Vol. 52, No. 2INFECTION AND IMMUNITY, May 1986, p. 364-3690019-9567/86/050364-06$02.00/0Copyright © 1986, American Society for Microbiology

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

JOSEPH 0. HILL

Trudeau Institute, Inc., Saranac Lake, New York 12983

Received 18 November 1985/Accepted 13 January 1986

A clear understanding of the etiology of the various forms of leishmaniasis will require knowledge of howphysiological 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 ofinoculation in the skin, their capacity to disseminate to visceral and distant cutaneous sites, and their capacityto multiply once there. This paper details the pattern of development of disseminated Leishmania majorinfection in susceptible BALB/c nu/+ and BALB/c nulnu mice. It was found that the parasite disseminates fromthe hind footpad to distant cutaneous sites soon after metastatic foci are established in the liver and spleen. Bothmononuclear 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 numberprogressively. L. major has the capacity to multiply at visceral and cutaneous sites at the same rate. Despite thepresence of viable parasites in a number of skin sites, cutaneous metastatic lesions developed almost exclusivelyon 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 ofhuman disease (8). Leishmania donovani is the etiologicagent of visceral leishmaniasis (kala azar). In this form of thedisease, only a small ulcer may develop at the site of the biteof the insect vector, yet the parasite disseminates to theliver, spleen, and bone marrow (31). If untreated, the diseaseis fatal. Leishmania mexicana, on the other hand, usuallyremains confined to a single, yet pronounced, cutaneouslesion. It is thought that the clinical type of leishmaniasis isa reflection of the properties of the specific parasite in-volved, such as the ability of the parasite to multiply at thetemperatures of the skin and the visceral organs (11, 16).Clearly, however, host immunity also influences, in a majorway, the pattern of development of leishmaniasis not only byrestraining the multiplication of the parasite at the site ofinoculation (6), but also by destroying parasites in metastaticfoci at distant visceral and cutaneous sites. It is the apparentfailure of these immune mechanisms that contributes to thedevelopment of the disseminated forms of leishmaniasis,such as kala azar, post-kala azar dermal leishmaniasis, anddiffuse cutaneous disease (11, 16).

Disseminated leishmaniasis has proved difficult to treatwith the antibiotics that are currently available becauserepeated courses of chemotherapy are neccessary (30).Furthermore, the parasite is often not completely eliminatedfrom the tissues, and relapses are common (30, 11). Al-though no vaccine has been developed, it is hoped thatimmunization (12) or immunotherapy (20) may succeedwhere chemotherapy has failed. This hope has stimulated a

great deal of interest in the immune mechanisms responsiblefor the destruction of the parasite (for a review, see refer-ence 16). However, very little is known about the etiology ofleishmaniasis, that is, how particular combinations of para-

site and host result in specific forms of the disease. Because

Leishmania spp. are obligate intracellular parasites in themammalian host, mononuclear phagocytes are possiblyother host cells may be involved in the transport of the

parasite through the blood and in its establishment in distantvisceral and cutaneous sites. Thus, a clearer understandingof the mechanisms of dissemination of Leishmania spp. inexperimental mammalian hosts may facilitate the develop-ment of effective chemotherapy and immunotherapy.A number of different mouse strains and species of Leish-

mania have been used in animal models of the various formsof human leishmaniasis (19). Visceral disease can be inducedby injecting L. donovani intravenously. However, in thismodel the early events in the development of disseminateddisease, such as the multiplication of the parasite in the skinand its dissemination to the viscera, cannot be studied. Onthe other hand, Leishmania major and L. mexicana domultiply at the cutaneous site of inoculation. However, inmost immunocompetent hosts the parasite disseminates tothe 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 boththe visceral and the disseminated cutaneous forms of thenatural disease in man. This is L. major in BALB/c mice.BALB/c mice are extremely susceptible to this parasite, aswell as to many other leishmania spp. that cause humancutaneous disease. It has been shown in other laboratoriesthat the cutaneous lesions increase progressively in size andvisceral disease and metastatic cutaneous lesions develop (2,4). Furthermore, as a result of the development of methodsto enumerate viable Leishmania parasites in tissue (5, 7),specific aspects of the dissemination of Leishmania spp. canbe examined. This paper reports the results of studies thatdocument the pattern of development of systemic disease inBALB/c mice and that provide a basis for formulatingspecific hypotheses about the mechanisms by which a Leish-mania sp. disseminates and establishes foci of infection invisceral and cutaneous metastatic sites. Furthermore,athymic (nude) mice were included in the study so that therole of certain physiological properties of the parasite in the

364

DISSEMINATED LEISHMANIASIS 365

disease could be examined in the absence of T-cell-mediatedimmunity.

