Leishmania enriettii: Radiation effects and evaluation of radioattenuated organisms for vaccination

EXPERIMENTAL

35,

PARASITOLOGY

161-169

(1974)

Leishmania enriettii: of Radioattenuated AKLILU

Radiation Effects and Evaluation Organisms for Vaccination l, 2

LEMMA

3 AND LEONARD

Stanford Research Institute, (Submitted

COLE

Menlo Park, California

for publication,

26 June

4

94025

1973)

LEMMA, AKLILU,AND COLE, LEONARD. 1974. Leishmunia enriettii: Radiation effects and evaluation of radioattenuated organisms for vaccination. Experimental Parasitology 35, 161-169. The effects of gamma radiation and ultraviolet light on motility, morphology, reproduction, ability to transform from amastigote to promastigote, infectivity, and vaccine potential of Leishmunia endettii were studied. Over 800,000 roentgens (R) was necessary to immobilize immediately the organisms, whereas only 25,000 R rendered them noninfective and 50,000 R made amastigotes unable to transform to promastigotes. Increasing degrees of morphologica abnormality were seen with increased radiation doses. Single, double, and triple vaccination with 25,000-100,000 R irradiated organisms had no protective value against L. emiettii. INDEX DESCRIPTOR.S: Leishmania enriettii; Immunity; Radiation attenuation; Gamma rays; Ultraviolet light; Skin tests.

Although research on the effect of radiation on protozoa has been on free-living genera, mainly Paramecium, some work has been done on parasitic forms. Ruff and Hansen ( 1970a, b ) studied Histomow, and among the many studies on trypanosomes,Halberstaedter (1914) reported that Trypanosoma brucei ‘and T. gambiense lose their

infectivity

after

irradiation

12,000 R, with 600,000 R being required to immobilize them. Similar results were later reported by Emmett (1950) for T. cruxi and by Duxbury and Sadun (1969) for T. rhodesiense. Martinez-Silva et al. (1969) found that irradiation of T. cruzi with 6250 or 12,500 R renders 90% and 99%, respectively, of the organisms noninfective. Various morphological aberrations and inhibition of multiplication due to radiation have also been reported. There have been many reports of vaccines consisting of irradiated helminth parasites, but only a few attempts have been made to use ionizing radiation to immunize against protozoan infections. Immunization by the use of irradiated malaria parasites has been accomplished in chickens (Ceithaml and Evans 1946) and rodents (Corradetti, Verolini, and Bucci 1966; Wellde and Sadun 1967). Duxbury and Sadun (1969) evaluated the vaccine po-

with

1 This study was supported in part by National Science Foundation Grant GB-27377 and International Atomic Energy Agency, Contract 656,’ Rl/RB. 2 The authors gratefully acknowledge the valuable technical assistance of Mr. Percy Yau in the experiments reported herein; the provision of laboratory space to the senior author by Dr. W. A. Skinner; and the valuable discussions they had with Dr. Gerald Brody. 3 Present address: Institute of Pathobiology, Haile Sellassie I University, Addis Ababa, Ethiopia. Reprint requests to be directed to this address. 4 Deceased before the completion of the study. 161 Copyright All

rights

0

1974 by of reproduction

Academic Press, in any

form

Inc. reserved.

162

LEMMA

tential of radioattenuated T. rhodesiense in rats and mice and obtained very promising results. In the present study, we attempted to determine the effect of radiation on motility, morphology, temperature tolerance, population growth, infectivity, and radioattenuation of Leishmania enriettii, a hemoflagellate closely related to trypanosomes. The primary objective of the study was to explore the potential of attenuating these organisms for possible use as vaccine.

