Pathophysiology of Experimental Leishmaniasis: Pattern of Development of Metastatic Disease in the Susceptible Host

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1 INFCTION AND IMMUNITY, May 1986, p /86/5364-6$2./ Copyright 1986, American Society for Microbiology Vol. 52, No. 2 Pathophysiology of xperimental Leishmaniasis: Pattern of Development of Metastatic Disease in the Susceptible Host JOSPH. HILL Trudeau Institute, Inc., Saranac Lake, New York Received 18 November 1985/Accepted 13 January 1986 A clear understanding of the etiology of the various forms of leishmaniasis will require knowledge of how physiological properties of the parasite and host immunity influence the pattern of development of the disease. Of particular importance are how these factors affect the growth rate of Leishmanua spp. at the site of inoculation in the skin, their capacity to disseminate to visceral and distant cutaneous sites, and their capacity to multiply once there. This paper details the pattern of development of disseminated Leishmania major infection in susceptible BALB/c nu/+ and BALB/c nulnu mice. It was found that the parasite disseminates from the hind footpad to distant cutaneous sites soon after metastatic foci are established in the liver and spleen. Both mononuclear phagocytes and neutrophils may be the vehicles for the transport of the parasite in the blood. Once visceral and cutaneous metastases are established, the parasites in those foci increase in number progressively. L. major has the capacity to multiply at visceral and cutaneous sites at the same rate. Despite the presence of viable parasites in a number of skin sites, cutaneous metastatic lesions developed almost exclusively on the feet and the tail. Furthermore, these lesions appeared to develop preferentially at sites near joints, suggesting that factors other than temperature may influence the development of cutaneous metastatic lesions. Species of the genus Leishmania cause different forms of human disease (8). Leishmania donovani is the etiologic agent of visceral leishmaniasis (kala azar). In this form of the disease, only a small ulcer may develop at the site of the bite of the insect vector, yet the parasite disseminates to the liver, spleen, and bone marrow (31). If untreated, the disease is fatal. Leishmania mexicana, on the other hand, usually remains confined to a single, yet pronounced, cutaneous lesion. It is thought that the clinical type of leishmaniasis is a reflection of the properties of the specific parasite involved, such as the ability of the parasite to multiply at the temperatures of the skin and the visceral organs (11, 16). Clearly, however, host immunity also influences, in a major way, the pattern of development of leishmaniasis not only by restraining the multiplication of the parasite at the site of inoculation (6), but also by destroying parasites in metastatic foci at distant visceral and cutaneous sites. It is the apparent failure of these immune mechanisms that contributes to the development of the disseminated forms of leishmaniasis, such as kala azar, post-kala azar dermal leishmaniasis, and diffuse cutaneous disease (11, 16). Disseminated leishmaniasis has proved difficult to treat with the antibiotics that are currently available because repeated courses of chemotherapy are neccessary (3). Furthermore, the parasite is often not completely eliminated from the tissues, and relapses are common (3, 11). Although no vaccine has been developed, it is hoped that immunization (12) or immunotherapy (2) may succeed where chemotherapy has failed. This hope has stimulated a great deal of interest in the immune mechanisms responsible for the destruction of the parasite (for a review, see reference 16). However, very little is known about the etiology of leishmaniasis, that is, how particular combinations of parasite and host result in specific forms of the disease. Because Leishmania spp. are obligate intracellular parasites in the mammalian host, mononuclear phagocytes are possibly other host cells may be involved in the transport of the 364 parasite through the blood and in its establishment in distant visceral and cutaneous sites. Thus, a clearer understanding of the mechanisms of dissemination of Leishmania spp. in experimental mammalian hosts may facilitate the development of effective chemotherapy and