Leishmania mexicana: Serial cultivation of intracellular stages in a cell-free medium

EXPERIMENTAL

PARASITOLOGY

Leishmania

58, 72-80 (1984)

mexicana:

Serial Cultivation of intracellular Cell-Free Medium ALFRED

Department

of Immunology

Stages in a

A. PANI

and Infectious Diseases, The Johns Hopkins Health, Baltimore, Maryland 21205, U.S.A.

(Accepted for publication

School

of Hygiene

and Public

1 November 1983)

PAN, A. A. (1984). Leishmania mexicana: Serial cultivation of intracellular stages in a cell-free medium. Experimental Parasitology 58, 72-80. A cell-free liquid medium has been devised for serial cultivation of Leishmania mexicana pifanoi amastigotes at 33 and 35 C. It consists of tissue culture Medium 199 fortified primarily with a high concentration of water-soluble vitamins, nucleotides, and inactivated fetal bovine serum. The initial pH of the medium is 7.2. Starting with a population of promastigotes as inoculum and serially cultured at 33 or 3.5 C at 4- to IO-day intervals, the proportion of amastigotes steadily increased and that of promastigotes gradually decreased during the first subculture. By the end of the incubation period in the second subculture, practically all (99%) of the organisms are amastigotes. The amastigotes thus obtained can be cultured indefinitely by serial transfers. In this medium, amastigotes may reach a density of 8 x lO’/ml after IO days of incubation at 33 C, and 5 x lO’/ml at 35 C. The medium was modified to have an initial pH of 8.0 by Hepes [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] buffer and higher concentrations of sodium bicarbonate. When amastigotes cultured in the original medium at 33 or 35 C are transferred into the modified medium and incubated at 26 C, the amastigotes entirely transformed into promastigotes after three serial passages. These promastigotes could be serially subcultured indefinitely in the modified medium at 4- to 12-day intervals. The promastigotes cultured at 26 C may reach a population density of 7 X lo’/ ml after 12 days of incubation. INDEX DESCRIPTORS: Leishmania mexicana pifanoi; Protozoa, parasitic; Hemoflagellate; Amastigote; Promastigote; Cultivation, serial, axenic; Transformation; Nutrition; Medium, cell-free.

macrophages (Chang and Dwyer 1976). These intracellular organisms have been cultured in vitro at body temperature in macrophages (Berens and Marr 1979; Chang 1980; Chang and Dwyer 1978; Herman 1966). However, attempts to serially culture amastigotes in cell-free media have met with difficulty (Lemma and Schiller 1964; Trager 1953; Wonde and Honiberg 1971). Extensive biochemical and metabolic studies have been carried out with promastigotes, as these organisms are readily obtained in cell-free media at temperatures below 28 C (Dwyer 1972; Evans 1978). Very little is known concerning the biochemical and metabolic properties of amastigotes,

INTRODUCTION

The Leishmania species have a simple life cycle which includes two distinct morphological and physiological forms. Promastigotes exist as extracellular motile parasites in the digestive tracts of phlebotomine insects, and can be grown at ambient temperature in various cell-free, complex media containing mammalian serum or in defined media (Evans 1978; Hendricks et al. 1978). Amastigotes are found in the phagolysosomes of vertebrate

’ Present address: Center for Parasitology, Zoology Department, Morrill Science Center, University of Massachusetts, Amherst, MA. 01003-0027, U.S.A. 72 0014-4894184 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.

Leishrnania

mexicana:

