Owings Charity G, Skaggs Christine, Sheriff Winyu, Manicke Nicholas, Picard Christine J
Department of Biology, Indiana University-Purdue University Indianapolis, Indianapolis, IN.
Department of Chemistry, Indiana University-Purdue University Indianapolis, Indianapolis, IN.
Environ Entomol. 2018 Jun 6;47(3):586-593. doi: 10.1093/ee/nvy027.
Filth flies are commonly implicated in pathogen transmission routes due to their affinity for vertebrate waste and their synanthropic associations. However, solidifying the link between flies and infected feces in the wild can be difficult, as interpretations made solely from microbial culturing or sequencing methods may represent an incomplete picture of pathogen acquisition. We present an analytical assay using high performance liquid chromatography tandem mass spectrometry (HPLC MS/MS) to detect vertebrate fecal metabolites (urobilinoids) in adult blow fly guts. Proof of concept experiments consisted of controlled feeding in which flies were grouped into three treatments (unfed, exposure to beef liver tissue, and exposure to canine feces; N = 20/treatment) using the black blow fly Phormia regina Meigen (Diptera: Calliphoridae). It was revealed that only feces-related samples exhibited peaks with an m/z of 591 and MS/MS spectra consistent with urobilinoids. These peaks were not seen for beef liver tissue, flies exposed to beef liver tissue, or unfed flies. Samples taken directly from beef liver tissue and from feces of several animals were also tested. To test this assay in wild flies, 216 flies were additionally analyzed to determine whether they had ingested vertebrate feces. About 13% of the wild flies exhibited these same peaks, providing a baseline measure of blow flies collected in urban and residential areas consuming feces from the environment. Overall, this assay can be used for P. regina collected in an applied setting and its integration with microbial culturing and sequencing methods will help to improve its use.
由于其对脊椎动物粪便的偏好以及与人类的共生关系，腐蝇通常被认为是病原体传播途径中的一环。然而，在野外确定苍蝇与感染粪便之间的联系可能很困难，因为仅通过微生物培养或测序方法做出的解释可能无法完整呈现病原体获取的情况。我们提出了一种使用高效液相色谱串联质谱法（HPLC MS/MS）来检测成年丽蝇肠道中脊椎动物粪便代谢物（尿胆素类）的分析方法。概念验证实验包括对照喂养，将黑丽蝇（Phormia regina Meigen，双翅目：丽蝇科）分为三组处理（未喂食、暴露于牛肝组织、暴露于犬粪便；每组N = 20）。结果显示，只有与粪便相关的样本出现了质荷比为591的峰，且串联质谱图与尿胆素类一致。牛肝组织、暴露于牛肝组织的苍蝇或未喂食的苍蝇均未出现这些峰。还对直接取自牛肝组织和几种动物粪便的样本进行了测试。为了在野生苍蝇中测试该分析方法，另外分析了216只苍蝇，以确定它们是否摄入了脊椎动物粪便。约13%的野生苍蝇出现了相同的峰，这为在城市和居民区收集的摄食环境中粪便的丽蝇提供了一个基线测量值。总体而言，该分析方法可用于在实际环境中收集的黑丽蝇，将其与微生物培养和测序方法相结合将有助于改进其应用。
