TRYPANOSOMA (HERPETOSOMA) RANGELI TEJERA, 1920: PRELIMINARY REPORT ON HISTOPATHOLOGY IN EXPERIMENTALLY INFECTED MICE
C e c i l i a d e S C O R Z A ( 1 ) , S e r v i o U R D A N E T A - M O R A L E S ( 2 ) & F e l i x T E J E R O ( 2 )
S U M M A R Y
Male mice (NMRI strain) of 3 and 5 g were inoculated i. p. with 8 x 106 and 9 x 104 metatrypomastigotes/g harvested from a 12-day-old LIT culture of
Try-panosoma rangeli of the "Dog-82" strain. At regular intervals after inoculation, the animals were sacrificed and portions of heart, liver, spleen, lung, thigh, kidney, stomach, intestine, brain, sternum, and vertebral column were embedded in paraffin, sectioned, and stained with haematoxylin-eosin and Giemsa colopho¬ nium. Pathology was encountered in the first five tissues cited above. The sub-cutaneous, periosteal, interstitial, and peribronchial connective tissues, and later
the muscle cells of the heart, were heavily parasitized by amastigotes and trypo¬ mastigotes. The possible reasons for the decrease in tissue parasitosis at the
same time that the parasitemia is reaching its peak, and for the low level of in-flammation in the parasitized tissues, are discussed. The observations of other workers, as well as the results described here, indicate that certain strains of T. rangeli under certain conditions may well cause pathological alterations in mammals.
KEY WORDS: Experimental trypanosomiasis — Trypanosoma (Herpetosoma) rangeli Tejera, 1920 — mice
I N T R O D U C T I O N
Ever since DE LEON'S7 report on the first human infections by Trypanosoma rangeli in Guatemala in 1934, many wild and domestic mammals have been identified as reservoir hosts for the parasite in Central and South America5
'1 7
. Most authors have considered T. rangeli to be non-pathogenic in humans URDA-NETA-MORALES and TEJERO2 9 have now ob-served amastigote stages of T. rangeli which multiply by binary fission in various tissues of experimentally infected with mice. This paper presents the preliminary observations on the histopathology caused by this flagellate in
suckl-ing mice intraperitoneally inoculated with me-tatrypomastigotes of T. rangeli harvested from LIT culture medium.
MATERIALS AND METHODS
The culture and the experimental valida-tion of the purity of the strain of T. rangeli used have been described by
URDANETA-MORA-LES AND TEJERO »>». Two groups of suckling male white mice (NMRI non-isogenic strain)
we-re inoculated. In the first group, 12 mice of 3 g average weight received 8 x 106
metatrypo-R e q u e s t s f o r r e p r i n t s s h o u l d b e a d d r e s s e d t o t h e s e c o n d a u t h o r .
( 1 ) F a c u l t a d d e C i e n c i a s , D e p a r t a m e n t o d e B i o l o g i a , U n i v e r s i d a d d e L o s A n d e s , M e r i d a , 5 1 0 1 , V e n e z u e l a
mastigotes/g i.p. from a 12 day-old LIT culture. One animal was sacrificed at 5, 18, and 24 hr post inoculation, and one other daily thereafter. Portions of heart, liver, spleen, lung, kidney, in-testine, stomach, brain, sternum, the vertebra) column -with adjacent tissues and the whole thigh were then fixed in 10% formalin, process-ed in paraffin, sectionprocess-ed at 5 it, and stained
with haematoxylin-eosin and Giemsa colopho-ny3.
A second group of six 5 g mice received 9 x 10* metatrypomastigotes/g i.p. and two each were sacrificed at 3, 4, and 5 days postinocula-tion, being processed as above. Parasitemia was followed, by daily examination of fresh preparations of tail blood4 and also of thin smears fixed in methanol and stained with Giemsa. Microphotographs were taken as al-ready described2 8.
RESULTS
In both experimental groups, the parasite-mias increased steadily up to the fourth day, at which they reachet their peak values.
Bloot films taken daily showed no signs whatever of trypomastigotes in division.
Table I shows the occurrence of T. rangeli in the various tissues at various intervals post-inoculation, omitting those tissues in which the parasites were never observed. In can be seen that the parasites were very abundant from 5 hr — 5 days post-inoculation in the sob-cotaneous connective tissue of the thigh, in the periosteal connective tissue of the femur, and in the interstitial connective tissue of the skele-tal muscles associated with the thigh, verte-bral column and the sternum (Pig. la). After 4 days, the parasites were fewer, and remained as such at least up to the tenth day. Enormous clusters of parasites were seen in the connec-tive tissue, some within macrophages but the majority free. Form and size were highly va-riable; amastigotes and intermediat forms were seen, many with double nuclei and/or ki-netoplasts (Fig. lb). Some had apparently doubled themselves around a large vacuole, these being similar to certain stages interme-diate between trypomastigote and amastigote seen by PAN1 8 (Fig. la, arrows) in the
intra-myocellular nests of T. cnizi infections in mi-ce. The cytoplasm of the parasites in the con-nective tissue was very weakly stained, but the nuclei and the kinetoplasts were strongly co-loret, often appearing free in the amorphous surrounding substance. At 5 and 18 hr. there were observed in some areas many
macropha-ges with vacuolated cytoplasm, and at 24 hr, somd accumulations of polymorphs, with a few monocytes, and also some degranulated mastocytes (Fig. lc). There were no parasites visible in the fibers of the femoral, paraferte-bral, or pectoral muscles, although focal dege-neration and destruction were observed in so-me of the fibers. Interstitial edema, fibroblastic proliferation, macrophages containing parasi-tes (Fig. Id), and discrete infiltration by poly-morphs and monocytes were present in some areas; at some sites many free parasites with weakly stained cytoplasm, a large vacuole, and strongly stained kinetoplast were seen.
In the bone marrow, amastigotes were seen, the majority rather weakly stained, free or within macrophages. They were most abun dant between 5 and 48 hr post-inoculation.
of the spleen was also discrete, being observed from 5-72 hr post-inoculation, amastigotes being seen in the sinusoids and macrophages of the red pulp- As in the liver, parasites were more strongly stained than in the connective tissue. In the lung, parasites were seen at 18 hr post inoculation, reaching a maximum at 48 hr. Amastigotes were seen in the alveolar macro-phages and some trypomastigotes were in the lumen of the alveoli. The greatest parasitic density was in the peribronchial connective tis-sue of the bronchi of medium caliber and in the adventitia of the blood vessels; the para-sites were free and similar to those in the sub-cutaneous connective tissue. Amastigotes were
also seen in highly vacuolated macrophages, and some of them appearing to be within the peribronchial smooth muscle fibers. Groups of trypomastigotes were also seen in the lumen of some of the veins.
abundant in many nests of varying size within the myocells in all parts of the heart (Fig. 3). The parasites within the individual nests were extremely uniform in aspect, amastigotes pre-dominant in some, while others contained main-ly trypomastigotes or intermediate forms. Amastigotes were well stained, with basophilic cytoplasm and sometimes a double kinetoplast. Ruptured nests and various stages of free pa-rasites, and amastigotes within macrophages were also seen. The frequent observation of amastigotes within the mesothelial cells of the epicardium and the endothelial cells of the en-docardium, and of the veins of medium caliber was very striking; they were also seen in the lumen of some capillaries and veins. No in-flammatory reaction was observed, only dis-crete foci of infiltration in the subendocardial region, with predominance of histiocytes and apparently free parasite nuclei and kineto-plasts.
DISCUSSION
Results indicate that, under the experimen-tal conditions described, the "Dog-82" strain of T. rangeli undergoes an early tisular repro-ductive cycle in the connective tissues (subcu-taneous, periosteal, and the interstitial connec-tive tissue of the skeletal muscles). Later, the liver, spleen, lungs, bone marrow, and finally the heart are invaded.
The heart is intensely, but very briefly pa-rasitized, the parasites being seen only between the second and fourth days post-inoculation. The presence of amastigotes in the mesothelial cells of the epicardium, and later, in the endo-thelium of the endocardium and of some intra-myocardial veins of medium caliber is an in-teresting phenomenon, not habitually seen in T. c r u z i infections.
