HEMOLYTIC ACTIVITY OF MONOCERCOMONAS SPP.
DA SILVA A.C.*, DE CARLI G.A.*, BRASSEUR P.**, TASCA T.*, CASTILHOS D.* & WENDORFF A.*
S u m m a r y :
The hemolytic activity of an isolate of Monocercomonas spp. from Tropidophis melanurus [snake:Boidae] was investigated. The isolate was tested against human erythrocytes of groups A, B, AB and O and against erythrocytes of six adult animals of different species (rabbit, rat, chicken, horse, bovine, and sheep). Results show that Monocercomonas spp. exerted an hemolytic activity against all erythrocytes tested.
KEY W O R D S : Monocercomonas spp., snake: Boidae, Tropidophis melanurus, hemolytic activity.
Résumé : ACTIVITÉ H É M O L Y T I Q U E D'UN ISOLAT DE MONOCERCOMANAS SPP.
L'activité hémolytique d'un isolat de Monocercomonas spp.
provenant de Tropidophis melanurus (serpent: Boidae) a été étudiée. L'isolât a été testé avec les hématies appartenant aux groupes sanguins humains A, B, AB et O et les hématies de six animaux adultes d'espèces différentes (lapin, rat, poulet, cheval, bœuf et mouton]. Les résultats ont montré que Monocercomonas spp. exerce une activité hémolytique sur toutes les hématies testées.
MOTS CLES: Monocercomonas spp., Serpent : Boidae, Tropidophis melanurus, hémolyse.
INTRODUCTION
onocercomonas Grassi, 1 8 7 9 is a flagellate M protozoan o f the large intestine o f squamate
reptiles. It is c o n s i d e r e d the most primitive k n o w n m e m b e r o f the order T r i c h o m o n a d i d a Kirby (Honigberg, 1 9 6 3 ) and limited information is currently available o n the pathogenicity o f this parasite. T h e p a t h o g e n i c m e c h a n i s m s o f several s p e c i e s o f tricho- monads: Trichomonas vaginalis, Trichomonas gallinae,
Tritrichomonas FOETUS and Tritrichomonas suis have b e e n widely studied in animal m o d e l s especially in m i c e since 1 9 3 4 ( B o s , 1 9 3 4 ) , providing valuable infor- m a t i o n s o n s p e c i f i c host-parasite interactions ( s e e review by Kulda et al, 1 9 9 0 ) . Interactions b e t w e e n T. vaginalis and cell culture monolayers have also b e e n studied and have demonstrated a contact d e p e n d e n t cytopathic effects o f the parasite. In addition, several studies h a v e s u g g e s t e d that m o l e c u l e s released b y T. vaginalis may exert contact i n d e p e n d e n t cytopathic effects ( s e e review o f Honigberg, 1 9 9 0 ) . Cytopatho- genicity induced in vitro b y other protozoan parasites h a v e also b e e n demonstrated for Entamoeba histoly-
tica, and Giardia lamblia (Lopez-Revilla & Said-Fer- nandez, 1 9 8 0 , Ravdin & Guerrant, 1 9 8 1 , Ortega et al., 1 9 8 7 ) .
Using in vitro methods, the hemolytic activity o f dif- ferent s p e c i e s o f Trichomonadida, such as T. vaginalis (Grys & Hernik, 1 9 7 3 ; Krieger et al., 1 9 8 3 ; D e Carli et al, 1989, 1 9 9 4 ; Dailey et al, 1 9 9 0 ; Potamianos et al, 1 9 9 2 ) , T. gallinae ( D e Carli et al, 1 9 9 6 a ) , T. foetus ( B u r g e s s etal, 1 9 9 0 ; D e Carli et al, 1 9 9 4 , 1 9 9 6 6 ) and
