EXPERIMENTALPARASITOLOGY57, 269-273(1984)

Leishmania major: Culture Media, Mouse Strains, and Promastigote

Virulence and Infectivity
R. A. NEAL
Department

of Medical Protozoology, London School of Hygiene and Tropical Medicine, Keppel Street,
London WCIE 7HT, England, U.K.
(Accepted for publication

25 January 1984)

NEAL, R. A. 1984. Leishmania major: Culture media, mouse strains, and promastigote
virulence and infectivity. Experimental Parasitology 57, 269-273. Promastigotes of Leishmania major were isolated from an infected mouse in two media, blood agar and Schneiders
medium + 30% fetal calf serum, and maintained continuously for over 1 year. Infectivity
studies in two strains of mice, outbred CD1 strain and inbred BALB/c strain, showed that
promastigotes grown in Schneiders medium maintained infectivity to BALB/c mice
throughout the period of cultivation. Infectivity to CD1 strain mice was progressively lost.
Promastigotes grown in blood agar medium, however, lost infectivity to both strains of mice
at a faster rate than promastigotes grown in Schneiders medium.
INDEX DESCRIPTORS:Leishmaniu major; Protozoa, parasitic; Promastigote; Culture in
vitro; Mice, CDl, BALB/c; Virulence; Infectivity.
INTRODUCTION

Previous studies on the chemotherapy of

cutaneous leishmaniasis using the promastigotes of Leishmania major showed that,
although the promastigotes lost infectivity
with continuous cultivation, the organisms
remained infective to mice up to the 10th
week of cultivation (Neal 1964).
With the recent development
of new
media for the growth of Leishmania species
based on insect cell tissue culture (Childs
et al. 1978; Hendricks et al. 1978), it was
decided to determine the duration of infectivity in the new media. Once established,
growth of L. major was equally good in
Schneiders medium or blood agar. Thus,
infectivity comparisons were made over a
period of more than a year using the original strain of mice used in previous studies
and the susceptible inbred mouse strain
BALB/c (Howard et al. 1980).
MATERIALS

ANDMETHODS

The strain previously known as Leishmoniu tropica

major has been maintained continuously since 1959,
and is now known as Leishmanin major (Liverpool

reference number, LV39). Its history is given in Neal

(1964).
Schneiders medium was prepared commercially
(GIBCO) and 30% heat-inactivated fetal calf serum
(Wellcome Diagnostics) was added before use. Five
milliliters of the complete medium was pipetted into
25-cm2 plastic tissue culture flasks (Coming).
The blood agar medium was prepared by adding
10% v/v defibrinated fresh rabbits blood to molten bacteriological
nutrient agar. The agar was cooled to
about 45 C before the addition of blood. About 5 ml
of the mixture was transferred to 5-0~ medical flat
screw-capped bottles, and solidified on the flat side of
the bottle. The blood agar medium was completed by
the addition of 5 ml of glucose saline. Blood agar medium was used within 2 weeks of preparation.
All
media were stored at 4 C.
The L. major cultures originated from the nonulcerated lesion, about 3 mm diameter, of a mouse inoculated 12 days previously with L. major promastigotes. Portions of the lesion containing amastigotes
were inoculated into blood agar medium and Schneiders medium + 30% fetal calf serum. The liquid
phase of these media contained about 10,000 I.U. or
pg of benzylpenicillin
(as sodium salt) and streptomycin (as sulphate salt), respectively. Cultures were
incubated at 26 C. After the primary cultures, no further antibacterial compounds were added during the
period of cultivation. At no time were the cultures
contaminated with fungi or bacteria. The cultures were
maintained by subcultivation at the end of the loga-

