Rev. Soc. Bras. Med. Trop. vol.5 número4

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THE AVIRULENCE OF THE CULTIVATED Y STRAIN OF

TRYPANO-

SO M A CRUZI

IV — THE EFFECT OF IMMUNOSUPPRESSIVE

AGENTS IN MICE.

H u m b er t o M en ezes *

Antiprolifer ative drugs (Azathioprine and M ethotrexate) were able to enhanoe a T. cruzi virulent in fectkm in mioe, but failed to induce evident in-fectibn in animais previously vaccinateã w ith th e live PF strain o f the same parasite.

Vinblastine (Vbl) had a similar effect w ith th e virulent Y strain, but pro-m oted th e appearance of positive blood eultures in anipro-m ais vaecinated and treated previously w ith th e d ru g . Negative results are obtained if the im m uno-supressive agent is used 15 days after the vaocine. The flagellates isolated from the vaccinateã Vbl treated mice remaineã avirulent for baby mice, after i.p. inoculation.

The im m u n ity induceã by th e PF strain is probably sterile i. e. does not im ply in th e persistence of a la ten t infection (prem unition).

INTRODUCTION

Sincé we have described th e avirulence of w h a t we suppose to be a m u ta n t (PF) of th e “Y ” s tra in of Trypanosoma cruzi,

(M enezes 3> 4- õ), we hav e trie d to dem ons- tr a te th ro u g h several te sts t h a t su eh avi rulen ce w as fixed an d n o t due to environ- m e n tal circu m stan ces (M enezes 7> 8> 9; M e nezes & A lbuquerque 10) .

T he o b serv atio n of p rim ary im m une- depression in ex p erim en tal try p an o so m ia- sis (W alker14; C am argo e t al. J; L uckins2), v iral (Squam e e t al.12; Z lotnik et al. lr>) a n d b a c te rial in fectio n s (T rip a th y & M a- ckaness 1!i) of an im ais tre a te d w ith a n ti p ro liferativ e drugs, induced us to use such ag e n ts in m ice v ac cin ated w ith T. cruzi

P P strain .

W ith su ch experim ents, we in ten d ed to produce fu rth e r evidence of th e avirulence of our T. cruzi live vaccine.

MATERIAL AND METHODS

1 — Sixty m ale albino m ice w ith lOg of m ean body w eight w ere divided into six groups o f te n an im ais each .

F o u r groups w ere tre a te d su b cu tan e- ously w ith 0,02 m g of é-am ino-N w -m ethyl ptero y lg lu tam ic acid sodium * (M tx).

T h ree days la te r two of th e four groups were v accin ated by subcutaneous route w ith 0.2 m l of a S aline suspension of T. cruzi, P F stra in , cu ltiv ated for 8 days in liquid m edium of Noller — (P F /M tx 1 &

* M e th o tr e x a te S o d iu m — L e d e rle L a b o ra to rie s.

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214 Rev. Scc. Bras. Med. Trop.

**Vol. V _ N * 4**

2 ). T h e e stim ated n u m b er of p a ra site s in th e inoculum w as 14 x 106/m l, 60% taeing mobile form s an d alm o st 4%, m etaeyclic form s. O ne group received th e sam e dose of th e vaccine an d becam e th e control group (PF) . O ne group of th e M tx tre a te d an im ais w as infected, in trap e rito n e ally , w ith blood form s of T. cruzi, Y v iru le n t s tra in (Y /M tx ), in a 5,000 p a ra site s /g body w eight ratio.

O ne m ore group w as in fected a s th e preceding one, a n d k ep t as co n tro l of th e in fectio n ( Y ) .

Finally, th e la s t group w as only M tx- tre a ted , a n d m ain ta in e d as control of th e d ru g (M tx ).

Ali th e an im ais of th e d iffe re n t M tx- tre a te d groups received every 3 days, for a 15 day period, a sub cu tan eo u s M tx in jectio n of 0.02 m g.

D uring th e su b seq u en t 15 days th ey re ceived, every 3 days, 0.05 m g of th e sam e d ru g .

P ara sitem ia a n d m o rta lity ra te s w ere recorded 8, 15 an d 30 days a f te r th e Y inocu latio n an d P F v accin atio n . T he a n i m ais of th e P F /M tx-1 group w ere sa c ri- ficed by ex sa n g u in a tio n (h e a rt p u n c tu r e ) : 5 a fte r 15 days a n d th e la s t surv iv in g 3 mice, on th e 30th d ay . In b o th cases ali th e blood d ra w n from th e an im ais w as cu ltiv ated in W a rre n ’s liquid m edium for trypanosom es.

F ra g m en ts of sev eral viscerae w ere fixed in 10% F o rm alin solution fo r histologic ex am ination.

Ali th e surviving an im ais of th e P F / M tx2 group w ere killed a n d th e blood cu l tiv ate d for trypanosom es, 30 days a fte r th e vaccination.

2 — S ixty albino m ale mice, w ith lOg of body w eight, w ere divided in to six groups of te n each .

F o u r groups received in tra p e rito n e a lly 0,12 m g of az ath io p rin e * (A Z T ). T his tre a tm e n t w as pursued, daily, fo r 33 days.

T h e suspension of th e d rug w as m ade in carboxy — m eth y l cellulose as reeom - m ended by O k u m u ra & D ecourt11.

Two of th e above groups (PF/A ZT 1 a n d 2), a f te r th re e daily in jec tio n s o f AZT, were v accin ated by su b cu tan eo u s ro u te

w ith 0.2 m l of a saline suspension of T. cruzi, P F stra in , c u ltiv a ted fo r 30 days in P a c k c h an ian m edium .

T he n u m b er of flag ellates w as a b o u t 2.5 x 10°/ml w ith 50% of m obile form s an d ab o u t 10% m etaeyclic p a ra site s.