T. suis ( D e Carli et al, 1 9 9 4 ) has b e e n studied. For T.
vaginalis, Fiori et al, in 1 9 9 3 s h o w e d that hemolysis w a s a c o n t a c t and temperature d e p e n d e n t p h e n o - m e n o n and hypothetized that cytopathic effects could b e related to pore-forming in the m e m b r a n e o f red b l o o d cells. H o w e v e r the contact o f T. vaginalis and red b l o o d cells is not a prerequisite for hemolysis w h i c h c o u l d also b e due to a pH d e p e n d e n t lytic pro- tein s e c r e t e d b y t h e p a r a s i t e (Fiori et al, 1 9 9 6 ) . Although attempts to demonstrate a hemolytic activity in d i f f e r e n t s p e c i e s o f Trichomonas h a v e b e e n c o n d u c t e d , the hemolytic activity o f Monocercomonas spp. have n e v e r b e e n studied.
MATERIAL AND METHODS
* Faculdadc de Farmacia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga, 2752, Porto Alegre 90610-000 RS, Brazil.
** Laboratoire de Parasitologic, Centre Hospitalier Universitaire, Hôpital Charles Nicolle, 76931 Rouen, France.
Correspondence : Philippe Brasseur.
Tel. : 33 2 32 88 80 15 - Fax : 33 2 32 88 80 17.
ORGANISMS
h e Monocercomonas spp. isolate ( R 1 8 3 ) used in this study w a s isolated by Kulda in Cuba in J L 1 9 6 5 from Tropidophis melanurus ( s n a k e : Boidae) and kindly provided by Dr B e n c h i m o l ( C B B -
Parasite, 1998, 5, 79-82
Note de recherche 79
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1998051079
DA SILVA A.C.. DE CARLI G.A., BRASSEUR P., TASCA T., CASTILHOS D. & WENDORFF A.
UENF, C a m p o s , RJ, Brazil). T h e flagellates w e r e cul
tured axenically in a Trypticase-Yeast extract-Maltose ( T Y M ) medium ( D i a m o n d , 1 9 5 7 ) without agar, pH 7.0, s u p p l e m e n t e d with 10 % ( v / v ) heat inactivated c o l d horse serum, in air, at a temperature o f 2 8 °C. Isolates w e r e subcultured every 4 8 hours in T Y M medium.
M o n o c e r c o m o n a d s in the logarithmic p h a s e o f growth and subcultured every 4 8 hours e x h i b i t e d m o r e than 9 5 % o f mobility and a normal morphology. T h e pro
t o z o a n s w e r e c o u n t e d with a h e m o c y t o m e t e r and adjusted to a concentration o f 1 x 106 living organisms per ml in T Y M medium. Isolates w e r e stored in liquid nitrogen ( - 1 9 6 ° C ) with 5 % dimethyl sulfoxide ( D M S O ) .
ERYTHROCYTES
Fresh h u m a n b l o o d o f groups A, B , A B and O was o b t a i n e d at the City E m e r g e n c y Hospital ( H P S ) b l o o d c e n t e r o f Porto Alegre (Brazil) from volunteer donors in an equal v o l u m e o f Alsever's solution (dextrose 10.5 g, sodium citrate 8.0 g, citric acid 0.55 g, sodium chloride 4.2 g, distilled water to o n e liter), and from six different adult animal species: rabbit, rat, c h i c k e n , horse, ovine, s h e e p . All erythrocytes w e r e harvested and w a s h e d three times using centrifugation ( 2 5 0 x g for 10 m i n ) in an equal v o l u m e o f Hank's b a l a n c e d salt solution ( H B S S ) (Bio-Merieux, F r a n c e ) . T h e super- natants w e r e discarded. Each e x p e r i m e n t was per
formed using fresh erythrocytes from human b l o o d donors and adult animals. Erythrocytes w e r e stored in HBSS at 4 °C.
HEMOLYSIS ASSAY
Parasites w e r e harvested from a 24 hours culture (via
bility > 9 5 % ) in T Y M medium, in air, at 2 8 °C, and w a s h e d three times in HBSS b y centrifugation ( 7 5 0 x g for 2 0 m i n ) . A v o l u m e o f 5 0 ml o f w a s h e d fresh undi
luted erythrocytes was m i x e d with 2.5 ml o f HBSS containing a total n u m b e r o f 1 x 1 06 trophozoites o f Monocercomonas spp. ( K r i e g e r et al, 1 9 8 3 ) . After 18 hours o f incubation at 2 8 °C, in air, without sha
king, the mixture was centrifuged ( 2 5 0 x g for ten min).
