小印度蒙古狼的异苯抗酸模拟分析(赫皮斯特奥罗普塔图斯)塞拉,用作口服狂犬病疫苗接种生物标记
Summary
我们提供以乙基或甲基硫磷酸为生物标志物的圈养驼鹿安慰剂口服狂犬病疫苗诱饵,并使用新型液相色谱法与串联质谱法(LC-MS/MS)方法验证诱饵的捕杀。
Abstract
小型印度驼鹿(赫皮斯特龙),是波多黎各狂犬病病毒(RABV)的宿主,每年报告的动物狂犬病病例占70%以上。控制野生生物水库的RABV循环通常是通过口服狂犬病疫苗接种(ORV)策略实现的。目前波多黎各不存在野生动物ORV项目。对口服狂犬病疫苗和各种诱饵类型的研究已经展开,取得了可喜的成果。监测ORV的成功依赖于估计目标物种的诱饵吸收,这通常涉及评估接种后RABV中和抗体(RVNA)的变化。在有活跃的野生动物或病毒计划的地区,或在RABV是动物的和在水库物种中存在RVNA背景水平的地区,这种策略可能难以解释。在这种情况下,与疫苗或诱饵基质结合的生物标志物可能很有用。我们提供16个圈养的驼鹿安慰剂ORV诱饵含有乙基-硫磷酸(et-IPA)的浓度在诱饵内和0.14%在诱饵内和0.14%的诱饵内。我们还提供了12个含有甲基-硫磷酸(me-IPA)的圈养驼鹿ORV诱饵,其浓度为0.035%、0.07%和0.14%。我们在提供诱饵之前收集了血清样本,然后每周在提供后八周内收集。我们从血清中提取硫磷酸到醋酸,并使用液相色谱/质谱法进行定量。我们通过液相色谱-质谱法分析了血清的et-IPA或me-IPA。我们发现,对于et和me-IPA,分别至少8周和4周的标记能力。两种IPA衍生物都适用于对驼鹿的ORV诱饵摄入量进行实地评估。由于在蒙古塞的标记的寿命,必须小心,不要混淆结果,使用相同的IPA衍生物在连续评估。
Introduction
狂犬病病毒(RABV)是一种负感单搁浅赖萨病毒,在食肉动物和奇罗普特拉的指令内的各种野生植物群中传播。多种驼鹿是RABV的储藏库,而小印度驼鹿是波多黎各和西半球其他加勒比岛屿中唯一的水库。1、2、3.控制野生生物水库的RABV循环通常是通过口服狂犬病疫苗接种(ORV)策略实现的。在美国(美国),这一管理活动由美国农业部/APHIS/野生动物服务局国家狂犬病管理计划(NRMP)4协调。目前波多黎各不存在野生动物ORV项目。对狂犬病疫苗和各种诱饵类型的研究已经进行,有希望的结果表明,一个ORV计划为驼鹿是可能的5,6,7,8。
监测ORV的影响依赖于估计目标物种的诱饵吸收,这通常涉及评估RV抗体血清患病率的变化。然而,在野生动物或病毒项目活跃的地区,或者在RV是动物的、RABV中和抗体(RVNA)的背景水平存在于储层物种的地区,这一策略可能具有挑战性。在这种情况下,诱饵或外部诱饵基质中包含的生物标志物可能很有用。
各种生物标记已经被用来监测诱饵的摄入在众多物种,包括熊(普罗西翁乐透)9,10, stoats(Mustela ermine)11,12,欧洲 (梅莱斯·梅莱斯)13、野猪(苏斯·斯克鲁瓦)14只,印度小猩猩15只,草原犬(西诺米卢斯多维丘斯)16只,17只,等等。在美国,操作的ORV诱饵通常包括1%四环素生物标志物在诱饵基质监测诱饵的捕杀18,19。然而,使用四环素的缺点包括越来越担心抗生素在环境中的分布,四环素的检测通常是侵入性的,需要拔牙或破坏动物才能获得骨骼样本20.罗丹胺B已被评估为各种组织中的标记物,可以用紫外线(UV)光和荧光在头发和胡须10、21中检测出来。
硫磷酸 (IPA) 是一种白色,结晶粉末,已用于评估在土狼(Canis latrans)22的诱饵消费, 北极狐 (Vulpes lagopus)23,红狐 (Vulpes vulpes)24,熊9,25、野猪14只、红鹿(塞弗乌斯·拉普胡斯)26只、欧洲12只野猪和雪铁龙(M.furo)27只,以及其他几种哺乳动物。 IPA的保留时间因物种而异,从一些动物不到两周28,29,到至少26周,在未顾胶26和超过52周的家庭狗(卡尼斯狼疮熟悉)30。保留时间也可能与剂量相关31。硫磷酸与血清白蛋白强结合,历史上通过测量血碘水平32来检测。这种间接方法被高性能液相色谱 (HPLC) 方法所取代,用 UV 检测33直接测量硫磷酸浓度,并最终使用液相色谱和质谱法 (LCMS)34,35.在这项研究中,开发了一种高度敏感和选择性的液相色谱与串联质谱法(LC-MS/MS)方法,利用多反应监测(MRM)来量化两种类似物的硫磷酸。我们的目标是使用此 LC-MS/MS 方法评估 2-(3-羟基-2,4,6-三硫多苯基)丙酸(甲基-IPA 或 me-IPA)和 2-(3-羟基-2、4、6-三硫多苯基)丁酸(乙基-IPA 或 et-IPA)的标记能力,并在 ORV 中交付诱饵圈养的蒙古人。
