SciELO - Scientific Electronic Library Online

 
vol.23 número3-4The distribution of Ascaris lumbricoides infection in humans from a rural community in Venezuela índice de autoresíndice de assuntospesquisa de artigos
Home Pagelista alfabética de periódicos  

Serviços Personalizados

Journal

Artigo

Indicadores

Links relacionados

Compartilhar


Parasitología al día

versão impressa ISSN 0716-0720

Parasitol. día v.23 n.3-4 Santiago jul. 1999

http://dx.doi.org/10.4067/S0716-07201999000300001 

TRABAJOS DE INVESTIGACION

Hemolytic activity of fresh isolates and clones of
Trichomonas gallinae

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

ABSTRACT

In in vitro assay the hemolytic activity of live isolates and clones of Trichomonas gallinae was investigated. The fresh isolates and clones were 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 showed that each of the isolates and clones tested lysed all human blood groups, as well as rabbit, rat, chicken, horse, bovine and sheep erythrocytes. No hemolysin released by the parasites could be identified. Parasite culture supernatants of hemolysis assays, tested in the presence of human erythrocytes did not induce any hemolytic activity. Trichomonads culture supernatants from 18, 24, and 48 hr extracts of sonicated parasites or cold killed organisms tested with human erythrocytes did not exhibit hemolysis. Although an hemolytic activity of T. gallinae was clearly demonstrated, a relationship between hemolysis and cell pathogenicity is not yet clearly established.
Key words: Trichomonas gallinae, hemolytic activity, isolates, clones.

*Instituto de Biociências, Curso de Pós-Graduação em Biociências-Zoologia, Pontifícia Universidade Católica do Rio Grande do Sul (IBC-PUCRS), Av. Ipiranga 6681, Porto Alegre 90619-900 RS, Brazil.
**Departamento de Análises Clínicas, Faculdade de Farmácia, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Av. Ipiranga 6681, Porto Alegre 90619-900 RS, Brazil.
+ Corresponding author: Fax (051) 332-2582, E-mail: gdecarli@portoweb.com.br

INTRODUCTION

Trichomonas gallinae parasitizes a variety of birds all over the world. This trichomonad occur in the upper digestive tract and in various organs of different avian groups, especially columbiformes (doves and pigeons).1 The domestic pigeon, Columba livia, is the primary host of this flagellate protozoan. Other columbi-formes hosts have been found to arbor it, as ave also galliform birds, Java sparrows, various raptors, and sea gulls. Most T. gallinae strains are nonpathogenic or only moderately pathogenic for their avian hosts. There are however, many virulent strains.2 A survey of the literature reveals that the pathogenicity of isolates have been reported previously, using subcutaneous mouse assay 3, intraperitoneal inoculation,4, 5 and histopathologic studies of the lesions.6, 7

Cytopathogenicity induced in vitro by other protozoan parasites have also been demonstrated for Entamoeba histolytica, and Giardia lamblia.8-10

Using an in vitro method, the hemolytic activity of different species of Trichomonadida, such as Trichomonas vaginalis,11-15 T. gallinae,16 Tritrichomonas foetus,17-19 and Tritrichomonas suis19 has been studied. In this paper we describe the results of lysis of red blood cells by fresh isolates and clones of T. gallinae.

MATERIALS AND METHODS

Organisms - Two strains of T. gallinae (TG1, TG2) were isolated from the upper digestive tract of domestic pigeons, C. livia. The throats of pigeons were swabbed and the swabbings inoculated into trypticase-yeast extract-maltose (TYM) medium20 for incubation. Samples were cultured axenically in vitro, without antibiotics,2 in a TYM medium without agar, pH 7.2, supplemented with 10% (v/v) heat inactivated horse serum, in air, at a temperature of 37°C (± 0.5). Isolates were subcultured every 48 hr in TYM medium. Some cultures were established without the use of antibiotics, others were isolated with 5,000 units of penicillin and 1,000 µg of dihydrostrepto-mycin per ml of the original culture. In no instance were antibiotics used in the subsequent serial transfers. Certain samples of the original isolations were frozen and maintained at -196°C with 5% (v/v) of dimethyl sulfoxide (DMSO) as previously described 18. Isolates were subcultured every 48 hr in TYM medium. Trichomonads in the logarithmic phase of growth, and subcultured every 48 hr exhibited more than 95% of mobility and a normal morphology. The protozoa were counted with a hemocytometer and adjusted to a concentration of 1 x 106 living organisms per ml in TYM medium.

