On-line version ISSN 0717-7712
Parasitol. latinoam. vol.57 no.1-2 Santiago Jan. 2002
Parasitol Latinoam 57: 5 - 8, 2002
Shape variations of Trichomonas vaginalis in presence
of different substrates
Shape variations of strains of Trichomonas vaginalis were demonstrated by scanning electron microscopy (SEM) following inoculation on McCoy cell monolayers and after interaction with human erythrocytes. The parasites adhered to both cells and presented amoeboid forms, with pseudopodia-like extensions. Several amoeboid organisms swam freely over the cell monolayers and produced aggregates. These unusual forms of the urogenital flagellate may be a response to the composition of the culture medium and may play an important role in the pathogenic process of T. vaginalis.
Key words: Trichomonas vaginalis, shape variations, scanning electron microscopy.
Trichomonas vaginalis is a very common cause of infection of the female genito-urinary tract, and trichomonois is recognized as a major sexually transmitted disease. Its clinical presentation ranges from a totally asymptomatic infection to a severe vaginitis. Women require drug intervention to eliminate this parasite1, illustrating an adaptation to survival in the constantly changing environment of the human vagina.2 Adherence to host vaginal epithelial cells is essential for initiation and maintenance of infection by this mucosal parasite, and four trichomonad surface proteins that mediate parasite cytoadherence to host cells have been identified.3,4
The adherence is accompanied by shape variations of the trophozoites. Abnormal forms of T. vaginalis, with variation in size and shape, have previously been reported.5-10 Giant forms measuring between 30 and 50 mm in diameter appeared in liquid medium 12 h after incubation.7 Previous studies with Trichomonas tenax also reported giant forms of a strain maintained in culture for two years.11 Actively swimming forms are ellipsoidal or ovoidal, sometimes spherical.12 All strains are able to form pseudopodia-like extensions, which are used in feeding, for attachment to stationary objects, but not for amoeboid movement. In the presence of higher concentrations of agar, of certain food particles, and of various substrate layers (cells or tissues in vivo and in vitro) trichomonads tend to be amoeboid.9 Here we report amoeboid forms of T. vaginalis observed using scanning electron microscopy (SEM) after inoculation on McCoy cell monolayers and after interaction with human erythrocytes.
MATERIALS AND METHODS
Parasites _ Two strains of T. vaginalis were used in this investigation. The VG strain was isolated from a woman with symptomatic vaginitis attending at the Venereal Disease Department of Charles Nicolle Hospital, Rouen, France. The 30,238 T. vaginalis strain is metronidazole-resistant, from the American Type Culture Collection (ATCC). The organisms were cultured axenically in vitro at 37 ºC in Diamond's medium13 trypticase-yeast extract-maltose (TYM), without agar, pH 6.0, supplemented with 10% heat inactivated bovine serum, penicillin (1000 UI ml-1) and streptomycin sulphate (1 mg ml-1). Isolates were subcultured every 48 h in TYM medium.
McCoy cell tissue culture - McCoy cell tissue was performed in a 24-well, flat bottom, cell tissue essential medium (MEM) (Eurobio, Paris) containing essential amino acid solution (10 ml ml-1) (Flobio, France), foetal calf serum (100 ml ml-1, Bio-Merieux, France) and netilmicin (10 ml ml-1) (Netromicine-Unicet, France). The cells were incubated at 37 ºC in the humid atmosphere of a CO2 incubator (5% CO2 in air). The cells were used at confluence, washed three times with the same medium. Each monolayer of McCoy cells was inoculated with T. vaginalis at 2 x 106 organisms ml-1. 14,15
Human erythrocytes - Peripheral blood was drawn from healthy human volunteers of O group, with equal volume of Alsever's solution. The plasma was discarded by centrifugation (250 x g for 5 min) and the erythrocytes were washed three times in sterile PBS buffer. The pellet containing the erythrocytes was subsequently used to investigate the interaction with T. vaginalis using a erythrocytes: protozoa ratio of 100:1.