immunotherapy. A number of different mouse strains and species of Leishmania have been used in animal models of the various forms of human leishmaniasis (19). Visceral disease can be induced by injecting L. donovani intravenously. However, in this model the early events in the development of disseminated disease, such as the multiplication of the parasite in the skin and its dissemination to the viscera, cannot be studied. On the other hand, Leishmania major and L. mexicana do multiply at the cutaneous site of inoculation. However, in most immunocompetent hosts the parasite disseminates to the liver, spleen, and distant cutaneous sites very slowly (1, 9), if at all (7, 23). There is one particular host-parasite combination that results in a disease that has components common the both the visceral and the disseminated cutaneous forms of the natural disease in man. This is L. major in BALB/c mice. BALB/c mice are extremely susceptible to this parasite, as well as to many other leishmania spp. that cause human cutaneous disease. It has been shown in other laboratories that the cutaneous lesions increase progressively in size and visceral disease and metastatic cutaneous lesions develop (2, 4). Furthermore, as a result of the development of methods to enumerate viable Leishmania parasites in tissue (5, 7), specific aspects of the dissemination of Leishmania spp. can be examined. This paper reports the results of studies that document the pattern of development of systemic disease in BALB/c mice and that provide a basis for formulating specific hypotheses about the mechanisms by which a Leishmania sp. disseminates and establishes foci of infection in visceral and cutaneous metastatic sites. Furthermore, athymic (nude) mice were included in the study so that the role of certain physiological properties of the parasite in the Downloaded from on October 7, 218 by guest

2 VOL. 52, 1986 disease could be examined in the absence of T-cell-mediated immunity. MATRIALS AND MTHODS Mice. Specific-pathogen-free, female BALB/c nul+ (normal) and BALB/c nulnu (nude) mice, 8 to 12 weeks old, were used. The mice were obtained from the Trudeau Institute Animal Breeding Facility, Saranac Lake, N.Y., and were maintained under barrier-sustained conditions in isolators. The colony was started in 1977 from founder stock carrying in nu mutation on a BALB/cAnN genetic background. The mouse colony is routinely monitored for pathogenic bacteria and mycoplasmas by standard bacteriological techniques and monitored for virus infections by serological tests (mouse virus profile 8-211; Microbiological Associates, Bethesda, Md.). numeration of Leishmania parasites. L. major 173 (WR173, LIV68) was used. The source and passage history of this human isolate have been previously described (6, 7). Primary, subcutaneous infections were initiated by injecting 15 amastigotes in 5,ul of Schneider Drosophila medium (GIBO Laboratories, Grand Island, N.Y.) into the left hind footpad (6, 7). In some experiments, systemic infections were established by injecting 15 amastigotes in 2 ixl into a lateral tail vein. Amastigotes were used to infect mice because a previous study from this laboratory (7) showed that compared with promastigotes, a larger proportion of amastigote inocula survives the first 24 h to initiate the infection. At predetermined intervals, four or five mice were anesthetized, and a.7- to 1.-ml sample of cardiac blood was taken. The blood sample was immediately transferred to a sterile plastic tube containing 5,ul of.1 M DTA. Peripheral blood smears were then prepared and stained with Giemsa. To document the pattern of development of disseminated disease, the number of viable parasites in the left hind footpad (primary lesion), in the draining lymph nodes (27), the blood, liver, spleen, the external ear (pinna), and in the right hind footpad (metastatic lesion) was determined by plating appropriately diluted aliquots of tissue homogenates on rabbit blood agar (5, 6). After 5 to 7 days of incubation at 26 C, the promastigote colonies were counted, and the numbers of parasites in the respective tissues were calculated. Measurement of lesion size. The progress of the infection was also followed by measuring changes in the thickness of the infected footpad as previously described (7). RSULTS Pattern of development of disseminated