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The effects of trans-chalcone and chalcone 4 hydrate on the growth of Babesia and Theileria
Get PDFapplication/pdfBACKGROUND: Chemotherapy is a principle tool for the control and prevention of piroplasmosis. The search for a new chemotherapy against Babesia and Theileria parasites has become increasingly urgent due to the toxic side effects of and developed resistance to the current drugs. Chalcones have attracted much attention due to their diverse biological activities. With the aim to discover new drugs and drug targets, in vitro and in vivo antibabesial activity of trans-chalcone (TC) and chalcone 4 hydrate (CH) alone and combined with diminazene aceturate (DA), clofazimine (CF) and atovaquone (AQ) were investigated. METHODOLOGY/PRINCIPAL FINDINGS: The fluorescence-based assay was used for evaluating the inhibitory effect of TC and CH on four Babesia species, including B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi, the combination with DA, CF, and AQ on in vitro cultures, and on the multiplication of a B. microti-infected mouse model. The cytotoxicity of compounds was tested on Madin-Darby bovine kidney (MDBK), mouse embryonic fibroblast (NIH/3T3), and human foreskin fibroblast (HFF) cell lines. The half maximal inhibitory concentration (IC50) values of TC and CH against B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi were 69.6 ± 2.3, 33.3 ± 1.2, 64.8 ± 2.5, 18.9 ± 1.7, and 14.3 ± 1.6 μM and 138.4 ± 4.4, 60.9 ± 1.1, 82.3 ± 2.3, 27.9 ± 1.2, and 19.2 ± 1.5 μM, respectively. In toxicity assays, TC and CH affected the viability of MDBK, NIH/3T3, and HFF cell lines the with half maximum effective concentration (EC50) values of 293.9 ± 2.9, 434.4 ± 2.7, and 498 ± 3.1 μM and 252.7 ± 1.7, 406.3 ± 9.7, and 466 ± 5.7 μM, respectively. In the mouse experiment, TC reduced the peak parasitemia of B. microti by 71.8% when administered intraperitoneally at 25 mg/kg. Combination therapies of TC-DA and TC-CF were more potent against B. microti infection in mice than their monotherapies. CONCLUSIONS/SIGNIFICANCE: In conclusion, both TC and CH inhibited the growth of Babesia and Theileria in vitro, and TC inhibited the growth of B. microti in vivo. Therefore, TC and CH could be candidates for the treatment of piroplasmosis after further studies.journal articl
Inhibitory effects of methanolic Olea europaea and acetonic Acacia laeta on growth of Babesia and Theileria
Get PDFapplication/pdfObjective: To evaluate the antipiroplasmic activities of methanolic extract of Olea europaea (MOE) and acetonic extract of Acacia laeta (AAL) against Babesia and Theileria parasites in vitro and evaluate the chemotherapeutic effects of these extracts against Babesia (B.) microti in vivo. Methods: Fluorescence assay using SYBR Green 1 nucleic acid stain was used to detect inhibitory effects of the two extracts as well as the combination effects of the two extracts with diminazene aceturate and atovaquone on four Babesia species and Theileria equi in vitro while for in vivo experiments, 8-weekold female BALB/c mice were injected intraperitoneally with 1 × 107B. microti-iRBCs and treated orally at a dose of 150 mg/kg of both extracts. Results: The half maximal inhibitory concentration (IC50) values of AAL against B. bovis, B. bigemina, B. divergens, B. caballi, and Theileria equi were lower than those of MOE extracts. Toxicity assay on Madin-Darby bovine kidney, mouse embryonic fibroblast (MH/3T3), and human foreskin fibroblast cell lines showed that MOE and AAL affected only the viability of Madin-Darby bovine kidney cell line with half maximal effective concentrations (EC50) of (794.7±41.9) and (873.9±17.5) μg/mL, respectively. The oral treatments of MOE and AAL at 150 mg/kg inhibited the growth of B. microti in mice by 80.4% and 64.4%, respectively. The MOE and diminazene aceturate combination showed a higher chemotherapeutic effect than that of monotherapy. Conclusions: MOE and AAL have the potential to be an alternative remedy for treating piroplasmosis. Furthermore, the combination therapy of MOE + DA was more potent against B. microti infection in mice than their monotherapies.journal articl
Association between mycoplasma pneumonia outbreaks in calves and mycoplasma mastitis in milking cows on dairy farms
