In vitro Hatching of Gastrophilus intestinalis Larvae

Abstract

A method is described by which Gastrophilus intestinalis eggs were incubated, disinfected externally, and artificially stimulated to hatch in vitro. The percentage of larvae hatched varied directly with increasing differences between incubation and stimulation temperatures. Over 80% of the larvae hatched from eggs that had embryonated for at least 8 days at 24 C, then were disinfected at 5 C, and stimulated to hatch by suddenly adding Tyrode's balanced salt solution at a temperature of 44 C. In roller-tube cultures containing horse serum, Tyrode's solution (with or without glucose), antibiotics, and an atmosphere of 5% CO-95% No, at least 73% of the first instar larvae were actively motile for 48 hr. The in vitro culture of parasitic organisms is one of the more challenging goals of parasitology. The usual point of departure for in vitro studies is an attempt to duplicate in the laboratory certain of the natural environmental situations of the parasite. Several authors (Lapage, 1958; USDA, 1959; Wells and Knipling, 1938) have outlined the life cycle of Gastrophilus intestinalis, the common botfly of the horse. In that part of the life cycle applicable to this research, the female botfly cements one to four eggs per hair for a total of about 500 eggs near the distal end of the hairs on the forelegs, mane, belly, neck, and flanks of the horse. The eggs are fully embryonated in about 76 hr at 33 to 34 C and are ready to hatch on the 5th day. Embryonation may be delayed by lower temperatures. The principal stimulus for the larvae to hatch is a sudden rise to a favorable temperature as provided by the warm moist lips of the horse. The newly hatched larvae promptly burrow into the tissues of the tongue and remain there for 24 to 28 days before continuing the parasitic phase of their life cycle. The practical application of the use of hot water as a hatching stimulus for Gastrophilus larvae has been reported (Wells and Knipling, 1938). The infested areas of the horse were sponged with ample water at 104 to 118 F on a day when the air temperature was 60 F (15.6 C) or below. The larvae thus stimulated Received for publication 15 February 1967. * From a general study on equine parasites initiated by Merck, Sharp and Dohme, Inc. and the Research Council of Texas A & M University and with continued financial support from the American Quarter Horse Association through the Morris Animal Foundation. to hatch died of exposure. The authors state that this method resulted in elimination of about 95% of the latent larvae. Wehr (1933) hatched larvae by vigorously stirring egg-bearing hairs submerged in warm water (100 to 105 F) in a Syracuse watch glass. This paper presents a method for disinfecting the eggs, then hatching and maintaining the first-instar larvae in vitro. MATERIALS AND METHODS Eggs of G. intestinalis were collected by clipping hairs from the forelegs and shoulders of an infested horse. The eggs were examined microscopically and only apparently normal eggs were included in these experiments.