disease. Within 48 h of inoculation into the left hind footpad of BALB/c nul+ mice, L. major 173 began to multiply progressively (Fig. 1). Parasites were detected in the popliteal and the left lumbar lymph node by day 3, in the liver by day 7, and in the spleen by day 14. Cutaneous metastases in the contralateral feet were first detected at week 4 of the infection, 2 to 3 weeks after dissemination to the visceral organs. However, macroscopic evidence of a primary or metastatic lesion was not seen until approximately 2 x 16 parasites were in the infected foot. Although small numbers of parasites (up to 13) were found in homogenates of the external ear, lesions on the pinnae were rarely found, and then only at terminal stages of the disease (week 15). The developing metastatic lesion differed from that of the DISSMINATD LISHMANIASIS 365 primary lesion macroscopically. dema in the metastatic lesions in the hind feet first appeared around the ankle rather than in the footpad (inserts, Fig. 1). Lesions on the front feet began as edema around the carpals ("wrists"), and with time the swelling progressed distally to involve the footpads and the toes. Metastatic lesions also developed on the tail. Like the development of cutaneous metastases in the feet, tail lesions began as edema around joints, and papules eventually appeared on the surface of the tail skin between the caudal vertebrae. Because it was not known whether the amount of edema at a site was a reflection of the number of parasites present, an additional experiment was performed to determine the number of parasites in specific areas of the hind feet. At 1 week after swelling was first detected in the left and right hind feet (weeks 3 and 1, respectively), four mice were sacrificed, and their hind feet were removed and cut into six pieces (A to F, Fig. 2b and c). ach section of foot was separately weighed and homogenized, and samples were plated on rabbit blood agar to enumerate parasites. It was found that 8% of the parasites in the primary lesion in the left hind foot were in the sections (C and D) containing the footpad. This was expected because the primary infection was initiated at that site. The majority (82%) of parasites in the metastatic lesion, by contrast, were found in the two sections of the foot which contained the tibiocalcar joint (ankle) and the tarsal bones (sections A and B). Lower concentrations of parasites were found in the sections of the foot that contained the footpad and the phalanges (sections C through F). Thus, not only are the contralateral feet and tails of mice predisposed as sites of cutaneous metastatic lesions, but the lesions appear to develop preferentially at sites near joints. In terms of changes in the number of viable parasites in the tissues, the pattern of development of disseminated disease in BALB/c nulnu mice was similar to that seen in BALB/c nul+ mice. L. major multiplied progressively in the footpad (Fig. 3), and although larger numbers of viable organisms were found in the liver and spleen of BALB/c nulnu mice at the late stages of the infection, the temporal relationship between the establishment of metastatic foci in visceral and in cutaneous sites was the same (data not shown). The absence of hair on the BALB/c nulnu mice allowed all cutaneous sites to be examined during the study for metastatic lesions. Nonetheless, cutaneous metastatic lesions developed only on the feet and-the tail. The only striking difference between normal and athymic, nude mice was that the primary lesions in the nude mice were always smaller, though they contained the same number of viable parasites (Fig. 3). For example, at week 6 of the infection, the mean sizes of the lesions in normal and nude BALB/c mice were 6.8 and 1.7 mm, respectively. After week 6, the feet became necrotic and did not further decrease in size. Presence and distribution of parasites in the blood. At week 3, when parasites were first detected in the contralateral foot, viable organisms were found in blood samples, and parasitemia increased nominally in subsequent weeks. xamination of Giemsa-stained smears of peripheral blood revealed that parasites were inside mononuclear phagocytes and polymorphonuclear leukocytes. At week 8 of the infection, for example, 56% of the 26 intact parasites identified in blood smears from five mice were in mononuclear phagocytes; 39% were in polymorphonuclear leukocytes. Although parasitized host cells usually contained 1 to 3 parasites, large mononuclear phagocytes containing 1 or more intact amastigotes were occasionally found in smears of blood taken from mice at late stages of the disease. Downloaded from on October 7, 218 by guest

