Comparative study of cultivation of feces in vermiculite or charcoal to obtain larvae of Strongyloides venezuelensis Steveen

Introduction: We compared feces culturing in charcoal or vermiculite to obtain Strongyloides venezuelensis larvae. Methods: Feces (5g) from infected rats was mixed with vermiculite (10g) or coal (10g) in plastic cups and incubated at 28°C for 48h. Larvae were recovered using Baermann-Moraes method. Results: Signifi cantly higher number of positive larval cultures were recovered from vermiculite than from charcoal (15/17 and 4/17, respectively; p < 0.001; 990.6 ± 307.5 and 215 ± 78.1 larvae, p = 0.027). Conclusions: Vermiculite yields more larvae and provides cleaner pellets, improving larvae identifi cation and facilitating their use for other purposes.

Cultivation of stool to obtain helminthic larvae is generally carried out with addition of different substrates or by spreading feces on plates containing solidifi ed agar nutrients 1,2 .The most commonly used culture medium is coal, which must be pure, lacking additives, and, in particular, being free from colloidal silver.Moreover, visualization of the larvae or adult worms is rendered diffi cult due to the presence of coal particles.Agar culture is laborious and requires appropriate growth medium, adequate glassware, and sterilization systems.
Vermiculite is a compound formed by hydration of basaltic mineral; it has been used as a substrate for fecal culture, particularly in veterinary parasitology, as it has the advantages of being similar to soil and costing less [3][4][5][6][7] .It has also been used for growing insect 8 or nematodes larvae in order to evaluate the larvicidal effects of fungi [9][10][11][12] .
Very few reports have compared the culturing of stools in vermiculite vs. that with addition of other media.Steffan et al. 13 compared the addition of vermiculite or polystyrene pellets to cultivate cattle feces and found that both signifi cantly improved growth of larvae of Ostertagia ostertagi and Cooperia oncophora as compared to the cultivation of feces without additives.
Agyei 14 compared the addition of vermiculite or sawdust to cultivate cattle feces to obtain L3 larvae of Haemonchus, Trichostrongylus, Cooperia, and Oesophagostomum and concluded that the number of L3 larvae obtained from sawdust cultures was signifi cantly higher than those obtained by other techniques; the former also did not require further elaborate apparatus.
As there have been no reports comparing the cultivation of feces in vermiculite and charcoal, we here compared vermiculite with charcoal as growth medium for larvae of Strongyloides venezuelensis.Feces of Wistar albino rats infected with S. venezuelensis (900 larvae, subcutaneously) were collected using an anticoprophagic cage.The eggs were quantifi ed with a McMaster chamber.Two identical samples (in terms of weight and eggs per gram) of feces were homogenized with vermiculite (Vermiculite Insulation Thermo-Acoustic Ltd., Belo Horizonte, MG; granulation 1) or coal (Tobasa Bioindustrial, Tocantinópolis, TO; granulation 4) in disposable plastic cups (200ml) at a ratio of two parts of vermiculite or charcoal to one part stool (e.g., 5g of feces and 10g of vermiculite or charcoal).The cultures were incubated at 28°C for 48h, and were humidifi ed daily with distilled water.The incubation time was based on the time used for cultivation of feces on coal for recovery of larvae of Strongyloides stercoralis 2 .Larvae were collected from the plastic cups using the Baermann-Moraes technique and counted in one aliquot of 50µL.The results of culturing in both media are summarized in Table 1.Of 17 cultures grown in vermiculite, 15 were positive, with recovery of fi larial larvae and, exceptionally, free-living forms of the worm.In contrast, among the 17 cultures on coal, only four were positive.The two negative cultures in vermiculite were also negative in coal.When comparing the positive cultures, the mean number of larvae recovered was signifi cantly higher in cultures containing vermiculite (Table 1).
In conclusion, data presented here demonstrated that fecal cultures in the presence of vermiculite, assessed