O ne AZT tre a te d g ro u p w as in fected w ith v iru le n t T. cruzi, Y stra in , (Y AZT) as in th e previous ex p erim en t an d th e o th er AZT group w as k ep t as co n tro l of th e d rug (A Z T ).

O ne m ore group w as in fected on th e sam e day w ith th e sam e dose a n d s tra in as th e Y/AZT group a n d re m a in ed as th e control of th e v iru le n t s tra in (Y) .

T he la s t group w as in jected su b -c u ta n e - ously w ith 0.2 m l of th e P F s tra in saline suspension an d k e p t as con tro l of th e v ac cine (PF) .

F o u r an im ais of th e PF/A Z T 1 group w ere sacrificed by ex sa n g u in a tio n a fte r a blood sea rch for p arasites, 8 days a fte r vaccination.

T h e blood w as c u ltiv ated in liquid m e dium for try p an o so m es.

Five m ore of th e sam e groupe w ere killed, a fte r blood sea rc h fo r p arasite s, 15 days follow ing th e vaccination.

Ali th e blood rem oved by h e a r t p u n c tu re w as cu ltiv ated in W arre n m edium .

Five of th e n in e surviving m ice of th e PF/AZT2 group w ere bled to d e a th on th e 30th day of v accin atio n a n d th e blood cul tiv ate d fo r try p an o so m es.

T he fo u r re m a in in g an im ais w ere sub- m ited to xenodiagnosis u n til to ta l e x sa n g u in atio n a n d d e a th (5 R. prolixus n y m p h s fo r ea ch m ice) .

F ra g m en ts of o rg an s a n d tissues w ere saved from ali an im ais for histologic ex a m in a tio n .

3 — Seventy m ale albino m ice w ith lOg of m ean body w eig h t w ere divided in to seven groups of te n each .

F o u r groups received in tra p e rito n e a lly O.Olmg of V in b lastin e ** (Vbl) .

T h e tre a tm e n t w as co n tin u ed by in jectio n s o f 0.02 m g, 0.03 m g a n d 0,06 mg, in th e second, th ird an d fo u rth w eek re s- pectively.

Two of th e V b l-tre a ted groups, th re e days a fte r th e firs t dose, w ere vaccin ated w ith 0.1 m l of a saline suspension of T.

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Rev. Soc. Bras. Med. Trop.

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cruzi, P F stra in , cu ltiv ated fo r 27 days in F a c k c h a n ia n m edium (PF/V bl 1 a n d 2 ).

T h e vaccine h a d ap p ro x im ately 2 x 107 p a ra site s/m l w ith 40% m obile fo rm s an d ab o u t 4.5% m etaeyclic fo rm s.

O ne Vbl group w as in je cte d w ith 5,000 parasites,/g body w eight, of blood form s of th e Y v iru le n t T. cruzi (Y /V b l). T h e la st Vbl group w as k e p t as control of th e d ru g (Vbl) .

One group of n o n -tre a te d an im ais w as in fected by th e sam e v iru le n t s tra in w ith a n id en tic al dose ( Y ) .

One group received only th e vaccine (PF) as th e P F /V bl group, a n d finally, th e la st n o n -tre a te d n o n -v a c cin a te d 10 m ice were k e p t as n o rm a l co n tro l ( N ) , receiving weekly in tra p e rito n e a l in je ctio n s of 0.2 m l saline solution.

S earch for p a ra site s in th e p e rip h e ra l blood w as done 8, 15 a n d 30 days a fte r th e v accin atio n or in fectio n in th e an im ais of th e groups PF/V bl2; P F ; Y a n d Y/Vbl.

T h e m ice of th e P F /V bl 1 group also h a d p a ra site blood se a rch 8 a n d 15 days a fte r th e use of th e vaccine.

F ive of th ese a n im a is w ere sacrificed by h e a r t p u n c tu re 8 days follow ing th e vaccination, a n d th e blood d raw n w as cu lti v ated in W arre n m edium .

T h e o th e r five m ice w ere killed in th e sam e w ay 7 days la te r, i. e., 15 days a fte r vaccin atio n . Ali th e PF/V bl2 surviving an im ais on th e 30th d ay of v a ccin atio n were bled an d th e blood w as cu ltiv ated for trypanosom es.

T h e e x p e rim e n t w as considered fin ish - ed 30 days a f te r th e v a ccin atio n an d in fection, an d ali surviving an im ais w ere killed a n d h a d fra g m e n ts of h e a rt, liver, spleen, esophagus, ly m p h nodes a n d ce n tra l nervous system saved for histologic exam in atio n .

4 — S ev en ty b aby m ice w ere divided in to seven groups of te n ea ch : th e group PF/V bl 1 w as v accin ated subcutaneously w ith 0.2 m l of a P F vaccine co n tain in g 2.7 x 107 p a ra site s m l, w ith alm o st 80% m o bile form s an d ab o u t 3% m etaeyclic form s. F ifte en days la te r th e an im ais w ere tre a te d in trap e rito n e ally , w ith w eekly in jectio n s of V in b lastin e (Vbl) in successive doses of 0.05 m g a n d 0.1 mg.

Five days a fte r th e firs t Vbl in jectio n a blood se a rch fo r trypanosom es w as m ade

a n d a fte r th is, fo u r m ice of th e group were bled to d e a th a n d th e blood cu ltiv ated in liquid m edium .

Six days a f te r th e second Vbl in jectio n th e p rocedure w as re p e ate d in th e re m ain - in g m ice.

A sim ilar group (PF/V bl2) was vaccin a te d as th e P F /V bl 1 an d tre a te d 15 days la ter, in tra p e rito n e a lly , w ith weekly in jec tio n s of Vbl in successive doses of 0.02 mg, 0.05 m g, 0.07 m g a n d 0.1 mg, p er mouse.

