Development of an in <i>vitro</i> culture method for harvesting the free-living infective larvae of <i>Strongyloides venezuelensis</i>

, Baek, B K; , Islam, M K; , Kim, J H

doi:1998.36.1.15

Korean J Parasito > Volume 36(1):1998 > Article

Original Article


Korean J Parasitol. 1998 Mar;36(1):15-22. English. Published online Mar 20, 1998. http://dx.doi.org/10.3347/kjp.1998.36.1.15
Copyright © 1998 by The Korean Society for Parasitology


Development of an in vitro culture method for harvesting the free-living infective larvae of Strongyloides venezuelensis
B K Baek,^*M K Islam and J H Kim
College of Veterinary Medicine, Chonbuk National University, Chonju 561-756, Korea.
^*Corresponding author (Email: baekbk@moak.chonbuk.ac.kr)

Received September 18, 1997; Accepted January 05, 1998.
Abstract
An in vitro culture technique was established for harvesting Strongyloides venezuelensis free-living infective larvae using a nutrient broth medium as a substitute for rat-feces in polyvinyl culture bags (10 × 12 cm). The egg hatch rate (Y) in sterile saline at different incubation temperatures (X) was expressed as the quadratic function, Y = -0.192X² + 8.673X - 19.550 (r = 0.901). The highest (100%) egghatch rate was observed at 25℃. A significant difference (p < 0.05) in development rate (Y) of free-living infective larvae was observed between different concentrations of nutrient broth (X) which was highest (20.6%) in 0.12% nutrient broth concentrations, incubated at 20 degrees C for 5 days [Y = -864.032X² + 245.995X - 0.560 (r = 0.875)]. Yields (Y) of infective larvae were observed relatively high when the culture medium was incubated at higher temperatures (X) which peaked at 25℃ (20.0%) than at lower temperatures, 15℃ (10.9%) and 20℃ (18.1%) [Y = -0.189X² + 8.387X - 72.795 (r = 0.981)]. The period (Y) required for the development of infective larvae decreased with higher incubation temperatures (X) [Y = 0.035X² - 2.025X + 32.375 (r = 0.995)]. The highest yield (19.2%) of infective larvae was obtained from culture bag inoculated with 15,000 eggs than with below and over 15,000 eggs in 0.12% nutrient broth and incubated at 25℃ for 4 days. The newly adapted culture method (from egg to third-stage larva) may be useful as a bio-bar/bioassay system for screening new chemical products, anthelmintics and pesticides, as well as for parasito-immunological studies with Strongyloides species.

Figures

Figs. 1-3
Fig. 1. Polyvinyl culture bags (10 × 12 cm) developed initially for the cultivation of Strongyloides venezuelensis free-living L₃ containing infected-rat feces and distilled water, showing dark coloration of the culture medium.

Fig. 2. Polyvinyl culture bag containing parasite eggs and a scanty of freshly passed uninfected-rat feces dissolved in sterile saline. Culture medium is relatively more clean.

Fig. 3. Polyvinyl culture bag containing parasite eggs in nutrient broth showing very clean culture medium.


	Fig. 4 Hatch rate of S. venezuelensis eggs in sterile-saline incubated at different temperatures for 48 hr. Hatch rate of eggs (Y) in relation to incubation temperature (X) is expressed by the following quadratic function, Y = -0.192X² + 8.673X - 19.550 (r = 0.901).


	Fig. 5 An egg of S. venezuelensis collected from freshly passed rat feces. Bar = 10µm.


	Fig. 6 Development of S. venezuelensis L₃ in different concentrations of nutrient broth medium in polyvinyl culture bags and incubated at 20℃. Development rate (Y) of the L₃ according to different concentrations of nutrient broth (X) is expressed by the following quadratic function, Y = -864.032X² + 245.995X - 0.560 (r = 0.875).

Fig. 7
Development rate and period requirement for the S. venezuelensis L₃ stage in 0.12% nutrient broth medium incubated at different temperatures. The relationship between larval development rate (Y) (-□-□-) to the L₃ stage and incubation temperatures (X) as well as period (Y) (-○-○-) required for the L₃ formation is expressed by the following quadratic function; Y = -0.189X2 + 8.387X² 72.795 (r = 0.981). for development rate and Y = 0.035X² - 2.025X + 32.375 (r = 0.995). for period. respectively.


	Fig. 8 Yield of the S. venezuelensis L₃ according to the number of eggs inoculated in polyvinyl culture bag and incubated at 25℃ for 4 days.


	Figs. 9-11 Fig. 9. A free-living L₃ of S. venezuelensis harvested from nutrien broth culture. Bar = 100µm. Fig. 10. A free-living adult female of S. venezuelensis recovered from the small intestine of rat. Bar = 200µm. Fig. 11. A parasitic female of S. venezuelensis recovered from the small intestine fo rat. Bar = 200µm.

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