AND COLE

was used. The radiation dose was determined with a UV intensity meter. Initial variations in response of the organisms to UV radiation were later determined to be due to the limited depth of penetration of UV light and to protective effects of different components of the culture media, including glucose. In an attempt to overcome this and to standardise the test, we adopted the following procedure. The organisms to be irradiated were washed three times in buffered saline. About 10 x lo6 of the washed organisms MATERIALSAND METHODS were suspended in 1 ml of buffered saline and were thinly spread over a S-cm-diameSource and Maintenance of Organisms ter sterile petri dish. The dish was then The L. enriettii strain used was obtained placed under the UV source, and the organfrom Dr. R. S. Bray, London School of isms were irradiated with predetermined Hygiene and Tropical Medicine, in 1967. doses. This procedure gave reproducible The organisms were routinely maintained results. in guinea pigs. For in vitro cultivation, we used modified blood-agar base with Locke’s Animals solution overlay (Lemma and Schiller In all the experiments reported herein, 1964). Freshly isolated promastigotes durDuncan-Hartley weanling guinea pigs, ing their exponential growth phase (thirdabout 7 days old, were used. Except when or fourth-day cultures) were used in all otherwise stated, all inoculations consisted radiation experiments. Organisms were of a standard dose of 1 x lo6 organisms in counted with a hemocytometer. 0.1 ml, which was inoculated on shaved skin on the rump just above the tail base. Radiation Sources and Methods The inoculation site was shaved once a X- and gamma-rays. A 100-kV constant week with an electric shaver. potential X-ray unit (supplied by Philips, Holland) was used in the earlier studies. RESULTS However, since this machine was primarily designed for superficial (skin) therapy, it Efect of Radiation on Motility of Promastigotes in Vitro did not provide uniform and reproducible radiation of the organisms in the suspendThe effect of gamma radiation on the ing media. This shortcoming was later rectimotility ‘of promastigotes in vitro at r0om fied by using a powerful cobalt-60 source temperature (22 C) was studied for 6 hr. of radiation at Stanford Research Institute. The results, summarized in Table I, show All radiations were made in lo-ml that radiation with l,OOO,OOO R immobilizes capacity, screw-capped test tubes contain- all organisms immediately, but lower doses ing 2 ml of about 20 x lo6 organisms sus- take a progressively longer time. The manipended in the liquid phase of the culture festation of radiation effects is time demedium. pendent; i.e., the longer the observation Ultraviolet light. A photochemical lamp time after irradiation, the greater the effect. capable of producing an intensity of 30,000 Within the first 6 hr of observation, organ1.1’117/crn2 of 2537 A short-wave UV light isms irradiated with 400,000 R completely

Leishmaniu lost their motility and 99% of those irradiated with 200,600 R were immobilized. The effects of UV radiation on the motility of promastigotes (Table II) were similar to those for gamma radiation. Radiation with l,OOO,OOO ergs/cm2 immobilized all organisms immediately, but 6 hr were required for 400,060 ergs/cm2 to have a similar effect. As with gamma radiation, lower doses had progressively slower and less severe eff e&s. In an attempt to determine whether the immobilizing effect of UV radiation is reversible, organisms irradiated with 50,000 ergs/cm2 were transferred into fresh media and observed for a week. All such organisms progressively died, and there was no detectable recovery. We also studied the possibility of photoreactivation of the UV effect on these organisms. Irradiated organisms were exposed to ordinary light of different intensities and were observed for a week. No detectable photoreactivation was seen. Effect of Radiation on Reproduction Promastigotes in vitro

of

The effect of gamma radiation on the reproduction of promastigotes is presented in Fig. 1. Organisms irradiated with 50,000100,000 R did not reproduce (the slight increase in population seen in organisms irradiated with 50,000 R may be due to completion of divisions that had started prior to radiation). In subsequent subcultures, however, organisms irradiated with 25,060 R or less reproduced and gradually achieved high population densities that were similar to normal. Irradiation with UV light gave comparable results. All organisms irradiated with 50,000 ergs/cm2 or more lost their ability to reproduce, but those irradiated with 25,000 or 12,560 ergs/cm2 gradually reproduced to attain a normal high population of anoarentlv active nromastigotes.

