Contractile activity of cultured adult Dirofilaria immitis

A method for long-term maintenance of adult heartworms (HW) in culture for use in contractile activity studies was developed. Culture conditions included Eagle's minimum essential medium (MEM) containing Earle's balanced salt solution and MEM vitamins and supplemented with 10% horse serum, pH 7.6, 37 degrees C, and humidified 5% CO2:95% room air atmosphere. Motility was observed for up to 91 days. Reducing the culture atmosphere from 20% oxygen to 5% oxygen reduced acid production and survival to 28 days or less. Spontaneous contractile activity of adult HW coils (1 cm diameter) was measured using an isometric force displacement transducer system. Activity had an arrhythmic pattern of good magnitude that could be recorded after up to 50 days in culture for male HW and after up to 40 days in culture for female HW. Analyses of contractile activity included determination of its amplitude, frequency, contraction index, and basal tension. Amplitude for males (3.4 +/- 1.2 g) (mean +/- S.D.) was significantly greater (P < 0.02) than that for females (3.0 +/- 1.1 g), whereas frequency for females (8.2 +/- 2.3 min) was significantly greater (P < 0.03) than that for males (7.5 +/- 2.3 min). The contraction index for females was 16.7 +/- 13.7 mm/min and for males, 14.4 +/- 9.0 mm/min. The difference was not significant. The contraction index was based on line integration of the record of contractile activity. Amplitude and frequency of contractile activity for anterior segments (2.5 cm), suspended lengthwise, from cultured adult female HW, were not significantly different from results for coils, but the contraction index (34.5 +/- 33.8) was significantly higher (P < 0.01) indicating that the pattern of activity was more uniform in the segments. An applied basal tension of about 4 g was suitable for the coils, while a suitable basal tension for segments was about 1.5 g. For coils, amplitude, frequency, and contraction index increased significantly (P < 0.02) with increase in basal tension. Coils were not sensitive to changes in ionic, glucose, oxygen, and osmol concentrations, and pH (6.8-7.6) of the bathing solution. A pH of 8.0 markedly increased basal tension and contractile activity. At 20 degrees C contractile activity stopped. These effects of pH and temperature were reversible. The HW appeared resistant to changes in their environment. This is probably related to an efficient cuticular barrier. The culture and recording methods used in this study open improved opportunities for quantitative evaluation of responsiveness of HW to a variety of physiological and pharmacological factors.