CELL-FREE

since it has not been possible to obtain large numbers in cell-free media (Dwyer 1976; Trager 1983). Intracellular amastigotes may be obtained by isolation from infected animals or from using in vitro-cultured macrophages as a substrate. The procedures for such isolation are usually time consuming and “harsh,” and may result in the collection of degenerating parasites with altered biochemical and physiological characteristics. In addition, contamination with hostcell debris cannot be avoided (Brazil 1978; Chang 1980; Childs et al. 1976; Hart et al. 1981a; Infante et al. 1980). In vitro cultivation of the intracellular stages of Leishmuniu species at or near the physiological temperature in cell-free medium could provide information leading to a better understanding of the nutritional requirements and metabolism of these organisms. This report describes serial cultivation of amastigotes of a strain of L. mexicunu pifunoi at 33 and 35 C. MATERIALS AND METHODS The Leishmania mexicana used in these experiments are provisionally identified as L. m. pifanoi (Convit et al. 1972) based on serological and pathogenic characteristics. It was obtained through the courtesy of Dr. S. C. Pan (Harvard University School of Public Health, Boston, MA, USA). The organism was originally isolated from a skin lesion of a Venezuelan patient suffering from disseminated cutaneous leishmaniasis, and was sent to Harvard in 1960 (Convit ef al. 1962). The strain was passed in golden hamster, Mesocricetus auratus, 19 times before it was received for experiments. Arriving in our laboratory in 1978, the strain was cloned by a modification of the Dwyer (1977) procedure for obtaining a genetically homogeneous population. A clone, having desirable growth characteristics, was selected for the following experiments. The compositions of two media, the cell-free medium (JH-30) and the modified cell-free medium (JH31), are shown in Tables I-V. The initial pH of medium JH-30 is 7.2 and that of JH-31 is 8.0. The osmotic pressure of both media is 357 mOsmo1. These media were variants of a medium (F-69) for the cultivation of Trypanosoma cruzi amastigotes (Pan 1978). The major modifications were (1) the final concentration of fetal bovine serum was raised to 25% for both media; (2) Hepes [4-(2-hydroxyethyl)-l-piperazine-

73

MEDIUM FOR SERIAL CULTURE

ethanesulfonic acid] was used as a buffer; (3) the concentration of sodium bicarbonate was increased in medium JH-3 1; and (4) a higher concentration of nucleotides was used in the stock solution (Table V). Cultures for various experiments were prepared in sterile screw-cap, disposable centrifuge tubes (15 ml capacity) (Coming Glass Works, Coming, NY, USA). Each tube contained 5 ml of medium, and received 1 ml of inoculum from a previous subculture which had been adjusted with fresh medium to a concentration of lo6 organisms/ml. The incubation temperatures (26, 33, or 35 C) fluctuated 2 1 C. Wright-Giemsa-stained smears were prepared according to Pan (1978) for morphologic studies under oil-immersion lens. Enumeration of organisms were made with a hemocytometer at 400X magnification after a sample of culture was diluted with 10% formalin in phosphate-buffered saline (PBS; pH 7.2). For serial cultivation, two culture tubes were prepared for each transfer. After an incubation period (usually 96 hr) at a designated temperature, cultures were pooled and subcultures were made. Measurements of organisms on stained smears were made by calibrated ocular micrometer under oil-immersion lens. For each growth curve studies, 28 culture tubes were prepared for each experiment and incubated appropriately. Tubes in duplicate were removed every 24 hr for enumeration, morphological studies, and pH determination. Several experiments were conducted at 26 and 33 C TABLE I Basal Solution for Leishmania

Sp. Medium

Glucose (8.0%) Ml99 (10x concentrated)

Trypticase (5.0%) Vitamin mix (Table III) Biotin” (4 mdlO0 ml) ’ Folic acidb (10 mg/lOO ml) Vitamin B,, (1 mg/lOOml) Sodium pyrnvate (0. I M) Methyl cellulose (4.0%) Penicillin ~100,000units/ml) Hemin’ (50 mg/100 ml) Nucleotide mix (Table IV) Fetal bovine serum (56 C/30’) (= 25%) L-Glutamine (3.0%) Note. Ifypticase,

methyl cellulose,

2.5 10.0

ml

10.0 2.0 2.0 5.0 2.0 3.0 5.0 0.1 5.0 10.0 33.5 2.0

and distilled water were

sterilized by autoclaving at 20 lb for 20 min. The glucose, vitamin mix, biotin, folic acid, vitamin B,,, sodium pyruvate, penicillin, hemin, nucleotide mix, and NaHCO, were sterilized by filtration (0.20~km pore size). Glucose, biotin, folic acid, vitamin B,,, penicillin, nucleotide mix, and L-Glutamine were stored at -20 C; and M199, trypticase, vitamin mix, sodium pyruvate, methyl cellulose, hemin, and NaHCO, were stored at 4 C. a Biotin isdissolved in 0.083% (w/v) HCl. ’ Folic Acid is dissolved in 0.008% (w/v) NaOH.

’ Hemin is dissolved in 0.04% (w/v) NaOH.