3 366 HILL INFCT. IMMUN _ '- -j ' 3. 2.' Downloaded from 1. I -T- T- a.,j x x N Days / mmmw ---~~~~~~~5~~~ i Weeks TIM OF INFCTION FIG. 1. Pattern of development of disseminated disease in BALB/c nul+ mice inoculated in the left hind footpad with 15 L. major amastigotes. At predetermined intervals, the numbers of viable parasites in the left hind foot (primary lesion), popliteal lymph node, liver, spleen, the blood (normalized to 1. ml), and the right hind foot (metastatic lesion) were determined. Data are expressed as the means of four or five mice per time point. The detection limit for L. major in this study was approximately 1 parasites (2. logs). Parasites were first detected in the left lumbar lymph node (27) at day 3 of the infection and increased progressively in number thereafter (data not shown). Asterisks indicate the time when an increase in the size of the infected foot was first detected. The top insert is a photograph of a left hind foot at week 4 of the infection. The other two inserts are photographs of right feet: one at week 4 (no edema) and the other at week 1. on October 7, 218 by guest In situ multiplication of L. major in the visceral organs. The increase in the number of parasites in the liver and spleen during a disseminating cutaneous infection (Fig. 1) could be caused by the accumulation in those organs of parasites disseminating from the primary lesion or draining lymph nodes. In this case, the parasites that establish foci in the visceral organs need not have the capacity to replicate in those sites. Alternatively, a relatively small number of parasites might have colonized the liver and spleen and multiplied progressively. Amastigotes were implanted in the liver and spleen of nude mice by an intravenous injection to determine directly the capacity of L. major 173 to multiply in the visceral organs. Amastigotes began to multiply progressively within 7 days (Table 1). Furthermore, linear regression analysis revealed that the slopes of the growth curves for L. major in the footpad and liver of nude mice were not significantly different. The doubling time for L. major 173 in vivo was 52 to 57 h. This means that L. major 173 has the capacity to multiply at the same rate in the visceral organs as it does in the footpad. DISCUSSION Analyses of the role of host immunity and parasite physiology in the development of the various forms of leishmaniasis require a clear understanding of the behavior of the respective etiologic agents in the mammaiian host. This includes the growth rate of the parasite in the primary lesion, whether it disseminates to distant visceral or cutaneous sites, and the capacity of the parasite to multiply in those metastatic foci. This paper describes in detail the pattern of development of experimental, disseminated leishmaniasis in the mouse. A previous study (7) from this laboratory showed

4 VOL. 52, 1986 DISSMINATD LISHMANIASIS 367 D3 U,._o 2-I a -j b + I j~.iti ntire A B C D F ntire A B C D F Foot Foot Section Foot Foot Section C U.._- 2 W FIG. 2. Distribution of viable parasites within the left hind foot (primary lesion) and right hind foot (metastatic lesion) of BALB/c nul+ mice at the time when each contained equivalent numbers of parasites. Panel a shows the right hind foot of a mouse at week 1 of the infection; specific areas that were analyzed for their content of viable parasites are marked. Note the edema in the ankle region. Panel b (primary lesion, left hind foot at week 3) and panel c (metastatic lesion, right hind foot at week 1) display the concentration of viable parasites found in specific sections of the foot (II) and the. percentage of the total parasites in the foot (_) that was found in those sections. Bars show the mean + standard deviation (n = 4). that in BALB/c mice, L. major multiplies progressively at the site of inoculation in the footpad and disseminates to the visceral organs. However, cutaneous metastatic lesions appear late in the infection (1, 4, 7, 9, 23), and the temporal relationship of the