R evista da S ociedade B rasileira de M ed icin a Tropical 21(3): 135-138, Jul-Set, 1988.
T R Y P A N O S O M A (H E R P E T O S O M A ) R A N G E L I
TEJERA, 1920:
IN FL U E N C E O F H O ST W EIG H T, SIZE OF IN O CU LU M , A N D RO UTE
OF IN FE C T IO N U P O N E X PE R IM E N T A L PA RASITEM IA.
F e l ix T e je r o , N a tie la G a lli a n d S e rv io U r d a n e t a -M o r a le s
T o s tu d y th e in flu e n c e o f h o s t a g e, in o c u lu m siz e , a n d ro u te o f in f e c tio n o n
T rypanosom a (H erpetosom a) rangeli, 1 2 lo ts o f 6 .0 g a lb in o m ic e ( N M R I s tr a in ) w e re in fe c te d up. w ith f r o m 2 5 x 1 0 1 to 2 5 x l ( f i tr y p o m a s tig o te s /g r a m b o d y w e ig h t h a rv e s te d f r o m L I T m e d iu m . T h e lo w e r in o c u la p r o d u c e d lo w b u t p e r s is te n t p a r a s ite m ia s , w h ile th e h ig h e r in o c u la p r o d u c e d h ig h le v e ls o f p a r a s i t e m i a th a t f e l l q u ic k ly , s u g g e stin g th e m o b iliz a tio n o f r e s is ta n c e m e c h a n is m s. I n o th e r e x p e r im e n ts , i.p. in o c u la tio n p r o d u c e d h ig h e r p a r a s i t e m i a s th a n s.c. in o c u la tio n , a n d 6 .0 g m ic e h a d h ig h e r p a r a s i t e m i a s th a n 1 6 .0 o r 2 6 . 0 g m ice. T hus, a s t a n d a r d m e th o d o lo g y w o u ld se e m to b e n e c e s sa ry in th e s tu d y o f th e v a r io u s s tr a in s a n d / o r sp e c ie s t h a t m a y m a k e u p th e T. rangeli
c o m p le x .
Key-words: T rypanosom a (H erpetosom a) rangeli. In o c u lu m siz e . In fe c tio n route. H o s t w e ig h t. I m m u n e re sista n c e .
The study o f experim ental infections of T r y p a n o s o m a ( H e r p e to s o m a ) r a n g e li Tejera, 1920 in
animal models has been hampered by the low virulence of the parasite6 and the lack o f precisely defined expe rim ental conditions. In a form er paper21 we have described a mouse model for obtaining high and persistent parasitem ias of T. ra n g e li. The present paper describes experiments with m ice of differing weights, varying numbers of inoculated parasites, and with different routes o f inoculation, in order to evaluate the influence o f variables such as these upon the course of infection and to determ ine the optim um methodo logy for this m ouse model.
M A T E R IA L S A N D M E T H O D S
The strain o f T ry p a n o s o m a r a n g e li employed and the details of its culture have been described in the previous paper21.
In th e fir st e x p e r im e n t, 1 2 lo t s o f e ig h t m a le
w h ite m ic e e a c h (N M R I stra in ) w e ig h in g 6 .0 g a v. w ere in o c u la te d i.p . w ith th e fo llo w in g n u m b ers o f c u ltu r e -d e r iv e d m e t a c y c lic t r y p a n o s o m e s /g b o d y w ig h t 2 5 , 2 . 5 x l 0 2 , 5 x l 0 2 , 2 5 X 1 0 2 , 5 0 x 1 0 2 , l x l O 4 , 2 . 5 x l 0 4 , 5 x l 0 4 , 1 x 1 0 5 , 2 . 5 x 1 0 5 , l x l O 6 , a n d 2 .5 x 1 0 ® .
T h is work w as su pp orted b y the C o n se jo d e D esa rro llo
C ie n tific o y H u m a n istic o de la U n iv ersid a d Central de V e n e zu ela (P ro ject N o . C - 0 3 .2 5 /8 5 ) .
In stituto de Z o o lo g ia T ro p ica l, F a cu lta d d e C ie n c ia s, U n iversidad Central de V enezu ela.