74

ALFRED TABLE II Composition of JH-30: Cell-Free Medium for Leishmaniu Sp. Amastigotes Component

Volume (ml)

Basal solution Hepes (1.0 M) NaHCO, (4.2%) Distilled water (2 x )

92.1 1.0 3.0 39.5

to determine whether the vitamin mix (Table IV) or the nucleotide mix (Table V) improved the growth of amastigotes. A modified JH-30 medium was prepared without the vitamin mix or without nucleotides. The starting inocula for experiments were obtained from lo-day-old cultures of amastigotes in complete medium JH-30. The organisms were washed three times in sterile PBS before inoculation. Culture tubes were set up similar to growth curve experiments and serially passaged for eight subcultures at 7-day intervals. RESULTS

In our routine cultures of Leishmania mexicana pifunoi in JH-30, we noticed that, during the early stages of incubation at 26 C when the pH is alkaline (pH 7.2), the majority of the organisms (81%) were usually promastigotes. However, by the end of the incubation period, when the medium became more acidic (pH 6.0), the proportion of promastigotes decreased and that of amastigotes increased. In an effort to maintain the culture primarily as promastigotes, the initial pH of the medium was adjusted to 8.0 by increasing the concentrations of sodium bicarbonate and Hepes. By serially passaging organisms in this medium (JH-3 1) they grew entirely as promastigotes. TABLE III Composition of JH-31: Cell-Free Medium for Leishmania Sp. Promastigotes Component

Volume (ml)

Basal solution Hepes (1 .O M) NaHCO, (4.2%) Distilled water (2 x )

92.1 2.5 10.0 29.5

A. PAN TABLE IV Vitamin Mix for Leishmania p-Aminobenzoic acid D-CdCiUIU pantothenate Choline chloride Isoinositol Nicotinamide Nicotinic acid Pyridoxal-HCl Pyridoxine-HCl Pyridoxamine-2HCl Riboflavin-5-phosphate-Na Thiamine-HCl Distilled water (2 x)

Sp. Media 30 mg 40 mg 30 mg 30 mg 50 mg 20 mg 20 mg 20 mg 20 mg 4 mg 20 mg 100 ml

* 2H,O

The kinetics of the growth of promastigotes in JH-31 were determined using organisms from the eighth passage as inoculum. The organisms increased over 400fold in 12 days, which resulted in a peak density of 7 x lO’/ml of culture, as shown in Fig. 1. The medium remained constant at pH 8.0 throughout the incubation period. Promastigotes were grown in JH-31 at 26 C, and 1 ml of this culture containing 6 x lo6 organisms was inoculated into 5 ml of medium JH-30. Thus, the culture contained a population density of lo6 organisms/ml at the start, and was subsequently serially subcultured at 96-hr intervals at 33 C. No prior acclimatization of organisms to successively higher temperatures was carried out so as to select temperature-adapted organisms. During the first subculture, 72% of the organisms were amastigotes after 96 hr of incubation, reaching a density of lO’/ml. In the second passage at 96 hr, 99% of the orTABLE V Nucleotide Mix for Leishmaniu ATP ADP AMP Glutathione (reduced) L-Cysteine-HCl Ascorbic acid Distilled water (2 x )

Sp. Media 200 100 100 20 20 20 100

mg mg mg mg mg mg ml

Leishmania mexicana: CELL-FREE MEDIUM FOR SERIAL CULTURE

I

2

3

18 *

4

5

PASSAGE

/ 4

I s

6

IO

12

4x) 14

FIG. 1. Fourteen-day growth of Leishmania mexipromastigotes in Medium JH-31 at 26 C. Each tube contained 5 ml of medium and received 1 ml of inoculum containing lo6 organisms (0); pH (A). cana pifanoi

ganisms were amastigotes. The number of amastigotes by the end of the incubation period in the subsequent passages increased further as the number of promastigotes diminished. Amastigotes thus obtained could be serially subcultured indefinitely (over 70 weekly continuous subpassages, in one experiment), yielding population densities of over 2.5 x 107/ml of medium in 4 days at 33 C. The results of serial cultivation at this temperature are summarized in Figs. 2 and 3. A typical growth curve at 33 C is shown in Fig. 4. The population density increased 500-fold, reaching 8.1 x lo7 amastigotes/ml in 10 days. The doubling time during logarithmic growth was approximately 24 hr. The parasites also grew as amastigotes at 35 C. At this temperature, the organisms

24

TIME

7

6

8

NO.

FIG. 3. Promastigote to amastigotetransformation of mexicana pifanoi during eight serial passages in JH-30 (Percentageof amastigotes at 96-hr passages from initial 100% promastigotes, 0).

