Parasitol Res (1991) 77:369-373 004432559100053E

Parasitelngy Research 9 Springer-Verlag1991

Original investigations

Studies on the lethal effect of ultraviolet light on Trichomonas vaginMis *' * * P. Karanis 1 W. Maier 1, D. Schoenen 2 and H.M. Seitz 1 1 Institut ffir Medizinische Parasitologie and 2 Hygiene-Institut der Universit/it Bonn, W-5300 Bonn, Federal Republic of Germany Accepted January 3, 1991 Abstract. Following cultivation in Asami medium, centrifugation and resuspension in saline or in water from a medicinal spring, Trichomonas vaginalis trophozoites were exposed to well-defined doses of ultraviolet (UV) light (254 nm). We used 24- and 48-h-old trichomonads at concentrations of I x 105 and 5 x 104 trophozoites/ml in a total volume of 20 ml for these studies. The apparatus for UV irradiation was especially constructed for batch experiments. After irradiation at doses ranging from 80 to 160 mJ/cm 2, the mobility of the parasites was reduced and morphological alterations appeared: rounding of the cells, vacuolization of the cytoplasm and even cytolysis. A dose of 401.7 mJ/cm 2 killed 99.8% of the 48-h-old trichomonads when irradiation occurred in saline at a cell density of 1 x 105 trichomonads/ml and 98.9% when irradiation was done at a cell density of 5 x 104 trichomonads/ml. A dose of 362.1 mJ/cm a killed only the more sensitive 24-h-old trichomonads. In mineral water, 24l mJ/cm 2 was sufficient to kill up to 99.5% of the 48-h-old trichomonads. When 48-h-old trichomonads that had been exposed to a radiation dose of 160-240 mJ/cm 2 were subcultured, they lost their ability to propagate. At a dose of 80 mJ/cm z, both the trichomonads that had been harvested during the log phase and the 48-h-old organisms suspended in mineral water lost their ability to propagate on subculture. These results indicate that 24-h-old trichomonads were more sensitive than 48-h-old organisms. Furthermore, the experiments demonstrated that a higher dose of UV radiation must be applied to T. vaginalis trophozoites than to the more sensitive bacterial strain Escherichia coli ATCC 11229 so as to achieve comparable killing results. At present drinking water is disinfected chemically, mainly by the use of chlorine or chlorine dioxide. Since * Dedicated to Prof. Dr. J. Eckert on the occasion of his 60th birthday ** Supported by the Bundesminister ffir Forschung und Technologie (02-wt 8720) Initiator: Prof. Dr. G.O. Schenck, Koordinator: Prof. Dr. H. Bernhardt Offprint requests to: P. Karanis

trihalomethanes are generated by chlorine disinfection (Rook 1974), alternatives must be found. One possibility for future disinfection of drinking water could be ultraviolet (UV) irradiation. The efficiency of UV light in making drinking water germ-free is currently being examined by studying its effects on bacteria (Zemke and Schoenen 1989; Zemke et al. 1990) and parasites. However, there is significantly more knowledge about the effects of UV rays on bacteria than about their influence on parasites, as documented extensively in the literature (Kiefer and Wienhard 1977; Harm 1980; Chang et al. 1985). Very little has been published on the effect of UV light on parasites. Data from previous investigations (Chamberlain and Vedder 1916; Stoll et al. 1945; Standen and Fuller 1959) are not reliable, as no exact irradiation doses were given. The available knowledge is restricted to data on the distance between the UV source and the sample vessel or on the duration of irradiation. More recent investigations dealing with UV light and cyst-producing parasites such as Acanthamoeba spp. (Chang et al. 1985) or Giardia lambIia (the agent of one of the most frequent water-borne diseases in the United States and Canada, Rice and Hoff 1981 ; Carlson et al. 1985) have been carried out exclusively in North America. The experiments described in the literature demonstrate that parasites are killed by UV rays but require a higher radiation dose than do bacteria. Herein we describe an examination of the effects of UV light on Trichomonas vaginalis, a flagellate that lacks the ability to produce cysts. It grows rapidly in simple Asami medium. T. vaginalis is an ubiquitous pathogen infecting the mucous membranes of the genitals of men and women and is sexually transmitted. Acquisition of infection from swimming-pool water is unlikely if the water is chlorinated (Piekarski and Saathoff 1973). In water with average chloride levels, such as tap or bath water (chlorine content, 0.44-0.56 mg/ml), few trichomonads can survive for periods ranging from 5 rain to 8 h (Piekarski et al. 1973). Survival is dependent on the density of trichomonads in the suspension. In the high mineral concentrations of medicinal spring water they can survive for up to 5 h (Piekarski and Saathoff 1973;

