OF WASHINGTON, VOLUME 59, NUMBER 1, JANUARY 1992 135 used: 1. Because the recovery after thawing is Literature Cited less than 1%, large numbers of L3's must be fro- Hawdon, J. M., and G. A. Schad. 1991. Long-term zen (> 100,000). 2. Upon thawing, the larvae storage of hookworm infective larvae in buffered should be incubated in BU at room temperature saline solution maintains larval responsiveness to for 20 to 24 hr, allowing the injured L3's to die. host signals. Journal of the Helminthological So- ciety of Washington 58:140-142. Thereafter, the living L3's are collected. 3. Al- Nolan, T. J., L. M. Aikens, and G. A. Schad. 1988. though it is not absolutely necessary, immuno- Cryopreservation of first-stage and infective third- suppression of the rat, before and after the sub- stage larvae of Strongyloides stercoralis. Journal cutaneous injection of the L3's, can be used to of Parasitology 74:387-391. Van Wyk, J. A., H. M. Gerber, and R. M. R. Alves. increase the number of adults maturing in the 1990. Cryopreservation of some common nem- small intestine. 4. Because few adults develop atodes of ruminants for up to 11.3 years. Onder- from cryopreserved L3's, it is necessary to am- stepoort Journal of Veterinary Research 57:1-6. plify the infection by passage through another rat , , and W. P. Van Aardt. 1977. Cryo- to obtain sufficient S. ratti for most experimental preservation of the infective larvae of the common nematodes of ruminants. Onderstepoort Journal purposes. of Veterinary Research 44:173-194. We acknowledge the help of Dr. Veena Bho- pale and Dr. Susan Niamatali during this study. This work was supported by NIH grant AI-22662. J. Helminthol. Soc. Wash. 59(1), 1992, pp. 135-140 Research Note Histochemical Observations on Cyathostoma lari (Strongyloidea: Syngamidae) W. THRELFALL Department of Biology, Memorial University, St. John's, Newfoundland, Canada A1B 3X9 ABSTRACT: The location and relative abundance of chemical components of adult female Cyathostoma lari were determined using a variety of histochemical methods. Carbohydrates, acid mucopolysaccharides, lipids, and proteins were widely distributed throughout the nematode. Host blood and hemoglobin were detected in the gut lumen, with hemoglobin also being demonstrated in the pseudocoel. Ribonucleic acid, mitochondria, succinic dehydrogenase, and acid and alkaline phosphatases were located in the digestive and reproductive systems. The data suggest that the anterior intestine plays the most important role in digestion. The role and importance of the various substances are discussed. KEY WORDS: histochemistry, Nematoda, Cyathostoma lari. Much work has been performed on the phys- concerned with determining the precise location iology and biochemistry of metazoan parasites, of various substances within nematodes (Sood particularly digeneans and cestodes (von Brand, and Kalra, 1977; Sood and Sehajpal, 1978; Maki 1979; Chappell, 1980; Barrett, 1981; Smyth and and Yanagisawa, 1980; Sharma and Singh, 1985). McManus, 1989). One of the least examined The distribution of the chemical components groups has been the nematodes, with species of of a parasite is a reflection of where biochemical medical and veterinary importance receiving the processes are occurring, with intensity and type most attention. Within this group larvae and eggs of reaction perhaps changing at different times have been the objects of greatest attention. In during the host's life cycle. Host physiology and examining the chemical constituents of nema- location of the parasite within the host will also todes, studies have to a large extent been per- affect the physiological status of the parasite. formed using homogenates of whole worms Phylogenetic differences among hosts and among (Singh and Sharma, 1981; Chopra, 1986; Rao parasite species might also be reflected in the and Rajlingam, 1989). Few studies have been parasite's physiology. As noted by Threlfall et al. CCooppyyrriigghhtt ©© 22001111,, TThhee HHeellmmiinntthhoollooggiiccaall SSoocciieettyy ooff WWaasshhiinnggttoonn 136 JOURNAL OF THE HELMINTHOLOGICAL SOCIETY (1990), data to support such a contention are with and without ribonuclease (Gurr, 1958). Mi- rare. The present histochemical study was un- tochondria were demonstrated in frozen sections dertaken to determine the distribution and rel- by the presence of succinic dehydrogenase using ative concentrations of various substances the NET technique (Pearse, 1972; Bancroft, 1975) throughout the body of adult female Cyathosto- and in Altmann's-fixed sections with Metzner's ma lari Blanchard, 1849, recovered from herring stain (Metzner and Krause, 1928). Acid phos- gull (Larus argentatus Pontoppidan, 1763) chicks phatase was detected using the Gomori lead ni- taken in both Wales and Newfoundland, and to trate, Burnstone's, and the modified Gomori compare the results with previous works. Cy- methods (Pearse, 1968), whereas alkaline phos- athostoma lari is an obligate, blood-sucking phatase was demonstrated using the calcium co- nematode that lives in the nasal cavities of a balt (Gomori, 1952) and modified Gomori meth- variety of birds including gulls (Larus spp.) (Ba- ods (Pearse, 1968). Tests for phosphatases were rus et al., 1978). A plug of host mucosa is drawn performed on material that had been fixed in cold into the buccal cavity of the worm and lacerated acetone (4°C), except in the case of the modified with cutting plates so that blood may flow freely Gomori test for alkaline phosphatase where through the worm (Threlfall, 1966; Colam, specimens were fixed in 90% ethanol. Controls 197la). This study also expands on the work of were performed as follows: acid phosphatase, in- Colam (1971 a) who studied the gut ultrastructure cubated as for the test but 0.01 M sodium flu- and digestive physiology of this species, as well oride was included in the reaction medium; as Rhabdias bufonis, R. sphaerocephala, and alkaline phosphatase, 3% sodium-b-glycero- Cosmocerca ornata (Colam, 1971b, c). phosphate in the medium was replaced by dis- Worms were fixed in situ by flooding the si- tilled water (Chayen et al., 1973). nuses of freshly killed gull chicks with a variety The distribution and amounts or activity of of fixatives, including Altmann's, cold acetone various chemical components in the digestive (4°C), 90% ethyl alcohol, Susa's, and Zenker's and reproductive tracts of C. lari are shown in depending on the tests to be performed. The Table 1. Glycogen comprises the principal car- worms were then removed from the sinuses, bohydrate reserve in nematodes, with the amount blocked at 54°C in paraffin wax, and sectioned varying from species to species (Fairbairn, 1958, at 5, 10, and 15 /mi. A small number of worms 1960; Barrett, 1981). A general orthochromasia were recovered alive, quick-frozen, and sec- was noted throughout the worm with the highest tioned at 10 and 15 /tm using a freezing micro- concentrations of carbohydrates being seen in the tome. Carbohydrates were detected in Zenker's- anterior intestine (Table 1). Sood and Sehajpal fixed worms, using Best's carmine, and the PAS (1978) noted similar results in Haemonchus con- reaction, with and without amylase treatment tortus. The ovaries of C. lari showed a similarly (Gurr, 1956, 1958; Pearse, 1968). The presence intense reaction. The cuticle of this helminth is of acid mucopolysaccharide was revealed in Su- extremely thin, particularly when compared to sa's-fixed specimens using the thionin, toluidine that of many intestinal inhabitants, e.g., Ascaris blue, and Male's dialysed iron methods outlined lumbricoides, and was negative in tests for car- by Gurr (1958). Lipids were stained using Sudan bohydrates, whereas the hypodermis and cyto- Black B in specimens fixed in Altmann's and plasmic portion of the muscle cells stained light- Zenker's or in frozen specimens, or copper ly. A similar situation was noted by Sood and phthalocyanin using methanol fast blue stain Kalra (1977) in Haemonchus contortus and (Pearse, 1968). Controls were with Sudan Black Xiphinema insigne. Acid mucopolysaccharide, B plus pyridine extraction. Mercuric bromphe- an ubiquitous substance in nematodes (Barrett, nol blue with and without deaminase was used 1981), was present in all the body tissues except to detect proteins in Zenker's-fixed worms (Ma- the cuticle and the eggshell/membranes. Sood zia et al., 1953; Pearse, 1968). The digestive tract and Kalra (1977) noted the presence of this sub- of the nematode contained host blood, and the stance in the inner cortical layer of the cuticle of pseudocoel was filled with a red pigment-con- H. contortus and X. insigne. Sood and Sehajpal taining fluid. To determine the distribution of (1978) were unable to demonstrate acid muco- hemoglobin within the worm, Zenker's-fixed sec- polysaccharide or glycogen in the brush border tions were stained with Giemsa stain and