Leishmania

DAYSOFCULTURE

v ,$5&d 0 :

75

72

doubled every 33 hr, and reached a final population density of 5 x 107/ml by Day 10. The pH of the medium dropped with increasing cell densities, in cultures grown at 33 or 35 C, from 7.2 at the start of incubation to 5.8 at the end (Day 14). Amastigotes obtained from the previous experiment were used to determine if these organisms would transform back into promastigotes upon lowering the incubation temperature and raising the pH of the medium. At the beginning of incubation, these amastigotes were adjusted to a population density of 106/ml, and were serially subcultured at 96-hr intervals at 26 C. Very few promastigotes were present at the end of the first 96-hr subculture (12.5%), and the total number of organisms reached only 7.2 x 106/ml. At the end of the second passage, the number of promas-

I

96

;$?I 2

FIG. 2. Promastigote to amastigote transformation of Leishmania mexicana pifanoi in JH-30. Numbers in parentheses represent the percentage of amastigotes during the first passage at 24-hr intervals from initial 100% promastigotes (Total cells/ml, 0).

4

6

8

10

12

14

DAYSOFCULTURE

FIG. 4. Fourteen-day growth of Leishmania mexiparasites in Medium JH-30 at 33 C. Each starting culture contained 6 ml of medium with 1.6 x lo5 organisms. (Amastigotes/ml, 0; pH, A). cana pifanoi

76

ALFRED

tigotes increased to 1.3 x 107/ml (99.2%) as the number of amastigotes decreased to 0.1 x 106/ml (0.8%) and, by the end of the third subculture, promastigotes were the only morphological type present. The total average population density in the third through eighth passages (2.6 x 107/ml) were double that of the second passage. The cultures could be serially maintained as promastigotes (over 60 serial passages) at 26 C. The results of serial cultivation at this temperature are summarized in Figs. 5 and 6. Many of the promastigotes in WrightGiemsa-stained smears appeared elongated and pointed especially, at the posterior end, as seen in Fig. 7. Most organisms showed an anteriorly directed flagellum exceeding the body length. At the base of the flagellum is seen a bar-shaped, deeply stained kinetoplast. The nucleus appeared broadly ellipsoidal or spheroidal and, occasionally, a central nucleolus surrounded by a clear zone was observed. Amastigotes in stained smears appeared oval or pyriform in shape. A bar-shaped, small kinetoplast is positioned anteriorly, with the long axis directed toward the nucleus or at a more acute angle to the nucleus. Many amastigotes were observed in various stages of division with two nuclei and two kinetoplasts (Fig. 8). The average size of 100 amastigotes from cell-free culture at 33 C was 3.5 t 0.2 pm, and 3.2 + 0.5 pm from impression smears IO’

.2

,06 0

ox, 24

TIME

48

72

96

(HR)

FIG. 5. Amastigote topromastigote transformationof Leishmania mexicana pifanoi in JH-31. Numbers in parentheses represent the percentage of promastigotes during first passage at 24-hr intervals from initial 100% amastigotes (Total cells/ml, 0).

A. PAN

’

2

3

4

PASSAGE

5

6

7

8

NO

FIG. 6. Amastigote topromastigote transformationof mexicana pifanoi during eight serial passages in JH-31 (Percentage of promastigotes at 96-hr passages from initial 100% amastigotes, 0). Leishmania

of infected hamster foot pads. Statistically, this difference is not significant. Furthermore, the amastigotes from axenic culture were indistinguishable in appearance by electron microscopy (unpublished data) from amastigotes in infected foot pads of hamsters. However, differences in the tine structure of amastigotes and promastigotes were noticed. Several experiments at 26 and 33 C, to determine whether the vitamin mix (Table IV) or the nucleotide mix (Table V) improved the growth of cultured amastigotes, were conducted. The number of organisms in medium without nucleotide solution steadily decreased during six serial subcultures from the 7th day of the first subculture (1 x 107/ml) to 6.2 x 106/ml at the 7th day of the sixth subculture, as compared to the average control value of 4.7 x 107/ml. Cultures in medium lacking the vitamin mix also decreased similarly. In another experiment, transformation from promastigotes to amastigotes by serial transfers could not be accomplished consistently. Complete JH-30 medium, however, gave more luxuriant growth (typical of Fig. 4), and peak density of cultures was reached l- to 3-days earlier (Pan 1982). DISCUSSION