Scanning electron microscopy - The McCoy cell tissue cultures inoculated with T. vaginalis were fixed for SEM 6 h after infection. The medium was carefully decanted and replaced with 2.5% (v/v) glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, at room temperature for 2 h 30 min. The cultures were then rinsed in PBS for 30 min, and then adhered to glass coverslips previously coated with 0.1% poly-L-lysine (Sigma Chemical Company, St Louis), and post-fixed in 1% osmium tetroxide in 0.1 M at room temperature for 2 h. Trichomonads were maintained with erythrocytes during 30, 60 and 90 min and the SEM fixation was the same as performed for infected McCoy cell tissue cultures. Fixed samples were dehydrated in ethanol and critically point dried with CO2. The coverslips were mounted on stubs and lightly coated with gold particles and examined with a Philips XL30 scanning electron microscope.
RESULTS AND DISCUSSION
The presence of a large number of adherent T. vaginalis in the McCoy cells presented an opportunity for a SEM study of morphological adaptations of trichomonads to the surface of a cell. Trichomonads presented amoeboid forms and in some instances, several amoeboid organisms were applied to one another by their pseudopods, forming groups of organisms. The parasites swam freely over the cell monolayers and formed aggregates consisting of numerous cells ("swarming") (Figure 1).
Figure 2 shows the pseudopodia-like extension appearing from the surface of the flagellate. The parasite typically applied itself to the cells by its ventral surface, that is, the surface opposite to that invested with the undulating membrane.
The amoeboid forms started to appear in the first or second passage, and were then seen persistently in the subsequent subcultures, but in variable number and always intermixed with pyriform trophozoites.
|Figure 1. Several amoeboid trophozoites of Trichomonas vaginalis are attached to one another by their pseudopods, forming a group of organisms. (M McCoy cell; T trichomonad). Figure 2. Pseudopodia-like extensions exhibited by Trichomonas vaginalis. Note the parasite attached to the McCoy cell by its ventral surface. (AF anterior flagella; M McCoy cell; P pseudopodia-like extensions; UM undulating membrane). Figure 3. Amoeboid trophozoite of Trichomonas vaginalis applied to an erythrocyte after 30 min of interaction between parasites and erythrocytes. (E erythrocyte; T trichomonad). Figure 4. Amoeboid trophozoite of Trichomonas vaginalis after 60 min of interaction between parasites and erythrocytes. (E erythrocyte; T trichomonad).|
After 30 and 60 min of incubation of T. vaginalis and erythrocytes, SEM showed amoeboid forms of the parasite applied to the erythrocytes (Figures 3 and 4).
Some authors claim that the shape of the organism varies according to the composition of the growth medium. Studies carried out in several cell cultures to investigate the pathologic process caused by T. vaginalis show that the more virulent strains tend to settle on and adhere to the culture cells; the less inherently virulent strains (perhaps as a result of prolonged cultivation) have less tendency for cytadherence. Moreover, the typically amoeboid virulent trichomonads apply themselves very closely to the cell culture elements.16
The urogenital trichomonads vary in size and shape. Physicochemical conditions (e.g., pH, temperature, oxygen tension, and ionic strength) affect the shape of trichomonads; however, shape tends to be more uniform among cells grown in nonliving culture media than among those observed in vaginal secretions and urine. In general, in axenic cultures grown in liquid media, for example, trypticase-yeast extract-maltose (TYM), with or without low concentrations of agar and without solid food particles, the organisms are usually ellipsoidal, ovoidal, or spheroidal.17
Mechanical action, or at least injury resulting from direct contact between the parasites and the culture cells, plays a very important role in pathogenesis associated with urogenital trichomonois.16 The significance of these shape variations is still to be assessed, and the influence of the constituents of the culture medium on their development should also be studied.
