Endothelial cells release soluble factors that support the long-term survival of filarial worms in vitro.

The inability to maintain filarial nematodes in long-term in vitro culture greatly limits research into the basic biology of these parasites and hinders in vitro screening of novel anti-filarial agents. In this study, we sought to characterize nutrients that promote the long-term survival of filarial worms in vitro. Using microfilariae (MF) obtained from gerbils infected with Litomosoides sigmodontis, a filarial parasite of rodents, we found that Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) resulted in MF survival of only 5 days. However, co-culturing MF with a mouse endothelial cell line (EOMA) enabled survival for 40 days. Culturing EOMA cells in transwell plates extended MF survival to the same degree as direct co-culture, suggesting that the factors microfilariae require are soluble in nature. Heat inactivation of EOMA conditioned media at 56 °C reduced MF survival by approximately 50%, and heat inactivation at 100 °C reduced survival to 3 days, demonstrating that both heat labile and heat stable factors are involved. EOMA cells require FBS to produce these factors, as conditioned media collected from EOMA cells grown in the absence of FBS failed to prolong survival. The removal of lipids also abrogated survival, indicating MF are likely utilizing lipid factors released by EOMA cells. Dialysis experiments demonstrate that at least some of the required factors are between 0.1 and 1 kDa in size. Importantly, L. sigmodontis adult worms also show significantly extended survival when cultured in EOMA conditioned media. Together, these results suggest that EOMA-produced factors include lipid-containing molecules, heat labile molecules (likely a protein), and micronutrients between 0.1 and 1 kDa in size. These studies have established a cell-free approach to maintaining MF and adult stage filarial worms in long-term in vitro culture and have taken important steps towards biochemically characterizing host-derived nutrients required for parasite survival.

The host-parasite relationships in pyridoxine (vitamin B6) deficient cotton rats infected with Litomosoides carinii (Nematoda: Filaroidea).

This paper demonstrates that the establishment and growth of the filarial nematode parasite, Litomosoides carinii, is reduced in pyridoxine-deficient cotton rats. Young cotton rats were assigned to one of three dietary: vitamin B6-deficient cotton rats (B6-AL) were fed a pyridoxine-free diet ad libitum; pair-fed controls (B6 + PF) were fed the same amount of pyridoxine-free diet as animals in the deficient group and given daily oral supplements of 100 micrograms pyridoxine; and pyridoxine-sufficient controls (B6 + AL) were fed the pyridoxine-free diet ad libitum and supplemented daily with 100 micrograms pyridoxine. Half of each group was infected with 50 L3 of L. carinii by subcutaneous injection 8 weeks after the start of the experimental feeding period. B6-deficient cotton rats ate less (P < 0.001) and gained less weight (P < 0.001) than B6-supplemented controls. The levels of microfilaraemia in deficient animals, measured weekly throughout the experiment by taking blood smears, was significantly lower than in supplemented animals (P < 0.001). The deficient rats became latent for L. carinii at 20 weeks post-infection, whereas there was patent microfilaraemia in rats in the other dietary groups until the end of the experiment. Smaller (P < 0.001) and fewer (P < 0.05) adult worms were recovered from the pleural and abdominal cavities of deficient animals than from either pair-fed or sufficient controls at autopsy 28 week post-infection.

Radiolabeling of Brugia malayi infective larvae in mosquitoes with 75Se-methionine and detection of these larvae in tissues of the Mongolian jird by autoradiography.

Through preliminary experiments, an effective method for radiolabeling Brugia malayi-infected mosquitoes in order to produce labeled infective Brugia larvae was developed. Starting on the 6th day after the infective blood meal, mosquitoes were fed a 7% sucrose solution containing 100 microCi/ml 75Se-L-methionine for 5 days. Infective larvae, retrieved 2 days after this labeling period, averaged 381 +/- 136 counts/min. Jirds were infected with these infective, labeled larvae either by allowing infected mosquitoes to feed on uninfected jirds for 30 min or by inoculating jirds subcutaneously in the groin with washed larvae recovered from mosquitoes. Jirds were killed at various times after infection and were sliced into approximately 0.5 mm thick sagittal sections, which were dried and placed on X-ray film. Autoradiograms were developed after 30-60 days at 5 degrees C. In a sample of 26 inoculated jirds, approximately 30% of the infecting larvae could subsequently be accounted for as Ag degrees foci on autoradiograms. The Ag degrees foci representing larvae were apparent up to 2.5 weeks after infection. In jirds infected by mosquito feeding, the Ag degrees associated with the feeding site persisted for more than 6 weeks after infection.