269
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270

R. A. NEAL

rithmic growth phase, every 3 to 4 days about lo6 promastigotes being transferred to fresh medium. By the
end of the series of experiments, the cultures had
reached subculture nos. 111 (blood agar) and 110
(Schneiders).
Male mice, weighing about 20 g, were obtained from
commercial breeders; outbred strain CD1 (Charles
Rivers) and inbred strain BALB/c
(Bantin
&
Kingman). After inoculation, the mice were divided
into groups of 10, and fed with commercial mouse food
and given water ad libirum. The animal room was not
air conditioned but was situated on the cooler north
side of the building and heated to maintain a room
temperature of about 23 C.
The inoculum was prepared from 72-hr cultures in
either blood agar or Schneiders medium. The promastigotes were pooled and counted with a haemocytometer, and the density was adjusted to give 10s
promastigotes/ml.
Rosettes of flagellates were dispersed by agitating on a laboratory shaker. One-tenth
milliliter of promastigote suspension was inoculated
subcutaneously on the dorsal body surface, about 10
mm. anterior to the root of the tail. This area was
closely clipped to remove hair before inoculation, but
was not clipped again during the experiment.
The inoculated mice were inspected for lesion development every 7 days, starting 15 to 18 days after
inoculation. When lesions were observed, each lesion
was measured in two directions at right angles to each
other. The mean of the two measurements was recorded. Observations were continued for about 3
months in each experiment.
In one experiment,
impression smears were prepared from the lesion or
from the skin at the site of inoculation. The impression
smears were fixed with methanol and stained with
Giemsa stain, Smears were examined microscopically
for the presence of amastigotes.

velopment are shown in Figs. 1, 2, 3, and

4. The lesions in BALB/c mice developed
quicker and to a larger size than those in
CD1 mice. With continued cultivation in
vitro, lesion development
was progressively slower with promastigotes grown in
both media. After 3 weeks cultivation (Fig.
l.), lesion development was that expected
for the two strains of mice, and no difference was seen between the two media.
After 11 weeks cultivation (Fig. 2.), lesion
development
from promastigotes
inoculated into BALB/c mice were identical, but
lesions in CD1 mice were not progressive.
Again no difference was seen between the
two media. However, after 23 weeks in
continuous cultivation, progressive lesions
were observed with BALB/c mice inoculated with promastigotes grown in Schneiders medium, while there was no signifi-

18 -

18 -

RESULTS

In the primary Leishmania

major cultures, heavy promastigote growth occurred
within 3 days in the blood agar medium,
whereas, in Schneiders medium, initial
promastigote growth occurred more slowly,
requiring 8 to 11 days to reach maximum
growth. After this initial period, the promastigote
growth was similar in both
media, reaching a maximum concentration
of between lo7 and lo8 flagellates/ml within
4 days.
The two strains of mice were inoculated
with promastigotes
from the two media
after 3, 11, 23, and 57 weeks of continuous
in vitro cultivation. The graphs of lesion de-

4-

CD1 mice, blood agar medium

CD1 mica, 8chndden medium
9alblc mica, blood a9m mdium
Bdbh mice, Sdmeiden mdium

2-

o-

I
18

1
25

1
32
DAY

1
39
AFTER

48
53
80
INOCULATION

87

FIG. 1. Development of mouse lesions due to inoculation of Leishmania major promastigotes grown in
two culture media for 3 weeks.

271

,?hhmaniu major: VARIATIONSIN VIRULENCE

18 -

16 -

CD1

mice.

blocd

-4

CD1

mice,

Schnsidar

agr

medium
mmdiim

OLdI
1

14

22

I*.#
26

11
42

DAY

AFTER

66

67

I
22

18

2os64s50
DAV

AFTER

67

64

71

76

64

INOCULATION

FIG. 2. As Fig. 1 but Leishmania

vitro for 11 weeks.

major grown in

cant lesion growth in CD1 mice inoculated

with blood agar-grown promastigotes. CD1
mice inoculated with Schneider+grown
promastigotes,
and BALB/c mice inocu16 r

16 -

14 -

4-

15

22

29

26
DAY

42
AFTER

50

68

74

81

95

INOCULATION

FIG. 3. As Fig. 1 but Leishmania

vitro for 23 weeks.

major grown in

71

76

66

INOCULATION

FIG. 4. As Fig. 1 but Leishmania

vitro for 57 weeks.