Address: A partad o 4 7 3 3 2 , C aracas 1 0 4 1 -A , V E N E Z U E L A
R e ce b id o para p u b lica ç ã o em 3 / 1 1 /8 6
Twenty-four hours post-infection and daily thereafter, half the anim als in each group were exam ined accor ding to the m ethod o f Brener3 to evaluate the intensity and persistence o f the parasitem ia.
A s explained in the results, an inoculum o f 100 000 metacyclic trypanosom es/g body weight pro duced parasitem ias that were m ost adaptable to experim ental purposes. Therefore, in the second ex periment, two lots o f eight male 6.0 gram white mice were inoculated with 100 0 0 0 parasites/g body weight, one group intraperitoneally and the other subcuta- neously. Parasitem ias were followed as above.
O n finding th at i.p. inoculation produced the highest parasitem ias (see Results), three lots of eight male white mice, weighing 6.0, 16.0 and 26.0 g respectively, were inoculated i.p. with 1x10s m eta cyclic trypanosom es/gram body weight. Parasitem ias were followed as above.
R E S U L T S
The results obtained are shown in Figs. 1 a, b, c; 2, and 3. A ll inocula, excepting the sm allest (25/g), produced parasitem ias th a t were p atent on the first exam ination (24 hr p.i.).
Concerning the size of the inoculum, Figs. 1 a, b, and c show that the larger inocula produced a higher level of parasitem ia, but the sm aller inocula produced a m ore persistent parasitem ia. All parasitem ias, w ha tever the size o f the inoculum, reached their peaks between the second and fourth day after injection a characteristic o f T. ra n g e li. Although the larger ino cula produced higher parasitem ias in all cases, there
Tejero F, G alli N, U rdaneta-M orales S. Trypanosom a (H erpetosom a) rangeli Tejera 1920: influence o f h o st weight, size o f incolum, a n d route o f infection upon experim ental parasitem ia. R evista da Sociedade Brasileira de M ed icin a Tropical 21: 135-138, Jul-Set, 1988
F i g, i - C o u r s e o f p a r a s i t e m i a in 6 . 0 g a l b in o m ic e i n o c u l a t e d i.p . w ith m e t a c i c l i c t r y p a m a s t i g o t e s o f
T . rangeli.
a ) I n o c u l a o f 0 . 2 5 , 2 . 5 , 5 .0 , a n d 2 5 x l 0 2 tr y p o
-m a s t i g o t e s / g b o d y w e ig h t.
b ) I n o c u la o f 5, 1 0 , 2 5 , a n d 5 0 x 1 0 s t r y p o
m a s t i g o t e s / g b o d y w e ig h t.
was no correlation between the size o f the inoculum and the parasitem ia level; rather there appeared to be a
m a s t i g o t e s / g b o d y w e ig h t.
stepwise effect, with wide ranges o f inoculum size pro ducing similar levels o f parasitemia. The inoculum of 1 x
105 metacyclics/g produced a peak o f parasitem ia esti m ated to be 7.9 times the original inoculum, and the parasitem ia persisted for 17 days, so that this inoculum appears to be particularly suited for experim ental purposes.
Fig. 2 shows that i.p. inoculation o f lx lO 5 trypomastigotes g produced parasitem ias far higher and more persistent in 6.0 g mice than in 16.0 or 26.0 g mice.
F ig . 2 - C o u r s e o f p a r a s i t e m i a in a l b in o m ic e o f 6 . 0 , 1 6 . 0 ,
a n d 2 6 . 0 g i n o c u l a t e d i.p . w ith 1 x 1 0 s m e t a c y c l i c
t r y p o m a s t i g o t e s o f T . rangelV g b o d y w e ig h t.
Tejero F, G alli N, U rdaneta-M orales S. Trypanosom a (H erpetosom a) rangeli Tejera 1920: influence o f host weight, size o f incolum, a n d route o f infection upon experim ental p arasitem ia. R evista da Sociedade Brasileira de M edicina Tropical 21: 135-138, Jul-Set, 1988
Fig. 3 shows that i.p. inoculation produced m uch higher parasitemias than s.c. inoculation.
F ig . 3 - C o u r s e o f p a r a s i t e m i a in 6 . 0 g a l b i n o m ic e in o c u
l a t e d w ith 1 x 1 05 m e t a c y c l i c t r y p o m a s t i g o t e s o f T . rangeU/g b o d y w e ig h t b y i.p . in o c u l a ti o n a n d
b y s.c . in o c u la tio n .