Leishmaniasis major protozoan

remains as one of the diseases in the tropical

&ishmUniU

meXiCUnU:

CELL-FREE

MEDIUM

FOR SERIAL

CULTURE

77

FIG. 7. F’romastigotes of Leishmania mexicana pifanoi in Medium JH-31 at 26 C. Many organisms showed one stout flagellum directed anteriorly, sometimes exceeding the length of the body. The kinetoplast was anterior to the nucleus. The nucleus appeared broadly ellipsoidal or spheroidal, in the midportion of the body (Giemsa, x 2300). FIG. 8. Leishmania mexicanapifanoi amastigotes in Medium JH-30 at 33 C. Arrows indicate dividing amastigotes with two nuclei and two kinetoplasts. Other amastigotes are in typical oval or pyriform shape (Giemsa, x 2100).

and subtropical regions of the world. Some hope for its control lies in the search for vaccines and better chemotherapeutic agents. These prophylactic and curative measures must be directed primarily against amastigotes in macrophages. Purified amastigotes isolated from cell cultures could provide the starting material for these types of studies. However, the original vitality and the contamination of host-cell debris may affect critical biochemical or immunological studies. We have demonstrated that amastigotes of a strain of Leishmuniu mexicunu pifunoi can be serially cultured in a cell-free medium in quantity at high temperature (33 or 35 C); that promastigotes can transform into amasti-

gotes and vice versa under appropriate alteration of certain conditions; and that these axenically grown amastigotes are morphologically similar to intracellular amastigotes. Studies are now being undertaken to compare in vitro-obtained amastigotes with intracellular amastigotes from infected animals and macrophage cell cultures . Attempts at cultivation of amastigotes in cell-free media have been carried out by Lemma and Schiller (1964) and Trager (1953). However, a controversy has arisen in that temperature produced a selection of mutant forms or a selective breeding of genetically altered parasites (Wonde and Honigberg 1971). Newton (1968) has ques-

78

ALFRED

tioned whether the loss of a flagellum represents an intracellular form and has advocated the biochemical detection of a cytochrome system (Krassner 1966; Simpson 1968) or the use of differential respiration (Hart er al. 1981b; Janovy 1967) as a basis for true differentiation. Several authors have recently reported on in vitro transformation of promastigotes to amastigotes of L. braziliensis panamensis or L. mexicana in response to elevated temperature. However, they could not serially maintain the transformed amastigotes indefinitely (Hendricks 1978; Hendricks and Childs 1980; Hendricks et al. 1978; Hunter et al. 1982). Our first series of experiments was designed to test the biological and nutritional aspects of axenic amastigotes of L. m. pifunoi. Experiments on growth curves at various temperatures indicated that promastigotes of this strain seem to favor a basic pH (-8.0) and a temperature of 26 C. Amastigotes seem to grow well in an acidic medium, with a pH range below 7.2 and a higher temperature (33 or 35 C). In our cellfree culture system for amastigotes, medium JH-30 contains an abundance of nutritional additives that may be present in an intracellular environment. These nutrients were also found to act favorably for promastigote to amastigote transformation. During construction of the various formulations of medium, it was noticed that some intermediate forms, with short flagella (although in round to ovoid shape), developed when the vitamin solution and nucleotide mix were kept for over 2 months. These forms transformed back into the more ovoid, pyriform-shaped amastigotes without a visible flagellum when fresh vitamin and nucleotide solutions were added. It may be advisable that fresh solutions of vitamin mix and nucleotides (Tables IV and V) be used in order to obtain uniform cultures of amastigotes. The apparent requirement by L. m. pifanoi amastigotes for nucleotides is interesting, in

