in animal hospitals than the BANA-degrading enzyme test and can be used to measure the enzymatic activity of trypsin-like enzyme specifically produced from P. gulae. The results showed that the degree of gingivitis, thiol concentration, and periodontal bacterial activity were significantly lower in the CPC and Pt-containing formulation group than in the untreated group, suggesting that the combination of CPC and Pt-containing formulations with dental care such as tooth brushing increases the effectiveness of the treatment. The combination of physical dental care such as tooth brushing and CPC and Pt-containing preparations after dental treatment proved to have sufficient preventive effect against periodontal disease.
In today's aging companion animal population, the incidence of periodontal disease is expected to increase. Detection of the fimA genotype of P. gulae and assessment of periodontal disease risk in dogs, as well as daily dental care instruction to prevent periodontal disease, may be useful tools in maintaining a good oral environment.歯周病は、犬や猫などの小型伴侶動物において、罹患率の最も高い感染症であり、その過程で認められる歯垢の付着、歯肉炎および歯周炎は、米国における犬31,484頭の調査において、最も重要な健康問題であると結論付けられている。また、2歳になるまでに猫の70%、犬の80%が歯周病を有しており、小型犬・トイ種では特に影響を受けやすいとされている。犬の歯周病は、歯周病原性細菌が歯周ポケットにバイオフィルムを形成し、歯肉の傷害および歯槽骨の融解を引き起こす慢性歯科疾患である。歯周病は多くの歯周病原性細菌が関連しあって形成される疾患であるが、ヒトではPorphyromonas gingivalis（以下、P. gingivalis ）、 Treponema denticola およびTannerella forsythiaの3菌種をRed complexと呼び、特に悪性度が高いとされている。一方、犬や猫では、P. gingivalis の近縁種であるPorphyromonas gulae（以下、P. gulae）が歯肉溝から分離され、歯周病の発症および増悪に重要な役割を有していることがわかっている。P. gulaeの菌体表面に存在する線毛を形成する病原蛋白質である分子量41kDaのフィンブリリンはFimAと呼ばれ、歯への付着や病原性に重要な役割を示すことが判明している。FimA蛋白質をコードするfimA遺伝子は、A型、B型、およびC型の3種類のサブタイプに分類され、これらのfimA遺伝子の中で、C型のP. gulaeが重度の歯周炎の犬の口腔内で優勢であるため、最も病原性が高いと考えられている。年齢によるfimA遺伝子型の推移、fimA遺伝子型と歯周病の重症度および歯周病の臨床的特徴との関連は不明である。また、歯周病は歯周炎および歯槽骨の融解が引き起こされると回復が難しい疾患であり、動物病院だけでなく、飼い主による自宅での予防的デンタルケアの重要度は高い。
そこで本研究では、第1章で日本の小型犬種におけるP. gulaeの各fimA遺伝子型の年齢別保有実態と歯周病態（歯周病スコア）と臨床的兆候（永久歯数）の関連に関する疫学調査を実施し、続いて第2章および3章では、犬の疫学調査に基づいた犬の歯周病予防製品CPC + Pt含有製剤（Q-nessマウスクリーナー®、株式会社QIX）の効果について、それぞれin vitroおよびin vivoで調べた。
第１章：犬におけるP. gulaeのfimA遺伝子型の保有率とfimA遺伝子型による歯周病悪性度との相関
プリモ動物病院相模原中央、厚木プリモ動物病院、相模大野プリモ動物病院および安田獣医科医院に来院した生後6〜211ヶ月齢の歯周病罹患犬225頭（過去6ヶ月以内に抗生物質を使用している症例は検討から除外）を月齢によって生後50ヶ月齢未満（n = 50）、50ヶ月齢以上100ヶ月齢未満（n = 60）、100ヶ月齢以上（n = 115）の3つのグループに分け、P. gulaeの各fimA遺伝子型の保有率、歯周病重症度および歯周病態を裏付ける臨床兆候として永久歯数を、fimA遺伝子型間で比較検討した。その結果、225頭全体におけるP. gulae陽性率は74.6%であり、その割合は加齢に伴って増加した。特に、C型のfimA遺伝子のP. gulaeを有する犬の割合が、他の遺伝子型と比較して年齢とともに著しく増加（生後50ヶ月齢未満の群で20%、50ヶ月齢以上100ヶ月齢未満および100ヶ月齢以上の群で40%以上）していた。このことからも、加齢に伴う歯周病の増加、悪化に関与しているのではないかと推測された。
