Abstract
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.
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Acknowledgements
The authors thank Rossella Panarese (Department of Veterinary Medicine of the University of Bari, Italy) for her help in the sample collection. D.O. was partially supported by EU funding, within the NextGenerationEU-MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases (Project no. PE00000007, INF-ACT).
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Sample collection and processing: Ranju Ravindran Santhakumari Manoj and Younes Laidoudi; conceptualization: Ranju R. S. Manoj, Oleg Mediannikov, Maria Stefania Latrofa, and Domenico Otranto; supervision: Oleg Mediannikov, Florence Fenollar, Maria Stefania Latrofa, and Domenico Otranto; methodology: Ranju R. S. Manoj, Meriem Louni, Oleg Mediannikov, and Florence Fenollar; investigation: Ranju R. S. Manoj, Meriem Louni, and Oleg Mediannikov; data curation: Ranju R. S. Manoj, Younes Laidoudi, Meriem Louni, Oleg Mediannikov, and Maria Stefania Latrofa; writing — original draft: Ranju R. S. Manoj, Maria Stefania Latrofa, and Younes Laidoudi; writing — review and editing: Ranju R. S. Manoj, Younes Laidoudi, Meriem Louni, Oleg Mediannikov, Maria Stefania Latrofa, Florence Fenollar, and Domenico Otranto. All the authors read and approved the final manuscript.
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Manoj, R.R.S., Latrofa, M.S., Louni, M. et al. In vitro maintenance of the endosymbiont Wolbachia of Dirofilaria immitis. Parasitol Res 122, 939–943 (2023). https://doi.org/10.1007/s00436-023-07789-4
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DOI: https://doi.org/10.1007/s00436-023-07789-4