T h e surviving an im ais w ere killed by ex sa n g u in a tio n on th e 3rd day a fte r th e fo u rth a n d la s t Vbl injection.

T h e to ta l blood w as cu ltiv ated for try panosom es. E ach an im al of th e Y group w as in fected w ith 2,500 (250/g) v iru len t blood form s of T. cruzi Y stra in .

P a ra site m ia s w ere perfo rm ed 8, 15, 20 a n d 30 days a fte r th e infection.

T h e Y /Vbl group w as in fected as th e Y grou.p a n d tre a te d 15 days la te r w ith suc cessive w eekly in tra p e rito n e a l in jectio n of 0.05 m g an d 0.1 m g of Vbl.

Blood search es fo r p a ra site s w ere done as fo r th e preceding group.

F in a lly th e la s t group of th e n o rm al (N) an im ais received weekly in tra p e rito n ea l in jectio n s of salin e so lu tio n (0.2 m l).

Ali blood c u ltu re s w ere exam ined a fte r 30 days of c u ltiv atio n a n d w hen negative a t th e u su al m icroscopy, th e m edium was cen trifu g ed a n d th e sed im en t exam ined fo r trypanosom es.

T h e xenodiagnosis w ere equally exam in ed a t th e 30th day.

T h e fra g m e n ts of o rg an s a n d tissues saved fo r histologic ex a m in atio n w ere fixed in 10% F o rm alin solution, im bedded in p a- ra ffin e a n d th e sectio n s s ta in e d by h em a- toxylin-eosin.

RESULTS

1 — As show n by T able 2 an d G ra p h I th e p a ra site m ia ,peak w as a tta in e d in groups Y a n d Y /M tx, 15 days a fte r in fe c t ion, w ith th e g re a te st p a ra site m ia in the co n tro l Y group.

T h e m o rta lity r a te w as slig h tly h ig h er am ong th e Y /M tx anim ais, a t th e end of th e 15th day, b u t w as th e sam e (100%) in b o th groups a t th e end of th e experim ent

(30th day) .

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216 Rev. Soc. Bras. Med. Trop.

Vol. V —

m

4

8 15 3 0

DAYS AFTER VACCINATION 0R IN FEC TIO N

c o n tra st as seen h isto lo g ically . T he n u m - b e r o f pseudocysts in th e h e a r t of th e Y / M tx m ice w as h ig h e r th a n in th e Y a n i m ais, a n d th e in fla m m a to ry re actio n w as scan ty as com pared w ith th a t of th is la tte r group (Figs. IA e 2C) .

T h e im m unodepressive activ ity of th e d ru g could be docum ented by th e g re at a tro p h y of th e spleen a n d ly m p h nodes.

Ali th e v accin ated PF an d P F /M tx mice h ad n egative blood c u ltu re s for try p a n o - somes (T able 1 ). T h e m o rta lity ra te in b o th P F groups w ere 10% a n d 20% res- pectively, less th a n in th e co n tro l M tx

(40%) .

T he histologic a lte ra tio n s in th e two v ac cin ated groups w ere sim ilar to those already described in P F v ac cin ated an im ais (M enezes5) .

Mice of th e M tx group p resen ted h e a rt a n d liver lesions sim ilar to those seen in th e v accin ated anim ais, b u t less freq u en - tly .

2 — T ables 3 a n d 4 an d G ra p h I I give us a su m m ary of w h a t o ccurred w ith th e AZT tre a te d an im ais.

T h e p a ra site m ia a n d m o rta lity ra te s w ere m u ch h ig h e r in th e Y/AZT group th a n in th e Y m ice a t th e 8th d ay of in- fectio n .

T he AZT a n d PF/A Z T an im ais su ffered th e sam e m o rta lity ra te w hile no an im al in th e P F group died.

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F IG U R E 1

A — M y o c a rd iu m — M o u se F R 6 0 /9 — Y /M T x D ie d 17 d a y s a f t e r i n f e c tio n . O rig . 100 x.

B — Id e m . — M o u se F R 6 0 /7 — P F /M T x 2 K ille d 30 d . a . v a c c in a t io n . O rig . 100 x.

C — Id e m . — M o u se F R 6 2 /9 — Y /A Z T D ie d 12 d . a . i . O rig . 100 x.

D — Id e m . — M o u se F R 6 2 /8 — P F /A Z T K ille d 15 d . a . v . O rig . 100 x.

F IG U R E 2

A — M y o c a rd iu m — M o u se F R 6 4 /1 — Y /V b l D ie d 14 d . a . i . O rig . 100 x.

B —■ Id e m . M o u se F R 6 4 /3 — P F /V b l-1 K ille d 8 d . a . v . O rig . 100 x. B lo o d c u l t u r e P O S IT IV E .

C — Id e m . M o u se F R 6 2 /3 — Y D ied 17 d .a .i. O rig . 100 x.

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GRAPH 1 PARASIT.

_A_ A-PF-AZT

AZT __________. Y

MOfiT. m Eü3 5 E3

A . A . É L & A . / I ▲

1 0 0

90

«0

70

. >o z

o

9 *o 1

♦ 0 H

3 0

20

t o

e u

DAYS AT TER VACCINATION OR INFECT1ÍN

T he histologic iesion of th e h e a r t a n d liver of th e Y/AZT an im ais (Fig 1 C) w ere less conspicuous th a n th o se seen in th e Y m ice, b u t we m u st p o in t o u t t h a t 60% o f th e an im ais of th a t group died before th e 9th d ay of infection, i. e. w hen th e h isto logic lesions in g en e ral are n o t y et very im p ressiv e.

T h e an im ais of th e Y /A ZT group th a t died a fte r th is period p resen ted lesions qu ite sim ilar to th o se of th e Y control grou.p w ith alm o st th e sam e age.