A b s o r b a n c e o f the supernatants and controls w e r e
measured at 5 4 0 nm ( D e Carli et al., 1989) with a s p e c trometer and was c o m p a r e d with a standard curve o b t a i n e d by o s m o t i c lysis o f erythrocytes o f e a c h s p e cies. Control tubes without parasites w e r e included in all assays to m e a s u r e the s p o n t a n e o u s hemolysis. A kinetic study o f Monocercomonas spp. hemolysis w a s c o n d u c t e d in the standard conditions described a b o v e , with h u m a n group O erythrocytes. Hemolysis w a s evaluated hourly from 1 to 10 hours, and at 14 and 18 hours. This study w a s performed three times in tri
plicate. Results w e r e e x p r e s s e d as p e r c e n t a g e s o f total hemolysis ( 1 0 0 % ) . T h e m e a n and standard error o f the hemolytic activity o f m o n o c e r c o m o n a d s with the different erythrocytes w e r e calculated after performing the assay at least 12 times and e a c h sample w a s d o n e in triplicate.
CELL LYSATES
Parasites harvested in late e x p o n e n t i a l p h a s e w e r e w a s h e d three times in P B S pH 7.2. T h e s u s p e n s i o n adjusted at 1 x 1 06 was sonicated (five cycles o f ten s e c at 50 watts in ice b a t h ) , centrifuged and supernatants filtered through a 0,22 um filter (Millipore).
Statistical analysis was performed using the Student's t-test.
RESULTS
R
esults s h o w that Monocercomonas s p p . isolate tested had a hemolytic activity ranging b e t w e e n 3 3 and 71 % with human erytrocytes and betw e e n 17 to 5 6 % with animal erythrocytes ( T a b l e 1 ) . T h e hemolytic activities o b s e r v e d with h u m a n ery
throcytes o f group O o r group A B w e r e significantly higher than thoses o f group A o r group B ( p < 0 . 0 0 1 ) . A m o n g animal erythrocytes, the lowest activities w e r e o b s e r v e d with b o v i n e or s h e e p erythrocytes ( 1 9 and 17 % respectively). Neither adhesion nor agglutination w a s m i c r o s c o p i c a l l y o b s e r v e d b e t w e e n Monocerco
monas spp. and erythrocytes at any p h a s e o f h e m o lysis assay.
M o n o c e r c o m o n a d s isolated at the e n d o f hemolysis assays w e r e alive and w e r e successfully cultured in
Percentages of hemolysis*
No of assays Human erythrocytes groups Animal erythrocytes
12
A B AB ()
3 3 ± 2.3 3 6 ± 2.7 71 ± 1.9 6 3 ± 1.4
rabbit rat chicken horse 28 ± 0.3 5 6 ± 1.5 4 3 ± 2.9 3 2 ± 1.5
bovine sheep 19 ± 0.8 17 ± 0.1
* Mean values + one standard deviation of triplicate experiments
Table I. — Hemolytic activity of R183 strain of Monocercomonas spp. on human erythrocytes of groups A, B, AB, O and six adult animal erythrocytes.
80 Note de recherche P a r a s i t e , 1 9 9 8 , 5, 7 9 - 8 2
TYM medium. Parasite culture supernatants o f h e m o - lysis assays, tested in the p r e s e n c e o f different s p e c i e s of erythrocytes did n o t i n d u c e a n y hemolytic activity.
A kinetic study o f hemolysis with human group O ery- throcytes s h o w e d that hemolysis w a s over 3 0 % after o n e hour, and r e a c h e d a plateau ( 6 3 % ) after 18 hours (Fig.
1).
Parasite culture supernatants, extracts o f s o n i c a t e d parasites or heat killed organisms tested with human or animal erythrocytes did not exhibit hemolytic acti- vity (details n o t s h o w n ) .