15

I
64

major grown in

lated with blood agar promastigotes, were

intermediate in the rate of lesion development. The last observations
were made
after 57 weeks of continuous cultivation
(Fig. 4). At this time, only promastigotes
grown in Schneiders medium and inoculated into BALB/c mice showed lesion development, though it was slower than in
previous experiments.
Two groups of 10 mice, of BALB/c and
CD1 strains, were separated from the batch
of mice used in the final experiment. These
mice were inoculated with freshly isolated
promastigotes grown for 3 weeks in blood
agar medium. The cutaneous lesion developed at a similar rate to those shown in Fig.
1, thus demonstrating that the failure to infect by long-term growth in vitro (seeFig.
4) was due to the parasites and not the host.
The differences between the three factors, mouse strain, culture medium, and
length of in vitro cultivation, is also shown
by the difference in infectivity (Fig. 5). This
shows that there was no loss of infectivity
by promastigotes maintained in Schneiders
medium and inoculated into BALB/c mice.
While the most rapid loss of infectivity was
with promastigotes grown in blood agar and
inoculated into CD1 mice, other combinations were intermediate in their infectivity.

212

It. A. NEAL

10

20
WEEKS

30

IN CONTlNOS

50

60

CLTIY*TION

FIG. 5. Proportion of mice infected by inoculation of

major promastigotes grown in vitro in two
culture media.
Leishmania

Metastatic spread of the parasites was not

observed during the period of observation.
DISCUSSION

One explanation
of the loss of Leishmania major infectivity could be a change
in growth rate in vitro, assuming that this
parallels the intracellular
growth rate of
amastigotes. Although no direct growthcurve observations were made, the yield of
promastigotes for inoculation was not noticeably different at any time. Therefore, if
it did vary, it is likely that any difference
was small.
Loss ,of virulence by in vitro growth of
microorganisms is a well-known phenomenon. Amongst the protozoa, it has been
well described for Entamoeba histolytica
(Vincent and Neal 1960; Martinez-Palomo
1982). Previous work showed that L. major
promastigotes had lost infectivity to mice
but were still infective to man (Adler 1961).
The earlier work, however,
employed
outbred strains of mice which may have
been more resistant and less uniform in
their response to inoculation with promastigotes. It may be argued that, the susceptibility to man may be more similar to infectivity of BALB/c mice although, in these
mice, the lesions are not self-limiting. More

recent work on infectivity of L. donovani

to hamsters has indicated a loss of infectivity by continuous cultivation of promastigotes in vitro (Semprevivo et al. 1981).
It is possible that the two media selected
different subpopulations,
but this requires
further
studies with cloned material.
Handman et al. (1983) showed the presence
of noninfective clones in their strain of L.
major. The present studies show that there
are at least two factors contributing to the
expression of virulence, the isolation and
culture in particular media and the susceptibility of the mouse.
The biochemical studies on the variation
of virulence has given conflicting results.
Using cloned material, Handman et al.
(1983) were unable to correlate the presence or absence of infectivity of L. major
with radiolabelled
cytoplasmic
or membrane proteins, lectin binding, binding to
monoclonal antibodies, or analysis of isolated kinetoplast DNA. There was evidence
suggesting a correlation with infectivity in
vitro to BALB/c macarophages. However,
Giannini et al. (1981) were unable to show
a difference in attachment of promastigotes
to macrophages between infective and noninfective promastigotes of L. donovani, and
postulated a difference in the post attachment stages. The situation on correlation of
infectivity with lectin binding to promastigotes may be different with L. bruziliensis,
since Dawidowicz et al. (1975) observed a
difference with binding of Ricinus commurk agglutinin (RCA). The degree of
RCA binding to L. donovani promastigotes
was shown by Doran and Herman (1981) to
be less with organisms cultured for a longer
period. It would be valuable to examine the
present L. major cultures for macrophage
infectivity and lectin binding.
The original purpose of these experiments was to check the length of time that
infectivity and virulence was maintained
during continuous cultivation in the new
culture medium. From the results, it is clear
that Schneiders medium can be substituted

Leishmania major: VARIATIONS IN VIRULENCE

for blood agar and the Leishmania major
promastigotesremain infective to BALBlc
mice for prolongedperiods.

273

ization of infective and non-infective clones of

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