T h e ly m phoid o rg an s of ali A Z T -treated m ice p re sen ted a p ronounced atro p h y , m orphological evidence of th e im m u n e- suppressive effec t of th e drug.

3 — T he ex p e rim en tal design of th is e x p e rim en t is show n in T ables 5 an d 6 a n d G ra p h III.

A nim ais tre a te d sim ultaneously w ith Vbl a n d vaccine h a d positive blood cu ltu re s 8 an d 30 days la ter, b u t th e m o rta lity r a te w as only 10%, th e sam e as th e co n tro l Vbl m ice.

H istological ex a m in atio n s of th e h e a rt (Fig. 2 B ), lung, liver, spleen, íym ph n o

-des, esophagus a n d c e n tra l nervous system of th o se an im ais p resen ted no evidence of infection.

A very im p o rta n t observation in th e positive v ac cin ated cases w as t h a t th e p a ra sites iso lated from blood cultures, w hen in o cu lated in baby mice, w ere incapable of ,prom oting in fec tio n even w hen th e inocu latio n s w ere successive as show n in T a- ble 7.

No v ac cin ated an im ais died d u rin g th e 30 days period w hile th e m o rta lity ra te w as 100% am ong ali in fected m ice (T ables 5 a n d 6 ).

T h e frequency of early m o rtality an d p a ra site m ia w as h ig h e r in th e Y/Vbl th a n in an y o th e r group (T able 6. G ra p h III) .

4 — As show n by T able 9 an d G rap h IV, th e an im ais v accin ated a n d tre a te d 15 days la te r w ith h ig h doses of Vbl h a d n e g ative p a ra site m ia s a n d blood cultures, 5, 13, 21 a n d 29 days a fte r th e first drug in - jectio n .

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/

T A B L E 1

Mice vaccinated an d vaccinated and treated w ith Mtx

G ro u p M o u se !

D A Y S A F T E R V A C C IN A TIO N

O b s e rv a tio n

8 15 30

P a r a s ite s 5 m m 3

b lo o d

B lo o d C u ltu r e

P a r a s ite s 5 m m 3 b lo o d

b lo o d

1 0 0 ( - ) K ille d 15 p v

2 0 0 (— ) K ille d 15 p v

s 3 0 X X _{D ie d} _{14 -pv}

4 0 0 (— ) K ille d 15 p v

+2

2 5₆ 0₀ 0 X 0 (— ) K ille d 30 p v

0 X 0 _{( — )} _{K ille d 30 p v}

7 0 0 X 0 _{( — )} _{K ille d 30 p v}

fa

₈ ₀ ₀

( — ) X X _{K ille d 15 p v}

9 X X X X X _{D ie d} _{3 p v *}

10 0 0 (— ) X X _{K ille d 15 p v}

1 0 0 0 (— ) K ille d 30 p v

2 0 0 0 ( — ) K ille d 30 p v

« 3 0 0 X X D ie d 26 p v

x 4 0 0 0 ( — ) K ille d 30 p v

- p

a

5 0 0 0 ( — ) K ille d 30 p v

i 6 0 0 0 ( _ ) K ille d 30 p v

i ₇ ₀ ₀ ₀

( — ) K ille d 30 p v

& 8 0 0 0 ( — ) K ille d 30 p v

9 0 0 X X _{D ie d} _{19 p v}

10 0 0 0 ( — ) K ille d 30 p v

M EDIAN 0 0 0 * E x c lu d e d

M o rta lity 0% 0% 20%

1 0 0 0 K ille d 30 p v

2 0 0 0 K ille d 30 p v

3 0 0 0 K ille d 30 p v

4 0 0 0 K ille d 30 p v

5 0 X X D ie d 10 p v

k ₆ ₀ ₀ ₀

K ille d 30 p v

7 0 0 0 K ille d 30 p v

8 0 0 0 K ille d 30 p v

9 0 0 0 K ille d 30 p v

10 0 0 0 K ille d 30 p v

M EDIAN 0 0 0

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221

A - A _ p F

PF-VBL.

V B L 1 ^ 3

v ' Ea

Y-**-!> ’

I ' :

p

i

-10 0

.«0 .«0

.70 2

a?

•60 >

r~

■so 1 ■to

■ 3 0

■ 2 0

,10

8 !5

D A Y S A F T E R IN F E C T IO N QR V A C C IN A T IO N

No an im ais died in th e v ac cin ated (PF) a n d n o rm a l (N) group, d u rin g th e days of th e ex p erim en t (T ables 9, 11).

T h e degree of p a ra site m ia an d th e m o rta lity ra te in th e Y /V bl g ro u p w ere still, h ig h e r in gen eral, th a n in th e Y group (T able 10) .

COMMENTS AND CONCLUSIONS

W ith th e aim of d em o n stratin g t h a t th e P F s tra in is a m u ta n t of th e o rig in al Y stra in t h a t h a s lost its v irulence fo r a n i m ais, we tried several know n parasitologic m ethods recom m ended to in crease th e vi rulence of lo w -v iru len t trypanosom e stra in s

(M enezes7. s. 9. 10) .

A n o th er w ay to a p p ro a ch th e problem w as sim ply to try to im p air th e im m u n e

response of lab o rato ry an im ais th ro u g h w ell-know n im m unosuppressive agents.

T h e cap acity of th ese drugs to enh an ce try p an o so m e in fectio n s h a s been dem ons- tra te d by W alk er11 in Trypanosome rhode-siense, L uckins2 in T. brucei a n d T. congo-lense, an d by C am argo e t al.1 in T. cruzi.

P rednisolone in very h ig h dosages w as n o t able to induce in fec tio n in mice vac cin a te d w ith th e live P F s tra in (M ene zes9) .