ben- of the gastrodermis of//, contortus. In the present zidine (Gurr, 1958). Ribonucleic acid was de- study, considerable amounts of both glycogen tected using the pyronine/methyl green method and acid mucopolysaccharide were present in the CCooppyyrriigghhtt ©© 22001111,, TThhee HHeellmmiinntthhoollooggiiccaall SSoocciieettyy ooff WWaasshhiinnggttoonn OF WASHINGTON, VOLUME 59, NUMBER 1, JANUARY 1992 137 Table 1. Histochemistry of the digestive and reproductive systems of adult female Cyathostoma lari. Body region Intestine Buccal Esoph- Ovum Egg- region agus Ant. Mid. Post. Rectum Ovary (in shell) shell Carbohydrates + + + + + + + + + + - Acid mucopolysaccharide + + + + + + + + — Lipids - - + + + ++ ++ - + + + + + Proteins + + Ribonucleic acid — — Mitochondria - - Succinic dehydrogenase — — Acid phosphatase — — Alkaline phosphatase — — + + + = strongly positive; + + = moderately positive; + = weakly positive; — = negative. gastrodermis. Monne (1959) showed the pres- masia with mercuric bromphenol blue. The cu- ence of acid mucopolysaccharide in the eggshell ticle, hypodermis, lateral cords, and muscles, and membranes of 3 species of lungworms, a particularly the myofilaments, stained lightly. The finding at odds with the present work. Helminths intestine and reproductive system were rich in such as C. lari that have access to a rich supply proteins. Many enzymes are proteinaceous in na- of oxygen via the host blood or live in aerobic ture. The present results suggest that the anterior conditions tend to have little glycogen in their intestine is most heavily involved in the digestive tissues. This is unlike the situation seen in an- process. This observation supports the work of aerobic or anoxybiotic organisms such as Ascaris Colam (197la), but refines our knowledge by lumbricoides and Porrocaecum decipiens (Bar- showing that enzymatic activity may vary along rett, 1981). the length of the intestine. The ovary is a region Lipids were detected in the cuticle, hypoder- of intense protein, DNA, and mitochondrial ac- mis, and muscle cell cytoplasm. This distribution tivity resulting in the formation of ova. The egg- was similar to that noted by Sood and Kalra shells stained quite intensely with mercuric (1977). The intestine was particularly rich in lip- bromphenol blue, probably as a result of their id globules as noted previously by Colam (1971 a). being composed of lipoproteins (Seesee, 1977). Sood and Sehajpal (1978) observed lipids in the RNA was detected in the intestine; the most esophagus and intestine of//, contortus and not- intense reaction was in the anterior portion, with ed that lipids may well form a major storage less intense reactions in the mid and posterior product in nematodes. No lipids were detected portions and rectum. The ovaries and ova were in the esophagus of C. lari, whereas the intestine also rich in this substance. The hypodermis and stained intensely for these substances. The con- hypodermal cords did not stain for RNA, which centration of lipid globules in the intestinal wall differs from the results of Sood and Kalra (1977), of C. lari may represent the end-product of di- who noted a reaction in the hypodermis, hypo- gestion in the gut lumen and the syncytium of dermal cords, and inner cortical layer of the cu- the gastrodermis. The greater concentration in ticle of//, contortus. Barrett (1981) noted the the anterior region of the intestine suggests that types of RNA found in nematodes and discussed this is where the greatest biochemical activity is their importance in protein building. The gut occurring. The ovaries were well supplied with contents of the nematode, host blood containing lipids, with lesser amounts being detected in the nucleated erythrocytes and leucocytes, stained ovum. The eggshell was rich in lipids, a phenom- intensely for RNA. enon previously reported in other nematode spe- Cyathostoma lari is an obligate hematophage, cies (Rogers, 1962; Seesee, 1977). Lipids were with its "normal" red coloration being due to confined to the outer and inner layers of the the presence of host blood in its gut and a red 3-layered eggshell. pigment in its pseudocoel. Tests revealed the Proteins form the major structural component presence of hemoglobin (Hb) in the intestinal of C. lari as reflected by the general orthochro- lumen, and in the protein-containing pseudo- CCooppyyrriigghhtt ©© 22001111,, TThhee HHeellmmiinntthhoollooggiiccaall SSoocciieettyy