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Therapeutic potential of ozone water treatment in alleviating atopic dermatitis symptoms in mouse models: Exploring its bactericidal and direct anti-inflammatory properties
Get PDFCurrently, ozone water is utilized for antibacterial and antiviral purposes without any reported safety concerns. Therefore, ozone water may have clinical applications in treating staphylococcal-specific cutaneous diseases, such as atopic dermatitis (AD) and pyoderma. This study aimed to verify the bactericidal effects of ozone water at different concentrations (3 and 11 mg/L) against staphylococcal species in vitro, as well as evaluate the anti-inflammatory effects of ozone water in a mouse model of AD and pyoderma. Initially, the bactericidal properties of several concentrations of ozone water were confirmed with Staphylococcus aureus and methicillin-resistant S. pseudintermedius. Both 3 and 11 mg/L of ozone water exhibited a significant bactericidal effect against staphylococci at less than 100 times dilution. We next examined the cellular cytotoxicity and cytokine production (Interleukin (IL)-6 and IL-8) induced by S. pseudintermedius pre-treated with ozone water, and our findings indicated that cytotoxicity and cytokine production induced by staphylococci were significantly inhibited after ozone water pre-treatment. In vivo experiments showed that ozone water-pre-treated S. pseudintermedius significantly inhibited the development of pyoderma in mice; however, limited effects were observed in a therapeutic setting. Interestingly, ozone water at concentrations of 3 and 11 mg/L exhibits dual bactericidal and anti-inflammatory effects in mice with AD. This observation was corroborated by the significant inhibition of cytokine production in interferon-γ/tumor necrosis factor-stimulated human epidermal keratinocyte cells exposed to ozone in vitro. These findings indicate that administering ozone can be a novel therapeutic approach for managing allergic skin diseases, such as AD.journal articl
Reducing Effects of Whey Protein Hydrolysate on Coloration of Cured Sausages
Get PDFCuring produces a characteristic pink color during meat processing through the production of nitrosyl myoglobin (NOMb), which requires nitric oxide (NO). Nitrites and nitrates in coloring agents are crucial NO sources; however, a reducing agent is necessary to facilitate their chemical conversion to NO. This study aimed to investigate the effect of the reducing properties of whey protein hydrolysate (WPH) on the reddening of cured meat products. Cured and cooked sausage models were treated with WPH, which enhanced the reddening of the meat color and increased the a* value in the models compared with that of the controls. Additionally, ethanol-extracted WPH induced Fe3⁺ reduction, lowered oxidation–reduction potential, and decreased nitrite (NO2−) levels. Moreover, ethanol-extracted WPH promoted the formation of NOMb in myoglobin solution. This effect was also observed when ethanol-extracted WPH treated with maleimide was used, implying that certain peptides rather than the thiol group of WPH are involved in promoting NOMb formation. Furthermore, the peptides that decreased NO2− levels were isolated from ethanol-extracted WPH, identified, and synthesized. These synthesized peptides, particularly the FFVAPFPEVFGK peptide, showed NO2−-reducing activity. Hence, WPH may promote the coloration of cured meat products through the reducing potential of the peptides contained within.journal articl
Long-term monitoring of huddling behavior in mice using online image processing
Get PDFMany animal species, including mice, form societies of numerous individuals for survival. Understanding the interactions between individual animals is crucial for elucidating group behavior. One such behavior in mice is huddling, yet its analysis has been limited. In this study, we propose a cost-effective method for monitoring long-term huddling behavior in mice using online image processing with OpenCV. This method treats a single mouse or a group of mice as a cluster of pixels (a ‘blob’) in video images, extracting and saving only essential information such as areas, coordinates, and orientations. This approach reduces data storage needs to 1/200000th of what would be required if the video were recorded in its compressed form, thereby enabling long-term behavioral analysis. To validate the performance of our algorithm, ~2000 video frames were randomly chosen. We manually counted the number of clusters of mice in these frames and compared them with the number of blobs automatically detected by the algorithm. The results indicated a high level of consistency, exceeding 90% across the selected video frames. Initial observations of both male and female groups suggested some variations in huddling behavior among male and female groups; however, these results should be interpreted cautiously due to a small sample. Group behavior is known to be disrupted in several neuropsychiatric disorders, such as autism. Various mouse models of these disorders have been proposed. Our measurement system, when combined with drug or genetic modification screening, could provide a valuable tool for high-throughput analyses of huddling behavior.journal articl
Exploratory study of volatile fatty acids and the rumen-and-gut microbiota of dairy cows in a single farm, with respect to subclinical infection with bovine leukemia virus
Get PDFBackground
Subclinical infection with bovine leukemia virus (BLV) in cows can cause economic losses in milk and meat production in many countries, as BLV-related negative effects. The volatile fatty acids (VFAs) and microbiota present in the digestive tracts of cows can contribute to cow health. Here, we exploratorily investigated the VFAs and microbiota in the rumen and gut with respect to subclinical BLV infection using cows housed at a single farm.