T h e occurrence of positive blood culture in an im ais tre a te d w ith Vbl previous to th e v accin atio n d em o n strate s t h a t it is a m ore efficien t im m unosuppressive ag e n t th a n th e o th ers alread y m entioned.

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4

I tjüY». Í I

CD 09 (D m

> > > >

T A B L E 2

Mice in fec ted an d in fected a n d tre a te d w ith M tx

G ro u p M o u se

N u m b e r o f p a r a s ite s 5 m m 3 o f b lo o d

O b s e rv a tio n I

8

)a y s a f t e r in fe c tio n

15 30

1 3.360 3.570 D ie d 17 p . i .

2 2.275 X _{D ie d 15 p . i .}

3 3.045 10.500 D ied 17 p . i .

4 X X D ie d 7 p . i .

5 2.170 10.500 D ie d 16 p . i .

6 X X D ie d 1 p . i . *

7 2.730 3.675 D ie d 26 p . i .

8 1.680 4.375 D ied 19 p . i .

9 4.900 17.500 D ie d 16 p . i .

10 3.010 35.000 D ie d 17 p . i .

M ED IA N 2.870 10.500

—

* E x c lu d e d

M o r ta lity 11% 22% 100%

1 X X _{x D ie d 1 p . i . *}

2 3.045 4.025 D ie d 17 p . i .

3 2.870 7.875 D ie d 20 p . i .

X

+3 4 3.780 7.175 D ie d 19 p . i .

3 5 17.500 X _{D ie d 15 p . i .}

1 6 2.590 X D ie d 15 p . i .

>* 7 X X D ie d 1 p . i . *

8 X X _{D ie d 4 p . i . *}

9 5.375 10.500 D ie d 17 p . i .

10 2.445 4.375 D ie d 17 p . i .

M ED IA N 3.045 7.175 X

(11)

Julho-Ago.,

Rev. Soc. Bras. Med. Trop.

223

very stro n g evidence of th e avirulence of th e P F s tra in .

T h e neg ativ e histologic fin d in g s re in - force th is sta te m e n t.

T h e Vbl acts u,pon th e h o st b u t seem s n o t to m odify th e p arasite .

O ur eonviction of th e P P avirulence becom es stro n g er w hen we an alise th e re - sults of th e la s t ex p erim en t.

W hen th e im m unosuppressive tr e a t m e n t is estab lish ed a fte r th e im m unologic in d u ctio n period, th e m odified cells seem to be re s is ta n t to th e ac tio n of th e d ru g an d able to destroy th e p arasite s as d e- m o n stra te d by th e negative blood cultures.

S tudies on a m ore efficien t im m uno- suppressor — ALS is in course an d will be rep o rted later.

RESUMO

Drogas imunossupressoras (Azathiaprina e M ethotrexato) que se mostraram capazes de agravar infecções experimentais por T rypanosom a cruzi virulento (cepa Y )t em camundongos, não conseguiram favorecer o aparecimento de qualquer sinal de infecção em animais vacinados com a cepa avirulenta PF do mesm o tripanosoma.

Vinblastine, quando ãaão 3 dias antes da vacina, não favoreceu parasite-mias positivas nem mortalidade significativam ente aum entada, mas induziu o aparecimento de hem oculturas positivas.

Os tripanosomas dessas culturas foram incapazes de infectar camundongos jovens. Quando o Vinblastine foi usado 15 dias após a vacinação dos animais, todos os testes foram negativos, inclusive as hem oculturas.

BIBLIOGRAPHY

1. CAMARGO, M . E .; KLOETZEL, J. & BACHELLA, T . — A n o te on th e h a - v erstin g of larg e n u m b er of T. cruzi

blood form s in a d u lts dogs. Rev. In st. Med. tro p . São Paulo, 12: 217-220, 1970.

2. LUCKINS, A. G . — T h e effect of an tily m p h o cy te seru m an d cyclophos- p h am id e on th e course of in fectio n of T. brucei an d T. congolense in ra ts a n d m ice. T ra n s. Roy. Soc. Trop. Med. Hyg„ 63: 423-424, 1969.

3. MENEZES, H . — P ro tectiv e effect of a n av iru le n t (cu ltiv ated ) s tra in of

Trypanosoma cruzi a g a in st ex p eri m e n ta l in fec tio n in m ice. Rev. In st. Med. tro p . São P aulo, 10: 1-4, 1968.

4. MENEZES, H . — Lesões histológicas em cam undongos “vacinados” com u m a cepa a v iru le n ta de Trypanosoma cruzi. Rev. B ras. Med., 25: 160-165, 1968.

5. MENEZES, H . — Active im m u n izatio n of m ice w ith th e a v iru le n t Y s tra in of Trypanosoma cruzi a g a in st h e te ro - logous v iru le n t stra in s of th e sam e p a ra s ite . Rev. In st. Med. trop. São

P aulo, 11: 335-342, 1969.

6. MENEZES, H . — Active im m unization of dogs w ith a n o n v iru len t s tra in of

Trypanosoma cruzi. Rev. In st. Med. trop. São Paulo, 11: 258-263, 1969. 7. MENEZES, H . — I — T h e avirulence

of th e cu ltiv ated Y s tra in of Trypa nosoma cruzi. Rev. In st. Med. trop. São P aulo, 12: 64-68, 1970.

8. MENEZES, H . — II — The avirulence of th e cu ltiv ated Y s tra in of Trypa nosoma cruzi. Rev. In st. Med. trop. S ão P aulo, 12: 129-135, 1970.

9. MENEZES, H . — III — T h e avirulence of th e cu ltiv ated Y s tra in of Trypa nosoma cruzi. Rev. In st. Med. trop. São P aulo, 13: 14-17, 1971.