370

P. Karanis et al. : Studies on the lethal effect of ultraviolet light on Trichomonas vaginalis

L i n d n e r 1975). I n fact, we f o u n d t h a t in water f r o m a special m e d i c i n a l spring chosen for its high m i n e r a l c o n t e n t , t r i c h o m o n a d s survived for a m i n i m u m o f 3 h. Localities with m e d i c i n a l springs serve as health resorts. T h e infection o f p e r s o n s u s i n g the pools at such resorts c a n n o t be completely excluded (Piekarski a n d S a a t h o f f 1973).

Materials and methods

Parasites The trichomonads (TV strain of the Institut fiir Medizinische Parasitologie, Bonn) were cultured at 37~ C in the medium created by Johnson et al. in 1945 and modified by Asami in 1952 (Saathoff 1982), with minor modifications (tissue-culture tubes, Greiner). For the production of 48-h-old trichomonads, one to three drops of the parent culture were inoculated into 5-ml Asami medium and incubated for 48 h. Following this incubation period, all trichomonads were viable and fully grown. A growth curve was established for the production of log-phase trichomonads. A total of 5 x 10s stock-culture trichomonads were innoculated into 5 ml medium and incubated for 24 h, which corresponded to the late log phase.

Methods Preparation of the samples for irradiation. At 24 and 48 h, respectively, the cultures were centrifuged (5 min, 1000 g, 4~ C) and the pellets were resuspended in sterile physiological saline, this procedure was repeated twice. The number of cells per volume was then determined by means of a Neubauer haemocytometer.

Table 1. Results of the chemical analysis of medicinal spring water pH 6.3 Nitrate (NO~) Nitrite (NO~) Ammonium (NH2) Phosphate (PO]-) Chloride (C1-)

3 rag/1 20.002 rag/1 20.03 mg/l 0.3 rag/1 1340 rag/1

Sulphate (SO4z-) Calcium ions (Ca 2+) Magnesium ions (Mg 2+) Ferric ions (Fe z +) Sodium (Na +)

362.2 17.9 69.4 12.8 1600

Potassium 88 Fluoride 0.48 Carbonate hardness 87.3~ Total hardness 87.3~ Non-carbonate hardness 0~ (M-) alkalinity 63 Oxidizability - KMnO 2 consumption 8 Electrical conductivity 6260

rag/1 rag/1 mg/l rag/1 mg/t rag/1 mg/l D D D 1N HC1/1 rag/1 IS

The mineral water was taken directly from the spring and used for the experiments because that is the way it is used in the pool of the health resort; the water is not chlorinated before being used by the patients trol and experimental groups were expressed as a percentage of the total number of trichomonads (1 x 106 or 2 x 106 trichomonads in 20 ml, as relevant) present in the irradiated suspension or in non-irradiated controls. This total number was expressed as 100%.

Irradiation apparatus andprocedure. For irradiation, a special apparatus for batch experiments was used. Physiological saline (0.9% NaC1) or water obtained from a medicinal spring in Bad Honnef, a health resort near Bonn, were used as suspensions for irradiation. Parasites were suspended at cell densities of 5 x 104 and 1 x 105 cells/ml water in a total volume of 20 ml. The distance between the UV lamp and the sample vessel was 13 cm in all experiments. The irradiation density of the lamp (mercury low-pressure gasdischarge lamp, Stefisol NN 30/89, Heraeus) was determined by means of a potassium ferrioxalate actinometer according to the method of Calvert and Pitts (1966) as described by Zemke and Schoenen (1989). Irradiation was determined for a constant distance of 11,5 cm and a cuvette filling volume of 20 ml (quartz glass; inner sizes: height, 14.9 cm; width, 2.9 cm; depth, 0.5 cm). The irradiation dose D (mJ/cm2) is equal to the irradiation density E (mJ s-1 cm 2) multiplied by the length of time t (s) for which the radiation is delivered. There was no danger of the individual sample's becoming overheated during the irradiation process, as the temperature never exceeded 28~ C.

