Ultrastructural Changes in Litomosoides carinii Microfilariae in Gerbils Treated with Diethylcarbamazine

Abstract

Ultrastructural changes in Litomosoides carinii microfilariae in the liver of gerbils following treatment with diethylcarbamazine are described. At 20 min posttreatment, some of the microfilariae were freely circulating in the sinusoids and others were localized within hepatic cells. Neither group of microfilariae had visible sheaths. By 4 hr, the extracellular unsheathed microfilariae were undergoing lysis in the sinusoids and in parenchymal inflammatory foci, where they were phagocytized by Kupffer cells and neutrophils. The sequence of events supports the concept that the drug exerts its effect on microfilariae through a process of lysis, perhaps through loss of the sheath, with subsequent removal by phagocytosis. Intracellular microfilariae appeared normal except for loss of the sheath and apparently escaped phagocytosis. These viable organisms could possibly be considered a source of continued infection. Diethylcarbamazine (DEC = Hetrazan? Lederle; N,N-diethyl-4-methyl-l-piperazine carboxamide) has been widely investigated since its discovery as an antifilarial agent (Hewitt et al., 1947). It is unique among chemotherapeutic agents in contrasting an extremely rapid action in vivo with little or no apparent effect in vitro (Bangham, 1955a). Following the intravenous injection of DEC into cotton rats infected with Litomosoides carinii, there is a rapid, almost total clearance of the circulating microfilariae from the peripheral blood, with a sharp increase in the microfilarial population in the liver (Hawking et al., 1950). The sequence of events in the liver is not clear, although the microfilariae may be immobilized in the hepatic sinusoids or capillaries and eventually cleared by phagocytosis (Hawking et al., 1950; Taylor, 1960). We studied the electron microscopic changes in L. carinii following administration of DEC to infected gerbils in an attempt to clarify the mechanism of drug action on the microfilariae. MATERIALS AND METHODS Litomosoides carinii infections were induced in Mongolian gerbils (Meriones unguiculatus, male weanlings) via cyclical transmission through tropical rat mites (Bdellonysus bacoti) by the tankexposure procedure of Hawking and Sewell (1948) for infecting cotton rats. The mites were infected by allowing them to feed on experimentally infected cotton rats for a period of 14 days. Four infected gerbils were selected at random Received for publication 6 December 1967. * Present address: Ralston Purina Research Farm, Parasitology Laboratory, Gray Summit, Missouri. 351 for study. A summary of the experimental data is given in Table I. Microfilarial counts were made using Giemsa-stained thick films of blood drawn from the retro-ocular sinus. Treated animals were given intravenous DEC in normal saline at a dosage of 25 mg/kg. Single animals were killed 20 min and 4 hr after treatment to compare effects on the microfilariae. Excised pieces of liver were immediately cut into 1-mm cubes and fixed in cold 2% osmic acid buffered with veronal acetate. They were dehydrated through a graded series of alcohols, placed in propylene oxide as an intermediate solvent, and embedded in Vestopal W (Ryter and Kellenberger, 1958). A blood cell pack was prepared by centrifugation to isolate the buffy coat which was then fixed in cold veronal acetate-buffered osmic acid, and centrifuged on an agar substrate (Flickinger, 1966). The cell pack was cut into 1-mm cubes and prepared as above. Sections were cut on a PorterBlum MT-1 ultramicrotome with glass knives. Thick (1 A) sections were stained with toluidine blue (Trump et al., 1961) and examined by light microscopy to select fields containing microfilariae or other areas of interest. Thin sections (600 to 900 A) were cut from the selected blocks, picked up on carbon-stabilized, collodion-coated grids, and stained with lead citrate or uranyl acetate-lead citrate double stain as modified by Stevens (1966). Observations were made with a Hitachi HS-7S electron microscope. Original magnifications were 3,000 to 10,500 X.