enriettii

163 TABLE

I

___;i of

Effect of Gamma Radiation OTZ Motility Promastigdes of Leishmania enriettii in Vitro (.% C) Radiation

dose

Motility

after

radiation

(RI During 1st hr Control (unirradiated) 50,000 100,000 200,000 400,000 800,000 1,600,OOO

At 6 hr

++++* ++++ ++++ +++ ++ + -

+++-+ ++ + f -

= very motile, almost all moving ; = about 257;, 5Ocj,, SO’%, and 99%, respectively, immobilized ; = 100% immobilized.

= ++++ +++,++,+,k

-

Effect of Radiation on Morphology mastigotes

of Pro-

Morphological abnormalities were observed in organisms irradiated with both gamma rays and UV light. The number of abnormalities was proportional to the radiation dose; the higher the dose, the more abnormalities. However, organisms irradiTABLE Ejects

II

of UV Radiation on fifotility of Promaslig0te.s in Vitro

Iladiat.ion dose (ergs/cmz)

74 Immobilization various times radiation 10 min

Control (unirradiated) 50,000 100,000 200,000 400,000 600,000 800,000 1,000,000

5 19 28 44 70 86 92 100

30 min i)25 40 52 70 90 97 100

at after

1 hr

6 hr

6 28 41 62 80 98 100 100

8 60 70 80 100 100 100 100

164

LEMMA

AND

COLE

flagella, multinucleation, vacuolation, and progressive loss of motility leading to eventual death and degradation.

Effect of Radiation on Temperature ance of Promastigotes in Vitro

25,000

Toler-

The effect of gamma radiation on temperature sensitivity of the promastigotes of L. enriettii was determined by incubating subcultures of the irradiated organisms at various temperatures. Table III shows that radiation significantly reduces the temperature tolerance of the organisms.

r 1

Effect of Radiation on Ability of Amastigotes to Transform and Reproduce as Proin Vitro

nzastigotes

2

0

4 DAYS

6

8

FIG. 1. Population growth of control and gammairradiated Leishmania enriettii at 22 C.

ated with 12,000 R or less did not show any apparent morphological abnormalities. The abnormalities seen included shrinkage, rounding, ballooning, loss of flagella, agglutination, possession of two or more TABLE

Efect of Gamma 1Cadiation on Teq~raturc: Radiation WI

dose

Amastigotes obtained from biopsied, homogenized skin tissue of heavily infected guinea pigs were suspended in tissue culture medium No. 199. Tubes containing approximately 10” Leishman-Donovan (L.D.) bodies in 1 ml of solution were irradiated and subsequently subcultured in fresh diphasic media. The cultures were left at room temperature (22 C ) and examined daily for transformation and reproduction. The results (Table IV) show that organisms irradiated with 75,000 or 100,000 R III

‘I’olPrance

Relative

of

Promastiyotcs of Leishmnnia

viability of organisms at different after irradiation and incubation

22 c .____

48 hr Control

25000 50,000

(unirradiated)

++++”

enriettii times

28 c

32 c __~

96 hr ++++

48 hr ++++

++++

++++

+++ ++

+++ ++

++ +

+ zt

100,000

++

++

+

-

200,000 400,000 800,000

++ + -

+ -

-

(1 + + + + represents growth of unirradiated control organisms + ++, + +, and + represent 757,, 507(,, and 2d070, respectively, - represents no organism seen.

-

48 hr

96 hr

; of control

;

96 ht ++++

+ +

+ f

-

-

-

-

Leishmaniu TABLE Efect

Amastigote

radiation (It)

dose

Population

and motility after radiation

6 days

++++” +++ ++ ++ i -

on Infectivity

TABLE of UV Irradiation to Transform

3 days (unirradiated)

0 As in Table

IV.