Subcultures after irradiation. Subcultures were established from every irradiated cell sampte (0.1 ml each of irradiated suspension and non-irradiated controls in 2 ml Asami medium) and incubated at 37~ C for 24 h. Cultures were harvested and centrifuged, following which the supernatant was examined macroscopically and the sediment microscopically for living trichomonads. Chemical analysis of medicinal spring water. The medicinal spring water was analysed by the Department of Water Examination of the Institute for Hygiene, Bonn, with regard to its mineral content. The water contained a high concentration of chloride and sodium ions and other elements (Table 1). For irradiation, water samples were taken directly from the spring. The water naturally flows directly and unaltered from the spring to the swimming pool of the health resort, where it is used by visitors to the spa.

Results

Evaluation of results

Morphological alterations immediately after U V irradiation

Procedure after irradiation. Following irradiation, the trichomonad samples were centrifuged and 19 ml supernatant was discarded. The pellet was resuspended in the remaining 1 ml and the numbers of living and dead cells were determined by enumeration in a Neubauer haemocytometer. Viability was established on the basis of the degree of mobility of the parasites: trichomonads capable of moving with their flagella were regarded as being alive. The percentage of the surviving trichomonads was plotted logarithmically against the dose of UV radiation. Surviving trichomonads in con-

I n a d d i t i o n to killing the t r i c h o m o n a d s , U V r a d i a t i o n also caused a b n o r m a l i t i e s in the parasites: r o u n d i n g o f the cells, v a c u o l i z a t i o n o f the c y t o p l a s m and, finally, cytolysis. T h e n u m b e r o f cells u n d e r g o i n g lysis increased with a s c e n d i n g i r r a d i a t i o n dose. This f r a c t i o n of cells was s u m m a r i z e d as losses c o n d i t i o n e d b y the m e t h o d used. T h e results o f the q u a n t i t a t i v e e v a l u a t i o n are s h o w n in Figs. 1-4.

P. Karanis et al. : Studies on the lethal effect of ultraviolet light on Trichomonas vaginalis 100

Survivat (%]

100

371

Survival (%~

---._...

10

10

Or1 0

i

i

80

160

i

240 Dose [mJ/cm 2]

Op~

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320

400

0

I

I

I

I

I

80

160

240

320

400

~__

480

Dose [mJ/cm2J

Fig. 1. Survival of Trichomonas vaginalis (48 h old) after UV irradiation in saline, I x 105 trichomonads/ml (means of 20 trials)

Fig. 3. Survival of Trichomonas vaginalis (48 tl old) after UV irradiation in water from a medicinal spring, 5 x 104 trichomonads/ml

(means of 10 trials)

100

Survival (%)

of 5 x 104 cells/ml, 3.6% survived, and no living trichomonads could be found in the suspension afterwards (Fig. 2). At a density of 5 x 104 cells/ml, trichomonads irradiated in mineral water could not tolerate radiation doses as high as those delivered to parasites in saline. After exposure to a dose of < 240 mJ/cm 2, survival of 0.5% of the parasites was determined (Fig. 3), which is equivalent to a killing rate of 99.5% at this radiation dose. The number of dead trichomonads (registered directly after irradiation) is not shown.