1.- MÜLLER M, MEINGASSNER J G, MILLER M A, LEDGER W J. Three metronidazole-resistant strains of Trichomonas vaginalis from the USA. Am J Obstet Gynecol 1980; 138: 808-12. [ Links ]
2.- ENGBRING J A, ALDERETE J F. Characterization of Trichomonas vaginalis AP33 adhesin and cell surface interactive domains. Microbiology 1998, 144; 3011-8. [ Links ]
3.- ALDERETE J F, GARZA W E. Identification and properties of Trichomonas vaginalis proteins involved in cytoadherence. Infect Immun 1988; 56: 28-33. [ Links ]
4.- ARROYO R, ENGBRING J, ALDERETE J F. Molecular basis of host cell epithelial cell recognition by Trichomonas vaginalis. Mol Microbiol 1992; 6: 853-62. [ Links ]
5.- POWELL W N. Trichomonas vaginalis Donné 1836: its morphologic characteristics, mitosis and specific identity. Am J Hyg 1936, 24: 145-169. [ Links ]
6.- TRUSSELL R E. Trichomonas vaginalis and trichomonas flagellates of man. J Parasitol 1947; 17: 117. [ Links ]
7.- WINSTON R M L. The relation between size and pathogenicity of Trichomonas vaginalis. J Obstet Gynaecol Br Comm 1974; 81: 399-404. [ Links ]
8.- OVCINNIKOV N M, DELEKTORSKIJ V V, TURANOVA E N, YASHKOVA G N. Further studies of Trichomonas vaginalis with transmission and scanning electron microscopy. Brit J Vener Dis 1975; 51: 357-75. [ Links ]
9.- JOHN J, SQUIRES S. Abnormal forms of Trichomonas vaginalis. Brit J Vener Dis 1977; 54: 84-7. [ Links ]
10.- HEATH J P. Behaviour and pathogenicity of Trichomonas vaginalis in epithelial cell cultures _ a study by light and scanning electron microscopy. Br J Vener Dis 1981; 57: 106-17. [ Links ]
11.- RIBAUX C L, JOFFRE A, MAGLOIRE H. Trichomonas tenax: ultrastructure of giant forms. J Biol Buccale 1988; 16: 19-23. [ Links ]
12.- WARTON A, HONIGBERG B M. Structure of trichomonads as revealed by scanning electron microscopy. J Protozool 1979; 26: 56-62. [ Links ]
13.- DIAMOND L S. The stablishment of various trichomonads of animals and man in axenic cultures. J Parasitol 1957; 43: 488-90. [ Links ]
14.- BRASSEUR P, SAVEL J. Evaluation de la virulence des souches de Trichomonas vaginalis par l'etude de léffet cytopathogéne sur culture de cellules. C R Soc Biol 1982; 176: 819-60. [ Links ]
15.- ROUSSEL F, DE CARLI G, BRASSEUR P. A cytopathic effect of Trichomonas vaginalis probably mediated by a mannose/N-acetyl-glucosamine binding lectin. Int J Parasitol 1991; 21: 941-4. [ Links ]
16.- HONIGBERG B M. Host Cell-Trichomonad interaction and virulence assays in vitro systems. In: Trichomonads Parasitic in Humans. (Honigberg, B. M., ed). pp 155-212. Springer-Verlag, New York, 1990. [ Links ]
17.- HONIGBERG B M, BRUGEROLLE G. Structure. In: Trichomonads Parasitic in Humans. (Honigberg, B. M., ed). Pp 5-35. Springer-Verlag, New York, 1990. [ Links ]
+Corresponding author. Fax: +55-51-3332.2582. E-mail: email@example.com
Acknowledgements: To Prof. Sérgio De Meda Lamb for support, Prof. Philippe Brasseur, Iveli Rosset, and Paola Tessele. To Centro de Microscopia e Microanálises (CEMM _ PUCRS) for technical assistance. This work was partially supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Commission des Communautés Europénnes (CCE), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS).