The clastogenic effect of adult Spirocerca lupi secretory products on murine fibroblasts

Abstract

Spirocerca lupi (S. lupi) is a nematode that parasitizes vertebrates, particularly canids. In 25% of Spirocerca infections in dogs, nodules progress from inflammatory to pre-neoplastic and eventually to sarcomatous neoplasia within a few months. Researchers have postulated that the parasite itself induces the sarcomatous transformation through the excretory / secretory products (ESPs) thought to contain growth factor-like substances, possibly proteins and/or chemicals. With the mechanism of the sarcomatous neoplastic transformation being incompletely understood, the objective of this study was to investigate whether adult S. lupi parasite ESPs could induce proliferation (carcinogenicity) in primary mammalian fibroblast cell cultures. The adult S. lupi parasite ESPs were also investigated by chromatography for presence of potential clastogens. The mammalian fibroblasts were harvested from Balb/c mice pinnae, prepared and maintained in-vitro using Dulbecco’s Modified Eagle’s Medium (DMEM) and supplemented with 10 % foetal bovine serum (FBS). Live adult S. lupi parasites were obtained from dogs at necropsy. The parasites were subsequently cultured in various media (RPMI 1640 Medium, Iscove’s Modified Dulbecco’s Medium, Dulbecco’s Modified Eagle’s Medium, Ham’s F12 Medium and saline) and maintained at 37 °C in an incubator in order to obtain worm ESPs. The adult S. lupi parasite ESPs obtained from the culture media were extracted and dissolved in organic solvents (Ethylacetate, Methanol, Acetone and Hexane) at different dilutions (10, 20 and 30 μl) and exposed to the cultured fibroblasts. The ESPs extracted from media did not induce an increase in mitosis compared to the controls. The ESPs were further analysed using thin layer chromatography (TLC) and liquid chromatography-coupled mass-spectroscopy (LC-MS/MS). Chromatography revealed the Iscove’s media to be richest in worm ESPs. LC-MS/MS revealed nine compounds (301.3625 m/z, 400.2112 m/z, 450.8195 m/z, 464.8737 m/z, 538.1112 m/z, 580.2783 m/z, 594.2576 m/z, 660.5320 m/z, 682.5770 m/z) in adult S. lupi parasite ESPs, for which library comparison revealed to be proteins similar to those isolated from Nematostella vectensis, Caenorhabditis brenneri and Sus scrofa. The protein Caebren was also identified. We conclude that the essential media (Iscove’s, DMEM, RPMI and Ham’s F12) do not contain the necessary nutrients required for the survival of the parasites. The media in which the parasites were incubated, whilst rich in compounds, were also unable to induce a direct clastogenic effect in cultured murine fibroblasts. As a result, it would appear that the neoplastic transformation induced by the parasite is not due to the excretion of simple clastogenic proteins or chemicals and more importantly, may actually be related to the parasite actively feeding. Further work is therefore required to ascertain the nutrient requirements of the S. lupi parasite, in order to study its clastogenic effect which seems likely to be of protein origin.