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In vitro culture of Dirofilaria immitis third- and fourth-stage larvae under defined conditions
Abstract:
The objective of the present study was to define culture conditions under which larval Dirofilaria immitis would molt, grow, and survive. Third-stage larvae (L3) survived for over 3 wk with a molt rate of up to 95% in a variety of media supplemented with fetal calf serum. Bovine albumin, added to several media at concentrations of 10-30 mg/ml, also proved to be an effective culture supplement for the induction of molting and for supporting larval survival. Two gas phases were tested, 5% CO2/95% N2 and 5% CO2/air; no differences were noted in larval development based on gas phase. Larvae, maintained in media with FCS or albumin for 48 hr, were capable of completing the molting process and growing in length in unsupplemented media. If the temperature at which cultures were maintained was changed from 37 C to 27 C, L3 did not molt but did survive for several weeks. Two factors required for larval D. immitis molting and growth have been identified, temperature of approximately 37 C and the presence of albumin in the culture medium. The defined culture system developed for D. immitis L3 may provide a source for collection of excretory-secretory antigens, which could prove useful in immunodiagnosis or immunoprophylaxis as well as provide a means of studying the process and requirements of filarial larval molting.
Insights
Researchers defined culture conditions for Dirofilaria immitis larvae (L3). Optimal conditions for molting and growth involve fetal calf serum or albumin at approximately 37°C, aiding future immunodiagnosis and research.
Area of Science:
- Veterinary Parasitology
- Molecular Biology
- Infectious Diseases
Background:
- Dirofilaria immitis, the causative agent of heartworm disease, requires specific conditions for larval development.
- Understanding larval molting is crucial for developing effective diagnostic and prophylactic strategies.
Purpose of the Study:
- To establish optimal in vitro culture conditions for Dirofilaria immitis third-stage larvae (L3).
- To identify key factors influencing larval molting, growth, and survival.
Main Methods:
- Culturing L3 in various media supplemented with fetal calf serum (FCS) or bovine albumin.
- Testing different gas phases (5% CO2/95% N2 and 5% CO2/air) and temperatures (37°C vs. 27°C).
- Assessing larval survival, molting rates, and growth over time.
Main Results:
- L3 survived over 3 weeks with up to 95% molting in media with FCS.
- Bovine albumin (10-30 mg/ml) effectively induced molting and supported survival.
- Larval development and molting were optimal at approximately 37°C with albumin present; temperature change to 27°C halted molting but allowed survival.
Conclusions:
- Defined culture conditions require approximately 37°C and the presence of albumin for D. immitis L3 molting and growth.
- This system provides a source for excretory-secretory antigens for immunodiagnosis and immunoprophylaxis.
- Facilitates further study into filarial larval molting processes and requirements.