Results
We analyzed a herd of 38 cows kept at one farm, which included 15 uninfected and 23 BLV-infected cows. First, the analysis of the VFAs in the rumen, gut, and blood revealed an absence of statistically significant differences between the uninfected and BLV-infected groups. Thus, BLV infection did not cause major changes in VFA levels in all tested specimens. Next, we analyzed the rumen and gut microbiota. The analysis of the microbial diversity revealed a modest difference between the uninfected and BLV-infected groups in the gut; by contrast, no differences were observed in the rumen. In addition, the investigation of the bacteria that were predominant in the uninfected and BLV-infected groups via a differential abundance analysis showed that no significant bacteria were present in either of the microbiota. Thus, BLV infection possibly affected the gut microbiota to a small extent. Moreover, bacterial associations were compared between the uninfected and BLV-infected groups. The results of this analysis suggested that BLV infection affected the equilibrium of the bacterial associations in both microbiota, which might be related to the BLV-related negative effects. Thus, BLV infection may negatively affect the equilibrium of bacterial associations in both microbiota.
Conclusions
Subclinical BLV infection is likely to affect the rumen and gut microbiota, which may partly explain the BLV-related negative effects.journal articl
Up- and Downregulated Genes after Long-Term Muscle Atrophy Induced by Denervation in Mice Detected Using RNA-Seq
Get PDFSkeletal muscle atrophy occurs rapidly as a result of inactivity. Although there are many reports on changes in gene expression during the early phase of muscle atrophy, the patterns of up-and downregulated gene expression after long-term and equilibrated muscle atrophy are poorly understood. In this study, we comprehensively examined the changes in gene expression in long-term denervated mouse muscles using RNA-Seq. The murine right sciatic nerve was denervated, and the mice were housed for five weeks. The cross-sectional areas of the hind limb muscles were measured using an X-ray CT system 35 days after denervation. After 28 d of denervation, the cross-sectional area of the muscle decreased to approximately 65% of that of the intact left muscle and reached a plateau. Gene expression in the soleus and extensor digitorum longus (EDL) muscles on the 36th day was analyzed using RNA-Seq and validated using RT-qPCR. RNA-Seq analysis revealed that three genes—Adora1, E230016M11Rik, and Gm10718—were upregulated and one gene—Gm20515—was downregulated in the soleus muscle; additionally, four genes—Adora1, E230016M11Rik, Pigh, and Gm15557—were upregulated and one gene—Fzd7—was downregulated in the EDL muscle (FDR < 0.05). Among these genes, E230016M11Rik, one of the long non-coding RNAs, was significantly upregulated in both the muscles. These findings indicate that E230016M11Rik could be a candidate gene for the maintenance of atrophied skeletal muscle size and an atrophic state.journal articl
Antimicrobial susceptibility of bovine clinical mastitis pathogens in Japan and development of a simplified agar disk diffusion method for clinical practice
Get PDFThis study aimed to examine the antimicrobial susceptibility of bovine mastitis pathogens in Japan and develop criteria for testing antimicrobial susceptibility using the simplified agar disk diffusion (ADD) method that is currently being used in clinical practice. Milk samples from 1,349 dairy cows with clinical mastitis were collected and cultured. The minimum inhibitory concentrations (MICs) of the antimicrobials were determined for 504 strains of 28 bacteria. Of the gram-positive bacteria, most Staphylococcus spp. were susceptible to penicillin G (PCG), kanamycin (KM), oxytetracycline (OTC), cefazolin (CEZ), pirlimycin, enrofloxacin, and marbofloxacin. Streptococcus spp. and Trueperella pyogenes showed resistance to OTC and KM. Most gram-negative bacteria were resistant to OTC and CEZ and particularly susceptible to fluoroquinolones. To develop the criteria for a disk diffusion test of the