establishment of metastatic foci in visceral and in cutaneous sites is not known. The present data clearly show that metastatic foci are established in the contralateral foot weeks before lesion development i.e., soon after the parasite disseminates to the liver and spleen. Not until the number of parasites in the foot approached 2 x 16 did a lesion appear. Although parasites were found in the liver and spleen by week 2 of the infection, parasites were not detected in blood samples until 2 weeks later. Leishmania spp. can disseminate from the skin to the visceral organs only via the blood. Therefore, either there were too few parasites in the circulation during the early stages of the disease to be detected, or parasitemia was intermittent, and no parasites were captured in the "grab samples" of the cardiac blood. It is well known that primary lesions in BALB/c mice eventually become necrotic (19), a progressive process that could release free parasites into the blood. In light of the capacity of the liver 1L I and spleen to remove Leishmania amastigotes from the blood (24), the release of small numbers of parasites into the peripheral circulation on an intermittent basis could result in the establishment of visceral metastatic foci. The present study shows that L. major 173 has the capacity to multiply in the liver and spleen. Within days after implantation in BALB/c nulnu mice, intravenously injected amastigotes began to multiply progressively. Thus, without the modulating influences of a protective immune response, the progressive increase in the number of parasites in the liver and spleen can be explained by multiplication in situ of a relatively small number of organisms that were taken up by those organs early during the development of systemic disease. It should be noted that many strains of L. major can cause visceral disease in susceptible mouse strains (2, 4, 7, 9, 23). It is possible, however, that not all cutaneous Leishmania strains posses the capacity to multiply at the same rate in visceral and cutaneous sites. Additional studies of other Leishmania strains in nude mice will be required to determine, in a systematic way, which cutaneous strains share this property. An interesting observation from the current studies was Downloaded from on October 7, 218 by guest

5 368 HILL INFCT. IMMUN. Site of TABL 1. Comparison of multiplication rate of L. major in liver and footpad of BALB/c nulnu mice Log1o of L. majoe infection 24 h wk 1 wk 2 wk 3 wk 4 r y-intercept Slopeb MDTc Liver 5.21 ± ± ± ± ± Footpadd 4.47 ± ± ± ± ± a Mean ± standard deviation of four animals. bslopes not significantly different (t =.174; P >.5) as determined by the Student t test for the equality of two slopes (26). ' MDT, mean doubling time in hours. d Data are those used in Fig. 3. that despite containing equivalent numbers of parasites, the infected footpads of nulnu mice were smaller than the corresponding footpads of their nul+ littermates. Studies by Handman et al. (4) and Mitchell et al. (12) have addressed the development of cutaneous disease in nude mice of various genotypes. These authors, however, do not report any differences between the size of cutaneous lesions in BALB/c nulnu mice and lesion size in BALB/c nul+ mice. It is possible that differences observed in the present study are related to the use of the footpad rather than the base of the tail as a primary lesion site and to the use of footpad thickness rather than a lesion score to monitor the progress of the infection. Nonetheless, our results do suggest that the host response to the parasite may determine, to a large extent, the pathology of the disease. The reasons for the smaller lesions in nude mice are not known. However, it is possible that if the host inflammatory response is greatly reduced in infected nude mice, the granulomas are smaller or fewer. This happens in nude mice infected with Mycobacterium bovis BCG (28). How do intracellular parasites such as Leishmania spp. establish foci of infection in extravascular cutaneous sites? The capillary endothelium and underlying basement membrane (1) seem to pose a formidable barrier to the passive movement of the parasite from the blood to the interstitium. It seems possible, therefore, that certain Leishmania strains disseminate to cutaneous sites inside mononuclear phagocytes or polymorphonuclear