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Parasitol Latinoam 57: 5 - 8, 2002FLAP

ARTÍCULO ORIGINAL

Shape variations of Trichomonas vaginalis in presenceof different substrates

ABSTRACT

Shape variations of strains of Trichomonas vaginalis were demonstrated by scanning electronmicroscopy (SEM) following inoculation on McCoy cell monolayers and after interaction withhuman erythrocytes. The parasites adhered to both cells and presented amoeboid forms, withpseudopodia-like extensions. Several amoeboid organisms swam freely over the cell monolayersand produced aggregates. These unusual forms of the urogenital flagellate may be a response tothe composition of the culture medium and may play an important role in the pathogenic processof T. vaginalis.

Key words: Trichomonas vaginalis, shape variations, scanning electron microscopy.

TIANA TASCA*/** and GERALDO A. DE CARLI*/+

*Laboratório de Parasitologia Clínica, Faculdade de Farmácia, Pontifícia Universidade Católica do Rio Grande do Sul,Av. Ipiranga 6681, 900619-900 Porto Alegre, RS, Brasil.**Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul,Rua Ramiro Barcelos 2600 – Anexo, 90035-003 Porto Alegre, RS, Brasil.+Corresponding author. Fax: +55-51-3332.2582. E-mail: gdecarli@portoweb.com.br

INTRODUCTION

Trichomonas vaginalis is a very commoncause of infection of the female genito-urinarytract, and trichomonois is recognized as a majorsexually transmitted disease. Its clinicalpresentation ranges from a totally asymptomaticinfection to a severe vaginitis. Women requiredrug intervention to eliminate this parasite1,illustrating an adaptation to survival in theconstantly changing environment of the humanvagina.2 Adherence to host vaginal epithelial

cells is essential for initiation and maintenanceof infection by this mucosal parasite, and fourtrichomonad surface proteins that mediateparasite cytoadherence to host cells have beenidentified.3,4

The adherence is accompanied by shapevariations of the trophozoites. Abnormal formsof T. vaginalis, with variation in size and shape,have previously been reported.5-10 Giant formsmeasuring between 30 and 50 µm in diameterappeared in liquid medium 12 h after incu-bation.7 Previous studies with Trichomonas

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tenax also reported giant forms of a strainmaintained in culture for two years.11 Activelyswimming forms are ellipsoidal or ovoidal,sometimes spherical.12 All strains are able toform pseudopodia-like extensions, which areused in feeding, for attachment to stationaryobjects, but not for amoeboid movement. Inthe presence of higher concentrations of agar,of certain food particles, and of various substratelayers (cells or tissues in vivo and in vitro)trichomonads tend to be amoeboid.9 Here wereport amoeboid forms of T. vaginalis observedusing scanning electron microscopy (SEM) afterinoculation on McCoy cell monolayers andafter interaction with human erythrocytes.

MATERIALS AND METHODS

Parasites – Two strains of T. vaginalis wereused in this investigation. The VG strain wasisolated from a woman with symptomaticvaginitis attending at the Venereal DiseaseDepartment of Charles Nicolle Hospital, Rouen,France. The 30,238 T. vaginalis strain is metro-nidazole-resistant, from the American TypeCulture Collection (ATCC). The organismswere cultured axenically in vitro at 37 ºC inDiamond’s medium13 trypticase-yeast extract-maltose (TYM), without agar, pH 6.0, supple-mented with 10% heat inactivated bovineserum, penicillin (1000 UI ml-1) and strep-tomycin sulphate (1 mg ml-1). Isolates weresubcultured every 48 h in TYM medium.

McCoy cell tissue culture - McCoy celltissue was performed in a 24-well, flat bottom,cell tissue essential medium (MEM) (Eurobio,Paris) containing essential amino acid solution(10 µl ml-1) (Flobio, France), foetal calf serum(100 µl ml-1, Bio-Merieux, France) andnetilmicin (10 µl ml-1) (Netromicine-Unicet,France). The cells were incubated at 37 ºC inthe 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 inoculatedwith T. vaginalis at 2 x 106 organisms ml-1. 14,15

Human erythrocytes - Peripheral blood wasdrawn from healthy human volunteers of Ogroup, with equal volume of Alsever’s solution.The plasma was discarded by centrifugation

(250 x g for 5 min) and the erythrocytes werewashed three times in sterile PBS buffer. Thepellet containing the erythrocytes was subse-quently used to investigate the interaction withT. vaginalis using a erythrocytes: protozoa ratioof 100:1.