Isolation of clones - The solid medium for the isolation of clones (C1, C2) of T. gallinae was a modification of TYM medium.20 Agar culture medium was prepared with 20 ml of the TYM warm, serum-free medium, containning 1.6% (w/v) of agar (Difco) and poured into a sterile 100 mm plastic petri dish. Each plate was then placed into airtight boxes containing a 5 g piece of solid CO2 and left for at least 15 min to harden and absorb the CO2. For the overlay 10 ml of the TYM containing 0.8% (w/v) of agar was melt and hold in a water bath at 40°C. It was quickly added 1 ml heat inactivated horse serum. The medium was cooled to 37°C, inoculate with 0.5 ml of T. gallinae suspension (adjusted at 1 x 104 trophozoites per ml), and immediately poured over the base layer. The dishes were set in the CO2 chamber to harden and absorb gas. As soon as the plates were hardened, they were transferred to a Anaerobac anaerobic jar (Probac) in which a 2 to 3 g piece of solid CO2 had been allowed to evaporate. Three hydrogen-generating envelopes (Anaerobac) were placed inside the jar. The jar was then quickly sealed and incubated at 37°C for 3 to 5 days.21 The clones were stored in liquid nitrogen (-196°C) with 5% of dimethyl sulfoxide (DMSO).18

Erythrocytes - Fresh human blood of groups A, B, AB, and O was obtained at the City Emergency Hospital (HPS) blood center from volunteer donors. The blood was taken in an equal volume of Alsever's solution (dextrose 20.5 g, sodium citrate 8 g, citric acid 0.55 g, sodium chloride 4.2 g, distilled water to 1 liter), and from six different adults animal species: rabbit, rat, chicken, horse, bovine, and sheep. All the erythrocytes were harvested, and washed three times by centrifugation (250 x g/10 min) in equal volume of TYM medium. The supernatants were discarded. Each experiment was performed using fresh erythrocytes from all human blood groups and adults animals. Whole human blood samples were previously examined and determined to be hepatitis B antigen (HBsAg) negative, and human immunodeficient virus (HIV-antibody) negative. The erythrocytes were stored in TYM medium at 4°C.

Hemolysis assay - The trophozoites were harvested from a 24 hr culture (viability > 95%) in TYM medium, without antibiotics, in air, at 37°C and washed three times in TYM medium by centrifugation (750 x g/20 min). A volume of 50 µl of washed fresh undiluted erythrocytes was mixed with 2.5 ml of Hank`s ba-lanced salt solution (HBSS) or with 2.5 ml of TYM medium containing a total of 1 x 106 trophozoites of T. gallinae originated from a 24 hr culture in TYM medium. After 18 hr of incubation at 37°C, in air, without shaking, the mixture was centrifuged (250 x g/10 min). Absorbance of the supernatants and controls were measured at 540 nm (A540) 12 with spectrometer, and was compared with a standard curve obtained by osmotic lysis of the erythrocytes of each species. Control tubes without parasites were included in all assays to measure the spontaneous hemolysis. This study was performed three times in triplicate. Results were expressed as percentage of total hemolysis (100%). The mean and the standard error of the hemolytic activity of trichomonads with the different erythrocytes were calculated after performing the assay at least 12 times, and each sample was done in triplicate.

Cell lysates - Parasites harvested in late exponential phase were washed three times in PBS pH 7.2. The suspension adjusted at 1 x 106 per ml in PBS was sonicated (5 cycles of 10 sec at 50 watts in ice bath), centrifuged and supernatants filtered through a 0.22 µm filter membrane (Millipore).

Statistical analysis: it was performed using the Student's t-test.