蒙古人被关在网箱陷阱中,诱饵上装有商业上可用的熏香肠和鱼油。蒙古被安置在单独的60厘米x60厘米x40厘米不锈钢笼子,并喂养每天口粮约50克商业干猫食品,每周两次补充与商业可用的鸡大腿。水是可用的。 我们向安慰剂或V诱饵中的圈养的驼鹿交付了IPA的两种衍生物,乙基IPA和甲基IPA。所有诱饵均由28毫米x20毫米x9毫米箔泡包组成,带有外部涂层(下同”诱饵基质”),含有粉状鸡蛋和明胶(材料表)。诱饵含有0.7 mL的水或IPA衍生物,重约3克,其中±2克为外部诱饵基质。
我们提供16个圈养的蒙古和IPA在三种浓度: 0.14% (2.8毫克和IPA在+2 g诱饵基质; 3男性[m],3雌性[f]),0.4%(0.7 mL水泡组体积为2.8毫克和IPA;3m,3f),和1.0%(0.7 mL起泡包体积为7.0毫克乙酰IPA;2m,2f)。总剂量为2.8毫克,相当于5毫克/千克27,36的剂量率,以波多黎各560克的平均驼鹿体重为基础。我们选择1%作为最高浓度,因为研究表明,某些生物标志物的味觉厌恶在某些物种37中可能发生浓度>1%。我们只提供水泡包中的1%剂量,因为絮凝剂防止溶质溶解在溶剂中充分,以均匀地融入诱饵基质。一个对照组(2米,1f)收到装满无菌水的诱饵,没有IPA。在喂食其日常维持配给期间或之前,我们于上午(上午 8 点)向驼鹿提供诱饵。大约24小时后,诱饵遗骸被移走。我们在治疗前采集血液样本,治疗后一天,治疗后每周8周。我们通过吸入亚福兰毒气来麻醉驼鹿,并通过颅面静脉穿刺收集高达1.0 mL的全血,如雪铁龙38所述。我们离心全血样本,将血清转移到冷冻液中,并将其储存在-80°C,直到分析。并非所有动物都对所有时间段进行取样,以尽量减少重复采血对动物健康的影响。对照动物在治疗后第0天进行采样,然后每周进行8周。
我们在三种浓度中交付了 me-IPA:0.035%(0.7 毫克)、0.07%(1.4 毫克)和 0.14% (2.8 毫克),全部纳入诱饵基质,每个治疗组有 2 名男性和 2 名女性。两名男性和两名女性接受充满无菌水的诱饵,没有IPA。提供时间和蒙古麻醉的诱饵如上所述。我们在治疗前第1天采集血液样本,然后在治疗后每周4周。
我们测试了血清浓度数据的正常性,并估计了不同治疗组血清IPA浓度的方法。我们使用线性混合模型来比较平均血清和IPA浓度汇集在个人之间。诱饵类型(矩阵/水泡包)是一个固定的效果,除了实验日,而动物ID是一个随机效应。所有程序均使用通用统计软件(材料表)运行,显著性在 ±0.05 处进行评估。
Protocol
Representative Results
Discussion
为研究开发的LC-MS/MS方法利用多反应监测的选择性,准确量化了ms-IPA和et-IPA中的驼鹿血清。MS/MS 检测的选择性还允许一个简单的清理方案,仅依靠醋酸酯在分析前从血清中沉淀蛋白质。
硫磷酸可溶于ACN,但几乎不溶于水。为了排除ACN萃取中的水,添加了氯化钠以迫使清水:ACN相分离,通过增加水(血清)相的离子强度。还添加了挥发性酸三氟化酸(TFA),以确保在提取过程中将硫磷酸进行质?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究部分得到了美国农业部、动植物卫生检验局、野生动物服务局、国家狂犬病管理项目和IDT Biologika(德国德绍-罗斯劳)的校内研究计划的支持。
Materials
| Acetonitrile, Optima grade | Fisher | A996 | |
| Analytical balance | Mettler Toledo | XS204 | |
