Manoj Ranju Ravindran Santhakumari, Latrofa Maria Stefania, Louni Meriem, Laidoudi Younes, Fenollar Florence, Otranto Domenico, Mediannikov Oleg
Department of Veterinary Medicine, University of Bari, Valenzano, Italy.
Aix Marseille University, IRD, AP-HM, MEPHI, 13385, Marseille, France.
Parasitol Res. 2023 Apr;122(4):939-943. doi: 10.1007/s00436-023-07789-4. Epub 2023 Feb 22.
Wolbachia has an obligatory mutualistic relationship with many onchocercid nematodes of the subfamilies Dirofilariinae and Onchocercinae. Till date, no attempts have been made for the in vitro cultivation of this intracellular bacterium from the filarioid host. Hence, the current study attempted cell co-culture method using embryonic Drosophila S2 and the LD cell lines to cultivate Wolbachia from Dirofilaria immitis microfilariae (mfs) harvested from infected dogs. Microfilariae (mfs = 1500) were inoculated in shell vials supplemented with Schneider medium using both cell lines. The establishment and multiplication of the bacterium were observed during the initial inoculation, at day 0 and before every medium change (from days 14 to 115). An aliquot (50 µl) from each time point was tested by quantitative real-time PCR (qPCR). Comparing the average of Ct values, obtained by the tested parameters (i.e., LD/S2 cell lines and mfs with/without treatment), the S2 cell line without mechanical disruption of mfs provided the highest Wolbachia cell count by qPCR. Despite the maintenance of Wolbachia within both S2 and LD-based cell co-culture models for up to 115 days, a definitive conclusion is still far. Further trials using fluorescent microscopy and viable staining will help to demonstrate the cell line infection and viability of Wolbachia. Use of considerable amount of untreated mfs to inoculate the Drosophilia S2 cell lines, as well as the supplementation of the culture media with growth stimulants or pre-treated cells to increase their susceptibility for the infection and development of a filarioid-based cell line system are recommended for the future trials.
沃尔巴克氏体与盘尾丝虫亚科和盘尾丝虫亚科的许多盘尾丝虫线虫存在 obligatory 互利共生关系。迄今为止，尚未尝试从丝虫类宿主中对这种细胞内细菌进行体外培养。因此，当前的研究尝试使用果蝇胚胎 S2 和 LD 细胞系的细胞共培养方法，从感染犬只体内采集的犬恶丝虫微丝蚴（mfs）中培养沃尔巴克氏体。使用这两种细胞系，将微丝蚴（mfs = 1500）接种到补充有 Schneider 培养基的贝壳瓶中。在初始接种时、第 0 天以及每次更换培养基之前（从第 14 天到第 115 天）观察细菌的建立和增殖。每个时间点的等分试样（50 μl）通过定量实时 PCR（qPCR）进行检测。比较通过测试参数（即 LD/S2 细胞系以及有/无处理的 mfs）获得的 Ct 值平均值，未对 mfs 进行机械破坏的 S2 细胞系通过 qPCR 提供了最高的沃尔巴克氏体细胞计数。尽管在基于 S2 和 LD 的细胞共培养模型中沃尔巴克氏体维持了长达 115 天，但仍远未得出明确结论。使用荧光显微镜和活细胞染色的进一步试验将有助于证明细胞系感染和沃尔巴克氏体的活力。建议在未来的试验中使用大量未处理的 mfs 接种果蝇 S2 细胞系，以及在培养基中添加生长刺激剂或预处理细胞，以提高它们对丝虫类细胞系系统感染和发育的敏感性。