RESULTS

As shown in Table 1 the fresh isolates and clones hemolyzed all human blood groups, as well as rabbit, rat, chicken, horse, bovine and sheep (Table 2). The degree of hemolysis varied greatly from one isolate or clone to another, and varies according to donors origin of erythrocytes. Hemolytic activity was maintained after a serial transfer in axenic culture, without antibiotics for six months.

 

Table 1. Hemolytic activity of two isolates and two clones of Trichomonas gallinae on
human erythrocytes groups
                   
   
Percentages of hemolysisa
   
Human erythrocytes groups

Organisms No. of assays
A
B
AB
O

TG1*

12 92 ± 1.5 96 ± 1.7 92 ± 1.7 91 ± 1.4

TG2

12 92 ± 0.9 97 ± 1.7 96 ± 0.8 97 ± 0.8

C1**

12 46 ± 2.3 55 ± 5.6 58 ± 3.8 41 ± 1.7
C2 12 65 ± 4.5 92 ± 0.2 68 ± 2.1 82 ± 1.8
                   
a Mean values ± one standard error of triplicate samples. * TG1, TG2 - strains of T. gallinae; ** C1, C2 - clones of T. gallinae.

 

Table 2. Hemolytic activity of two isolates and two clones of Trichomonas gallinae on six adult
animals erythrocytes

   
Percentages of hemolysis a
   
   
Animal erythrocytes
   
Organisms N° of assays
rabbit
rat
chicken
horse
bovine
sheep

TG1* 12 88 ± 3.1 72 ± 1.3 91 ± 1.5 71 ± 2.0 91 ± 2.2 79 ± 2.7
TG2 12 94 ± 0.9 81 ± 1.0 91 ± 1.6 91 ± 1.0 91 ± 1.0 72 ± 2.3
C1** 12 34 ± 1.2 82 ± 3.3 80 ± 2.0 94 ± 1.9 84 ± 7.1 82 ± 1.5
C2 12 40 ± 1.2 72 ± 1.5 72 ± 1.5 92 ± 2.3 88 ± 2.4 66 ± 1.3
 
a Mean values ± one standard error of triplicate samples. * TG1, TG2 - strains of T. gallinae;** C1, C2 - clones of T. gallinae.

Results show that T. gallinae isolates tested had a hemolytic activity ranging between 91% and 97% with human erythrocytes (Table 1), and ranging between 71% and 94% with animal erythrocytes (Table 2). Experiments with T. gallinae clones presented a hemolysis from 41% and 92% with human erythrocytes (Table 1), and from 34% and 94% with animal erythrocytes (Table 2).

In the course of all experiments neither adhesion or agglutination was observed between T. gallinae and erythrocytes at any phase of hemolysis assay using light microscopy. T. gallinae isolates and clones at the end of hemolysis assays were alive and were successfully cultured in TYM medium.

Parasite culture supernatants of hemolysis assays, tested in the presence of human erythrocytes did not induce any hemolytic activity. It was demonstrated in these experiments that the presence of antibiotics (penicillin and streptomycin or gentamycin) in TYM medium results in a rapid decrease of hemolytic activity of isolates and clones. T. gallinae TG2 lysed 2% of the human group O erythrocytes after incubation for one hour, and 97% after 18 hr. However, the same isolate, subcultured every 2 days in TYM medium supplemented with antibiotics for three nonths lysed 2% of erythrocytes in one hour, and reached a plateau (14%) after 18 hr under the same conditions.

Trichomonads culture supernatants from 18, 24, and 48 hr extracts of sonicated parasites or cold killed organisms tested with human erythrocytes did not exhibit hemolysis.

DISCUSSION

The data obtained from all the experiments demonstrate a hemolytic activity of T. gallinae in vitro cultivation. In this work, the fresh isolates and clones did not show the same degree of hemolysis.

This suggests that the lysis of red blood cells varies according to donors origin of erythrocytes. Nevertheless, our results are not in opposition with the finding 2 that the presence of antibiotics in the culture medium affects the pathogenicity levels of T. gallinae isolates. It was demonstrated in this study that the hemolytic activity of T. gallinae decrease when the isolates were grown and subcultured every two days in TYM medium supplemented with antibiotics.