Scanning electron microscopy - The McCoycell tissue cultures inoculated with T. vaginaliswere fixed for SEM 6 h after infection. Themedium was carefully decanted and replacedwith 2.5% (v/v) glutaraldehyde in 0.1 Mcacodylate buffer, pH 7.2, at room temperaturefor 2 h 30 min. The cultures were then rinsed inPBS for 30 min, and then adhered to glasscoverslips previously coated with 0.1% poly-L-lysine (Sigma Chemical Company, St Louis),and post-fixed in 1% osmium tetroxide in 0.1M at room temperature for 2 h. Trichomonadswere maintained with erythrocytes during 30,60 and 90 min and the SEM fixation was thesame as performed for infected McCoy celltissue cultures. Fixed samples were dehydratedin ethanol and critically point dried with CO2.The coverslips were mounted on stubs andlightly coated with gold particles and examinedwith a Philips XL30 scanning electron micros-cope.

RESULTS AND DISCUSSION

The presence of a large number of adherentT. vaginalis in the McCoy cells presented anopportunity for a SEM study of morphologicaladaptations of trichomonads to the surface of acell. Trichomonads presented amoeboid formsand in some instances, several amoeboidorganisms were applied to one another by theirpseudopods, forming groups of organisms. Theparasites swam freely over the cell monolayersand formed aggregates consisting of numerouscells (“swarming”) (Figure 1).

Figure 2 shows the pseudopodia-likeextension appearing from the surface of theflagellate. The parasite typically applied itselfto the cells by its ventral surface, that is, thesurface opposite to that invested with theundulating membrane.

The amoeboid forms started to appear inthe first or second passage, and were then seenpersistently in the subsequent subcultures, but

Shape variations of Trichomonas vaginalis – T. Tasca and G. A. De Carli

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in variable number and always intermixed withpyriform trophozoites.

After 30 and 60 min of incubation of T.vaginalis and erythrocytes, SEM showedamoeboid forms of the parasite applied to theerythrocytes (Figures 3 and 4).

Some authors claim that the shape of theorganism varies according to the compositionof the growth medium. Studies carried out inseveral cell cultures to investigate the pathologicprocess caused by T. vaginalis show that themore virulent strains tend to settle on and adhereto the culture cells; the less inherently virulentstrains (perhaps as a result of prolongedcultivation) have less tendency for cytadhe-rence. Moreover, the typically amoeboid

virulent trichomonads apply themselves veryclosely to the cell culture elements.16

The urogenital trichomonads vary in sizeand shape. Physicochemical conditions (e.g.,pH, temperature, oxygen tension, and ionicstrength) affect the shape of trichomonads;however, shape tends to be more uniform amongcells grown in nonliving culture media thanamong those observed in vaginal secretionsand urine. In general, in axenic cultures grownin liquid media, for example, trypticase-yeastextract-maltose (TYM), with or without lowconcentrations of agar and without solid foodparticles, the organisms are usually ellipsoidal,ovoidal, or spheroidal.17

Mechanical action, or at least injury resulting

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 byTrichomonas vaginalis. Note the parasite attached to the McCoy cell by its ventral surface. (AF anterior flagella; MMcCoy cell; P pseudopodia-like extensions; UM undulating membrane). Figure 3. Amoeboid trophozoite of Trichomonasvaginalis applied to an erythrocyte after 30 min of interaction between parasites and erythrocytes. (E erythrocyte; Ttrichomonad). Figure 4. Amoeboid trophozoite of Trichomonas vaginalis after 60 min of interaction between parasitesand erythrocytes. (E erythrocyte; T trichomonad).