leukocytes, cells that actively move from blood to extravascular sites. This possibility was raised more than 2 years ago by Stauber (25), who suggested that the capacity of these types of host cells to breach -nu/ nu/nu ' ~~~~~~~~~~~~6. r -J z 5. ~~~~~~~ ~~~56 hrs WK OF INFCTION FIG. 3. Comparison of the changes, against time, in lesion size (-) and the number of L. major () in the primary lesions of BALB/c nul+ and BALB/c nulnu mice. ach point represents the mean ± standard deviation of four to five mice. the capillaries may aid the dissemination of the parasite. In this regard, the present study shows that parasites could be found inside host blood cells when metastatic foci appeared in the contralateral foot. Although our data do not establish a cause-and-effect relationship between the presence of parasitized host cells in the blood and the initiation of cutaneous metastatic lesions, they are consistent with the hypothesis. Furthermore, the presence of infected neutrophils in the blood of animals (this study and [29]) and humans (21) with disseminated disease supports the suggestion made by others (3, 15, 25) that this cell type may also be an important component in the host response to Leishmania infection. It is well known that in BALB/c mice, metastatic lesions almost invariably appear on the feet (1, 7, 17, 23). By enumerating parasites at selected sites within the foot, we have extended those earlier observations by showing that parasites predominate in the region of the ankle. Furthermore, it is possible that the edema that develops distal to the ankle is caused by the progressive increase in the number of parasites in other foci around the metatarsophalangeal and other arthrodial joints. The predilection of cutaneous Leishmania strains for the feet and the tail may be a reflection of a tropism for cooler anatomical sites (11, 31). In addition, in vitro studies (22) have suggested that the microbicidal mechanisms of macrophages may be impaired at the lower temperatures of the extremities. In this regard, however, it is important to note that lesions on the ear were rare, despite viable parasites at that site. Furthermore, despite their lack of hair, athymic nude mice developed no metastatic lesions at cutaneous sites other than the feet and tail. Thus, factors other than temperature, such as the potential for constant, low-grade inflammation in tissues near joints, could contribute to the development of metastatic foci in cutaneous sites. Numerous studies have shown that immunity to Leishmania spp. is probably expressed by mononuclear phagocytes that have been activated to a state of enhanced microbicidal potential by lymphokines released from specifically sensitized T lymphocytes (13, 14, 17). It is clear, however, that we know very little about the pathophysiology of the various forms of leishmaniasis, including how mononuclear phagocytes (18, 25) and possibly other host cells may aid the dissemination of the parasite. The mechanisms involved are undoubtedly complex, and the physiological properties of the parasite and the specific immunological competence of the host may modulate the pattern of development of disease. However, the capacity to enumerate the parasite in host tissues should make it possible to identify the factors that influence the outcome of Leishmania infection in the mammalian host. ACKNOWLDGMNTS This work was supported by Public Health Service grants AI and AI from the National Institute of Allergy and Infectious Diseases, by Biomedical Research support grant RR-575 from the Downloaded from on October 7, 218 by guest

6 VOL. 52, 1986 Division of Research Resources, National Institutes of Health, and by grants to the Trudeau Institute by the J. M. and Leon Lowenstein Foundations. I acknowledge the technical assistance of Kathy Crowder and Mike Zemany and thank Mary Durett for typing the manuscript. LITRATUR CITD 1. Barral, A.,. A. Petersen, D. A. Sacks, and F. A. Neva Late metastatic leishmaniasis in the mouse: a model for mucocutaneous disease. Am. J. Trop. Med. Hyg. 32: Bjorvatn, B., and F. A. Neva A model in mice for experimental leishmaniasis with a West African strain of Leishmania tropica. Am. J. Trop. Med. Hyg. 28: Chang, K.