MATERIALS AND METHODS

Mice. Specific-pathogen-free, female BALB/c nul+ (nor-mal) and BALB/c nulnu (nude) mice, 8 to 12 weeks old, wereused. The mice were obtained from the Trudeau InstituteAnimal Breeding Facility, Saranac Lake, N.Y., and weremaintained under barrier-sustained conditions in isolators.The colony was started in 1977 from founder stock carryingin nu mutation on a BALB/cAnN genetic background. Themouse colony is routinely monitored for pathogenic bacteriaand mycoplasmas by standard bacteriological techniques andmonitored for virus infections by serological tests (mousevirus profile 80-211; Microbiological Associates, Bethesda,Md.).Enumeration of Leishmania parasites. L. major 173

(WR173, LIV680) was used. The source and passage historyof this human isolate have been previously described (6, 7).Primary, subcutaneous infections were initiated by injecting105 amastigotes in 50 ,ul of Schneider Drosophila medium(GIBO Laboratories, Grand Island, N.Y.) into the left hindfootpad (6, 7). In some experiments, systemic infectionswere established by injecting 105 amastigotes in 200 ixl into alateral tail vein. Amastigotes were used to infect micebecause a previous study from this laboratory (7) showedthat compared with promastigotes, a larger proportion ofamastigote inocula survives the first 24 h to initiate theinfection.At predetermined intervals, four or five mice were anes-

thetized, and a 0.7- to 1.0-ml sample of cardiac blood wastaken. The blood sample was immediately transferred to asterile plastic tube containing 50 ,ul of 0.1 M EDTA. Periph-eral blood smears were then prepared and stained withGiemsa.To document the pattern of development of disseminated

disease, the number of viable parasites in the left hindfootpad (primary lesion), in the draining lymph nodes (27),the blood, liver, spleen, the external ear (pinna), and in theright hind footpad (metastatic lesion) was determined byplating appropriately diluted aliquots of tissue homogenateson rabbit blood agar (5, 6). After 5 to 7 days of incubation at26°C, the promastigote colonies were counted, and thenumbers of parasites in the respective tissues were calcu-lated.Measurement of lesion size. The progress of the infection

was also followed by measuring changes in the thickness ofthe infected footpad as previously described (7).

RESULTSPattern 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 lumbarlymph node by day 3, in the liver by day 7, and in the spleenby day 14. Cutaneous metastases in the contralateral feetwere first detected at week 4 of the infection, 2 to 3 weeksafter dissemination to the visceral organs. However, macro-scopic evidence of a primary or metastatic lesion was notseen until approximately 2 x 106 parasites were in theinfected foot. Although small numbers of parasites (up to103) were found in homogenates of the external ear, lesionson the pinnae were rarely found, and then only at terminalstages of the disease (week 15).The developing metastatic lesion differed from that of the

primary lesion macroscopically. Edema in the metastaticlesions in the hind feet first appeared around the ankle ratherthan in the footpad (inserts, Fig. 1). Lesions on the front feetbegan as edema around the carpals ("wrists"), and with timethe swelling progressed distally to involve the footpads andthe toes. Metastatic lesions also developed on the tail. Likethe development of cutaneous metastases in the feet, taillesions began as edema around joints, and papules eventu-ally appeared on the surface of the tail skin between thecaudal vertebrae.Because it was not known whether the amount of edema at

a site was a reflection of the number of parasites present, anadditional experiment was performed to determine the num-ber of parasites in specific areas of the hind feet. At 1 weekafter swelling was first detected in the left and right hind feet(weeks 3 and 10, respectively), four mice were sacrificed,and their hind feet were removed and cut into six pieces (Ato F, Fig. 2b and c). Each section of foot was separatelyweighed and homogenized, and samples were plated onrabbit blood agar to enumerate parasites. It was found that80% of the parasites in the primary lesion in the left hind footwere in the sections (C and D) containing the footpad. Thiswas expected because the primary infection was initiated atthat site. The majority (82%) of parasites in the metastaticlesion, by contrast, were found in the two sections of thefoot which contained the tibiocalcar joint (ankle) and thetarsal bones (sections A and B). Lower concentrations ofparasites were found in the sections of the foot that con-tained the footpad and the phalanges (sections C through F).Thus, not only are the contralateral feet and tails of micepredisposed as sites of cutaneous metastatic lesions, but thelesions appear to develop preferentially at sites near joints.