10. MENEZES, H . & ALBUQUERQUE, R . — Im u n ização de cam undongos com “v ac in a” viva a v iru le n ta de Trypano soma cruzi. II — V ariação do meio de c u ltu ra . Rev. Soc. B rasil. Med. Trop., 4: 69-74, 1970.

11. OKUMURA, M . & DÉCOURT, L. V. — E studo de efeitos d a ad m in istração de drogas im unodepressoras sôbre a M oléstia de C hagas ex p erim en tal. Rev. Hosp. Clin. Fac. Med. S. Paulo,

(12)

T A B L E 3

Mice vaccinated an d vaccinated and treated w ith AZT

G ro u p M o u se

D A Y S A F T E R V A C C IN A T IO N

O b s e rv a tio n

8 15 30

b lo o d

B lo o d C u ltu r e X e n o

1 0 X 0 ( — ) K ille d 15 p v

2 0 ( — ) X X K ille d 8 p v

£ 3 0 X 0 ( —) K ille d 15 p v

fn 4 0 X 0 _{(— )} K ille d 15 p v

Cs] 5 0 _{(— )} X X K ille d 8 p v

6

X X X X D ie d 6 p v

1

7 0 (— ) X X K ille d 8 p v

Ph 8 0 ( —) X X K ille d 8 p v

9 0 X 0 ( — ) K ille d 15 p v

10 0 X 0 ( — ) K ille d 15 p v

1

0 0 0 X (—) K ille d 30 p v

2 0 0 0 ( —) X K ille d 30 p v

cg 3 0 0 0 X (— ) K ille d 30 p v

E_< 4 0 X X X X _{D ie d} _{9 p v}

N 5 0 0 0 X X _{K ille d 30 p v}

< ₆ ₀ ₀ ₀ _{(— )} _{(— )} _{K ille d 30 p v}

1 7 0 0 0 (— ) X K ille d 30 p v

h

&i 8 0 0 0 (— ) X K ille d 30 p v

9 0 0 0 (— ) X K ille d 30 p v

10 0 0 0 X _{(— )} _{K ille d 30 p v}

M E D IA N 0 0 Ú

M o r ta lity 0% 10% 10%

1 0 0 0 K ille d 30 p v

2 0 0 0 K ille d 30 p v

3 0 0 0 K ille d 30 p v

4 0 0 0 K ille d 30 p v

5 0 0 0 K ille d 30 p v

cu ₆ 0 0 ₀ _{K ille d 30 p v}

7 0 0 0 K ille d 30 p v

9 0 0 0 K ille d 30 p v

10 0 0 0 K ille d 30 p v

M E D IA N 0 0 0

(13)

T A B L E 4

Mice infected and infected and treated w ith AZT

G r o u p M o u se

N u m b e r c

I

8

>f p a r a s it e s 5 m m

>ays a f t e r in f e c tio

15

3 o f b lo o d

n

30

O b s e rv a tio n

1

t

X X X D ie d 7 p . i .

2 X X X D ie d 7 p . i .

3 8.750 2.835 X D ie d 17 p . i .

4 10.500 X X D ie d 11 p . i .

5 4.900 X X D ie d 12 p . i .

6 4.375 X X D ie d 13 p . i .

7 5.505 X X D ie d 11 p . i .

8 4.025 X X D ie d 14 p . i .

9 4.550 X X _{D ie d 11 p . i .}

10 X X X _{D ied 8 p . i .}

M E D IA N 4.900 2.835 X

M o r ta lity 30% 90% 100%

1 X X X D ied 7 p . i .

2 10.500 X X D ied 12 p . i .

3 X X X D ied 8 p . i .

H

Êsl 4 4.325 X X D ied 10 p . i .

< ₅ _5.250 _3.220 _X _{D ie d 19 p . i .}

+ 6 X X X D ie d 8 p . i .

í* 7 X X X D ie rd 7 p . i .

8 X X X D ied 6 p . i .

9 8.750 X X D ie d 12 p . i .

10 X ' X X D ied 6 p . i .

M E D IA N 7.000 3.220 X

(14)

T A B L E 5

Mice vaccinated and vaccinated and treated previously w ith Vbl

G ro u p M o u se

DA Y S A F T E R V A C CIN A TIO N

O b s e rv a tio n

8 15 30

b lo o d

1 B lo o d C u ltu r e

1 0 — _{K ille d 8 p v}

2 0 _— _{K ille d 8 p v}

3 0 + <* ) K ille d 8 p v

s 4 0 — K ille d 8 p v

> 5 0 _— _{K ille d 8 p v}

1 6 0 0 — K ille d 15 p v

Ph 7 0 0 — K ille d 15 p v

Ph 8 0 ₀

— K ille d 15 p v

9 0 0 — K ille d 15 p v

10 0 0 — K ille d 15 p v

1 0 0 0

_

K ille d 30 p v

2 0 ₀ ₀

_

_{K ille d 30 p v}

3 0 0 0 + < * * ) K ille d 30 p v

3 4 0 0 X X K ille d 30 p v

> 5 0 ₀ ₀ _— _{D ie d 24 p v}

+ 6 0 0 0 + ( * * ) K ille d 30 p v

7 0 0 0 + ( * * ) K ille d 30 p v

8 0 0 0 + ( * * > K ille d 30 p v

9 0 0 0 + < * * ) K ille d 30 p v

10 0 0 0 — K ille d 30 p v

M ED IA N 0 0 0

M o rta lity 0% 0% 10%

1 0 0 0 K ille d 30 p v

2 0 0 0 K ille d 30 p v

3 0 0 0 K ille d 30 p v

4 0 0 0 K ille d 30 p v

ÊH

fc 5 0 0 0 K ille d 30 p v

6 0 0 0 K ille d 30 p v

7 0 0 0 K ille d 30 p v

8 0 0 0 K ille d 30 p v

9 0 0 0 K ille d 30 p v

10 0 0 0 K ille d 30 p v

M EDIAN 0 0 0 | i

M o rta lity 0% 0% 0% ₁ ₁

(15)

T A B L E 6

Mice infected an d infected and treated w ith Vbl

N u m b e r o f p a r a s i te s 5 m m 3 o f b lo o d

G r o u p M o u se D a y s a f t e r in fe c tio n . O b s e rv a tio n

30 15 8

1 28.000 X X _{D ie d 12 p . i .}

2 22.400 X X _{D ied' 15 p . i .}

3 X X X _{D ie d 6 p . i .}

4 21.700 X X D ie d 13 p . i .

