Obihiro University of Agriculture and Veterinary Medicine
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Molecular epidemiology and genotypic diversity of equine piroplasma parasites
Get PDF帯広畜産大学博士（獣医学）Doctor of Veterinary Medicine2023application/pdf馬ピロプラズマ症（EP）は、Theileria equiとBabesia caballiによる2種類のマダニ媒介性赤血球内寄生性原虫によって引き起こされる馬の重篤な感染症である。これらの原虫に感染すると血管内で溶血が起こり、感染馬に発熱、貧血、黄疸、血色素尿などの臨床症状を引き起こし、最終的には死に至る場合がある。EPに関連する経済的損失には、治療費、流産や死亡による生産性損失、国際的な馬の移動の制限などが挙げられる。そのため、馬産業の利益を維持・確保するためにはEPの疾病制御が極めて重要となる。現在のEPの治療・予防戦略には、化学療法、マダニ駆除、馬の移動制限などが必要となる。しかし、治療薬の効果の低さや副作用、薬剤耐性マダニの出現、診断テストの不確かさなどにより、これらの戦略はあまり有効ではない。
EPは、T. equiとB. caballiを媒介できる特定マダニ種の生息分布と一致して世界的に広く発生が見られる。感染馬は持続的に原虫を保有することが知られており、T. equiは終生、またB. caballiは最長4年間持続感染する。これらの持続感染馬は重要な感染源となり、マダニの媒介により他の馬に感染を広げていく。このことから、EP清浄国である日本、オーストラリア、カナダ、アイスランド、ニュージーランド、グリーンランドでは馬を輸入する際に厳しい規制を設けてT. equiおよびB. caballiの持ち込みを監視している。一方で、EPの発生状況が依然不明な国が多く存在する。これらの国々の状況は、その国内および世界の馬産業に甚大な経済的損失をもたらす危険性がある。そのため、EPの発生が不明な国々におけるT. equiとB. caballiの感染疫学調査は重要である。
本研究では、スリランカとパラグアイで疫学調査が行われた。農業を基盤する両国は家畜動物の数も多い。また、その気候がマダニの活動に適しており、かつ大規模な牛群を飼育していることから、マダニの蔓延とマダニ媒介性疾患の発生は家畜動物で一般的と考えられている。しかしながら、媒介可能なマダニ種が存在するスリランカとパラグアイではEPに関する疫学調査が実施されていなかった。そこで、スリランカではロバを、パラグアイでは馬におけるT. equiおよびB. caballiの感染疫学調査を行った。
第1章では、スリランカで野生化した計111頭のロバから血液を採取し、血液塗抹標本を作製するとともに、そのDNAも抽出した。血液塗抹標本は顕微鏡検査に供試し、DNAサンプルを用いてT. equiとB. caballi-特異的PCR法によるスクリーニング診断を行った。その結果、顕微鏡検査とPCR診断によるT. equiの陽性率は、それぞれ57.7％と85.6％となった。一方で、すべてのロバは顕微鏡検査とPCR診断の両方でB. caballiは検出されなかった。T. equi陽性DNAサンプルから分離された18S rRNA配列の系統学的解析から、スリランカのロバはT. equiの遺伝子型CとDに感染していることが判明した。本研究の成果から、スリランカで減少しつつあるロバ個体群を保全する取り組みにおいてEPに対処することの重要性が明らかとなった。
第2章では、パラグアイで飼育されている計545頭の馬を対象にT. equiとB. caballiの感染について疫学調査を行った。その結果、PCR診断による両種の検出率はそれぞれ32.7％と1.5％となった。そのうち2頭（0.4％）の馬がT. equiとB. caballiの両方に共感染していた。T. equiの感染率は、馬の品種間、雌雄間、1～3歳と3歳以上の年齢層間で有意差は認めれらなかった。また、T. equiまたはB. caballiに単独感染した馬の血液学的指標の平均値は正常範囲内であり、非感染馬のそれと同等であった。一方で、2頭の共感染馬はその血液学的指標で明らかな貧血を示し、EPは貧血馬の鑑別診断項目に含めるべきであることが示唆された。系統学的解析の結果から、パラグアイの馬はT. equi 18S rRNA遺伝子型AとC、またB. caballi rap-1遺伝子型Bに感染していたことが明らかとなった。
第3章では、5つのT. equi遺伝子型（A～E）に対する遺伝子型特異的PCR法を開発し、スリランカのロバおよびパラグアイの馬から採取した計270のT. equi陽性血液DNAサンプルについて解析を行った。その結果、開発された遺伝子型特異的PCR法を用いることで、スリランカのロバサンプルから4つの遺伝子型（A、C、D、E）が、またパラグアイのサンプルから5つの遺伝子型すべてが検出されることが示された。さらに、スリランカでは全サンプル、パラグアイでは93.3％のサンプルで少なくとも1つの遺伝子型の感染が確認され、このPCR法の感度の高さが証明された。本PCR法は、スリランカとパラグアイのそれぞれ90.2％と22.5％のサンプルにおいて、様々な組み合わせの共感染も検出した。また、遺伝子型特異的PCR法で得られたアンプリコンの遺伝子配列から、本PCR法の特異性も証明された。これらの結果から、本遺伝子型特異的PCR法は、T. equiの遺伝子型を区別して検出できる有益なツールであることが確認された。
一連の調査研究の結果は、EPの発生状態が不明であっても媒介可能なマダニ種が蔓延している国であれば、T. equiおよびB. caballi感染が流行している可能性が高いことを示唆している。EPの状態が不明な地域でこれらの感染疫学調査を積極的に行うことは、効果的な疾病制御を促進する上で重要となる。また、T. equi用に開発された遺伝子型特異的PCR法を用いて、その遺伝子型の多様性を考慮した防疫戦略を策定すれば、EPに対する効果的な疾病制御に繋がる。結論として、本研究の成果は世界的なEPの制御に役立つものとなった。博士学位論文大学院畜産学研究科 獣医学専攻Doctoral Program of Veterinary Sciencedoctoral thesi