-P Leishmanicidal mechanisms of human polymorphonuclear phagocytes. Am. J. Trop. Med. Hyg. 3: : 4. Handman,., R. Ceredig, and G. F. Mitchell Murine cutaneous leishmaniasis: disease patterns in intact and nude mice of various genotypes and examination of some differences between normal and infected macrophages. Aust. J. xp. Biol. Med. Sci. 57: Hill, J Quantitation of Leishmania tropica major by its ability to form distinct colonies on agar-based media. J. Parasitol. 69: Hill, J Resistance to cutaneous leishmaniasis: acquired ability of the host to kill parasites at the site of infection. Infect. Immun. 45: Hill, J. O., R. J. North, and F. M. Collins Advantages of measuring changes in the number of viable parasites in murine models of experimental cutaneous leishmaniasis. Infect. Immun. 39: Hommel, M The genus Leishmania: biology of the parasites and clinical aspects. Bull. Inst. Pasteur 75: Leclerc, C., F. Modabber,. Deriaud, and L. Chedid Systemic infection of Leishmania tropica (major) in various strains of mice. Trans. R. Soc. Trop. Med. Hyg. 75: Luft, J. H Capillary permeability. I. Structural considerations, p In B. W. Zweifach, L. Grant, and R. T. McCluskey (ed.), The inflammatory process. Academic Press, Inc., New York. 11. Marsden, P. L Current concepts in parasitology: leishmaniasis. N. ngl. J. Med. 3: Mitchell, G. F., R. F. Anders, C. B. Chapman, I. C. Roberts- Thompson,. Handman, K. M. Cruse, M. D. Richard, M. W. Lightowlers, and. G. Garcia xamination of strategies for vaccination against parasitic infection or disease using animal models. Contemp. Top. Immunobiol. 12: Murray, H. W., B. Y. Rubin, and C. D. Rothermel Killing of intracellular Leishmania donovani by lymphokine-stimulated human mononuclear cells: evidence that interferon-y is the activating lymphokine. J. Clin. Invest. 72: Nacy, C. A., M. S. Meltzer,. J. Leonard, and D. J. Wyler Interacellular replication and lymphokine-induced de- DISSMINATD LISHMANIASIS 369 struction of Leishmania tropica in C3H/HeN mouse macrophages. J. Immunol. 127: Pearson, R. D., and R. T. Steigbigel Phagocytosis and killing of the protozoan Leishmania donovani by human polymorphonuclear leukocytes. J. Immunol. 127: Pearson, R. D., D. A. Wheeler, L. H. Harrison, and H. D. Kay The immunobiology of leishmaniasis. Rev. Infect. Dis. 5: Perez, H., F. Labrador, and J. W. Torrealba Variations in the response of five strains of mice to Leishmania mexicana. Int. J. Parasitol. 9: Poulter, L. W Mechanisms of immunity to leishmaniasis. II. Significance of the intramacrophage localization of the parasite. Clin. xp. Immunol. 4: Preston, P. M., and D. C. Dumonde Clinical and experimental leishmaniasis, p In S. Cohen and. H. Sadun (ed.), Immunology of parasitic infections. Blackwell Scientific Publications, Ltd., Oxford. 2. Reed, S. G., M. Barral-Netto, and J. A. Inverso Treatment of experimental visceral leishmaniasis with lymphokine encapsulated in liposomes. J. Immunol. 132: Rohrs, L. C Leishmaniasis in the Sudan Republic. XVIII. Parasitemia in kala-azar. Am. J. Trop. Med. Hyg. 13: Scott, P Impaired macrophage leishmanicidal activity at cutaneous temperature. Parasite Immunol. 7: Scott, P. A., and J. P. Farrell xperimental cutaneous leishmaniasis: disseminated leishmaniasis in genetically susceptible and resistant mice. Am. J. Trop. Med. Hyg. 31: Stauber, L. A Host resistance to the Khartoum strain of Leishmania donovani. Rice Inst. Pamphlet 45: Stauber, L. A The origin and significance of the distribution of parasites in visceral leishmaniasis. Trans. N.Y. Acad. Sci. 28: Steel, R. G. D., and J. H. Torrie Principles and procedures of statistics: with special reference to the biological sciences, p McGraw-Hill Book Co., New York. 27. Tilney, N. L Patterns of lymphatic drainage in the adult laboratory rat. J. Anat. 19: Ueda, K., S. Yamazaki, S. Yamamoto, and S. Someya Spleen cell transfer induces T cell-dependent granulomas in tuberculous nude mice. RS J. Reticuloendothel. Soc. 31: Van Joost, K. S., and J. F. Sluiters Appearance of Leishmania donovani (Kenya strain) in the blood of experimentally infected golden hamsters (Mesocricetus auratus). Trop. Geogr. Med. 24: World Health Organization Leishmaniasis. Annual report of the Special Program for Research and Training in Tropical Diseases. TDL/ARC5/81.7-LISH. World Health Organization, Geneva. 31. Zuckerman, A Current status of the immunology of blood and tissue protozoan. I. Leishmania. xp. Parasitol. 38: Downloaded from on October 7, 218 by guest