10

Secondary cultures

0,1

I

~

I

I

I

80

160

240

320

400

Dose [mJ/cm21

Fig. 2. Survival of Trichomonas vaginalis (a, 48 h old; b, 24 h old) after UV irradiation in saline, 5 x 104 trichomonads/ml (means of 15 and 10 trials, respectively)

It appears that at a cell density of 1 x 105 cells/ml in sterile physiological saline, only 0.2% o f the trichomonads survived (Fig. 1) following irradiation with a dose of 401,7 mJ/cm 2. At a density o f 5 x 104 cells/mI in sterile saline after the same irradiation dose, 1,1% of the trichomonads remained alive (Fig. 2). When trichomonads derived from the log-growth phase were irradiated with a dose of 289,7 mJ/cm 2 at a cell density

Survival was ascertained by quantitative evaluation (Figs. 1-4). At 24 h after subculture in Asami medium, trichomonads that had been exposed to a radiation dose of 240 mJ/cm 2 in saline had produced no progeny. On subculture, propagation was also inhibited in parasites that had been derived from the log phase and irradiated in saline, as well as in those that had been irradiated in water from a medicinal spring, at a dose of 80 mJ/cm 2.

Chemical analysis of medicinal spring water In addition to other ions, the medicinal spring water used in these studies contained a high concentration of Na + and CI- ions (Table 1). Discussion

Trichomonas vaginalis is a suitable model organism on which to base a test system for our assays, since cultivation of the trophozoite stage of flagellate is easy. The

P. Karanis et al. : Studieson the lethal effectof ultravioletlight on Trichornonasvaginalis

372 Survival (%1 100

80

60

40

20

0

'

Controls

Fig. 4. Survival of trichomonadsin non-irradiatedcontrols. A (cf. Fig. 1), B (cf. Fig. 2, a) and D (cf. Fig. 3), 48-h-old trichomonads; C (cf. Fig. 2, b) 24-h-old trichomonads

ability of the parasite to survive in tap water, sterile saline and mineral water has previously been determined (unpublished observations). In tap water, as expected, T. vaginalis died rapidly as a result of cytolysis. In sterile saline, viability was sustained for a minimum of 6 h, whereas in mineral water the parasites survived for a minimum of 3 h. The high concentrations of chloride and sodium ions appeared to play a significant role in the prolonged survival of trichomonads in mineral water. Further studies were therefore conducted using sterile saline and mineral water. It was established that UV doses of 400 and 240 mJ/cm 2, respectively, are needed to kill trichomonads. On examination of the multiplication rate in vitro, it became evident that the ability to propagate was abolished in 48-h-old organisms at 240 mJ/cm 2 and in 24-hold parasites at 80 mJ/cm 2 following irradiation in mineral water. T. vaginalis tolerates higher doses of UV irradiation in saline than in mineral water. It may be that oxidative products built up after irradiation of mineral water cause an acceleration in mortality. The percentage of non-irradiated parasites in saline or mineral water is given in Fig. 4. Trichomonads irradiated with 80 mJ/cm 2 showed similar survival (except controls from the log-phase trichomonads). Controls always continued to propagate on subculture, whereas 24and 48-h-old organisms exposed to radiation doses of 80 and 240 mJ/cm 2 in mineral water and saline, respectively, did not. Because of the observed failure of trichomonads to regenerate despite optimal growth conditions, we suggest that infectivity is lost along with the ability to propagate. The survival capacity appears to increase with age, i.e. 24-h-old organisms were more sensitive to irradiation than were their 48-h-old counterparts (Figs. 1, 2). Simi-