simplified ADD method, the relationships between MICs and diameters of inhibition zones (DIZs) were analyzed and compared with the conventional method. The susceptibility breakpoints of several antimicrobials were lower for both gram-positive and gram-negative bacteria. Particularly for gram-positive bacteria, the application of the new criteria lowers the breakpoint for PCG, suggesting that the use of PCG instead of CEZ may increase. The results suggest that use of these criteria for the simplified ADD method may lead to appropriate antimicrobial choice and consequently the appropriate use of antimicrobials in clinical practice.journal articl
Research for outbreaks and virulence of Erysipelothrix rhusiopathiae variant in swine
Get PDF麻布大学博士(獣医学）Erysipelothrix rhusiopathiae causes swine erysipelas (SE), which results in considerable economic loss on pig farms and recognized as one of the most important diseases in pig farm industry. In the 1970s, the spread of live vaccines in Japan decreased the occurrence of SE but it still occurs in around 2000 pigs annually, which cannot be ignored in the pig farms. Especially during SE outbreaks that occurred sporadically from 2008 to 2011 in Japan, new E.rhusiopathiae strains were isolated with a specific surface protective antigen (Spa)A protein characterized by methionine at position 203 and isoleucine at position 257 (M203/I257-SpaA type).
Based on these backgrounds, we investigated the subsequent trend of the E.rhusiopathiae M203/I257-SpaA type, which was first discovered in the survey from 2008 to 2012, with the aim of clarifying the factors of the epidemic. This research paper consists of 3 chapters: investigation of field samples affected by E.rhusiopathiae, bacteriological characterization by serotypes of E.rhusiopathiae, pathogenicity investigations in mice and pigs, the efficacy of inactivated vaccines against E.rhusiopathiae in pigs and obtained the following results.
In the first chapter, we isolated 79 strains of E.rhusiopathiae from pigs in Japan between 2012 and 2019 and identified their serovars and SpaA types. Genetic lineages of some serovar 1a strains were also identified. The pathogenicity of representative E.rhusiopathiae isolates in mice was also evaluated in comparison with the Fujisawa reference strain. Our epizootic investigation showed that serovar 1a was the most common serovar among the isolated strains (59/79) during the study period. None of the 59 serovar 1a strains had the SpaA type of the Fujisawa reference strain, while three strains were the I257-SpaA type and 56 strains were the M203/I257-SpaA type. This indicated that the M203/I257-SpaA type of E.rhusiopathiae was the prevalent strain in Japan, and that the outbreak of the M203/I257-SpaA type from 2008 to 2011 has continued until the time of this writing. Furthermore, the lineage Ⅳb-1 and Ⅳb-2 strains were isolated in the Kyushu and Honshu regions of Japan respectively, but the region-specific distribution of the E.rhusiopathiae lineages Ⅳb-1 and Ⅳb-2 were not clear in 2013, and both strains eventually co-circulated in Japan because they were isolated in the same prefecture in the same year. The disappearance thus seemed to occur within these few years from 2013 to 2019. In addition, the serovar 1a M203/I257 SpaA type of E.rhusiopathiae were more pathogenic than other serovars in the virulence confirmation test of using mice, and it was suggested that there is a high tendency to be isolated from acute symptoms pigs. On the other hand, no significant difference in virulence was found in a mice infection experiment with the Fujisawa reference strain which is the same serovar 1a. Based on these results, the epidemics of SE that have been observed in Japan in recent years are not due to strains in which a new mutation has been inserted into the base sequence of the SpaA gene of E.rhusiopathiae, and since in 2008, M203/I257 SpaA type of E.rhusiopathiae continues to infiltrate, and although it tends to be isolated from pigs with acute symptoms, its pathogenicity is not significantly different from that of the serovar 1a Fujisawa reference strain, at least in mice.