++++” ++-t++ zk -

30 days

++++ +++ ++ + -

++++ +++ + -

V

and motility after radiation

4 days +++l+++ ++ -

21 days

radiation with 25,00&100,000 R renders promastigotes and amastigotes completely noninfective. Organisms irradiated with 10,000 or 12,500 R, however, remained infective; although the lesions appeared late, they eventually developed into ulcerative sores comparable to those in control animals inoculated with unirradiated organisms. The results of animal inoculations with UV light-irradiated organisms followed the same pattern (Table VII). All organisms irradiated with 50,000 ergs/cm2 or more were noninfective, whereas those irradiated with 25,000 ergs/cm2 or less resulted in progressively developing ulcerative sores. comparable to those seen in control animals.

on Ability of Amastigotes of Leishmania and Reproduce as Promastigotes In Vitro Population

dose

++++ +++ ++ + -

times

as 100C;;i base lines;

The effect of gamma rays on the infectivity of promastigotes and amastigotes of L. enriettii to guinea pigs was determined. The animals were inoculated with IO6 irradiated organisms a few hours after the radiation. The results (Table VI ) show that

Effect

14 days

++++ +++ ++ + -

failed to transform and reproduce as promastigotes. About 15% of the L.D. bodies irradiated with 50,000 R, 30% of those irradiated with 25,000 R, and 60% of those irradiated with 12,500 R transformed and reproduced as promastigotes. Similar results were obtained with UV irradiation (Table V).

Effect of Radiation

enriettii

of promastigotes at various (average of five tests)

8 days

++++ +++ ++ + -

(L All estimations are based on using the controls therefore, all values are relative to the control - no viable organisms seen.

Control 12,500 25,000 50,000 100,000

IV

Radiation on Ability of Amastigotes of Leishmania to Transform and Reproduce as Promastigotes In Vitro

Control (unirradiated) 12,500 2.5,OOO 50,000 75,000 100,000

radiation (ergs/cm2)

165

of Gamma

4 days

Amastigote

enriettii

of promastigotes (average of five

5 days ++++ +++ + -

10 days ++++ +++ + -

enriettii

at various tests) 16 days ++++ +++ + -

times

30 days ++++ ++ f -

166

LEMMA

AND

COLE

TABLE VI Effect of Gamma Radiation on Infectivity of Leishmania Kadiation dose 0% Control

No. of guinea Pigs

(unirradiated)

Start of lesion (days after inoculat,ion)

24

10,000

20

12,500

28

23,000 50,000

27 24

100,000

Description

Well-developed

of lesion

ulcerative sores

26 (20-36) Lesions started late but 32 developed fully (30-38) No lesion developed; at 90 days, smear, culture, and skin tests were negative

4

Evaluation of the Protective Value of Radioattenuated L. enriettii in Guinea Pigs Results of experiments to determine the vaccine value of organisms rendered noninfective by gamma radiation are summarized in Table VIII. The lowest dose that renders the organisms nonpathogenic while they are still active and can reproduce in vitro is 25,000 R. Therefore, 25,000100,000 R was considered to be the attenuating dose range. However, none of the guinea pigs that were given single, double, or even triple vaccinating doses at various intervals were able to withstand a challenge infection given 6 weeks after the immunization attempt. Correlation between Time of Protective Antibody Production and Skin-Test Positivity In an attempt to determine whether skintest positivity correlated with the presence

of protective immunity, and to determine the time it takes the infected animal to reach tan immune state, groups of guinea pigs were given an initial inoculation with viable organisms in the right ear and subsequently, at weekly intervals, given challenge infections in the left ear. Skin tests were made weekly. The results (Table IX) show that skin-test positivity correlates with the development of protective immunity and that, under normal conditions, the animals attain such a state about 4 weeks after infection. DISCUSSION

Although it is generally recognized that there are differences in radiation sensitivity between different bacteria and protozoa and even between different species and strains of trypanosomes, the effect of radiation on L. enriettii appears to be very simi-

