Brannelly Laura A, Roberts Alexandra A, Skerratt Lee F, Berger Lee
One Health Research Group, College of Public Health, Medical and Veterinary Sciences, James Cook University; Department of Biological Sciences, University of Pittsburgh;
One Health Research Group, College of Public Health, Medical and Veterinary Sciences, James Cook University.
J Vis Exp. 2018 May 16(135):57345. doi: 10.3791/57345.
Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which infects and disrupts frog epidermis; however, pathological changes have not been explicitly characterized. Apoptosis (programmed cell death) can be used by pathogens to damage host tissue, but can also be a host mechanism of disease resistance for pathogen removal. In this study, we quantify epidermal cell death of infected and uninfected animals using two different assays: terminal transferase-mediated dUTP nick end-labelling (TUNEL), and caspase 3/7. Using ventral, dorsal, and thigh skin tissue in the TUNEL assay, we observe cell death in the epidermal cells in situ of clinically infected animals and compare cell death with uninfected animals using fluorescent microscopy. In order to determine how apoptosis levels in the epidermis change over the course of infection we remove toe-tip samples fortnightly over an 8-week period, and use a caspase 3/7 assay with extracted proteins to quantify activity within the samples. We then correlate caspase 3/7 activity with infection load. The TUNEL assay is useful for localization of cell death in situ, but is expensive and time intensive per sample. The caspase 3/7 assay is efficient for large sample sizes and time course experiments. However, because frog toe tip biopsies are small there is limited extract available for sample standardization via protein quantification methods, such as the Bradford assay. Therefore, we suggest estimating skin surface area through photographic analysis of toe biopsies to avoid consuming extracts during sample standardization.
两栖动物在全球范围内正经历着生物多样性的巨大损失，其中一个主要原因是传染病壶菌病。这种疾病由真菌病原体蛙壶菌（Batrachochytrium dendrobatidis，简称Bd）引起，它会感染并破坏青蛙的表皮；然而，其病理变化尚未得到明确的表征。细胞凋亡（程序性细胞死亡）可被病原体用来损害宿主组织，但也可以是宿主抵抗疾病以清除病原体的一种机制。在本研究中，我们使用两种不同的检测方法对受感染和未受感染动物的表皮细胞死亡进行量化：末端转移酶介导的dUTP缺口末端标记法（TUNEL）和半胱天冬酶3/7检测法。在TUNEL检测中，我们使用腹部、背部和大腿皮肤组织，观察临床感染动物表皮细胞的原位细胞死亡情况，并通过荧光显微镜将细胞死亡情况与未感染动物进行比较。为了确定表皮中的细胞凋亡水平在感染过程中如何变化，我们在8周的时间内每两周采集一次趾尖样本，并使用提取的蛋白质进行半胱天冬酶3/7检测，以量化样本中的活性。然后我们将半胱天冬酶3/7活性与感染负荷相关联。TUNEL检测对于原位细胞死亡的定位很有用，但每个样本成本高且耗时。半胱天冬酶3/7检测对于大样本量和时间进程实验很有效。然而，由于青蛙趾尖活检样本较小，通过蛋白质定量方法（如Bradford检测法）进行样本标准化时，可用的提取物有限。因此，我们建议通过对趾部活检样本的照片分析来估计皮肤表面积，以避免在样本标准化过程中消耗提取物。