ooff WWaasshhiinnggttoonn 138 JOURNAL OF THE HELMINTHOLOGICAL SOCIETY coelomic fluid. No attempt was made to char- in areas where intense biosynthesis is occurring. acterize the Hb in the pseudocoel. Colam (1971 a) The role, and distribution, of phosphatases in noted Hb in the gut lumen of C. lari and as small cestodes is somewhat better understood than in granules below the brush layer of the gastroder- nematodes (Threlfall et al., 1990). Arme and Read mis after hemolysis. Sood and Sehajpal (1978) (1970) and Mayberry and Tibbitts (1972) sug- noted Hb in the same location in the intestine gested that alkaline phosphatase is involved in of//, contortus, whereas Sood and Kalra (1977) active transport and/or digestion. Other workers identified Hb in the cuticle of//, contortus. Rose (Sood and Kalra, 1977; Sood and Sehajpal, 1978; and Kaplan (1972), working on the closely re- Maki and Yanagisawa, 1980) have shown a more lated species Syngamus trachea, noted that the general distribution of phosphatases in nema- Hb from the worm was composed of only 1 com- todes than was seen in C. lari. In the present ponent and had a different molecular weight than work the cuticle, hypodermis, hypodermal cords, that extracted from the host blood. This result and muscles appeared to be free of phosphatases. differs from that of van Grembergen (1954) who Cyathostoma lari is typical of many strongy- showed that the Hb of Heterakis gallinae had loids in possessing a limited number of cells characteristic alpha and beta absorption bands (Chitwood and Chitwood, 1974) and a syncytial that were similar to those shown by the Hb from intestinal wall (Colam, 197la). The presence of host blood. Hemoglobins in nematodes seem to a syncytial intestine, which has a well-developed have a very high affinity for oxygen, even at very bacillary (microvillar) layer, may be an adapta- low partial pressures. Oxyhemoglobin will, tion to hematophagy. Differences noted in the therefore, only dissociate at very low tissue con- amounts of various substances along the length centrations of oxygen, and it seems improbable of the intestine suggest differential enzymatic ac- that the Hb in an aerobic species, such as C. lari, tion along its length and are worthy of further will be used for oxygen transport. The differences study. noted above in the types of Hb found in nema- It became obvious that marked differences do todes, as well as the function of Hb, are deserving occur in the presence and distribution of sub- of further investigation. stances in different nematode species. Our Mitochondria, indicated by the presence of knowledge of such differences is at present ru- succinic dehydrogenase, were detected in greatest dimentary. A more complete understanding of amounts in the anterior part of the intestine. the physiological and biochemical processes oc- Lesser amounts were present in the remainder curring in nematodes will be aided by further of the digestive tract (except for the wall of the histochemical studies, including a comparison of buccal cavity and esophagus where they were male and female worms. A preliminary study of absent). The reproductive tract was also positive. male C. lari, which are much rarer and smaller These locations all correspond to sites of great than the females (Burt and Eadie, 1958), revealed biochemical activity. Colam (197 la) showed that chemical substances and distributions similar to digestion and absorption of blood cells occurred those noted above. It is possible that similarities in the gastrodermis of C. lari, and that numerous or differences among nematode species might be multicristate mitochondria were present in spe- partially explained by differing habitats of the cific regions of the tissue. Monne (1959) dis- parasites, stage of development, host phylogeny, cussed the mitochondria of developing lung- and/or host physiological differences. worm ova, and noted that they are numerous Thanks are due to the Natural Sciences and both at this stage and in adult worms. Engineering Research Council of Canada for the Acid and alkaline phosphatases were widely Operating Grant (NSERCC A3500) that funded distributed in the digestive and reproductive this work. tracts, regions where intense biochemical activity might be expected. Again the anterior intestine appeared to be the region where most activity Literature Cited occurred, with a decline of activity in the pos- Arme, C., and C. P. Read. 1970. A surface enzyme terior regions. Both these substances are asso- in Hymenolepis diminuta (Cestoda). Journal of ciated with digestion (Colam, 197 la; Riley, 1973). Parasitology 56:514-516. Bancroft, J. D. 1975. Histochemical Techniques. The presence of acid phosphatase has been used Butterworths, London. 