A. PAN

view of results obtained by other investigators that intracellular parasites must obtain ATP from their host cells (Moulder 1974; Pan 1978; Trager 1971, 1974b). In vitro-obtained amastigotes at 2 x 106/ 0.1 ml inoculated into golden hamster hind foot pads produced swellings (1.8 x 1.O x 1.6 cm) in approximately 3 months, while promastigotes with the same inoculum produced smaller-sized swellings (1.7 x 0.7 x 0.9 cm) in the same time period. Axenically obtained amastigotes are also capable of infecting a high proportion of macrophages from a 5774 continuous cell line (Chang 1980). We have already tested several different species of Leishmania to determine whether transformation from promastigotes to amastigotes can be accomplished. We are now able to serially subculture three other strains of the genus Leishmania, in the amastigote form, in cell-free medium. Monoclonal antibody techniques have typed these as a L. braziliensis ssp. and two L. mexicana ssp. from Venezuela (McMahon-Pratt, personal communication). Only the testing of various strains under the described culture conditions can determine if transformation, and subsequent serial cultivation to amastigote forms, can succeed. One additional suggestion, since preparation of the manuscript, is to reduce or delete the antibiotic solution. The possibility now exists for several types of biological, immunological, biochemical, and pharmacological studies. Axenic amastigotes can be used for investigating the mechanism of drug action, for a source of antigen in serological tests, and for monoclonal antibody techniques which might possibly lead to the production of a vaccine. It has been stated that morphological criteria alone are insufficient to characterize amastigotes obtained in vitro, and that the ability to grow at high temperatures without prior adaptation to successively higher tem-

Leishmaniu

mexicana:

CELL-FREE

peratures would be an additional criterion (Pan 1978; Trager 1974a). The amastigotes cultured in JH-30 meet at least two of these requirements; morphologically, the size and shape, and physiologically, the ability to grow and be serially subpassaged at elevated temperatures. In addition, the ability to grow, multiply, and transform to each stage at will should be an additional criterion (Pan 1978). We would also like to suggest, as another standard, that amastigotes of Leishmuniu species obtained in vitro must be able to grow and multiply in macrophage cultures and be highly infective to animals, so as to maintain their intracellular life cycle. Leishmuniu mexicunu pifunoi meets all of these requirements. ACKNOWLEDGMENTS This paper represents research conducted at the Johns Hopkins School of Hygiene and Public Health under the direction of Dr. M. Gottlieb. An abstract of this work was published and some results were presented at the Society of Protozoology Meeting, Pace University, New York, NY, USA; 20-24 June 1983. This study was supported in part by Training Grant 5 T32 AI07109-04 from the U.S. National Institutes of Health to Dr. B. M. Honigberg (University of Massachusetts, Amherst, MA, USA). REFERENCES R. L., AND MARR, J. J. 1979. Growth of Leishmania donovani amastigotes in a continuous macrophage-like cell culture. Journal of Protozoology 26, 453-456. BRAZIL, R. P. 1978. Isolation of the intracellular stages of Leishmania mexicana amazonensis using cellulose column. Annals of Tropical Medicine and Parasitology 72, 579-580. CHANG, K.-P. 1980. Human cutaneous Leishmania in a mouse macrophage line: Propagation and isolation of intracellular parasites. Science 209, 1240-1242. CHANG, K.-P., AND DWYER, D. M. 1976. Multiplication of a human parasite (Leishmania donovani) in phagolysosomes of hamster macrophages in vitro. Science 193, 678-680. CHANG, K.-P, AND DWYER, D. M. 1978. Leishmania donovani. Hamster macrophage interactions in vitro: Cell entry, intracellular survival, and multiplication of amastigotes. Journal of Experimental Medicine 147, 515-530. CHILDS, G. E., MCROBERTS, M. J., AND FOSTER,

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A. PAN SIMPSON, L. 1968. The leishmania-leptomonad transformation of Leishmania donovani: Nutritional requirements, respiration changes and antigenic changes. Journal of Protozoology 15, 201-207. TRAGER, W. 1953. The development of Leishmania donovani in vitro at 37 C. Effects of the kind of serum. Journal of Experimental Medicine 97, 177189. TRAGER, W. 1971. Malaria parasites (Plasmodium lophurae) developing extracellularly in vitro: Incorporation of labeled precursors. Journal of Protozoology 18, 392-399. TRAGER, W. 1974a. Nutrition and biosynthetic capabilities of flagellates: Problems of in vitro cultivation and differentiation. In “Trypanosomiasis and Leishmaniasis with Special Reference to Chagas’ Disease,” pp. 225-245. Ciba Foundation Symposium No. 20 (New Series). Elsevier, North-Holland. TRAGER, W. 1974b. Some aspects of intracellular parasitism. Science 183, 269-273. TRAGER, W. 1983. In vitro growth of parasites. In “Molecular Biology of Parasites” (J. Guardiola, L. Luzzatto, and W. Trager, eds.), pp. 39-51. Raven Press, New York. WONDE, T., AND HONIGBERG,B. M. 1971. Morphology and infectivity of Leishmania donovani cultivated in nonliving media at elevated temperatures. American Journal

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