Get PDFapplication/pdfTo clarify the possibility of Mycoplasma mastitis in milking cows from Mycoplasma pneumonia outbreaks in calves, Mycoplasma bovis was isolated over time from nasal swabs of calves and bulk tank milk at two dairy farms where no cases of Mycoplasma mastitis occurred. Isolates were genotypically analyzed using pulsed-field gel electrophoresis. At both farms, Mycoplasma bovis was first detected in bulk tank milk after pneumonia outbreaks in calves, and the genotypes of the isolates from nasal swabs and milk were consistent. These findings indicate that outbreaks of Mycoplasma pneumonia in calves is one of the possible etiologies of Mycoplasma mastitis in dairy herds.journal articl
Pilot study on serum C-reactive protein in pet rabbits: Clinical usefulness
Get PDFapplication/pdfObjectives The present study was performed to evaluate the usefulness of serum C-reactive protein (CRP) as an acute phase reactive protein in pet rabbits in clinical practice. Methods The CRP level using a rabbit CRP ELISA and white blood cell (WBC) count in pet rabbits (30 healthy controls and 62 with various diseases) were measured in the clinical practice setting. The CRP level and WBC count were measured before and after ovariohysterectomy of a healthy rabbit and a rabbit with uterine adenocarcinoma. The association between the CRP level and mortality in rabbits with various diseases was assessed. Results The CRP level in healthy controls was 0.52±0.82 mg/dl (mean±SD). No age and sex-related differences in neither the CRP level nor WBC count were observed in the healthy control rabbits. The CRP levels in rabbits with gastrointestinal disease (n=22, 11.74±22.89 mg/dl), reproductive and urinary system disease (n=20, 21.19±49.68 mg/dl), dental disease (n=6, 4.87±5.47 mg/dl) and musculoskeletal disease (n=4, 85.66±107.28 mg/dl) were significantly higher than those in healthy controls. The CRP levels in rabbits with neurological disease (n=7, 2.55±1.79 mg/dl) and dermatological disease (n=3, 8.84±7.71 mg/dl) were higher than those in healthy controls, but no significant difference was observed. The WBC counts were not significantly different between rabbits with diseases and healthy controls. Serum samples were collected from two rabbits before and after ovariohysterectomy. In both rabbits, the CRP peaked on postoperative day 1, but no obvious WBC peak was observed. The mortality rate increased as the CRP level increased; the mortality rate was significantly higher in rabbits with a CRP level of ≥100 mg/dl than of <10 mg/dl. Conclusions This study indicates that the serum CRP level is useful to determine the disease status, monitor the treatment course and evaluate the prognosis in pet rabbits in clinical practice.journal articl
５品種プラムの果皮と果肉に含まれるポリフェノール組成と機能性
Get PDFapplication/pdfプラム5 品種（プレジレント、ソルダム、ベイラー、パープルアイ、ケルシー）の果皮と果肉に含まれるポリフェノール組成、DPPH ラジカル消去活性および還元力を検討した。ソルダム果皮のポリフェノール含量が最も高く100g 乾物重量当たり17.4g で、プレジデント、ベイラーおよびケルシー果皮のそれは6 ～ 7g、パープルアイは最も低く2g であった。また、5 品種のプラム共に、果皮のポリフェノール含量は果肉のそれよりも高い値を示した。果皮と果肉の両者において、ポリフェノール含量とDPPH ラジカル消去活性および還元力との間に、正の相関が認められた。プレジレント、ベイラー、パープルアイおよびケルシーの主なモノマー型ポリフェノールは、ケルセチン、ルチン、クロロゲン酸類で、3 - カフェオイルキナ酸の割合が最も高かった。最も機能性の高かったソルダム果皮は、オリゴマー型ポリフェノールを最も多く含んでいた。主成分分析の結果は、ソルダム果皮ポリフェノールの高い抗酸化活性とプロシアニジン、アントシアニン類およびケルセチンの含量と関連のあることを示している。また、ソルダム果肉ポリフェノールは、果皮のそれよりも強いリパーゼ阻害活性を示した。In the present study, we investigated polyphenols, DPPH radical scavenging activity and reducing power in peel and flesh of 5 plum varieties (Purple eyes, Soldum, President,Valor, Kelsey). The peel of Solidum showed the highestpolyphenol content with 17.4 g/100 g dry weight, and those of President, Valor and Kelsey were 6-7 g/100 g dry weight.The polyphenol content of Purple eye was the 2 g/100 g dry weight, it is the lowest among 5 plum varieties. Moreover,polyphenol content in peel was higher than that of fresh in 5 plum varieties. There were high positive correlations between polyphenol content and DPPH radical scavenging activity,reducing power both in peel and flesh. For President, Valor,Purple eyes and Kalsey, quercetin, rutin, and chlorogenicacid were the main monomer polyphenols, and 3-CQA as a kind of chlorogenic acid,it had the largest proportions in all the polyphenol compounds. The highest functionalities of polyphenols were found in Soldum peel, which also contained the most oligomeric polyphenols. The result of principal component analysis showed that anthocyanins, procyanidin and quercetin were related to antioxidant activity in Soldum peel. Moreover, polyphenols of Soldum flesh exhibited stronger lipase inhibitory activity than that of peel.departmental bulletin pape
Improvement of SYBR Green I−based fluorescence assay reading procedure for anti−babesial drugs screening in vitro