Shape variations of Trichomonas vaginalis – T. Tasca and G. A. De Carli

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from direct contact between the parasites andthe culture cells, plays a very important role inpathogenesis associated with urogenitaltrichomonois.16 The significance of these shapevariations is still to be assessed, and theinfluence of the constituents of the culturemedium on their development should also bestudied.

REFERENCES

1.- MÜLLER M, MEINGASSNER J G, MILLER MA, LEDGER W J. Three metronidazole-resistantstrains of Trichomonas vaginalis from the USA.Am J Obstet Gynecol 1980; 138: 808-12.

2.- ENGBRING J A, ALDERETE J F. Characterizationof Trichomonas vaginalis AP33 adhesin and cellsurface interactive domains. Microbiology 1998,144; 3011-8.

3.- ALDERETE J F, GARZA W E. Identification andproperties of Trichomonas vaginalis proteinsinvolved in cytoadherence. Infect Immun 1988; 56:28-33.

4.- ARROYO R, ENGBRING J, ALDERETE J F.Molecular basis of host cell epithelial cell recognitionby Trichomonas vaginalis. Mol Microbiol 1992; 6:853-62.

5.- POWELL W N. Trichomonas vaginalis Donné1836: its morphologic characteristics, mitosis andspecific identity. Am J Hyg 1936, 24: 145-169.

6.- TRUSSELL R E. Trichomonas vaginalis andtrichomonas flagellates of man. J Parasitol 1947;17: 117.

7.- WINSTON R M L. The relation between size andpathogenicity of Trichomonas vaginalis. J ObstetGynaecol Br Comm 1974; 81: 399-404.

8.- OVCINNIKOV N M, DELEKTORSKIJ V V,TURANOVA E N, YASHKOVA G N. Furtherstudies of Trichomonas vaginalis with transmissionand scanning electron microscopy. Brit J Vener Dis1975; 51: 357-75.

9.- JOHN J, SQUIRES S. Abnormal forms ofTrichomonas vaginalis. Brit J Vener Dis 1977; 54:84-7.

10.- HEATH J P. Behaviour and pathogenicity ofTrichomonas vaginalis in epithelial cell cultures – astudy by light and scanning electron microscopy. Br JVener Dis 1981; 57: 106-17.

11.- RIBAUX C L, JOFFRE A, MAGLOIRE H.Trichomonas tenax: ultrastructure of giant forms. JBiol Buccale 1988; 16: 19-23.

12.- WARTON A, HONIGBERG B M. Structure oftrichomonads as revealed by scanning electronmicroscopy. J Protozool 1979; 26: 56-62.

13.- DIAMOND L S. The stablishment of varioustrichomonads of animals and man in axenic cultures.J Parasitol 1957; 43: 488-90.

14.- BRASSEUR P, SAVEL J. Evaluation de la virulencedes souches de Trichomonas vaginalis par l’etude deléffet cytopathogéne sur culture de cellules. C R SocBiol 1982; 176: 819-60.

15.- ROUSSEL F, DE CARLI G, BRASSEUR P. Acytopathic effect of Trichomonas vaginalis probablymediated by a mannose/N-acetyl-glucosamine bindinglectin. Int J Parasitol 1991; 21: 941-4.

16.- HONIGBERG B M. Host Cell-Trichomonadinteraction and virulence assays in vitro systems. In:Trichomonads Parasitic in Humans. (Honigberg, B.M., ed). pp 155-212. Springer-Verlag, New York,1990.

17.- HONIGBERG B M, BRUGEROLLE G. Structure.In: Trichomonads Parasitic in Humans. (Honigberg,B. M., ed). Pp 5-35. Springer-Verlag, New York,1990.

Acknowledgements: To Prof. Sérgio De Meda Lamb forsupport, Prof. Philippe Brasseur, Iveli Rosset, and PaolaTessele. To Centro de Microscopia e Microanálises(CEMM – PUCRS) for technical assistance. This workwas partially supported by grants from Conselho Nacionalde Desenvolvimento Científico e Tecnológico (CNPq),Commission des Communautés Europénnes (CCE),Fundação de Amparo à Pesquisa do Estado do Rio Grandedo Sul (FAPERGS).

Shape variations of Trichomonas vaginalis – T. Tasca and G. A. De Carli