Upon comparing the levels of tissue para-sitosis with the parasitemias reported above and also by URDANETA-MORALES and TEJE-R O2 9 for this experimental model, we may sug-gest the progressive passage of tissue forms of the parasite toward the blood stream. Parasi-temia attains its maximum between the fourth and eighth day post inoculation, while tissue parasitosis diminishes between the fourth an<J sixth davs nost-inoculation. There is no indi.
cation of reinvasion of the tissues, and the pa-rasitemia progressively diminishes until the 17th day post-inoculation.
The scanty inflammation in the parasitized tissues may be due to the immature immuno-logical system of the very young hosts and/or to the short time since infection in the animals. A weak, non specific immune response at the tissue level, with little destruction of parasi-tes, is suggested by the presence of macropha-ges, occasionally abundant and containing pa-rasites, by the sporadic foci of polymorphs in the connective tissues (subcutaneous ,intersti-tial of the skeletal muscles, and the subendo-cardial region), and by groups of free parasi-te nuclei and kinetoplasts. The reasons for the disappearance of the parasite from the blood are unknown, though AÑEZ et al.1 have sug-gested antibody activity.
In our experiments, we have inoculated suckling mice i. p. with metacyclic trypomasti-gotes of T. rangeli harvested from a modified. LIT medium. This is in accord with recommen-dations by WHO3 0 for inducing the multiplica-tion of strains of trypanosomes in the labora-tory by the use of susceptible hosts, particular-ly suckling mice, and different culture media.
Although T. rangeli has generally been con-sidered to be non-pathogenic for vertebrate hosts5, the observation of tissue multiplication described above allows speculation on possi-ble tissue alterations by the strain used by us, under certain conditions HOARE1 5 has suggest-ed that T. rangeli is a complex of strains, and DEANE6 postulates that these may be evolving toward tissue multiplication. The major inci-dence of this trypanosomiasis is in children 25
-2 7
,
and it may be that very young children, infect-ed with particular strains of T. rangeli would show invasion and damage in the heart and other organs. HERNANDEZ DE PAREDES y PAREDES 14 and De LEON s,9,io had suspected
that pathological manifestations in humans (particularly children) might have been due to T. rangeli, which in some cases9 was the only infective organism detected.
TORREAL-BA 2 3 , 2 4 , 2 5 and TORREALBA, PÍFANO and
lymphocy-tosis, and occasionally hepatosplenomegaly) characteristic of infectious disease. However, PÍFANO'9 reported that certain cases, origi-nally attributed to T. rangeli, showed associat-ed infections with T. cruzi on later
PICAÑO20, although stating that no patho-genic symptoms were found in 842 cases of T, rangeli infections he studied, noted that in 224 cases of established T. rangeli infection, infection, electrocardiograms showed discrete myocardial damage in 6.7%. In an epidemiolo-gical survey of rural areas in Venezuela ende-mic for Chagas* disease, the control group of 5251 persons negative both xenodiagnostically and immunologically for T. cruzi showed a ve-ry nearly equal percentage of electrocardiogra-phically detectable damage to the heart.
HERBIG SANDREUTER13 found no histo-pathological changes in 130 white mice and two monkeys inoculated s.c. with culture forms of T. rangeli, concluding that the parasite is of little importance as a human pathogen.
TONN 2 2
has questioned whether T. rangeli might modify the pathology due to T. cruzi in mixed infections or whether the pathological picture is a separate entity. ZELEDON32 has speculated on the possible effects of repeated exposure to T. rangeli antigens (brought about by the bites of infected triatomids) upon the development of pathology in mixed cases. GUHL and MARINKELLE a suggest that fre-quent reinfections in areas endemic for T. ran-geli may result in antibody titers high enough to be mistaken for T. cruzi. GROGL and KUHN ii state that the influence of concurrent or sequential T. rangeli infections on the pa-thogenesis of Chagas' disease has not been es-tablished.
The frequent finding of mixed infections of T. cruzi and T. rangeli in areas endemic for Chagas' diseases, plus the crossed serum reac-tions between the two parasites observed by ANTHONY et al?, GUHL and MARINKELLE u
and SCHECHTER et al.21, together with the
observations described in the present paper, underline the necessity for further study of the immunobiology of T. rangeli in the mammal host, above all with an open mind.