5 X X X D ie d 1 p . i . *

6 18.900 X X D ie d 13 p . i .

7 21.000 1.050 X D ie d 16 p . i .

8 44.800 X X D ie d 9 p . i .

9 X X X D ie d 1 p . i . *

10 18.200 X X D ie d 13 p . i .

M E D IA N

M o r ta lity 21.70012%

1.050 90%

X

100% * E x c lu d e d

1 46.900 X X D ie d 14 p . i .

2 44.800 X X _{D ie d 13 p . i .}

3 35.700 X X _{D ie d 13 p . i .}

4 43.400 X X _{D ie d 14 p . i .}

i 5 44.800 X X D ied 14 p . i .

< 6 35.700 X X D ie d 14 p . i .

ã 7 52.500 X X D ied 12 p . i .

8 48.300 X X D ied 14 p . i .

9 44.800 X X D ie d 14 p . i .

10 46.900 X X D ie d 13 p . i .

M E D IA N M o ta U ty

44.800 0%

X

100%

X

(16)

T A B L E 7

Mice inoculated w ith th e positive blood cu ltu re s from th e anim ais described in th e Table 5

G roup

Mouse Blood (*)

C u ltu re Positive

l st In o cu latio n

M aterial In oculated

2n<1 In o cu latio n

M aterial In o cu la ted

3rd In o cu latio n

Mouse P a ra sit. 8th

p. inoc. Mouse P arasit. 8th p. inoc.

Mouse

30th p .i.

P ara sitem i

15th p. i . a

8th p .i.

l-H

rO 1 C entrifuged 1 —

'T s 2 — Blood 2 —

& w 3 3 — Serum 3 —

4 — (Pool) 4 —

3 1 1 1 _

5 2 — 2 — 2 — — —

6 3 — 3 — 3 — ' — —

rH ₇ ₄ _— _{C entrifuged} ₄ _— _{Pool of} ₄ _— _— _—

> ₈ ₅ _— _Blood ₅ _— _{T otal} ₅ _— _— _—

1 S 9 6 —- Serum 6 — C itrated 6 — — —

fn Pool 7 — (Pool) 7 — Blood 7 — — —

Ph _of(**) ₈ _— ₈ _— ₈ _— _— _—

12 tubes 9 — 9 — — —

10 -- 10 *——

(17)

T A B L E 8

Mice Controls of th e vaccine (PF) an d V in b lastin (V bl).

D A Y A F T E R V A C C IN A T IO N

G r o u p M o u se

blo o d

15

b lo o d

30

blo o d

O b s e rv a tio n

1 0 0 0 K ille d 30 d p v

2 0 0 0 K ille d 30 d p v

3 0 0 0 K ille d 30 d p v

4 0 0 0 K ille d 30 d p v

5 0 0 0 K ille d 30 d p v

6 0 0 0 K ille d 30 d p v

7 0 0 0 K ille d 30 d p v

8 0 0 0 K ille d 30 d p v

9 0 0 0 K ille d 30 d p v

10 0 0 0 K ille d 30 d p v

M E D IA N 0 0 0

M o r ta lity 0% 0% 0%

W E E K L Y V b l IN JE C T IO N S

.o >

M o r ta lity

1 2 3 5 4

6

7 8 G 10

0,01 m g 0,02 m g

a liv e a liv e

a liv e d ie d a liv e

0% 10%

0,03 m g

a liv e

10%

0,06 m g

a liv e

10%

K ille d D ied K ille d K ille d K ille d K ille d K ille d K ille d K ille d K ille d

30_{th d a y}

(18)

T A B L E 9

Mice vaccinated and vaccinated an d treated 15 days late r w ith Vbl

G R O U P M O U SE

DAYS A F T E R l s t V bl IN JE C T IO N

O B S E R V A T IO N

5 13 21 29

P a r a s ite s 5 m m 3 B lo o d

B lo o d

C u ltu r e P a r a s ite s5 m m 3 B lo o d

B lo o d

C u ltu r e P a r a s ite s5 m m s B lo o d

B lo o d

C u ltu r e P a r a s ite s5 m m 3 B lo o d

1 __ ___ _{K ille d 5 d . p . l s t V bl}

»H 2 — — _{K ille d 5 d . p . l s t V bl}

3 — — _{K ille d 5 d . p . l s t V bl}

A 4 — — K ille d 5 d . p . l s t V bl

> 5 X X D ie d 11 d . p . v .

1 6 —

1

— _{K ille d 5 d . p . l s t V bl}

7 — X X _{D ie d} _{5 d . p . 2nd V b l}

fo

8 — — — _{K ille d 6 d . p . 2nd V bl}

Ph 9 — — _— _{K ille d 6 d . p . 2nd V bl}

1.0 — — — _{K ille d 6 d . p . 2nd V bl}

.Q > 1 2 3 4 5 6 7 8 9 10 D ied K ille d D ie d D ied K ille d K ille d D ied D ied K ille d K lU ed

3 d . p . 5 d . p . 3 d . p . 2 d . p . 5 d . p . 5 d . p . 4 d . p . 8 d . p . 5 d . p . 5 d . p .