D IS C U S S IO N
In general, the published literature on expe rimental infections o f T. ra n g e li in the m am m al host has little to say on the factors which influence the course of the parasitem ia, such as host age, size of inoculum, or source of inoculated parasites (blood, culture, or vector-derived). If these factors were to be standardized, it would be possible to establish the particular characteristics o f a given strain of the parasite. M cH ardy13 has studied the influence of host sex on resistance to T. c ru zi. H anson8 has demons trated differences o f susceptibility to T. c r u z i in different strains o f host mice. T he influence o f host age2 5; size o f inoculum4 11 19; route o f inoculation12 and source of infecting parasites16 have been deter mined in a variety o f T r y p a n o s o m a spp.
Phillips17 em phasizes the im portance of deter mining the inoculum which induces the greatest viru lence o f the parasite; the inoculation o f an insufficient num ber o f parasites m ay explain the attenuation or failure of infections by T. r a n g e li1 7918 and by other
H e r p e to s o m a 110 15. This m ay also explain the failure to invade tissues o f those species which have intra cellular m ultiplication10’ 15.
In the m ouse m odel which we have been deve loping for T. ra n g e li, we have inoculated trypom as tigotes from culture, since these forms, and those produced in the vectors o f the H e r p e to s o m a , are more infective than the bloodstream forms, the former being those transm itted in natural infections15. W e m ust
emphasize the im portance o f using re c e n tly is o la te d
strains of the parasite, rather than strains which have been long in culture, since long-cultured strains m ay have few or no m etatrypom astigotes21. A ttem pts at infecting mice in our laboratory, using the identical regimen described above, with tw o strains of T. ra n g e li, one m aintained in v itro for 30 years and the other for four years, have been uniformly unsuccessful (unpublished observations).
O ur results indicate the convenience of using low numbers (0.25 - 5 x l0 3 m etacyclics/g) for obtai ning long-lasting parasitem ias. H igh levels o f blood parasites persisting for an appreciable time m ay be obtained with an inoculum o f lx lO 5 m etatrypom as- tigotes/g body weight i.p. into 6.0 g mice. This inoculum multiplies itself nearly eight times in the mouse. W hile larger doses multiply to 1.6-6 tim es the original inoculum.
The inverse relationship betw een peak level and persistence o f the parasitem ia, plus the tendency o f the parasitem ias produced by inocula varying greatly in magnitude to fall into rather well-defined groups m ay indicate the successive mobilization o f different mechanisms o f resistance to the parasite, or mobili zation of the same m echanism s at different levels of activity.
It has been suggested that T. ra n g e li is a complex o f different strains or species6 10 20. To investigate this possible heterogeneity, comparative studies would be needed on mammal hosts with flagellates from a variety o f m am m alian reservoirs and insect vectors. The behavior o f the organisms, studied under speôific conditions. Should be predictable, with the aim o f stabilizing the parasite. A standardized methodology is vital to such a study.
R E S U M E N
P a r a e s tu d ia r la in flu e n c ia d e la e d a d d e l h o s v e d a d o r . d e l ta m a n o d e l in o c u lo y su ru ta d e a d m in is tr a c ió n s o b r e la in fe c tio n p o r Trypanosom a (H erpetosom a) rangeli, fu e r o n in o c u la d o s i.p . 1 2 lo te s d e r a to n e s a lb in o s (c e p a N M R I ) d e 6 . 0 g d e p e s o con 2 5 . 0 - 2 .5 x 1 o 6 m e ta tr ip o m a s tig o te s /g o b te n id o s d e l m e d io L I T . L o s in ó c u lo s m a s b a jo s p r o d u je r o n p a r a s ite m ia s b a ja s p e r o p e r s is te n te s ; lo s in ó c u lo s m a s e le v a d o s o rig in a ro n n iv e le s a lto s d e p a r a s i te m ia s q u e c a y e ro n p r o n ta m e n te , su g ir ie n d o la m o b i l i z a t i o n d e m e c a n is m o s d e re sis te n c ia a n iv e le s d if e r e n te s d e a c tiv id a d . E n o tr o s e x p e r im e n to s , la in o c u la tio n i.p. y e l u so d e r a to n e s d e 6 .0 g d ie ro n p a r a s ite m ia s m a s e le v a d a s q u e c u a n d o se u sa ro n in o c u la c io n e s s.c . o a n im a te s d e 1 6 .0 y 2 6 . 0 g. E s t o s r e s u lta d o s in d ic a n la n e c e s id a d d e e m p le a r u n a m e to d o lo g ia u n ifo rm e c u a n d o s e in v e s tig u e e l p o s i b l e c a r á c te r h e te ro g é n ic o d e l c o m p le jo d e T . rangeli.