In the second chapter, to find the reason why the M203/I257-SpaA type of E.rhusiopathiae is currently the major causative agent in outbreaks, four recent E.rhusiopathiae isolates comprising of two serovar 1a with M203/I257-SpaA type strains, one serovar 1b strain, and one serovar 2a strain were compared with each other and with the serovar 1a Fujisawa reference strain regarding in vitro phenotypes and in vivo virulence in mice and pigs. The serovar 1b and 2a strains, which are the less prevalent strains in the field in Japan, showed lower growth in liquid culture and lower virulence in animals than the serovar 1a variants. Adhesion of the serovar 2a strain to porcine endothelial cells was weaker than that of the serovar 1a and 1b strains. Several advantages of serovar 1a strains were found, but no plausible cause of the M203/I257-SpaA type variants to be selected for the most prevalent strains among serovar 1a strains was identified in this study. Other feasible phenotypic assay such as the antimicrobial susceptibility test, or the studies on phagocytosis resistance and intracellular persistence may resolve this issue. We think these approaches as the further study and will continue this research from the viewpoints of antigenicity and vaccine-induced immunity as the next step.
In the third chapter, we used SER-ME containing E.rhusiopathiae Tama-96 (serovar 2a) as a representative of current inactivated vaccines to investigate whether it was effective for protecting pigs against infection with the Fujisawa reference strain or the M203/I257-SpaA type variant. Sixteen pigs were used in this study. Half of pigs (8/16) were vaccinated, and the rest were unvaccinated and used as control. Challenge tests were performed using the Fujisawa reference strain or the M203/I257-SpaA type 2012 Miyazaki variant, both of which belong to serovar 1a of E.rhusiopathiae. Vaccinated pigs in groups 1 and 3 showed no apparent clinical signs regardless of the E.rhusiopathiae strain used during the challenge, indicating that vaccination protected pigs not only from the Fujisawa reference strain but also from the newly emerged M203/I257-SpaA type 2012 Miyazaki variant. However, all the unvaccinated pigs in group 2 and 4 developed SE. In group 2, two of the four pigs died (including the humane endpoint, the same shall apply hereafter) on the third day after Fujisawa strain challenge, one pig died on the fourth day, and the remaining pig died on the ninth day. In group 4, all four pigs died on the third day after challenge. The sum of the clinical scores were not significantly different (p<0.01) between unvaccinated pigs challenged with the Fujisawa strain (group 2) versus the Miyazaki variant (group 4). Therefore, it was clarified that the cause of the domestic epidemic of M203/I257-SpaA type of E.rhusiopathiae was not caused by the vaccine failure.