In terms of changes in the number of viable parasites in thetissues, the pattern of development of disseminated diseasein BALB/c nulnu mice was similar to that seen in BALB/cnul+ mice. L. major multiplied progressively in the footpad(Fig. 3), and although larger numbers of viable organismswere found in the liver and spleen of BALB/c nulnu mice atthe late stages of the infection, the temporal relationshipbetween the establishment of metastatic foci in visceral andin cutaneous sites was the same (data not shown). Theabsence of hair on the BALB/c nulnu mice allowed allcutaneous sites to be examined during the study for meta-static lesions. Nonetheless, cutaneous metastatic lesionsdeveloped only on the feet and-the tail. The only strikingdifference between normal and athymic, nude mice was thatthe 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 meansizes of the lesions in normal and nude BALB/c mice were6.8 and 1.7 mm, respectively. After week 6, the feet becamenecrotic and did not further decrease in size.

Presence and distribution of parasites in the blood. At week3, when parasites were first detected in the contralateralfoot, viable organisms were found in blood samples, andparasitemia increased nominally in subsequent weeks. Ex-amination of Giemsa-stained smears of peripheral bloodrevealed that parasites were inside mononuclear phagocytesand polymorphonuclear leukocytes. At week 8 of the infec-tion, for example, 56% of the 260 intact parasites identified inblood smears from five mice were in mononuclear pha-gocytes; 39% were in polymorphonuclear leukocytes. Al-though parasitized host cells usually contained 1 to 3 para-sites, large mononuclear phagocytes containing 10 or more

intact amastigotes were occasionally found in smears ofblood taken from mice at late stages of the disease.

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INFECT. IMMUN.

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TIME OF INFECTIONFIG. 1. Pattern of development of disseminated disease in BALB/c nul+ mice inoculated in the left hind footpad with 105 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 1.0 ml), and the right hind foot (metastatic lesion) were determined. Data are expressed as the means of fouror five mice per time point. The detection limit for L. major in this study was approximately 100 parasites (2.0 logs). Parasites were firstdetected 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 hindfoot 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 10.

In situ multiplication of L. major in the visceral organs. Theincrease in the number of parasites in the liver and spleenduring a disseminating cutaneous infection (Fig. 1) could becaused by the accumulation in those organs of parasitesdisseminating from the primary lesion or draining lymphnodes. In this case, the parasites that establish foci in thevisceral organs need not have the capacity to replicate inthose sites. Alternatively, a relatively small number ofparasites might have colonized the liver and spleen andmultiplied progressively. Amastigotes were implanted in theliver and spleen of nude mice by an intravenous injection todetermine directly the capacity of L. major 173 to multiply inthe visceral organs. Amastigotes began to multiply progres-

sively within 7 days (Table 1). Furthermore, linear regres-

sion analysis revealed that the slopes of the growth curvesfor L. major in the footpad and liver of nude mice were not

significantly different. The doubling time for L. major 173 invivo was 52 to 57 h. This means that L. major 173 has thecapacity to multiply at the same rate in the visceral organs asit does in the footpad.

DISCUSSIONAnalyses of the role of host immunity and parasite phys-

iology in the development of the various forms of leishma-niasis require a clear understanding of the behavior of therespective etiologic agents in the mammaiian host. Thisincludes the growth rate of the parasite in the primary lesion,whether it disseminates to distant visceral or cutaneoussites, and the capacity of the parasite to multiply in thosemetastatic foci. This paper describes in detail the pattern ofdevelopment of experimental, disseminated leishmaniasis inthe mouse. A previous study (7) from this laboratory showed

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366 HILL

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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 10 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 10) display the concentration of viable parasites found in specificsections of the foot (EII) 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 atthe site of inoculation in the footpad and disseminates to thevisceral organs. However, cutaneous metastatic lesions ap-pear late in the infection (1, 4, 7, 9, 23), and the temporalrelationship of the establishment of metastatic foci in vis-ceral and in cutaneous sites is not known. The present dataclearly show that metastatic foci are established in thecontralateral 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 x106 did a lesion appear.Although parasites were found in the liver and spleen by

week 2 of the infection, parasites were not detected in bloodsamples until 2 weeks later. Leishmania spp. can dissemi-nate from the skin to the visceral organs only via the blood.Therefore, either there were too few parasites in the circu-lation during the early stages of the disease to be detected, orparasitemia was intermittent, and no parasites were capturedin the "grab samples" of the cardiac blood. It is well knownthat primary lesions in BALB/c mice eventually becomenecrotic (19), a progressive process that could release freeparasites into the blood. In light of the capacity of the liver