A hemolytic activity has been demonstrated in several protozoan parasites including Trypanosoma congolense,22 and T. brucei,23 E. Histolytica,8 T. vaginalis,11-15 and T. gallinae.16 However, all the species of trichomonads are not similar since T. foetus,19 and T. suis19 have no hemolytic activity against human blood groups. The mechanism of hemolysis is not yet well established, and might be different for each species. For T. congolense, hemolysis has been found related to fatty acids released from endogenous phosphatidyl choline by a phosphalipase A.24 For T. gallinae no activity could be detected in culture supernatants suggesting that hemolytic activity was not related to an hemolysin or soluble metabolites released by the parasite.

A relationship between adhesion and cytopathogenicity was demonstrated for T. vaginalis in cell culture,25 and Fiori et al.26 showed that T. vaginalis lysed human erythrocytes by poreforming in their membrane. Although adherence of parasite on the target cell surface has been considered for long time as a prerequisite to cell damage, and particularly hemolysis of erythrocytes. Fiori et al.,27 showed that a contact independent hemolysis was mediated by a proteins of more than 30 kDa released by T. vaginalis under triggerig conditions.

In our experiments, neither adhesion or agglutination was observed by light microscopy between T. gallinae and erythrocytes at any phase of hemolysis assay, and contact independent hemolytic activity could not be exhi-bited using supernatant of hemolysis assays, culture medium or sonicated parasite extracts (details not shown).

Although an hemolytic activity of T. gallinae was clearly demonstrated, a relationship between hemolysis and cell pathogenicity is not yet clearly established.

Acknowledgements: To the Pharmacy´s Students Miss. Karine Rigon Zimmer (BPA-PUCRS) and Daniela Meneghini (FAPERGS).

REFERENCES

1.- STABLER R M. Trichomonas gallinae: a review. Exp Parasitol 3: 368-402, 1954.         [ Links ]

2.- STABLER R M, HONIGBERG B M, KING V M. Effect of certain laboratory procedures on virulence of the Jone`s strain of Trichomonas gallinae for pigeons. J Parasitol 50: 36-41, 1964.         [ Links ]

3.- HONIGBERG B M. Comparative pathogenicity of Trichomonas vaginalis and Trichomonas gallinae to mice. I. Gross pathology, quantitative evaluation of virulence and same factors affecting pathogeni-city. J Parasitol 47: 545-571, 1961.         [ Links ]

4.- BOS A. Ueber trichomoniasis bei tauben. IV. Mitteilung. pathogenität von Trichomonas columbae für Mäuse. Zentralbl Bakteriol Parasitenkd InfecionskrHyg (A) 132: 453-458, 1934.         [ Links ]

5.- HONIGBERG B M, STABLER R M, LIVINGSTON M C, KULDA J. Further observations on the effects of various laboratory procedures on the virulence of Trichomonas gallinae for pigeons. J Parasitol 56: 701-708, 1970.         [ Links ]

6.- FROST J, HONIGBERG B M. Comparative pathogenicity of Trichomonas vaginalis and Trichomonas gallinae for mice. II. Histopathology and subcutaneous lesions. J Parasitol 48: 898-918, 1962.         [ Links ]

7.- PEREZ-MESA C R, STABLER M, BERTHRONG M. Histopathological changes in the domestic pigeon infected with Trichomonas gallinae (Jones`s Barn strain). Avian Dis 5: 48-60, 1961.         [ Links ]

8.- LOPEZ-REVILLA R, SAID-FERNANDEZ S. Cytopathogenicity of Entamoeba histolytica hemolytic activity of trophozoite homogenates. Am J Trop Med Hyg 29: 200-212, 1980.         [ Links ]

9.- ORTEGA Y R, GILMAN R H, MIRANDA E et al. Cytopathic effects induced in vitro by Trichomonas vaginalis: A comparison with Entamoeba histolytica and Giardia lamblia. Abstracts of 36th Annual Meeting of American Society for Tropical Medicine and Hygiene, Los Angeles, Laurence, Kansas. p 117, 1987.         [ Links ]