The possible role of haematophagous flies in the incidence of bovine teat papillomatosis
Get PDFapplication/pdfThe relationship between the incidence of bovine teat papillomatosis and the activity of haematophagous flies was investigated in Japan. A total of 15,737 flies consisting of 33 species were collected by dry ice-baited mosquito net (DMN) trap and a sweep net from udders of cattle. Simulium aokii (Takahasi) of Simuliidae (black flies) was the predominant species, followed by S. tobetsuense Ono and S. iwatense (Shiraki). Simulium aokii had the highest peak in October, followed by September. Numbers of blood spots from the bites per teat in nulliparous cattle were significantly correlated with numbers of S. aokii collected by DMN trap. Numbers of teats with warts and spots of blood from the bites per teat were significantly more abundant in anterior teats than posterior teats. The average incidence of teat papillomatosis in nulliparous cattle was significantly higher than that in parous cattle, and the highest incidence by month was in May, followed by April. Although bovine papillomavirus (BPV) DNA was not detected in flies examined, the presence of black flies and blood spots from their bites were associated with subsequent high incidence of growing warts. In particular, it would pay to give attention to species such as S. aokii that severely attack udders in the present locality. Further investigations for the detection of BPV DNA from flies parasitizing on teats are needed.journal articl
Acaricidal activity of Erigeron acer L. root against Haemaphysalis longicornis and phytochemical profiling by liquid chromatography-tandem mass spectrometry
Get PDFapplication/pdfThe present study is focused on evaluating acaricidal activity and chemical compositions of Erigeron acer root, which was identified as a promising candidate among fifteen Mongolian plant extracts tested for acaricidal activity. The acaricidal effect was evaluated against Haemaphysalis longicornis, assessed for toxicity to normal human skin fibroblast, and analyzed for its chemical constituents. The acetone extract of E. acer root showed significant activity against H. longicornis, with a lethal concentration (LC50) of 5.31 mg/mL and low toxicity, evidenced by a cytotoxic concentration (CC50) of 267.00 µg/mL. Using liquid chromatography-tandem mass spectrometry and molecular networking, thirteen natural compounds were identified, including pyrrolidines, alkaloids, fatty acids, and flavonoids, highlighting the efficacy of E. acer root extract as an effective acaricide against H. longicornis and offering insights for developing new tick control solutions.journal articl
Mechanisms of interleukin-10 induction in murine spleen and RAW264 cells by Latilactobacillus curvatus K4G4 isolated from fermented Brassica rapa L.