348 pp. as an indicator of lysosomal activity (Duve, 1963; Barrett, J. 1981. Biochemistry of Parasitic Hel- Novikoff, 1963) and would be expected to occur minths. University Park Press, Baltimore. 308 pp. CCooppyyrriigghhtt ©© 22001111,, TThhee HHeellmmiinntthhoollooggiiccaall SSoocciieettyy ooff WWaasshhiinnggttoonn OF WASHINGTON, VOLUME 59, NUMBER 1, JANUARY 1992 139 Barus, V., T. P. Sergeeva, M. D. Sonin, and K. M. Mazia, D., P. A. Brewer, and M. Alfert. 1953. The Ryzhikov. 1978. Helminths of Fish-eating Birds cytochemical staining and measurement of protein of the Palaearctic Region 1. Nematoda. Dr. W. with mercuric bromphenol blue. Biological Bul- Junk Publ., The Hague, Netherlands. 318 pp. letin 104:57-67. Brand, T. von. 1979. Biochemistry and Physiology Metzner, R. von, and R. Krause. 1928. DieMethoden of Endoparasites. Elsevier/North-Holland Bio- zur Darstellung der Stoffwechsel-organellen der medical Press, Amsterdam. 447 pp. tierischen Zelle im fixierten Praparat. Pages 325- Burt, D. R. R., and J. M. Eadie. 1958. Cyathostoma 436 in E. Abderhalden, ed. Handbuch der biolo- lari Blanchard, 1849 [Nematoda, Strongyloidea]: gischen Arbeits-methoden. Urban and Schwar- its anatomy, intraspecific variation and hosts, with zenberg, Berlin. a re-definition of the genus. Journal of the Linnean Monne, L. 1959. On the formation of the egg enve- Society of London, Zoology 43:575-585. lopes and the early development of the lungworms Chappell, L. H. 1980. Physiology of Parasites. John Dictyocaulus viviparus, D. filaria, and Metastron- Wiley and Sons, New York. 230 pp. gylus elongatus. Arkiv for Zoologi 12:99-122. Chayen, J., L. Bitensky, and R. G. Butcher. 1973. Novikoff, A. B. 1963. Lysosomes in the physiology Practical Histochemistry. John Wiley and Sons, and pathology of cells: contributions of staining London. 271 pp. methods. Pages 36-73 in A. V. S. de Reuck and Chitwood, B. G., and M. B. Chitwood. 1974. Intro- M. P. Cameron, eds. CIBA Foundation Sympo- duction to Nematology. University Park Press, sium on Lysosomes. Little Brown and Co., Boston. Baltimore. 334 pp. Pearse, A. G. E. 1968. Histochemistry, Theoretical Chopra, A. K. 1986. Acid phosphatase activity in and Applied. Vol. 1. Williams and Wilkins Com- Bunostomum trignocephalum (Nematoda). Rivis- pany, Baltimore. 759 pp. ta di Parassitologia 47:225-230. . 1972. Histochemistry, Theoretical and Ap- Colam, J. B. 197la. Studies on gut ultrastructure and plied. Vol. 2. Churchill Livingstone, London. Pages digestive physiology in Cyathostoma lari (Nem- 753-1518. atoda: Strongylida). Parasitology 62:273-283. Rao, R., and G. Rajlingam. 1989. Phosphatase activ- . 1971b. Studies on gut ultrastructure and di- ity in avian parasites. Indian Journal of Helmin- gestive physiology in Rhabdias bufonis and R. thology 41 (Supplement): 1-5. sphaerocephala (Nematoda: Rhabditida). Parasi- Riley, J. 1973. Histochemical and ultrastructural ob- tology 62:247-258. servations on digestion in Tetrameres fissispina -. 1971c. Studies on gut ultrastructure and di- Diesing, 1861 (Nematoda: Spiruridea) with special gestive physiology in Cosmocerca ornata (Nem- reference to intracellular digestion. International atoda: Ascaridida). Parasitology 62:259-272. Journal for Parasitology 3:157-164. Duve, C. de. 1963. The lysosome concept. Pages 1- Rogers, W. P. 1962. The Nature of Parasitism. The 35 in A. V. S. de Reuck and M. P. Cameron, eds. Relationship of Some Metazoan Parasites to Their CIBA Foundation Symposium on Lysosomes. Lit- Hosts. Academic Press, New York. 287 pp. tle Brown and Co., Boston. Rose, J. E., and K. L. Kaplan. 1972. Purification, Fairbairn, D. 1958. Trehalose and glucose in hel- molecular weight, and oxygen equilibrium of he- minths and other invertebrates. Canadian Journal moglobin from Syngamus trachea, the poultry of Zoology 36:787-795. gapeworm. Journal of Parasitology 58:903-906. . 1960. The physiology and biochemistry of Seesee, F. M. 1977. The morphology and histochem- nematodes. Pages 267-296 in J. N. Sasser and W. istry of eggshell formation in Cephaluris colora- R. Jenkins, eds. Nematology. Fundamentals and densis, a parasite of pikas. Journal of Parasitology Recent Advances with Emphasis on Plant Para- 63:511-514. sitic and Soil Forms. University of North Carolina Sharma, S., and K. Singh. 