Summarizing these results, we indicated three conclusions. First: the epidemic of M203/I257-SpaA type of E.rhusiopathiae continues to dominate of the isolates in Japan, Second: there was no significant difference of the growth in liquid culture, adherence of porcine endothelial cells, and the virulence in mice and pigs between M203/I257-SpaA type and Fujisawa reference strain, The third: current vaccines in Japan, including SER-ME, suggest that outbreaks in Japan are unlikely caused by vaccine failure. Since it remains unclear why the M203/I257-SpaA type variant of E. rhusiopathiae is currently the major causative agent in outbreaks, we will further analyze the pathogenicity of the variants. In the future, it will be necessary to examine the superiority of the M203/I257-SpaA type of Erysipelas swine, for example, in animals that can infect and spread E. rhusiopathiae, or in sewage and soil where E. rhusiopathiae can exist. We hope that the series of findings obtained in this study will be of help in the future control of Erysipelas swine in the pig farming industry.豚丹毒菌（Erysipelothrix rhusiopathiae）はグラム陽性の桿菌である。豚に主たる病原性を示し、世界中の養豚業界において経済的損失を与える重要な人獣共通感染症であり、わが国では届出伝染病に指定されている。豚における豚丹毒の症状は主に3つに分類され、急性・亜急性・慢性症状がある。急性症状では敗血症による突然死、亜急性症状では発熱や食欲不振に加えて最も特徴的とされる蕁麻疹（ダイヤモンド・スキン）の出現、慢性症状では関節炎、リンパ節炎および心内膜炎などを引き起こし、その症状は多岐に渡る。疾病対策には他の疾病との類症鑑別が重要となる。豚丹毒菌の菌体表層に発現するSpa（Surface protective antigen）Aタンパク質は、感染防御ならびに病原性に関わる抗原として、多くの研究が報告されている。
日本における豚丹毒菌の発生は1970年代に生ワクチンが普及してから減少しているものの、未だに年間2000頭前後の発生があり、養豚業界において決して無視できない疾病である。特に2008年から20011年にかけては血清型1aのSpaAタンパク質の203位と257位のアミノ酸がそれぞれメチオニンとイソロイシンに変化したM203/I257-SpaA変異株が新たな変異株として流行した。この他にも、少数ながら同じ血清型1aにおけるSpaAタンパク質の203位と242位のアミノ酸が、それぞれメチオニンとアラニンに置換したM203/A242-SpaA変異株および203位のアミノ酸のみがメチオニンに置換したM203-SpaA変異株も発見された。このように、これまで分離されてこなかった豚丹毒菌血清型1aのSpaA変異株の台頭が近年では見受けられている。
これらの背景をふまえ、本研究は2008年から2011年の調査で初めて見つかった豚丹毒菌SpaA変異株のその後の動向および2012年以降、我が国で流行している株の病原性について検討した。本研究は3章から構成される。野外サンプル調査、血清型別、SpaA変異株と血清型別の細菌学的特性調査並びにマウス及び豚での病原性調査、不活化ワクチンの豚丹毒菌SpaA変異株に対する豚での有効性を中心に検討し、以下のような成果を得た。
第一章：2012年から2019年に分離した豚丹毒菌株を中心に血清型、SpaA遺伝子の表現型を調査し、SpaA変異株の国内分離状況および、マウスにおける病原性を検討した。2012年から2019年に日本各地から集めた79株の豚丹毒菌の血清型、SpaA遺伝子塩基配列の変異部分の解析およびLineage型別を行った。調査した79株のうち、59株が血清型1aに属しており、その59株のうち56株が2008年から2011年の調査で初めて見つかった株と同じ血清型1aのM203/I257-SpaA変異株であった。2012年以降も血清型1aのM203/I257-SpaA変異株の流行の継続が明らかとなった。さらには、血清型1aのM203/I257-SpaA変異株のLineage型別をすると、これまで主に九州地域（LineageⅣb-1）と本州地域（LineageⅣb-2）で分離した株が、2012年以降では日本全国に分布し、地域性が消失していることが確認できた。また、マウスを用いた病原性確認試験において血清型1aのM203/I257-SpaA変異株は血清型2aの株と比較して病原性が強いことが示唆された。一方、同じ血清型1aで病原性比較対照株である藤沢株とM203/I257-SpaA変異株においては大きな病原性の違いはないことが明らかとなった。これらの結果より、わが国における2012年以降の豚丹毒菌の流行は豚丹毒菌のSpaA遺伝子の塩基配列に新たな変異が挿入された株などによるものではなく、2008年から引き続いて血清型1aのM203/I257-SpaA変異株の地域性が混在し、流行が拡大していることが明らかとなった。また血清型1aのM203/I257-SpaA変異株はマウスを用いた病原性試験では藤沢株と同様な病原性を示す強毒株であることが判明した。
第二章：2012年以降の分離株から血清型1aのM203/I257-SpaA変異株を2株、血清型1b、2a株をそれぞれ1株ずつ選択した合計4株を、病原性比較対照株として広く用いられている血清型1aの藤沢株とin vitro の細菌学的特性およびin vivoの病原性の違いを比較検討し、M203/I257-SpaA変異株が国内で流行拡大を続ける原因を検索した。国内での分離数が少ない血清型2a株は血清型1a株と比較してin vitroでの増殖性が低く、豚血管内皮細胞との細胞接着性が低く、マウスや豚におけるin vivo病原性も低いことが明らかとなった。豚を用いた動物試験では、病原性を臨床症状スコアで評価すると、血清型1a株と血清型2a株の間、血清型1a株と血清型1b株の間、さらには血清型1b株と血清型2a株の間においても病原性の臨床症状スコアに有意差が認められた。以上のことより、血清型1a株と1b株、2a株の間に病原性の違いを見出すことが出来た。さらに血清型1b株と血清型2a株は第一章より分離数が少ないことも踏まえると、我が国で分離数が最も多い血清型1aのM203/I257-SpaA変異株は藤沢株同様に、豚へも強い病原性をもち、我が国に広く浸潤していることを裏付ける結果となった。
第三章では豚丹毒菌不活化ワクチンの血清型1aのM203/I257-SpaA変異株に対する豚での有効性を検討した。豚を4頭ずつ4つの群に振り分け、群1と群3に豚丹毒菌不活化ワクチンSER-ME（スワインテクトSER-ME、日生研株式会社）を投与し、群2と群4を非接種対照群とした。接種菌として群1、2に病原性比較対照株である血清型1aの藤沢株を、群3、4に血清型1aのM203/I257-SpaA変異株（2012年宮崎株）を用いた。非接種対照群である2つの群は菌接種後、臨床観察期間中に全頭が死亡した。一方でワクチンを投与した2つの群は臨床観察を示すことなく試験終了まで生存した。以上より、豚丹毒菌不活化ワクチンは豚で血清型1aのM203/I257-SpaA変異株に対しても従来株同等に有効であることを示した。従って、血清型1aのM203/I257-SpaA変異株の流行拡大の原因はワクチン投与後のブレイクスルー感染によるものではないことが示唆された。
以上の結果より、（１）血清型1aのM203/I257-SpaA変異株の流行拡大は現在も続いていること（２）最も流行している血清型1aのM203/I257-SpaA変異株はマウスや豚への病原性が強いこと（３）血清型1aのM203/I257-SpaA変異株の流行拡大の原因は豚での感染・防御試験によりワクチン投与後のブレイクスルー感染によるものではないことが明らかとなった。
豚丹毒菌は豚のみならず、家禽や多くの動物、自然環境から分離されている。今後は血清型1aのM203/I257-SpaA変異株が野生動物を含めた他の宿主や自然環境中に分布している可能性などを考慮し、広範囲な調査を続けることが、豚の豚丹毒の予防と制御に必要であると思われる。本研究で得られた一連の知見が今後の養豚業界における豚丹毒菌制御の一助になれば幸いである。doctoral thesi
Development of the novel quantification for bacterial toxins in cow’s milk
Get PDF麻布大学博士(獣医学)Cow’s milk is a highly nutritious food and has supported human health. However, because the cow’s milk is an animal-derived food, it is not completely free from bacterial toxins. Cow’s milk contaminated by cereulide (CRL) produced by Bacillus cereus and staphylococcal enterotoxin type A (SEA) produced by Staphylococcus aureus caused extensive outbreaks of gastroenteritis. The individual methods for identifying and quantifying CRL and SEA had their own problems and needed to be improved. Furthermore, there is a need for an analytical method capable of measuring two bacterial toxins that require differentiation. The aim of this study was to reliably identify the presence of CRL and SEA in cow’s milk under identical conditions using liquid chromatography-tandem mass spectrometry (LC-MS/MS). An accurate quantitative method was also developed with easy and simple sample preparations.
The presence of CRL in cow’s milk was identified and quantified using our validated method with LC-MS/MS. CRL was concentrated using protein acid-precipitation and extracted from the precipitate by using acetonitrile twice. The combination of protein acid-precipitation and extraction sufficiently eliminated the matrix compounds from the milk and a further clean-up step utilising solid-phase extraction could be omitted. For robustly measuring the samples and keeping the MS devices clean, the extraction solution was diluted 10-fold using methanol. Owing to the minimisation of the interferences caused by fragmentation patterns, multiple reaction monitoring information-dependent acquisition-enhanced product ion spectra enabled the characterisation and identification of CRL. Besides the matrix effect (−4%), an external solvent calibration curve was adapted for accurate quantification. The method was validated using fortified recovery tests, at two concentrations (10 and 50 ng/g), using three samples daily on five different days based on the Japanese guidelines. This new method exhibited good accuracy ranging from 91% to 94%. The relative standard deviations (RSD) of repeatability ranged from 2% to 5%, and the RSD of within-laboratory reproducibility ranged from 5% to 6%. These standard deviations satisfied the criteria for the Japanese validation guidelines. The limit of quantification (LOQ) was estimated to be 2 ng/g. On the product ion spectra at the LOQ level, the library match was satisfactory with a purity fit value of >70%. Thus, this study developed a practical screening method for quantifying CRL and proposed an operation model with conventional individual methods for verifying the trueness.
SEA contaminant was quantified in cow’s milk by LC–MS/MS with the use of a stable isotope-labelled peptide of SEA as an internal standard. SEA was cleaned up in a two-step process that included pH control and trichloroacetic acid (TCA) precipitation. The pH control phase eliminated other proteins. TCA precipitation cleaned up SEA without special equipment. An appropriate enzyme-to-protein ratio maximised tryptic digestion. A desalting process guaranteed the stable retention of SEA-digested peptides. The coverage of amino-acid sequences (>10%) clearly identified the toxin’s presence. SEA was accurately quantified using LC-MS/MS based on a multiple-reaction monitoring mode. The developed method was validated based on spiked recovery tests at 50 and 100 ng/g conducted with three samples collected on a daily basis for five days based on Japanese validation guidelines. The new method exhibited good accuracy which ranged from 80% to 82%. The RSD of repeatability were 13–14% and the RSD of within-laboratory reproducibility were 13–18%. These standard deviations satisfied the criteria of the Japanese validation guidelines. The LOQ was estimated to be 10 ng/g. By optimizing a sample preparation method for peptide analysis by LC-MS/MS, this study established an accurate reliable method for SEA quantification according to the criteria of the guideline.
To verify the applicability of the developed methods to raw milk, a fortified test using a special milk sample as an alternative was tested. In the fortified tests using the milk samples with 10 ng/g CRL (n = 3), the recovery and RSD were 81.1% and 1.6%, respectively. In fortified tests using the milk samples with 50 ng/g SEA (n = 3), the recovery and RSD were 78.8% and 9.1%, respectively. The suitability of the methods was tested and gave satisfactory results. Additionally, a survey of the actual residues of CRL and SEA was conducted. The developed method was applied to 14 raw milk and 8 milk samples pasteurised using the low-temperature, long-time process and collected in Tokyo. None of the samples was found to contain the target toxin.
In this study, an analytical method that can measure the two bacterial toxins, CRL and SEA, was developed using the same condition via LC-MS/MS. An accurate and reliable quantitative method was also developed with easy and simple sample preparations. The developed method was applied to a survey in Tokyo, and it was confirmed that there were no residues of CRL and SEA in cow’s milk samples. The developed method is expected to contribute to the identification of causative agents of food poisoning and the prevention of health hazards. Furthermore, it is expected that this study has contributed to the quality assurance of cow’s milk and the strengthening of the inspection system in food hygiene.牛乳は古くから高栄養食品として人々の健康を支えてきた。しかしながら、牛乳は動物由来食品であるため、細菌性毒素の汚染を完全に防除することができず、セレウス菌が産生するセレウリド（CRL）や黄色ブドウ球菌が産生するエンテロトキシンA（SEA）が牛乳に残留したことで、大規模な集団食中毒事件が発生してきた。CRLとSEAを同定かつ定量する個別試験法にはそれぞれの課題があり、改良が必要であった。特に、鑑別が必要な2つの細菌性毒素を測定できる信頼性の高い分析法が求められていた。本研究の目的は、液体クロマトグラフ-タンデム型質量分析計（LC-MS/MS）を用いて、牛乳中CRLとSEAを同一の条件で連続して測定し、再現性高く定量するとともに、簡単かつシンプルな前処理法を構築することとした。