RESUMEN
Trypanosoma (Herpetosoma) rangeli Tejera, 1920: nota prévia sobre a histopatologia em camundongos infectados experimentalmente
Ratones machos (cepa NMRI) de 3 y 5 gr, inoculados i.p. con 8 x 106 y 9 x 104 metatripo¬
mastigotes/gr cosechados de cultivo LIT de 12 días de repique de la cepa "Perro-82" de Try-panosoma rangeli fueron procesados, em deter-minados períodos postinoculación, para seccio-nar y teñir con hematoxilina-eosina y Giemsa-colofonio trozos de corozón, hígado, bazo, pul-mones, fémur, riñon, intestino, estómago, cere-bro, cerebelo, esternón y columna vertebral.
Se detallan los resultados preliminares so-bre la histopatologia provocada por el T. ran-geli en los cinco primeros órganos citados. Los tejidos conjuntivos subcutáneo, periosteal, in-tersticial y peribronquial, así como las miocé¬ lulas del corazón fueron altamente parasitados por amastigotes y tripomastigotes.
Se discuten las posibles razones de la dis-minución del parasitismo tisular cuando la pa-rasitemia se encontró en sus niveles máximos, así como la escasa inflamación obtenida en los tejidos parasitados.
Las observaciones de varios autores sobre el comportamiento del protozoario en humanos y en animales de laboratorio así como los re-sultados descritos en este trabajo, plantean la posibilidad de que determinadas cepas de T. rangeli, en condiciones particulares, podrían causar alteraciones patológicas en el mamífero.
ACKNOWLEDGEMENTS
The authors wish to thank Mr. Ian McLure for assistance, both technical and in preparing the manuscript, and for the English translation. Mrs. Carmen E. Bermudez and Mr. J. V. Scorza for histological preparations; the Consejo de Desarrollo Cientifico y Humanistico de la Uni-versidad Central de Venezuela (Project No C05.4/81) for financial support. Special thanks are due to Dr. Nestor Añez (Universidad de Los Andes, Venezuela) for supplying us with the strain of the parasite.
R E F E R E N C E S
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V e n e z u e l a r e s p e c t o a l a e t i o l i g i a c h a g a s i c a d e l a s m i o ¬ c a r d i o p a t i a s c r o n i c a s r u r a l e s . A r c h . v e n e z . M e d . t r o p . , 5 : 1 0 7 - 1 1 5 , 1 9 7 3 .
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2 6 . T O R R E A L B A . J . F . ; P I F A N O , F . & R O M E R , M . — i n v e s t i g a c i o n e s s o b r e E n f e r m e d a d d e C h a g a s y T r i -p a n o s o m i a s i s r a n g e l i e n e l D i s t r i t o R o s c i o , E s t a d o Guarico, Venezuela. Gac. méd. Caracas, 58: 85-98, 1950.
27. TORRES, R. A — Trypanosoma rangeli (Tejera, 1920) e n l a G u a j i r a v e n e z o l a n a . E s t u d i o d e l p a r a s i t o e n e l
h u m a n o . K a s m e r a , 7: 4 1 - 6 3 , 1 9 7 9 .
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c e l l u l a r a m a s t i g o t e s t a g e s o f r e p r o d u c t i o n i n w h i t e m i c e . R e v . I n s t . M e d . t r o p . S . P a u l o , 2 8 : 1 6 6 - 1 6 9 , 1 9 8 6 .
3 0 . W H O R e p o r t S e c o n d M e e t i n g H u m . C o m p . S c . W o r k . G r o u p C h a g a s ' D i s . U N D P / W H O S p . P r o g . R e s . T r a i n i n g T r o p . D i s . , 1 9 8 0 .
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3 2 . Z E L E D O N , R . — E p i d e m i o l o g y , m o d e s o f t r a n s m i s s i o n a n d r e s e r v o i r h o s t s o f C h a g a s ' d i s e a s e . I n : T R Y P A N O S O M I A S I S A N D L E I S H M A N I A S I S W I T H S P E C I A L R E F E R E N C E T O C H A G A S ' D I S E A S E . A m s t e r d a m , E l s e -v i e r , 1 9 7 4 . p . 5 1 - 8 5 . ( C i b a F o u n d a t i o n S y m p o s i u m 20 — n e w s e r i e s ) .