TABLE E$ect of GV Radiation dose (ergs/cm*) Control

enriettii in guinea pigs

(unirradiated)

Radiation

VII

on I1LjectiOitU of Leishmania No. of guinea pigs

Start of lesion (days after inoculation)

enriettii

in Guinea Pigs

Description

of lesion

6 (1021)

25,000

6

50,000 100,000

6 6

Well-developed

ulcerative sores

(1220) 1 No lesion developed; at 90 days, smear and culture were negative

Leishmaniu TABLE Evaluation Radiation dose (RI

of the Protective

No. of guinea pigs

Control

No. of vaccinating injectionsa

Value

enriettii VIII

of Radioattenuated

Leishmania

Sores at site of original inoculation

12 12 12

1 2 3

Ulcerative Ulcerative Ulcerative

25,000

12 12 12

1 2 3

50,000

12 12 12

100,000

6 6 6

in Guinea Skin

Positive Positive Positive

No lesion No lesion so lesion

Active Active Active

lesion lesion lesion

Negative Negative Negative

Positive Positive Positive

1 2 3

No lesion No lesion No lesion

Active Active Active

lesion lesion lesion

Negative Negative Negative

Positive Positive Positive

1 2 3

No lesion No lesion No lesion

Active Active Active

lesion lesion lesion

Negative Negative Negative

Positive Positive Positive

the two

Skin test immediately before left ear inoculation

Time and Skin-Test in Guinea Pigs” Status

Right

after

ear

ear

4

5 weeks

Positive

Ulcerative

sore

4

6 weeks

Positive

Ulcerative

sore

and left ears.

Positivi@

of infection 5 weeks inoculation

sore sore sore sore sore

for right

was 2 weeks.

IX

Ulcerative Ulcerative Ulcerative Ulcerative Ulcerative

per inoculation

injections

tion, but 25,000 R is sufficient to render the same organisms noninfective to guinea pigs. As would be expected, manifestations of the effects of radiation are directly proportional to both dose and postirradiation time.

Between Protective Antibody Production Due to Leishmania enriettii Znfection

Interval between 1st and 2nd (left ear) inoculation

between

Negative Negative Negative Negative Positive

bodies

6 weeks after challenge

Positive Positive Positive

Same day 1 week 2 weeks 3 weeks 4 weeks

a lo6 L.D.

test

No lesion No lesion No lesion

TABLE

4 ‘4 4 4 4

Pigs

At time of challenge

sore sore sore

lar to that reported for T. cruxi (MartinezSilva et al. 1969). A standard 4-day-old 10T/ml L. enriettii promastigote population will need at least one million roentgens of gamma radiation for immediate and complete immobiliza-

No. of guinea pigs given initial inoculation in right ear

enriettii

Sores at site of challengea

Q One tenth milliliter of lo6 organisms per injection. The interval With three injections, the interval between each was 1 week. b Infection given 6 weeks after last vaccinating injection.

CorrelatimL

167

Left

Ulcerative sore Ulcerative sore Ulcerat.ive sore Ulcerative core No sore, but hard nodule No sore, but hard nodule No sore, but hard nodule

168

LEMMA

With the standard tests we conducted, it appears that gamma and UV radiation produce comparable effects on L. enriettii. However, gamma radiation penetrates through glass and media and gives uniform radiation of organisms, but only a small amount of UV light can penetrate through ordinary glass and culture media containing protein or glucose. Thus, the organisms to be irradiated have to be washed several times in buffered saline and exposed to the UV source in quartz glassware; also, population density of the organisms and volume of the buffered saline in which they are suspended must be standarized. Photoreactivation of UV light damage is a well-known phenomenon. However, in the present study, reversion of the radiation damage could not be detected. This may be partly due to the rather high doses of radiation used. Perhaps with lower doses and better detection methods, the degree of reactivation of such organisms can be measured. We have observed that organisms irradiated with 25,000 R normally lose their infectivity if inoculated immediately after radiation; however, if the same organisms are kept in fresh media for about three subcultures, they become infective, This may be due to postradiation selection of viable and infective organisms. Results of our attempts to evaluate the potential use of radioattenuated organisms for immunization purposes were very disappointing. Single, double, and even triple vaccination regimens did not provide the guinea pigs with any protection against challenge infections. These results are similar to those of Coutinho (1954, 1955) and Kretschmar (1965) who tried and failed to vaccinate guinea pigs with heat-killed promastigotes of L. enrietiii. From our own observations and other supporting data (Bray and Bryceson 1968; Bryceson et al. 1970) it appears that the organisms must survive and react with the host cells for at least 4 weeks before the host acquires de-