Tejero F, G a lli N, U rdaneta-M orales S. Trypanosom a (H erpetosom a) rangeli Tejera 1920: influence o f h o st weight, s ize o f incolum, a n d route o f infection upon experim ental parasitem ia. R evista d a Sociedade B rasileira de M ed icin a T ropical 21: 135-138, J u l-Set, 1988
A C K N O W L E D G E M E N T S
The authors wist to thank M r. Ian M cLure for technical assistance and the English translation, and M rs. N u r ia Teje?o for preparing the graphs.
R E F E R E N C E S
1. A fie z N . S tu d ies o n T r y p a n o s o m a r a n g e l i T ejera, 1 9 2 0 . 1. D e p o sitio n , m igration, and grow th o f T. r a n g e l i
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8 . H a n so n W L . Im m u n e resp on se and m ech a n ism s o f r esistan ce in T r y p a n o s o m a c r u z i. In: C h a g a s’D is e a s e . P a n -A m erica n H ea lth O rgan ization S cien tific P u b lica tion 3 4 7 , p. 2 2 - 3 4 , 1 9 7 7 .
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12. M a rsd en P D . T r y p a n o s o m a c r u z i in fe ctio n s in C F I m ice. II. In fec tio n s in d u ced b y different rou tes. A n n a ls o f T rop ical M ed ic in e and P a ra sito lo g y 61: 6 2 - 6 7 , 1 9 6 7 .
13. M cH a rd y N . E ffe c t o f s e x o f m ice in relatio n to their resp on se to im m u n izatio n w ith v a c c in e s prepared from
T r y p a n o s o m a c r u z i . T ra n sa ctio n s o f th e R o y a l S o c ie ty o f T rop ical M e d ic in e and H y g ie n e 72: 2 0 1 - 2 0 2 ,1 9 7 8 . 14. M o ly n e u x D H . T h e m orp h ology a n d life-h istory o f
T r y p a n o s o m a ( H e r p e t o s o m a ) m i c r o t i o f the field -v o le
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1 6 . M sh e lb w a la A S , O rm erod W E . M easu rem en ts o f the
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17. P hillip s N R . E xp erim en tal stu d ies o n the quantitative tran sm issio n o f T r y p a n o s o m a c r u z i: C on sid eration s regarding the stan dardization o f m aterial. A n n a ls o f T rop ical M e d ic in e a n d P a ra sito lo g y 54: 6 0 - 7 0 , 1 9 6 0 . 18. P ifa n o F . E l e sta d o a ctu al d e la trip an osom iasis rangeli en V e n e z u e la . A r c h iv o s V e n e z o la n o s de M ed ic in a T rop ical y P arasito lo g ia M e d ic a 5: 1 8 5 - 1 9 2 , 1 9 7 3 . 19. S ilva L H , N u sse n z w e ig V . S ob re u m a c e p a d e T r y p a
n o s o m a c r u z i altam en te viru lenta para o c am u n d on go branco. F o lia C lin ica e t B io lo g ic a 20: 1 9 1 - 2 0 7 , 1 9 5 3 . 2 0 . T o b ie E J . E x p erim en ta l tran sm issio n an d b io lo g ic a l com p arison o f strains o f T r y p a n o s o m a r a n g e li. E x p e rim ental P a ra sito lo g y 11: 1 - 9 ,1 9 6 1 .
2 1 . U rd a n eta -M o ra les S , T ejero F . T r y p a n o s o m a ( H e r
-p e t o s o m a ) r a n g e l i T ejera, 1 9 2 0 : M o u se m o d e l for high, su stained p arasitem ia . Jou rn al o f P a ra sito lo g y 7 1 : 4 0 9 -4 1 -4 , 1 9 8 5 .