lar results were reported by Daly et al. (1980) for T. vaginalis and T. gaIlinae. This phenomenon may be explained by the improved efficacy of DNA-repair mechanisms with age. Little is known about these mechanisms in protozoa, especially parasitic ones. The first evidence that replication-repair mechanisms function after UV irradiation not only in prokaryotic but also in eukaryotic cells was demonstrated in Tetrahymena pyriformis by Brunck and Hanawalt (1967). In Paramecium aurelia it has been shown that the photoreactivation that occurs in many eukaryotic cells is based on the same enzymatic cleavage of pyrimidine dimers ~that operates in prokaryotic cells (Sutherland et al. 1967). Electron microscopy of UV-irradiated Crithidiafasciculata parasites provided evidence that in secondary cultures, alterations in the structure of kinetoplasts and, more rarely, in the matrix of mitochondria appear after the log phase (Barros and Andrade 1988). The authors discuss the existence of a DNA-repair mechanism. Calkins and Griggs (1969) reported on a UVreactivation mechanism in T. pyriformis and claim that this mechanism represents an induction or activation of the repair system following damage by irradiation. In addition, changes in cell shape, e.g. rounding, vacuolization of cytoplasm and, finally, cell lysis occurred. The rate of cell lysis was a function of the radiation dose delivered. Morphological changes in cells immediately after irradiation have also been reported by Giese (1938) for a range of protozoa with different sensitivities to UV irradiation. In addition, Molan and A1-Harmni (1989) have described changes in the mobility, morphology, reproduction and infectivity of irradiated Leishmania donovani promastigotes. The aim of the latter authors' work was to induce immunological resistance in rodents after inoculation with irradiated Leishmania. An inhibition of growth caused by UV radiation was observed in C. fasciculata (trypanosomidae) by Barros and Andrade (1988). The effect of UV light on the lag period showed dose dependence. The survival index of the population declined to J% after irradiation with 200 m J/ cm 2. The greater efficiency of killing in suspensions with higher cell densities (Figs. 1, 2) is explained by the scattering of UV radiation (Kiefer and Wienhard 1977). UV irradiation normally leads to inhibition of cell division, which is reversible at low doses. If cells do continue to grow, larger cell bodies are observed (Kiefer and Wienhard 1977). Our findings support these observations, i.e. cells that had been irradiated and cultured for 24 h survived but showed rounded and abnormal forms. Evidence that surface alterations also occur in UV-irradiated Tritrichomonas foetus has been put forward using an electrophoretic mobility technique (Silva Filho et al. 1986). A comparison of these irradiation results is difficult, if not impossible, since different systems and test conditions were used by the various authors. We used a system, that enabled us to apply well-defined radiation doses and to evaluate the effect of irradiation reliably. The use of UV irradiation for the elimination of parasites from drinking water was first examined by Chamberlain and Vedder (1916) using amoebae. Subse-

P. Karanis et al. : Studies on the lethal effect of ultraviolet light on Trichomonas vaginalis q u e n t efforts were m a d e by Stoll et al. (1945) using Entamoeba histolytica a n d b y S t a n d e n a n d F u l l e r (1959) emp l o y i n g c e r c a r i a e o f Schistosoma mansoni. H o w e v e r , findings a b o u t sensitivity to U V r a d i a t i o n valid for one p a r a s i t e m a y n o t be v a l i d for a n o t h e r . To ensure t h a t m o r e definitive results a r e o b t a i n e d , a l a r g e r v a r i e t y o f p a r a s i t e s m u s t be investigated. W e c o u l d d e m o n s t r a t e the existence o f i n t r a s p e c i f i c differences w i t h i n p a r a s i t e s , e.g. h i g h e r U V d o s e s h a d to be a p p l i e d to v e g e t a t i v e (T. vaginalis, Acanthamoeba quina/lugdunensis, A. rhysodes) as c o m p a r e d w i t h d o r m a n t stages ( K a r a n i s et al. 1990). It a p p e a r s t h a t p a r a s i t e s r e q u i r e h i g h e r doses o f U V r a d i a t i o n t h a n d o b a c t e r i a such as Escherichia coli A T C C 11229 ( Z e m k e a n d S c h o e n e n 1989; Z e m k e et al. 1990). I r r a d i a t i o n m e t h o d s m u s t be i m p r o v e d b e f o r e b e t t e r d i s i n f e c t i o n results c a n be o b tained. I t is nevertheless p o s s i b l e to foresee the successful use o f U V i r r a d i a t i o n for the e l i m i n a t i o n o f p a r a s i t e s such as T. vaginaIis, Acanthamoeba spp., Naegleria spp. a n d Giardia lamblia f r o m p r i v a t e s w i m m i n g p o o l s o r nonchlorinated baths.

Acknowledgement. We wish to thank G. Meier-Klodt for critically reading the manuscript.

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