10.- RAVDIN J I, GUERRANT R L. Role of cytoadherence in cytopathogenic mechanisms of Entamoeba histolytica: Study with mammalian tissue culture and human erythrocytes. J Clin Invest 68: 1305-1313, 1981.         [ Links ]

11.- DAILEY D C, CHANG T, ALDERETE J F. Characterization of Trichomonas vaginalis haemolysis. Parasitology 101: 171-175,1990.         [ Links ]

12.- DE CARLI G A, BRASSEUR P, SAVEL J. Activité hemolitique de differentes souches et clones de T. vaginalis. Bull Soc Fr Parasitol 7: 13-16, 1989.         [ Links ]

13.- GRYS E, HERNIK A. Hemolysis of human and rabbit erythrocytes by T. vaginalis. Wiad Parazytol 19: 399-400, 1973.         [ Links ]

14.- KRIEGER J N, POISSON M A, REIN M F. Beta-hemolytic activity of Trichomonas vaginalis correlates with virulence. Infect Immun 41: 1291-1295, 1983.         [ Links ]

15.- POTAMIANOS S, MASON P R, READ JS, CHIKUNGAUWO S. Lysis of erythrocytes by Trichomonas vaginalis. Biosc Rep 12: 387-385, 1992.         [ Links ]

16.- DE CARLI G A, DA SILVA A C, WENDORFF A, ROTT M. Lysis of erythrocytes by Trichomonas gallinae. Avian Dis 40: 228-230, 1996.         [ Links ]

17.- BURGESS D E, KNOBLOCK K F, DAUGHERTY T, ROBERTOSON NP Cytotoxic and hemolytic effects of Tritrichomonas foetus on mammalian cells. Infec Immun 58: 3627-3632, 1990.         [ Links ]

18.- DE CARLI GA, BRASSEUR P, DA SILVA A C, et al . Hemolytic activity of Trichomonas vaginalis and Tritrichomonas foetus. Mem Inst Osw. Cruz 97: 107- 110, 1996.         [ Links ]

19.- DE CARLI G, BRASSEUR P, ROTT M et al. Determination of hemolytic activity of different strains of trichomonads of genus Trichomonas Donné, 1836 and Tritrichomonas Kofoid, 1920. J Protozool Res 4: 58-163, 1994.         [ Links ]

20.- DIAMOND L S. The establishment of various Trichomonas of animals and man in axenic cultures. J Parasitol 43: 488-490, 1957.         [ Links ]

21.- LINSTEAD D. Cultivation of trichomonads parasitic in humans. p. 91-111. In B M Honigberg. Trichomonads parasitic in humans, Springer-Verlag, New York, 1989.         [ Links ]

22.- TIZARD I R, HOLMES W L, YORK D A, MELLORS A. The generation and identification of the hemolysin of Trypanosoma congolense. Experientia 33: 901-902, 1977.         [ Links ]

23.- TIZARD I R, SHEPPARD J, NIELSEN K. The characterization of a second class of hemolysins from Trypanosoma brucei. Trans R Soc Trop Med Hyg 72: 198-200, 1978.         [ Links ]

24.- TIZARD I R, HOLMES W L. The generation of toxic activity from Trypanosoma congolense. Experientia 32: 1533-1534, 1976.         [ Links ]

25.- BRASSEUR P, SAVEL J. Evaluation de la virulance des souches de Trichomonas vaginalis par l`étude de l`effet cytopathogène sur culture de cellules. C R Soc Biol 176: 849-860, 1982.         [ Links ]

26.- FIORI P L, RAPPELLI P, ROCCHIGIANI A M, CAPPUCCINELLI P. Trichomonas vaginalis haemolysis: Evidence of functional pores formation on red cell membranes. FEMS Microbiol Lett 109: 13-18, 1993.         [ Links ]

27.- FIORI P L, RAPPELLI P, ADDIS M F et al. Trichomonas vaginalis haemolysis: pH regulates a contact-independent mechanism based on pore-forming proteins. Microb Pathog 20: 109-118, 1996         [ Links ]

 

 

Creative Commons License Todo o conteúdo deste periódico, exceto onde está identificado, está licenciado sob uma Licença Creative Commons