Get PDFapplication/pdfLactic acid bacteria (LAB) are commonly used in fermented foods, and some LAB modulate the immune response. We aimed to investigate the mechanism by which LAB isolates from fermented Brassica rapa L. induce the production of anti-inflammatory interleukin (IL)-10 by the murine spleen and RAW264 cells. Spleen cells from BALB/c mice or the mouse macrophage cell line RAW264 were cultured with heat-killed LAB isolated from fermented B. rapa L., and the IL-10 level in the supernatant was measured. Latilactobacillus curvatus K4G4 provided the most potent IL-10 induction among 13 isolates. Cell wall components of K4G4 failed to induce IL-10, while treatment of the bacteria with RNase A under a high salt concentration altered K4G4 induction of IL-10 by spleen cells. In general, a low salt concentration diminished the IL-10 induction by all strains, including K4G4. In addition, chloroquine pretreatment and knock down of toll-like receptor 7 through small interfering RNA suppressed K4G4 induction of IL-10 production by RAW264 cells. Our results suggest that single-stranded RNA from K4G4 is involved, via endosomal toll-like receptor 7, in the induction of IL-10 production by macrophages. K4G4 is a promising candidate probiotic strain that modulates the immune response by inducing IL-10 from macrophages. © 2024 BMFH Pressjournal articl
Supplementation of rumen-protected lysine during the close-up period improves vaginal discharge clearance in Holstein dairy cows
Get PDFapplication/pdfWe aimed to evaluate the effects of rumen-protected lysine (RPL) supplementation during the close-up period on uterine involution and the resumption of ovarian function in dairy cows. Fifty-two multiparous Holstein cows were categorized based on parity and expected calving date and randomly assigned to the RPL or control (CON) groups. The RPL group received 80 g of RPL daily from day 21 before the expected calving date until parturition. Blood samples were obtained twice weekly from pre-supplementation to 6 weeks postpartum. The onset of luteal activity postpartum was determined via ultrasonography twice weekly for up to 6 weeks postpartum. Uterine involution was tracked at 3 and 5 weeks postpartum through the vaginal discharge score, percentage of polymorphonuclear cells (PMN) in endometrial cytology samples, presence of intrauterine fluid, and gravid horn diameter via ultrasonography. Before supplementation, the RPL group showed amino acid imbalance, which was improved by RPL supplementation. There were no significant differences in the onset of luteal activity, percentage of PMN, intrauterine fluid, or the diameter of the uterine horn between the two groups. The vaginal discharge score in the RPL group decreased from 3 to 5 weeks postpartum, whereas that in the CON groups did not decrease. The number of cows with clinical endometritis was lower in the RPL group. Overall, RPL supplementation during the close-up period enhanced vaginal discharge clearance, potentially averting clinical endometritis, but did not affect the first ovulation in dairy cows.journal articl
Effect of Raw and Cooked Green Bananas on Cecal Fermentation Characteristics in Rats
Get PDFapplication/pdfRaw and cooked (autoclaved) green bananas are fed to rats for 4 weeks and their effects on cecal microbiota and biomarkers of fermentation are evaluated and compared with those of potato starch, which is rich in RS. Relative abundances of genera Lachnospiraceae_NK4A136_group and Roseburia, and species Bifidobacterium pseudolongum in rat cecum are higher in the raw green banana group with higher RS consumption than in the control group. The raw green banana group shows higher cecal acetate production and similarly high levels of cecal n-butyrate production than that in the potato starch group. In the autoclaved green banana group, the cecal microbial composition is altered and the relative abundance of family Ruminococcaceae increased. Additionally, the molar proportion of n-butyrate in the cecum of the autoclaved green banana group is higher than that in the control group. The levels of cecal mucin and IgA in the raw green banana and potato starch groups are found to be higher compared to those observed in the control and autoclaved green banana groups. Therefore, raw and autoclaved green bananas can alter cecal microbial composition and thus exhibit beneficial intestinal fermentation characteristics.journal articl
Morphological variation of tail bone among two chicken breeds and their F1 progeny
Get PDFapplication/pdfFancy breeds of Japanese indigenous chicken display extensive morphological diversity, particularly in tail feathers. Although marked differences in tail and bone traits have been reported between Tosa-jidori (wild type) and Minohikichabo (rich type) breeds, little is known about the pattern of genetic inheritance in cross experiments. Therefore, this study aimed to investigate the strain and sex effects, and inheritance patterns, in the morphometric variation of pygostyle bones among Tosa-jidori, Minohikichabo, and their F1 hybrids. Five morphological traits, angle of the apex of the pygostyle, pygostyle length, margo cranialis length, tail feather number, and body weight, were evaluated at the adult stage. A significant strain difference was detected in all traits, whereas significant sex differences were observed in only three traits, but not in the angle of the apex of the pygostyle and tail feather number. In F1 hybrids, the angle of the apex of the pygostyle was significantly different to that of Tosa-jidori but not that of Minohikichabo, whereas the pygostyle length and tail number of F1 hybrids were significantly different from those of Minohikichabo but not those of Tosa-jidori. A significant heterosis effect was found in the margo cranialis length and body weight. All five traits showed nonadditive inheritance patterns but varied in each trait between partial dominance (angle of the apex of pygostyle), full dominance (pygostyle length and tail feather number), and over-dominance (margo cranialis length and body weight). Interestingly, different patterns of genetic inheritance in the F1 hybrid were observed at different locations, even within the same pygostyle bone. Using the Japanese indigenous chicken model, these results provide a substantial step toward understanding the genetic architecture of morphology in chickens. © 2024 Wiley Periodicals LLC.journal articl
Genetic parameters for novel mastitis traits defined by combining test-day somatic cell score and differential somatic cell count in the first lactation of Japanese Holsteins
Get PDFapplication/pdfIn this study, we aimed to improve current udder health genetic evaluations by addressing the limitations of monthly sampled somatic cell score (SCS) for distinguishing cows with robust innate immunity from those susceptible to chronic infections. The objectives were to (1) establish novel somatic cell traits by integrating SCS and the differential somatic cell count (DSCC), which represents the combined proportion of polymorphonuclear leukocytes and lymphocytes in somatic cells and (2) estimate genetic parameters for the new traits, including their daily heritability and genetic correlations with milk production traits and SCS, using a random regression test-day model (RRTDM). We derived 3 traits, termed ML_SCS_DSCC, SCS_4_DSCC_65_binary, and ML_SCS_DSCC_binary, by using milk loss (ML) estimates at corresponding SCS and DSCC levels, thresholds established in previous studies, and a threshold established from milk loss estimates, respectively. Data consisted of test-day records collected during January 2021 through March 2022 from 265 herds in Hokkaido, Japan. From these records, we extracted records between 7 to 305 d in milk (DIM) in the first lactation to fit the RRTDM. The model included the random effect of herd-test-day, the fixed effect of year-month, fixed lactation curves nested with calving age groups, and random regressions with Legendre polynomials of order 3 for additive genetic and permanent environmental effects. The analysis was performed using Gibbs sampling with Gibbsf90+ software. The averages (ranges) of the daily heritability estimates over lactation were 0.086 (0.075–0.095) for SCS, 0.104 (0.073–0.127) for ML_SCS_DSCC, 0.137 (0.014–0.297) for SCS_4_DSCC_65_binary, and 0.138 (0.115–0.185) for ML_SCS_DSCC_binary; the heritability curve for SCS_4_DSCC_65_binary was erratic. Genetic correlations within the trait decreased as the DIM interval widened, especially for those integrating DSCC, indicating that these traits should be analyzed using RRTDM rather than repeatability models. The averages (ranges) of genetic correlations with milk yield over lactation were 0.01 (−0.22 to 0.28) for SCS, −0.05 (−0.40 to 0.13) for ML_SCS_DSCC, −0.08 (−0.17 to 0.09) for SCS_4_DSCC_65_binary, and −0.08 (−0.22 to 0.27) for ML_SCS_DSCC_binary. Compared with SCS, the newly defined traits exhibited slightly stronger negative genetic correlations with milk yield. Especially in late lactation stages, the genetic correlation between ML_SCS_DSCC and milk yield was significantly below zero, with a posterior median of −0.40. Furthermore, the new traits showed positive correlations with SCS, having estimates varying from 0.68 to 0.85 for ML_SCS_DSCC, 0.14 to 0.47 for SCS_4_DSCC_65_binary, and 0.61 to 0.66 for ML_SCS_DSCC_binary, depending on DIM. Considering that ML_SCS_DSCC and ML_SCS_DSCC_binary have relatively high heritability (compared with SCS) and favorable genetic correlations with milk production traits and SCS, their incorporation into breeding programs appears promising. Nevertheless, their genetic relationships with (sub)clinical mastitis require further investigation.journal articl