Observations on the Survival In vitro of Bacteria-Free Adult Common Liver Flukes, Fasciola hepatica Linn., 1758

Abstract

Although Fasciola hepatica Linn., 1758, has been studied in vitro previously, earlier investigators were dealing with contaminated, dying flukes. The previous work has been well reviewed by von Brand (1952) and Bueding and Most (1953). Only Stephenson (1947) was able to keep the fluke alive for more than a few hours, but he, too, had to contend with bacterial contamination. Stephenson (1947) was able to keep the adult fluke alive for 75 hours in a borax-buffered saline containing 45 mM fructose per liter. The borax, in addition to being a buffer, acted as a bacteriostatic agent. Epps, Weiner and Bueding (1950) demonstrated a method for producing bacteria-free parasitic helminths when they used antibiotic solutions to produce bacteria-free Ascaris lumbricoides. Subsequent to the present investigation, Dawes (1954) reported that sterile F. hepatica adults could be obtained by cleaning the surface of the liver with absolute alcohol and then dissecting the livers in a Dettol-sprayed chamber. Dawes was able to maintain sterile flukes for up to 16 days in Hedon-Fleig solution. The present paper describes a method for obtaining and maintaining bacteriafree flukes in vitro, the survival in vitro in the presence of simple carbohydrates, and the survival under aerobic and anaerobic conditions. It confirms Stephenson's (1947) data that this fluke apparently absorbs simple carbohydrates through its cuticle. An attempt is made to correlate the data presented with data obtained by other investigators in order to obtain a better understanding of the in vivo physiology of F. hepatica. MATERIALS AND METHODS The procedures outlined by Stephenson (1947) were used in the early stages of this investigation. Flukes, while still in the bile ducts, were brought from the packing plant into the laboratory where they were removed from the livers. After being washed with Stephenson's saline III (Table I), the flukes were placed in flasks containing this saline. It was noticed that the longer the flukes remained in the bile ducts following the host's death, the shorter the time the flukes remained alive in vitro. Therefore, arrangements were made to permit removal of the parasites from the infected bovine livers immediately after the slaughter of the animals. All flukes used were from bovine sources. The livers were dissected on the killing floor; therefore, aseptic techniques could not be used, but gross contamination was held to a minimum. As