| C18 column, 2.1 x 50 mm, 2.5-µm particle size | Waters Corp. | 186003085 | |
| ESI Source | Agilent | G1958-65138 | |
| Ethyl-iophenoxic acid, 97 % | Sigma Aldrich | N/A | Lot MKBP5399V |
| Formic acid, LC/MS grade | Fisher | A117 | |
| LCMS software | Agilent | MassHunter Data Acquisition and Quantitative Analysis | |
| Methyl-iophenoxic acid, 97 % (w/w) | PR EuroChem Ltd. | N/A | Lot PR0709514717 |
| Microanalytical balance | Mettler Toledo | XP6U | |
| Microcentrifuge | Eppendorf | 5415C | |
| MS/MS | Agilent | G6470A | |
| N-Evap | Organomation | 115 | |
| Oral Rabies Vaccine Baits | IDT Biologika, Dessau Rossleau, Germany | N/A | |
| Propyl-iophenoxic acid, 99 % (w/w) | PR EuroChem Ltd. | N/A | Lot PR100612108RR |
| Repeat pipettor | Eppendorf | M4 | |
| Screw-top autosampler vial caps, PTFE-lined | Agilent | 5190-7024 | |
| Sodium chloride, Certified ACS grade | Fisher | S271 | |
| Statistical Software Package | SAS Institute, Cary, North Carolina, USA | N/A | |
| Trifluoroacetic acid, 99 % | Alfa Aesar | L06374 | |
| UPLC | Agilent | 1290 Series | |
| Vortex Mixer | Glas-Col | 099A PV6 | |
| 0.2-mL pipettor tips | Eppendorf | 30089.413 | |
| 0.5-mL pipettor tips | Eppendorf | 30089.421 | |
| 1.5-mL microcentrifuge tubes | Fisher | 14-666-325 | |
| 1250-µL capacity pipette tips | GeneMate | P-1233-1250 | |
| 1-mL pipettor tips | Eppendorf | 30089.43 | |
| 2-mL amber screw-top autosampler vials | Agilent | 5182-0716 | |
| 5-mL pipettor tips | Eppendorf | 30089.456 | |
| 80-position microcentrifuge tube rack | Fisher | 05-541-2 | |
| 8-mL amber vials with PTFE-lined caps | Wheaton | 224754 | |
| 70 % (v/v) isopropanol | Fisher | A459 | |
| 100-1000 µL air displacement pipette | Eppendorf | ES-100 |
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