Get PDFapplication/pdfDrug susceptibility of the Babesia parasite in vitro has been used to assess the anti-babesial activities of new candidates. Recently, the in vitro drug susceptibility assay has been performed by SYBR Green I (SG I) -based fluorescence assay. Herein, we investigated the validity of a fluorescence assay for the shipping process under various culture conditions. The highest fluorescence signal was observed in freeze-thaw of Babesia and Theileria parasites cultures incubated with lysis buffer containing SG I for a half hour. This result indicates the ability of this additional step to lysis of infected RBCs completely with releasing of parasite nucleic acid. Such that, the nucleic acid can interact with SG I stain. All tested drugs showed potential anti-babesial activity and slightly high selective index against the in vitro growth of the Babesia and Theileria parasites. The IC50 values generated using the fluorescence assay with additional freeze-thaw step is comparable with those of IC50 values obtained from the unmodified fluorescence assay and microscopy method.journal articl
Functional characterization of dense granule protein 9 in Toxoplasma gondii
Get PDF帯広畜産大学博士（畜産衛生学）2019application / PDF博士学位論文
大学院畜産学研究科博士後期課程 畜産衛生学専攻
Doctoral Program in Animal and Food Hygiene
Graduate School of Animal Husbandrydoctoral thesi
Development of unstable resistance to diminazene aceturate in Babesia bovis
Get PDFapplication/pdfDiminazene aceturate (DA) is commonly used in the treatment of bovine babesiosis caused by Babesia bovis. In this study, we attempted to develop resistance in B. bovis in vitro to DA and clofazimine (CF, a novel antibabesial agent) using short- and long-term drug pressures. In the short term, we found that 6.7 ± 2 (0.54 ± 0.16 μM)-, 12.9 ± 8.6 (1.05 ± 0.7 μM)-, and 14 ± 5.9 (1.14 ± 0.48 μM)-fold increases in the half-maximal inhibitory concentration (IC 50 ) of DA were demonstrated on B. bovis cultivated with 0.04 μM of DA pressure for 4, 8, and 12 days, respectively, as compared to that on parental culture (0.08 ± 0.0065 μM) before drug pressure was initiated. However, in B. bovis cultivated with 0.04 μM of DA pressure after 16 days, the parasites could not tolerate 0.8 μM of DA. In the long term, 7.6 ± 3.5-, 20.5 ± 0.1-, and 26.8 ± 5.5-fold increases in the IC 50 of DA were demonstrated on parasites from subcultures at days 8, 3, and 5 post-cultivation, respectively, in a drug-free medium, where these subcultures were obtained from B. bovis cultivated with DA pressure with changing doses for 30, 60, and 90 days, respectively. However, the second and third times, no increase was demonstrated on B. bovis from these subcultures at days 15 and 30 post-cultivation in a drug-free medium. In addition, in B. bovis cultivated with drug pressure after 90 days, the parasites tolerate up to 0.64 μM DA. All findings demonstrated that DA resistance in B. bovis is unstable and lost within 15 days of drug withdrawal. However, treatment with subtherapeutic doses of DA in cattle might result in the development of resistance in B. bovis, which may not even respond to subsequent treatments with high doses of DA. Thus, if the bovine babesiosis caused by B. bovis is unresponsive to DA, treatment with other antibabesial agents might be recommended. © 2019journal articl
Specific increase of Fusobacterium in the faecal microbiota of neonatal calves infected with Cryptosporidium parvum
Get PDFapplication/pdfThe faecal microbiota plays a critical role in host health, with alterations in the human faecal microbial composition associated with various conditions, particularly diarrhoeal diseases. However, little is known about microbial changes during cryptosporidiosis, one of the most important diarrhoeal diseases caused by protozoa in cattle. In this study, alterations in the faecal microbiota of neonatal calves as a result of Cryptosporidium parvum infection were investigated on a C. parvum-positive farm. Comparisons were made among groups of C. parvum-infected, rotavirus-infected, and the pathogen-negative calves. A specific increase in the abundance of Fusobacterium was observed in the faecal microbiota of C. parvum-infected animals. Diarrhoea severity increased in accordance with the abundance of C. parvum and Fusobacterium. Moreover, the specific increase of Fusobacterium appeared to be a universal feature of C. parvum infection, since neonatal calves from geographically separated areas showed the same result. These observations indicated that the growth of Fusobacterium may be an important aggravating factor of cryptosporidiosis.journal articl