次に、P. gulae の有無での歯周病の重症度スコアを比較すると、すべての年齢層でP. gulae非保有犬と比較し、P. gulae 保有犬で歯周病の重症度が高くなっていた。また、遺伝子型間での歯周病の重症度を比較すると、生後50ヶ月齢未満の群において、P. gulae 陰性の犬と比較して、A型およびC型のP. gulae fimA保有犬で有意な歯周病の重症度の悪化が観察された。50ヶ月齢以上100ヶ月齢未満の群では、P. gulae 陰性の犬と比較して、C型のP. gulae fimA保有犬で有意な歯周病の重症度の悪化が観察された。100ヶ月齢以上の群では、P. gulae 陰性の犬と比較して、全遺伝子型のP. gulae fimA保有により、有意な歯周病の重症度の悪化が観察された。そして、C型のP. gulae fimA保有により、どの月齢においても歯周病重症度を有意に悪化させることが確認された。
次に遺伝子型間での永久歯数を比較したところ、50ヶ月齢以上100ヶ月齢未満の群および100ヶ月齢以上の群において、完全な歯列（42本）を持つ犬の数はP. gulae陽性群では年齢とともに減少した。また、100ヶ月齢以上の群において、B型およびC型のP. gulae fimA保有犬で完全な歯列を持つ犬の数はP. gulae 陰性の犬と比較して有意に減少した。これらの結果は、P. gulae の存在と永久歯の数との間に有意な関連性があることを示していた。傷害を受けた歯周靭帯や歯槽骨は不可逆的であることからも、P. gulae、特にC型のfimAを保有するP. gulaeの感染防御は歯周病を防ぐために重要であることが強く示唆された。
第２章：塩化セチルピリジニウム、プラチナナノコロイドのP. gulae に対する効果検討
ヒト用の歯周病予防薬として利用されているプラチナナノコロイド（以下、Pt）および塩化セチルピリジニウム（以下、CPC）に着目した犬の歯周病予防薬の薬効解明を行った。
Pt製剤の作用機序は、炎症部位での活性酸素を除去し、局所の酸化ストレスを緩和・解消する。このことにより、活性酸素による歯周組織障害および炎症反応が緩和・抑制され、治癒が促進されると考えられる。またバイオフィルム溶解作用も認められているため、組織の修復過程で過剰な異物侵入や白血球浸潤、バイオフィルム下の細菌の定着を抑制する効果も人歯科医療では期待されてきた。また人歯科領域において、CPCは0.05%CPC溶液を歯周ポケット内の洗浄することで運動性桿菌、スピロヘータを減少させ、口腔洗浄液（含嗽剤）として使用することでプラークの蓄積の抑制や歯肉炎の改善に効果があるとの報告がある。
そこで、Pt、CPCのP. gulaeに対する増殖抑制効果、P. gulaeから産生される口臭原因物質（硫化水素およびメチルメルカプタン）に対する抑制効果、マクロファージ細胞における炎症反応に対する効果を試験管内で検証した。CPC+Pt含有製剤としてQ-nessマウスクリーナーを使用し、その推奨使用濃度とされる１％を実使用濃度として効果を評価した。CPC + Pt含有製剤の実使用濃度（1%）のP. gulaeに対する増殖抑制効果の検証では、培養後4時間で陰性対照群と比較して有意なP. gulaeの増殖抑制作用が認められた。CPC + Pt含有製剤の実使用濃度（1%）のP. gulaeに対する曝露によって、細菌からの硫化水素産生が陰性対照群と比較して有意に抑制された。さらに、P. gulaeを曝露したマクロファージ細胞（J774.1細胞）に実使用濃度（1%）のCPC + Pt含有製剤を処置した際のサイトカイン（IL-1β, IL-6, TNFα）産生量を測定した結果、CPC+Pt含有製剤曝露により陰性対照群と比較してTNFα産生の有意な減少が認められた。以上の結果より、CPC+Pt含有製剤は実使用濃度において、P. gulaeに対する増殖抑制、硫化水素産生抑制および抗炎症作用を有していることが示された。
第３章：CPC, Ptの一般飼育犬の歯周病に対する効果検討
歯周病および口臭の程度が軽度から中等度の犬15頭に対して、CPC+Pt含有製剤を1ヶ月間、自由飲水投与した。製剤を適用した前後での口臭スコア、歯垢付着度、歯周病スコア、口腔内総菌数、歯周病菌活性および口腔内チオール濃度の測定を実施した。歯周病菌活性は、BANA分解活性酵素検査（BANAテスト）により評価した。BANA分解活性酵素とは、歯周病菌（P. gulae、P. gingivalis、Treponema denticola、Tannerella forsythensis）をはじめとする嫌気性細菌から産生される酵素であり、この酵素を測定することで歯周病菌活性を評価した。その結果、CPC+Pt含有製剤投与の前後で歯周病態および口臭に大きな変化は認められず、すでに歯周病が進行している犬に対するCPC+Pt含有製剤の効果は低いことが示唆された。
次にCPC+Pt含有製剤の歯科処置に対する補助効果を調査する目的で、プリモ動物病院相模原中央に来院したスケーリング等の麻酔下歯科処置を実施した犬50頭について、歯みがきとCPC+Pt含有製剤を飲水投与した群（n=15）、歯みがきのみを実施した群（n=16)、CPC+Pt含有製剤のみを飲水投与した群（n=8)、無処置群（n=11）の4群に分け、歯科処置前、処置後7日、1および3ヶ月での歯肉炎指数、歯石指数、口臭、口腔内チオール濃度、歯周病菌活性を比較した。歯周病菌活性はN-ベンゾイル-DL-アルギニルペプチターゼの酵素活性をADplit®（アドプリット：共立製薬株式会社）により測定した。ADplitによる歯周病菌活性測定は、BANA分解活性酵素検査よりも動物病院において簡易的に実施ができ、P. gulaeから特異的に産生されるトリプシン様酵素であるN-ベンゾイル-DL-アルギニルペプチターゼの酵素活性を測定することができる。その結果、歯肉炎指数、チオール濃度および歯周病菌活性がCPC+Pt含有製剤投与群で無処置群と比較して有意に低下し、歯みがき等のデンタルケアと併用することでその効果は増大することが示唆された。歯科処置後に歯みがき等の物理的なデンタルケアとCPC+Pt含有製剤を組み合わせることによって、歯周病に対する十分な予防効果がみられることが証明された。伴侶動物の高齢化が進む現代において、歯周病罹患率はますます増加することが予想される。犬における歯科医療においてP. gulaeのfimA遺伝子型の検出と歯周病リスクの評価、および歯周病を予防するために日々のデンタルケア指導は良好な口腔内環境を維持する上で有用な手段であると思われる。doctoral thesi
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