Optimization of culture and analysis methods for enhancing long-term Brugia malayi survival, molting and motility in vitro

Abstract

Lymphatic filariasis is a neglected tropical disease caused by roundworm parasites such as Brugia malayi that spread via a mosquito vector. In vitro culture of these parasites provides controlled conditions to understand parasite biology and provides a cheaper way to screen potential micro-and macrofilaricides. Published studies have used a wide array of approaches and metrics regarding in vitro cultures of B. malayi; as a result, drawing comparisons and identifying the reasons why inability to reproduce outcomes are difficult. This study sought to determine conditions that ensure reproducible outcomes and used evaluation metrics that are easily measured and can be automated to ensure objectivity. We found culturing B. malayi third-stage larvae (L3) in endothelial basal media supplemented with 20% fetal bovine serum and 75 µM ascorbic acid in a temperature-and humidity-controlled incubator produced better survival and molting rates as well as longer and more motile parasites than previously reported. The benefit of ascorbic acid seemed to be unique to L3 parasites, as the addition of ascorbic acid to adult parasites had no significant impact on survival or motility. The methods reported in this study will help in designing experiments for both parasite behaviour studies and drug screening applications for disease eradication.

A mouse infection model and long-term lymphatic endothelium co-culture system to evaluate drugs against adult Brugia malayi

“…Excised B. malayi derived from experimentally infected Meriones unguiculatus (Mongolian gerbils) exhibit rapid loss of viability in serum-supplemented media which is reflected by upregulation of a number of stress-response genes over the first five days in culture [42]. A recent study utilising image-based motion analysis demonstrated decline in female B. malayi PLOS NEGLECTED TROPICAL DISEASES motility (a late-stage indicator of in vitro fitness) begins at 5-7 days in optimised serum supplemented medium culture [43]. Due to this rapid demise in vitro, moderate automated throughput screening utilising motility phenotype as a readout of Brugia female macrofilaricidal activity has been limited to a three-day exposure window [44].…”

Particles in the Eluate from Double Filtration Plasmapheresis—A Case Study Using Field Emission Scanning Electron Microscopy/Energy-Dispersive X-ray Spectroscopy (FE-SEM/EDX)

“…Preparation of the eluate samples were conducted under a laminar flow cabinet to avoid contamination with particles and filaments in the air and a specific protocol was used to perform the FE-SEM/EDX analysis in order to avoid any contamination of the sample and instrument. The thread-like object in Figure 4d, however, has a similar morphology and size as a threadlike parasitic nematode (roundworm) of the superfamiliy Filarioridea [56,57]. Different Filarioridea nematodes exist and can infect humans, including Wuchereria bancrofti, Brugia malayi, Brugia timori, Onchocerca volvulus, Loa loa, Mansonella ozzardi, Mansonella perstans, Mansonella streptocerca and Brugia pahangi [58].…”

Preliminary evaluations of 3-dimensional human skin models for their ability to facilitate in vitro the long-term development of the debilitating obligatory human parasite Onchocerca volvulus

“…Traditional 2-dimensional (2-D) culture systems were shown to support the early stages of development for few filarial nematodes in vitro , with some of these parasitic worms the cultures could be maintained long-term. The reported in vitro survival rates ranged from 33% after 77 days for Mansonella perstans L3 [ 17 ], 69% after 30 days for Brugia malayi L3 [ 18 ], and 60–90% for Loa loa L3 over 15 to 17.8 days in culture [ 19 , 20 ]. Recently, we described a novel 2-D in vitro culturing system based on a feeder layer of human umbilical vein endothelial cells (HUVEC) that has successfully supported the development of O .…”