1985. Distribution of acid Press, Chapel Hill. and alkaline phosphomonoesterases in the para- Gomori, G. 1952. Microscopic Histochemistry. Prin- sitic nematode Setaria cervi. Angewandte Parasi- ciples and Practice. University of Chicago Press, tologie 26:237-240. Chicago. 273 pp. Singh, K., and S. Sharma. 1981. 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Helminthol. Soc. Wash. 59(1), 1992, pp. 140-144 Research Note Experimental Fascioliasis in Llamas LORA G. RlCKARD1 AND WILLIAM J. FOREYT2 1 College of Veterinary Medicine, Oregon State University, Corvallis, Oregon 97331 and 2 Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164 ABSTRACT: Three llamas and 2 domestic sheep were and were supplemented with hay when needed. inoculated orally with metacercariae of liver flukes, All pastures used were known fluke-free pastures Fasciola hepatica. The prepatent period in llamas and and none of the animals had any history of liver sheep was 8-12 wk. Sizes of fluke eggs passed in feces fluke infections prior to experimental infection. were similar between llamas and sheep. At necropsy, the percentages of original inoculum recovered from All llamas were clinically normal. Two of the the llamas and sheep were 24% and 22%, respectively. llamas (nos. 2 and 3) were also given larvae of Sizes of flukes recovered from livers were similar be- meningeal worm, Parelaphostrongylus tenuis, on tween llamas and sheep. The gross appearance of the day 18 of this experiment. Because P. tenuis in livers from the llamas varied from slight discoloration with some bile duct thickening to marked fibrosis and llamas is confined to the neurologic system scarring. Llama livers were similar histologically. Bile (Baumgartneretal., 1985; Krogdahl et al., 1987), duct hyperplasia, portal fibrosis, and granulomas, often it was considered that it would not directly affect containing degenerated trematode eggs and necrotic the liver fluke infection. Two healthy domestic debris, were hallmarks of infection. These changes re- sheep (Ovis aries), 1.5-year-old wethers, were sembled chronic fascioliasis in sheep. The data indicate that llamas, like domestic sheep, have low resistance purchased as lambs from a known F. hepatica- to liver fluke infection. free area, and were housed on pasture until the KEY WORDS: Fasciola hepatica, liver flukes, exper- start of the experiment when they were moved imental infection, llama, Lama glama, sheep, Ovis ar- indoors and housed on concrete. ies. On day 0, 250 (llama 1) or 500 (all other an- imals) metacercariae of F. hepatica (Baldwin En- Fasciola hepatica is a prevalent and econom- terprises, Monmouth, Oregon) were adminis- ically important trematode parasite of cattle and tered to each animal orally either by stomach sheep in the United States. In endemic areas goats, tube (llamas) or gelatin capsule (sheep). Rectal rabbits, swine, horses, and man may also become fecal samples were collected and animals were infected (Leathers et al., 1982; Soulsby, 1982; weighed at approximately 2-wk intervals Malone, 1986; Wescott and Foreyt, 1986). In the throughout the trial. Animals were observed dai- United States, natural infections of F. hepatica ly for signs of clinical parasitism. have been reported in 1 llama in Texas and 1 Five grams of feces was examined at each sam- llama in Oregon (Cornick, 1988; Rickard and pling period for eggs of F. hepatica with a sedi- Bishop, 1991). The purpose of this study was to mentation technique. For llama 1, the samples determine the prepatent period of F. hepatica in were scored as negative or positive, and for the llamas, describe the lesions associated with ma- other animals, actual numbers of fluke eggs per ture infections, and compare them with those in gram of feces were determined. A minimum of domestic sheep. 20 eggs from each positive sample were mea- Three healthy adult female llamas (Lama gla- sured using a microscope equipped with an oc- ma), 5-7 years old, were donated for research ular micrometer. purposes because of reproductive or conforma- On day 157 postinfection, llama 1 was eu- tional problems. All were maintained on pasture thanized for reasons unrelated to parasitism. On CCooppyyrriigghhtt ©© 22001111,, TThhee HHeellmmiinntthhoollooggiiccaall SSoocciieettyy ooff WWaasshhiinnggttoonn