and spleen to remove Leishmania amastigotes from theblood (24), the release of small numbers of parasites into theperipheral circulation on an intermittent basis could result inthe 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 afterimplantation in BALB/c nulnu mice, intravenously injectedamastigotes began to multiply progressively. Thus, withoutthe modulating influences of a protective immune response,the progressive increase in the number of parasites in theliver and spleen can be explained by multiplication in situ ofa relatively small number of organisms that were taken up bythose organs early during the development of systemicdisease. It should be noted that many strains of L. major cancause visceral disease in susceptible mouse strains (2, 4, 7, 9,23). It is possible, however, that not all cutaneous Leishma-nia strains posses the capacity to multiply at the same rate invisceral and cutaneous sites. Additional studies of otherLeishmania strains in nude mice will be required to deter-mine, in a systematic way, which cutaneous strains sharethis property.An interesting observation from the current studies was

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TABLE 1. Comparison of multiplication rate of L. major in liver and footpad of BALB/c nulnu mice

Site of Log1o of L. majoeinfection 24 h wk 1 wk 2 wk 3 wk 4 r y-intercept Slopeb MDTc

Liver 5.21 ± 0.14 6.68 ± 0.21 7.35 ± 0.42 8.67 ± 0.37 9.30 ± 0.23 0.99 5.26 1.02 57Footpadd 4.47 ± 0.09 5.40 ± 0.21 6.95 ± 0.15 7.40 ± 0.15 8.36 ± 0.33 0.99 4.71 0.93 52

a Mean ± standard deviation of four animals.bSlopes not significantly different (t = 0.174; P > 0.5) 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, theinfected footpads of nulnu mice were smaller than thecorresponding footpads of their nul+ littermates. Studies byHandman et al. (4) and Mitchell et al. (12) have addressedthe development of cutaneous disease in nude mice ofvarious genotypes. These authors, however, do not reportany differences between the size of cutaneous lesions inBALB/c nulnu mice and lesion size in BALB/c nul+ mice. Itis possible that differences observed in the present study arerelated to the use of the footpad rather than the base of thetail as a primary lesion site and to the use of footpadthickness rather than a lesion score to monitor the progressof the infection. Nonetheless, our results do suggest that thehost response to the parasite may determine, to a largeextent, the pathology of the disease. The reasons for thesmaller lesions in nude mice are not known. However, it ispossible that if the host inflammatory response is greatlyreduced in infected nude mice, the granulomas are smaller orfewer. This happens in nude mice infected with Mycobacte-rium 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 mem-brane (10) seem to pose a formidable barrier to the passivemovement of the parasite from the blood to the interstitium.It seems possible, therefore, that certain Leishmania strainsdisseminate to cutaneous sites inside mononuclear pha-gocytes or polymorphonuclear leukocytes, cells that activelymove from blood to extravascular sites. This possibility wasraised more than 20 years ago by Stauber (25), who sug-gested that the capacity of these types of host cells to breach

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(-) and the number of L. major (0) in the primary lesions ofBALB/c nul+ and BALB/c nulnu mice. Each point represents themean ± standard deviation of four to five mice.

the capillaries may aid the dissemination of the parasite. Inthis regard, the present study shows that parasites could befound inside host blood cells when metastatic foci appearedin the contralateral foot. Although our data do not establisha cause-and-effect relationship between the presence ofparasitized host cells in the blood and the initiation ofcutaneous metastatic lesions, they are consistent with thehypothesis. Furthermore, the presence of infected neutro-phils in the blood of animals (this study and [29]) and humans(21) with disseminated disease supports the suggestion madeby others (3, 15, 25) that this cell type may also be animportant component in the host response to Leishmaniainfection.

It is well known that in BALB/c mice, metastatic lesionsalmost invariably appear on the feet (1, 7, 17, 23). Byenumerating parasites at selected sites within the foot, wehave extended those earlier observations by showing thatparasites predominate in the region of the ankle. Further-more, it is possible that the edema that develops distal to theankle is caused by the progressive increase in the number ofparasites in other foci around the metatarsophalangeal andother arthrodial joints. The predilection of cutaneous Leish-mania strains for the feet and the tail may be a reflection ofa tropism for cooler anatomical sites (11, 31). In addition, invitro studies (22) have suggested that the microbicidal mech-anisms of macrophages may be impaired at the lower tem-peratures of the extremities. In this regard, however, it isimportant to note that lesions on the ear were rare, despiteviable parasites at that site. Furthermore, despite their lackof hair, athymic nude mice developed no metastatic lesionsat cutaneous sites other than the feet and tail. Thus, factorsother than temperature, such as the potential for constant,low-grade inflammation in tissues near joints, could contrib-ute to the development of metastatic foci in cutaneous sites.Numerous studies have shown that immunity to Leishma-