Fig. 1. — Kinetic study of hemolytic activity of Monocercomonas spp. on human erythrocytes o f group O.
DISCUSSION
A
hemolytic activity has b e e n demonstrated in several protozoan parasites including Trypa- nosoma congolense (Tizard et al., 1 9 7 7 ) a n d T. brucei (Tizard, 1 9 7 8 ) , E. histolytica (Lopez-Revilla& Said-Fernandez, 1 9 8 0 ) , T. vaginalis (Grys & Hernik, 1 9 7 3 ; Krieger et al., 1 9 8 3 ; D e Carli et al, 1 9 8 9 , 1 9 9 4 ; Dailey etal, 1 9 9 0 ; Potamianos et al., 1 9 9 2 ) and T.gal- linae ( D e Carli et al., 1 9 9 6 a ) . T. foetus ( D e Carli et al., 1994, 1996fr), and 77. suis (De Carli etal., 1994) have n o hemolytic activity against human erythrocytes. T h e mechanism o f hemolysis is not yet well established and vary from o n e s p e c i e s to another. For 77. congolense, hemolysis has b e e n found related t o release o f fatty acids from e n d o g e n o u s phosphatidyl c h o l i n e b y a phosphalipase A (Tizard & Holmes, 1 9 7 6 ) . For T. vagi- nalis a n d T. gallinae n o activity c o u l d b e detected in culture supernatants, suggesting that hemolytic activity w a s n o t related to a hemolysin or soluble metabolites released b y t h e parasite ( D e Carli et al.,
1996a,
b). A relationship b e t w e e n adhesion a n d cytopathogenicity w a s d e m o n s t r a t e d for 77. vaginalis in cell cultures ( B r a s s e u r & Savel, 1 9 8 2 ) , and Fiori et al. in 1 9 9 3 , s h o w e d that T. vaginalis lysed human erythrocytes b ypore-forming in their m e m b r a n e . Proteins involved in c y t o a d h e r e n c e and pathogenesis o f T. vaginalis have b e e n identified (Fiori et al., 1 9 9 3 ; Alderete et al., 1995). Although adherence o f parasite on the target cell surface has b e e n c o n s i d e r e d for long as a prerequisite to cell damage, a n d particularly hemolysis o f erythro- cytes, Fiori et al. in 1996, s h o w e d that a contact inde- pendent hemolysis w a s mediated b y a protein o f m o r e than 3 0 k D a released b y T. vaginalis under triggering conditions. Such a cytopathic effect d u e to pore-for- ming protein has b e e n previously postulated for E. his- tolytica ( Y o u n g etal., 1 9 8 2 ; Ravdin, 1 9 8 6 ) . Although pore-forming activity w a s high in E. histolytica c o m - pared to n o n p a t h o g e n i c Entamoeba, it w a s found irrespective o f the virulence o f strains (Keller et al, 1 9 8 8 ) .
In o u r e x p e r i m e n t s , a d h e s i o n b e t w e e n Monocerco- monas spp. and erythrocytes w e r e not o b s e r v e d and contact independent hemolytic activity c o u l d not b e exhibited using supernatants o f hemolysis assays, cul- ture medium or sonicated parasite extracts. Although a h e m o l y t i c activity o f Monocercomonas s p p . w a s clearly demonstrated, a relationship b e t w e e n hemolysis and cell pathogenicity is not yet clearly established.
ACKNOWLEDGEMENTS
T
he authors thank Miss Luciane Hypolito, Miss Rosana M o n t e n e g r o and Mr G e r m a n o D e Carli for the technical assistance. This work w a s supported b y C o n s e l h o Nacional de D e s e n v o l v i m e n t o Cientifico e T e c n o l o g i c o (CNPq), Brazil, research grant:4 0 . 1 6 0 6 / 9 2 - 9 .
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Reçu le 10 juin 1997 Accepté le 14 septembre 1997
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DA SILVA A.C., D E CARLI G.A., BRASSEUR P., TASCA T., CASTILHOS D . & W E N D O R F F A.