AND

COLE

tectable protective cellular immunity. Both the heat-killed and radioattenuated organisms may die out before the host has enough time to build up protective immunity. REFERENCES A. D. hl. 1968. R. S., AND BRYCESON, Cutaneous leishmaniasis of the guinea pig. Action of sensitised lymphocytes on infected macrophages. Lance& 2, 898-899. BHYCESON, A. D. hf., BRAY, R. S., WOLSTEIXROFT, R. A., AND DUMONDE, D. C. 1970. Immunity in cutaneous leishmaniasis of the guinea pig. Clinical and Experimental Immunology 7, 301-341. CEITHASIL, J., ANI EVASS, E. A., JR. 1946. The biochemistry of the malaria parasite, IV. The in vitro effects of X-rays upon Plasmodium Journal of Infectious Diseases gallinaceum. 78, 190-197. CORRADETTI, A., VEROLIXI, F., ASD Buccr, A. 196G. Resistanza a Pla.smodium berghei da parte di ratti albini precedentemente immunizzati con Plasmodium berghei irradiate. Parassitologia 8, 133-145. COUTIXHO, J. 0. 1954. Preventive vaccination with dead leptomonads against leishmaniasis of guinea pigs due to Leishmania enriettii. Folia Clinica Biologica (Sao Paulo) 21, 321-326. COUTINHO, J. 0. 1955. Studies of Leishmania enriettii experimental infections. F&U Clinica Biologica (Sao Paulo) 23, 91-102. DUXBURY, R. E., AND SADUN, E. H. 1969. Resistance produced in mice and rats by inoculation with irradiated Trypanosoma rhodesiense. Journal of Parasitology 55, 859-865. E~~METT, J. 1950. Effect of X-radiation on Trypanosoma cruzi. ]ournal of Parasitology 36, 4547. HALBERSTAEDTER, L. 1914. Experimentelle Untersuchungen an Trypanosomes iiber die biologische Strahlenwirkung. Berlirwr Klinische Wochenschrift 252-253. KRETSCHMAR, W. 1965. Immunity in Leishmania enriettii infection of guinea pigs. Zeitschrift fiir Tropenmedizin und Parasitologic 16, 277283. LEXIMA, A. AND SCHILLER, E. L. 1964. ExtracelIular cultivation of the leishmanial bodies of species belonging to the protozan genus Leishmania. Experimental Parasitology 15, 503-513. MARTINEZ-SILVA, R., LOPEZ, V. A., COLON, J. I., BRAY,

Leishmania AND CHIRIBOGA,

J. 1969. Trypanosomu cruzi: Effect of gamma radiation on growth and infectivity. Experimental Parasitology 25, 162170. RUFF, M. D., AND HANSEN, M. F. 1970a. Histomonas meleagridis: Effect of gamma radiation on in vitro growth. Experimental Parasitology, 28, 450464.

enriettii RUFF, M. D., AND HANSEN, M. F. 1970b.

169

Effect of gamma radiation on the pathogenicity of Histomonas meleagridis. Avian Diseases 14, 646-653. WELLDE, B. T., AND SADUN, E. H. 1967. Resistance produced in rats and mice by exposure to irradiated Plasmodium berghei. Experimental Parasitology 21, 310-324.