nia spp. is probably expressed by mononuclear phagocytesthat have been activated to a state of enhanced microbicidalpotential by lymphokines released from specifically sensi-tized T lymphocytes (13, 14, 17). It is clear, however, thatwe know very little about the pathophysiology of the variousforms of leishmaniasis, including how mononuclearphagocytes (18, 25) and possibly other host cells may aid thedissemination of the parasite. The mechanisms involved areundoubtedly complex, and the physiological properties ofthe parasite and the specific immunological competence ofthe host may modulate the pattern of development of dis-ease. However, the capacity to enumerate the parasite inhost tissues should make it possible to identify the factorsthat influence the outcome of Leishmania infection in themammalian host.

ACKNOWLEDGMENTSThis work was supported by Public Health Service grants AI-14383

and AI-22964 from the National Institute of Allergy and InfectiousDiseases, by Biomedical Research support grant RR-05705 from the

INFECT. IMMUN.368 HILL

DISSEMINATED LEISHMANIASIS 369

Division of Research Resources, National Institutes of Health, andby grants to the Trudeau Institute by the J. M. and Leon LowensteinFoundations.

I acknowledge the technical assistance of Kathy Crowder andMike Zemany and thank Mary Durett for typing the manuscript.

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13. Murray, H. W., B. Y. Rubin, and C. D. Rothermel. 1983. Killingof intracellular Leishmania donovani by lymphokine-stimulatedhuman mononuclear cells: evidence that interferon-y is theactivating lymphokine. J. Clin. Invest. 72:1506-1510.

14. Nacy, C. A., M. S. Meltzer, E. J. Leonard, and D. J. Wyler.1981. Interacellular replication and lymphokine-induced de-

struction of Leishmania tropica in C3H/HeN mouse macro-phages. J. Immunol. 127:2381-2386.

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16. Pearson, R. D., D. A. Wheeler, L. H. Harrison, and H. D. Kay.1983. The immunobiology of leishmaniasis. Rev. Infect. Dis.5:907-927.

17. Perez, H., F. Labrador, and J. W. Torrealba. 1979. Variations inthe response of five strains of mice to Leishmania mexicana.Int. J. Parasitol. 9:27-32.

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19. Preston, P. M., and D. C. Dumonde. 1976. Clinical and experi-mental leishmaniasis, p. 167-202. In S. Cohen and E. H. Sadun(ed.), Immunology of parasitic infections. Blackwell ScientificPublications, Ltd., Oxford.

20. Reed, S. G., M. Barral-Netto, and J. A. Inverso. 1984. Treatmentof experimental visceral leishmaniasis with lymphokine encap-sulated in liposomes. J. Immunol. 132:3116-3119.

21. Rohrs, L. C. 1964. Leishmaniasis in the Sudan Republic. XVIII.Parasitemia in kala-azar. Am. J. Trop. Med. Hyg. 13:265-271.

22. Scott, P. 1985. Impaired macrophage leishmanicidal activity atcutaneous temperature. Parasite Immunol. 7:277-288.

23. Scott, P. A., and J. P. Farrell. 1982. Experimental cutaneousleishmaniasis: disseminated leishmaniasis in genetically suscep-tible and resistant mice. Am. J. Trop. Med. Hyg. 31:230-238.

24. Stauber, L. A. 1958. Host resistance to the Khartoum strain ofLeishmania donovani. Rice Inst. Pamphlet 45:80-96.

25. Stauber, L. A. 1966. The origin and significance of the distribu-tion of parasites in visceral leishmaniasis. Trans. N.Y. Acad.Sci. 28:635-643.

26. Steel, R. G. D., and J. H. Torrie. 1960. Principles and proce-dures of statistics: with special reference to the biologicalsciences, p. 161-193. McGraw-Hill Book Co., New York.

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28. Ueda, K., S. Yamazaki, S. Yamamoto, and S. Someya. 1982.Spleen cell transfer induces T cell-dependent granulomas intuberculous nude mice. RES J. Reticuloendothel. Soc. 31:469-478.

29. Van Joost, K. S., and J. F. Sluiters. 1972. Appearance ofLeishmania donovani (Kenya strain) in the blood of experimen-tally infected golden hamsters (Mesocricetus auratus). Trop.Geogr. Med. 24:292-297.

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