LC-MS/MSによる牛乳中のCRLの同定および定量法を開発した。酸性化によるタンパク質沈殿法によって牛乳中のCRLを濃縮し、アセトニトリルによる2回の抽出で回収した。これらの組み合わせにより、牛乳から夾雑成分を除去し、固相精製を用いたクリーンアップステップを省略した。さらに、再現性の良い測定と検出器を清浄に保つために、抽出溶液をメタノールで10倍に希釈した。その結果、フラグメンテーションへの干渉を最小限に抑え、プロダクトイオンスペクトルによるCRLの同定が可能となった。加えて、夾雑成分の影響を-4%とすることで、より簡便な外部標準検量線を採用し、正確な定量値を得た。開発した本法について、牛乳を対象試料に妥当性評価を行った。CRLを2濃度（10、50 ng/g）で添加し、1試験者が1日3併行5日間の回収試験を実施した。その結果、本法は充分な選択性を有しており、真度91-94%、併行精度2-5%、室内精度5-6%であった。定量下限値（LOQ）は2 ng/gに設定できた。各パラメータは農薬等の試験法開発に使用される厚生労働省ガイドライン基準に適合し、本法が試験検査に適応できる分析性能を有していることを確証した。さらに、LOQのプロダクトイオンスペクトルにおいて，標準品との一致率は70%以上であり、LOQまで確実かつ精確に同定・定量する本法の有用性を確証した。本研究は再現性の高いCRLスクリーニング法を開発し、従来の個別法と併用することで真度の検証といった運用を提案した。
(Koike, H., et al. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 35:2424-2433. 2018)
SEAの安定同位体標識ペプチドを内部標準物質として用い，LC-MS/MSにより牛乳中のSEA定量法を開発した。本研究ではトリプシン消化したSEA由来ペプチドを測定することで、牛乳中のSEAを確実かつ精確に同定・定量する。まず、SEA由来ペプチドを3つ選定し、SEA全体の10%以上のアミノ酸配列を網羅することで、LC-MS/MSを使用して未知タンパク質を同定するための基準を満たす同定性能を確保した。次に、CRLと同じLC条件の適用を検証した結果、これらペプチドを十分に分離できることから、2つの細菌性毒素を効率よく迅速に鑑別できることとなった。前処理では、牛乳中のSEAをpH調整とトリクロロ酢酸（TCA）沈殿法の2段階のプロセスで採集した。pH調整によってカゼインを取り除き、SEAを選択的に濃縮した。TCA沈殿法では、特別な器具を使用せずにSEAと他のタンパク質を分別した。また、酵素とタンパク質の比率を最適化することで、トリプシン消化効率を最大かつ安定化した。さらに、脱塩操作によって、分離を阻害する夾雑成分を取り除き、SEA由来ペプチドの保持時間を保証し再現性を高めた。開発した本法について、牛乳を対象試料に妥当性評価を行った。SEAを2濃度（50、100 ng/g）で添加し、1試験者が1日3併行5日間の回収試験を実施したところ、充分な選択性を有しており、真度80-82%、併行精度13-14%、室内精度13-18%であった。定量下限値は10 ng/gに設定できた。各パラメータは農薬等の試験法開発に使用される厚生労働省ガイドライン基準に適合し、本法が試験検査に適応できる分析性能を有していることを確証した。本研究はLC-MS/MSによるペプチド測定に特化した前処理法を開発することで、先行研究よりも高精度かつガイドライン基準を満たす信頼性の高いSEA定量法を確立した。
(Koike, H., et al. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 36:1098-1108. 2019)
開発した分析法を生乳に応用するため、生乳の代替となる特別牛乳を用いて添加回収試験を実施した。CRL 10 ng/gを添加した結果（n=3）では、回収率81.1%、RSD1.6%であった。SEA 50 ng/gを添加した試験（n=3）では、回収率およびRSDはそれぞれ78.8%および9.1%であった。本法の妥当性を検証した結果、十分な結果が得られた。さらに、CRLおよびSEAの実残留量の調査を実施した。東京都内で採取または購入した生乳14検体および低温殺菌牛乳8検体に本法を適用した。いずれの試料からも対象毒素であるCRLとSEAを検出しなかった。
以上のことから、本研究はCRLとSEAの2つの細菌毒素を同一のLC-MS/MS条件で連続して測定できる分析法を開発した。また、簡便な前処理法による精確かつ再現性の高い定量が可能となった。開発した本法を東京都内の実態調査に適用し、流通する牛乳類に定量下限値以上のCRLとSEAの残留がないことを確証した。開発した本法が食中毒の病因物質の同定や健康被害の防止に寄与するとともに、本研究が牛乳の品質保証や食品衛生における検査体制の強化に貢献することが期待される。doctoral thesi
Negative Influence of Aging on Differentiation and Proliferation of CD8+ T-Cells in Dogs
Get PDFImmunosenescence is an age-related change in the immune system characterized by a reduction in naïve T-cells and an impaired proliferative capacity of CD8+ T-cells in older individuals. Recent research revealed the crucial impact of immunosenescence on the development and control of cancer, and aging is one of the causes that diminish the therapeutic efficacy of cancer immunotherapies targeting CD8+ T-cell activation. Despite dog cancer being defined as an age-related disease, there are few fundamental understandings regarding the relationship between aging and the canine immune system. Therefore, we aimed to elucidate the characteristics of immunosenescence in dogs and analyzed the effects of aging on the differentiation status and proliferation of canine CD8+ T cells using T-cell specific stimulation with anti-canine CD3/CD28 antibody-coated beads and interleukin-2. As a result, we found that older dogs have a lower proliferative capacity of CD8+ T-cells and a reduction in the naïve subset in their peripheral blood. Further analysis showed that older dogs had attenuated proliferation of the effector and central memory subsets. These results indicate the importance of maintaining less differentiated subsets to expand CD8+ T-cells in dogs and provide helpful insight into the development of dog immune therapies that require T-cell expansion ex vivo.journal articl