4th V bl 4th V bl 3rd V bl 3rd V bl 4th V bl 4th V bl 4 th V bl 2sd V bl 4th V bl 4th V bl

MEDTAN M o rta lity

0

0% 100% 30%0 50%0

D A Y S A F T E R V A C C IN A T IO N

CU

15 30 45

K ille d K ille d K ille d K ille d K ille d K ille d K ille d K ille d

d . p . v . d . p . v . d . p . v . d . p . v . d . p . v . d . p . v . d . p . v . d . p . v .

M E D IA N

(19)

T A B L E 10

Mice in fected w ith v iru le n t y s tra in an d in fec ted w ith th e sam e s tra in an d tre a te d 15 d ay s la te r w ith Vbl

G R O U P M O U SE

P A R A S IT E S 5 m m 3 B L O O D

O B S E R V A T IO N S B e fo re V b l A f te r V b l

8 d a y s 15 d a y s 5 d a y s 10 d a y s

1 560 12.600 8.680 X D ie d 5 d a y s p o s t 2nd V bl

2 1.120 7.980 X X D ied 3 d . p . l s t V bl

3 2.380 15.680 X X D ie d 4 d . p . l s t V bl

4 2.030 14.490 X X D ie d 4 d . p . l s t V bl

P

> 5 595 10.500 X X D ied 4 d . p . l s t V bl

| 6 3.780 27.020 X X D ie d 3 d . p . l s t V bl

7 490 10.220 X X D ie d 3 d . p . l s t V bl

8 1.575 13.440 X X D ie d 3 d . p . l s t V bl

9 1.260 6.580 X X D ied 5 d . p . l s t V b l

10 2.590 16.800 X X D ied 4 d . p . l s t V bl

M E D IA N 1.417 13.020 8.680 X

M o r ta lity 0% 0% 90% 100%

D A Y S A F T E R IN F E C T IO N

8 15 20 30

1 2.590 18.200 X X D ie d 17 d a y s p o s t in íe c .

2 1.680 4.760 4.760 X D ie d 23 d . p . i .

3 1.890 6.720 6.720 X _{D ie d 25 d . p . i .}

4 3.570 5.740 X X _{D ie d 20 d . p . i .}

5 1.330 7.840 2.800 560 D ie d 30 d . p . i . >*

6 4.135 15.260 X X D ie d 18 d . p . i .

7 1.785 8.260 X X D ie d 20 d . p . i .

8 1.680 12.320 X X D ie d 20 d . p . i .

9 910 14.980 X X D ie d 19 d . p . i .

10 630 13.160 X X D ie d 20 d . p . i .

1.737 10.290 2.800 560

M E D IA N 0% 0% 70% 100%

(20)

:, V - V- ^ t J% r - * ,

,

T A B L E 11

A nim ais Controls of th e exp erim en ts described in th e T ables 7 a n d 8.

D A Y S A F T E R l s t IN JE C T IO N V bl

G R O U P M O U SE O B S E R V A T IO N

5 i s 21 29

1 a liv e a liv e a liv e d e a d D ie d 2 d a y s p o s t 4th V bl 2 » ” _{a liv e} K ille d 5 d . p . 4th V b l 3 »» » ” d e a d D ie d 2 d . p . 4th V bl

4 ♦> 99 _{d e a d}

X D ie d 5 d . p . 2nd V bl

s

5 >9 _{a liv e} _{d e a d}

D ied 4 d . p . 4th V b l > 6 ” a liv e K ille d 5 d . p . 4th V b l 7 99 _” _{d e a d} _{D ie d} _{2 d . p . 3rd V b l}

8 »» i> _” _”

D ie d 4 d . p . 3rd V b l'

9 n 99

9

_”

D ie d ; 3 d . p . 4th V bl 10 M ” K ille d 5 d . p . 4th V b l

M o r ta lity 0% 0% 10% 70%

D A Y S A F T E R l s t SAXjIN E SOL. IN JE C T IO N

1 a liv e a liv e a liv e a liv e K ille d 5 d a y s p o s t 4 th i n j . 2 *» ” » M _{K ille d 5 d a y s p o s t 4 th in j.} 3 ” ” ” »» K ille d 5 d a y s p o s t 4th i n j. 4 ” 9) _{K ille d 5 d a y s p o s t 4 th in j.}

5 ** ” K ille d 5 d a y s p o s t 4th i n j . 6 ” ” K ille d 5 d a y s p o s t 4 th i n j . 7 ” ” ” K ille d 5 d a y s p o s t 4 th in j. 8 ” ” ” » K ille d 5 d a y s p o s t 4 th in j. 9 ” ” ” M K ille d 5 d a y s p o s t 4th in j. 10 ” » 9» J» K ille d 5 d a y s p o s t 4th in j.

(21)

Julho-Ago.,

Rev. Soc. Bras. Med. Trop.

233

12. SQUAME, G.; PANE, G; VISCONTI, M. & PIAZZA, M . — A ttiv itá di u n fa rm ac o im m unosoppressore (azatio - p rin a ) su lla evoluzione d elia e p a tite sp erim en ta le de virus MHV3. R iv. In st. S ieroter. Ital., 43: 251-256, 1968.

13. TRIPATHY, S . P . & MACKANESS, G . p . — T h e effec t of cytotoxic ag e n ts on th e p rim a ry im m u n e re s ponse to Listeria m onocytogenes. J . Exp. Med., 13: 1-16, 1969.

14. WALKER, P . J . — T h e virulence of in fectio n s of P. berghei a n d T. rho-desiense in an im ais w hose im m une response h a s been im p aired by ra d ia - tion, d ru g s Or an tily m p h o cy te serum . J. Protozool. 15: 33-, 1968 - Supl.